DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION AND RESEARCH
This transcript has not been edited or corrected, but appears as
received from the commercial transcribing service. Accordingly the Food and Drug Administration makes no
representation as to its accuracy.
BLOOD PRODUCTS ADVISORY COMMITTEE
MEETING
76th Meeting
Thursday, March 13, 2003
8:00 a.m.
Hilton Gaithersburg
620 Perry Parkway
Gaithersburg, Maryland
PARTICIPANTS
Kenrad E. Nelson, M.D., Chairman
Linda A. Smallwood, Ph.D., Executive Secretary
Members
James R. Allen, M.D.
Kenneth Davis, Jr., M.D.
Michael G. Fitzpatrick,
Ph.D.
Jonathan C. Goldsmith,
M.D.
Harvey G. Klein, M.D.
Suman Laal, Ph.D.
Judy F. Lew, M.D.
Paul H. Schmidt, M.D.
Consumer Representative
Robert J. Fallat, M.D.
Temporary Voting Member
Liana Harvath, Ph.D.
Non-voting Industry Representative
D. Michael Strong, Ph.D.
C O N T E N T S
Page
Welcome, Statement of Conflict of
Interest, Announcements 5
Committee Updates
CBER Update: Mark A.
Elengold 11
Medical Device User Fee
and Modernization
Act of 2002:
Mary Elizabeth Jacobs,
Ph.D. 19
CLIA Waiver for the
OraQuick Rapid HIV-1
Antibody Test: Elliot Cowan, Ph.D. 25
TRANS-Net Pilot Program:
Alan Williams, Ph.D. 31
Open Public Hearing 67
I.
West Nile Virus (WNV) Donor Testing
Introduction and Update
of Previous
Activities: Hira Nakhasi,
Ph.D. 67
Industry Presentations
Update on NAT Testing:
James L. Gallarda,
Ph.D., Roche 77
Christina Giachetti,
Ph.D., Gen-Probe 94
Bruce Phelps, Ph.D.,
Chiron 104
Andrew Conrad, Ph.D.,
NGI 113
John Callaghan, MS,
Tetracore 129
George Dawson, Ph.D.,
Abbott Labs 141
Charles Tackney, Ph.D.,
Ortho
Diagnostics 157
Wallace Narajowski,
Focus Technologies 167
CDC Update on
Investigations of West Nile
Virus
Transfusion-Transmitted Cases
Lyle Petersen, M.D.,
CDC 173
Donor Serologic Studies
of WNV 2001 Outbreak
Susan Stramer, Ph.D.,
ARC 200
Regulatory Pathway for
WNV Testing
Guidance for Industry:
Martin Ruta, J.D.,
Ph.D. 229
Approval Criteria:
Robin Biswas, M.D. 238
Clinical Study Design,
Unit and Donor
Management:
Indira Hewlett, Ph.D. 244
C O N T E N T S (Continued)
Page
Panel Development and
In-House Testing:
Maria Rios, Ph.D. 263
Blood Supply Management
and Triggers for
West Nile Virus Testing
Alan Williams, Ph.D. 280
Testing Source Plasma
Donations and Clearance
of West Nile Virus in
Plasma-Derived Products
Mahmood Farshid, Ph.D.
PPTA Presentation: 304
Dominique Pifat, Ph.D. 311
Open Public Hearing
Dr. Roger Y. Dodd 326
Dr. Michael Busch 336
Questions for the Committee, Discussion
and Recommendations 339
P R O C E E D
I N G S
Welcome, Statement of Conflict of
Interest,
Announcements
DR.
SMALLWOOD: Good morning. Welcome to the 76th meeting of the Blood
Products Advisory Committee.
I
am Linda Smallwood, the Executive Secretary of this committee. At this time, I will read the statement of
conflict of interest that applies to the proceedings of this meeting.
This
announcement is part of the public record for the Blood Products Advisory
Committee meeting on March 13th and 14th, 2003.
Pursuant
to the authority granted under the Committee Charter, the Director of FDA's
Center for Biologics Evaluation and Research has appointed Dr. Liana Harvath as
a temporary voting member.
Based
on the agenda, it has been determined that there are no products being approved
at this meeting. The committee
participants have been screened for their financial interests. To determine if any conflicts of interest
existed, the agency reviewed the agenda and all relevant financial interests
reported by the meeting participants.
The
Food and Drug Administration has prepared general matter waivers for the
special government employees participating in this meeting who required a
waiver under 18 United States Code, Section 208.
Because
general topics impact on so many entities, it is not prudent to recite all
potential conflicts of interest as they apply to each member. FDA acknowledges that there may be potential
conflicts of interest, but because of the general nature of the discussions
before the committee, those potential conflicts are mitigated.
We
would like to note for the record that Michael Strong is participating in this
meeting as the Non-Voting Industry Representative acting on behalf of regulated
industry. Dr. Strong's appointment is
not subject to Title 18, United States Code, Section 208.
He
is employed by the Puget Sound Blood Center and Program and thus has a
financial interest in his employer. He
also is a researcher for Roche Molecular Diagnostics. In addition, in the interest of fairness, FDA is disclosing that
his employer Puget Sound Blood Center has associations with regional hospitals
and medical centers that store platelets and could be affected by today's
committee discussions.
With
regard to FDA's invited guests, the agency has determined that the services of
these guests are essential. There are
interests that are being made public to allow meeting participants to
objectively evaluate any presentation and/or comments made by the guests.
For
the discussions on West Nile virus, Dr. Michael Busch is the Scientific
Director, Blood Centers of the Pacific.
He receives consultant and speaker's fees from firms that could be
affected by the discussions. He also
has a financial relationship and/or a professional relationship with several
other firms that could be affected by the discussions.
Dr.
Lyle Petersen is the Deputy Director for Science, Division of Vector Borne
Infectious Diseases, CDC, in Fort Collins, Colorado.
For
the discussions on the storage period for pooled platelets, Dr. Mark Brecher is
employed as the Director, Transfusion Services, University of North
Carolina. He has various interests in
firms that could be affected by the committee discussions.
Dr.
Ruby Pietersz is employed as the Manager of the Department of Research and Education,
Sanquin Blood Bank, Northwest Region, Amsterdam, The Netherlands.
Dr.
Edward Snyder is employed as the Director, Pheresis Transfusion Service, Yale
New Haven Hospital. He has various
interests in firms that could be affected by the committee discussions.
In
addition, there are speakers making industry presentations and speakers giving
committee updates from regulated industry and other outside organizations. These speakers have financial interests
associated with their employer and with other regulated firms. They were not screened for these conflicts
of interest.
If
there are any other declarations to be made regarding proceedings concerning
this meeting, please do so at this time.
Hearing
none, then, we will proceed and this conflict of interest statement is
available for review.
At
this time, I would like to introduce to you the members of the Blood Products
Advisory Committee. As I call your
name, would you please raise your hand.
The
Committee Chairperson Dr. Kenrad Nelson.
Dr. James Allen. Dr. Kenneth
Davis. Dr. Michael Fitzpatrick. Dr.
Harvey Klein. Dr. Suman Laal. Dr. Judy Lew. Dr. Paul Schmidt. Dr.
Robert Fallat. Dr. Liana Harvath.
I
would also like to introduce to you two new members of the Blood Products
Advisory Committee, Dr. Jonathan Goldsmith and Dr. Michael Strong.
Members
absent for this meeting are: Dr.
Cunningham-Rundles, Dr. Donna DiMichele, Dr. Samuel Doppelt, Dr. Daniel McGee,
Dr. Sherri Stuver, Dr. Lori Styles, and Dr. Mary Chamberland.
Before
we move into our proceedings, I would like to ask Dr. Katherine McComas from
the University of Maryland to come forward.
Dr. McComas is involved with a project with the FDA regarding a survey,
and she will explain that to you at this time.
DR.
McCOMAS: Thank you and good
morning. My name is Katherine McComas
and I am an Assistant Professor at the University of Maryland. I am here today conducting research on
Public Perceptions of the Conflict of Interest Procedures that the FDA uses to
monitor real or potential conflicts of interest of its Advisory Committee
members.
So,
I am responsible for all those surveys that are sitting in your chairs of
people in the audience, and I also have a separate questionnaire which I will
be asking Advisory Committee members to fill out if they have a chance.
What
I would like to ask you to do is voluntarily, of course, is to take about 15
minutes sometime today or tomorrow and to complete the questionnaire. There is a box outside of this room where
you can drop it in if you have a chance to do it while you are here.
If
you don't have a chance to do it while you are here, there is an envelope that
is addressed to me, and you can just drop it in the mail also.
I
will be here today and tomorrow, so if you have any questions, please feel free
to ask me. I would be happy to talk to
you more about the survey. Again, I am
grateful for this opportunity to hear your opinions. Your opinions are important and they matter to us.
Thank
you very much for your time.
DR.
SMALLWOOD: Thank you.
I
would like to bring to your attention that we have a very full agenda today,
that we have designated time allotments for speakers, that we will adhere to
those time frames.
At
this time, I will turn the proceedings of the meeting over to the Committee
Chair, Dr. Kenrad Nelson.
DR.
NELSON: Thank you, Dr. Smallwood.
The
first items are a series of committee updates. First, is Mark Elengold, who
will talk about CBER reorganization or plans or whatever.
Committee Updates - CBER Update
Mark A. Elengold
MR.
ELENGOLD: Thank you. My name is Mark Elengold. I am the Deputy Director for Operations for
the Center.
The
first order of business, I would like to apologize for Dr. Jesse Goodman, who
is the relatively new Director of the Center, for not being here to do this
update himself, but he had a meeting he had to attend with the Commissioner
this morning, so I get the honor of doing it.
I
see Dr. Smallwood start the clock on me and rather than anger her, I will talk
fast.
[Slide.]
It
is pleasure to be here. I haven't been
at one of these for a few years, and I would like to introduce myself to the
new committee members and say hello to some of the old ones. A lot of things are happening at CBER as you
may have known, and a full update would probably throw the agenda into
disarray, so I will cover several of the high points. I don't see anything, so I will say next slide, please.
[Slide.]
The
first thing I would like to cover as we have a new Center Director, we also
have a new Commissioner. The Commissioner
has established five priorities for the agency over the next few years, and we
are in the process of developing a strategic framework for initiating them.
Number
one is a strong FDA. Number two, risk
management. Three, decreasing medical
errors and adverse events. Fourth,
better informed consumers and, fifth, countering terrorism.
All
of these have groups that are working on it.
I personally serve on the Strong FDA group and on the Human Capital
subgroup of that organization. Over the
next few months, you will be seeing more and more.
We
are holding, in the next few weeks, a Risk Management Workshop Seminar. For those of you on the committee and in the
audience who have an interest in that, I urge you to check on that and attend
and participate in it.
[Slide.]
What
do we see as challenges in CBER over the next year? Well, number one is organizational changes, and we will talk
about that a little bit. New
performance goals, I will talk about that as well. New technologies, international harmonization, e-business,
countering terrorism, something that is taking increasing amounts of our
efforts, and a strong regulatory research program, something that has been a
keystone of CBER's work since it was founded over 100 years ago.
[Slide.]
This
is our organization chart. The first
thing I will talk about is, number one, the little red box, which you probably
can't read, but is in the handouts.
That is the Office of Therapeutics Research and Review.
As
you probably know, a decision was made last summer to transfer responsibility
for therapeutic biologic products from the Center of Biologics to the Center
for Drug Evaluation and Research.
[Slide.]
These
are the key questions everybody immediately asks, what's going? There are monoclonal antibodies, cytokines
and growth factors, interferons including recombinant versions that mimic those
found in nature, proteins intended for therapeutic use that are extracted from
animals or microorganisms except clotting factors, which is of key interest to
this group, and other therapeutic immunotherapies.
[Slide.]
What's
staying and despite some of the things that you may have read in the trade
press, the majority of our products are staying as evidenced by the fact there
are two slides of what's staying.
Monoclonal
antibodies, cytokines, growth factors, other proteins that are used only in the
preparation of other biologic products, so products that are not intended for
use as therapies, but used to expand cells, expand vectors, create other
things, we will regulate as part of the products they ultimately are
incorporated in, viral-vectored gene insertions, what the lay press likes to
call gene therapy, products composed of human or animal cells or from physical
parts of those cells.
[Slide.]
Plasma
expanders, allergen patch tests; one of my favorite group of products,
allergenics; antitoxins, antivenins, and venoms; in vitro diagnostics used in
the screening of the blood supply or for the diagnosis of HIV; vaccines, and
toxoids and toxins intended for immunization.
Now,
those are the categories established in this transfer. It can get a little confusing. For example, one of our most popular
products in the lay press, the botulinum toxin, the cosmetic or other treatment
Botox is going because it is used as the treatment, not intended for
immunization.
Over
the next few months, as we work through this process, there will be a date
which will occur relatively soon where these products will be transferred for
review of responsibility to the Center for Drug Evaluation and Research.
To
minimize confusion on the budget side of the agency, for the first few months
of that, the people will continue to be employees of our Center, but be
detailed to CDER, and at the start of the next fiscal year, which is October
1st, the actual official transfer will take place.
We
will be using various means to notify sponsors of a way to tell whether their
product is being transferred, and there should be an announcement on the
details of that in the next few weeks.
[Slide.]
Another
big organizational change that is of interest to the committee members and to
those following the committee is the creation of the Office of Cellular Tissue
and Gene Therapies. That is the bottom
red box. Phil Noguchi is the Acting
Director of that.
[Slide.]
Why
did we do this? Well, an increase in
regulatory activities in these products and the consolidation of these products
which were spread among our other product offices into one place to leverage
the expertise and provide consistent regulatory authority.
[Slide.]
The
mission of it is to review things and regulate things, such as tissues, cell
and tissue products, gene therapies, xenotransplantation, unique assisted
reproductive therapies, and combination products including those that have
living cells and tissues.
Of
course, the ultimate goal is to assure the safety, identity, purity, and
potency of novel products.
[Slide.]
This
is the organizational structure. As you
can see, everyone is Acting for the time being. We will be in the process of recruiting for these positions.
[Slide.]
For
those of you who aren't familiar, I will go over these very quickly. These are our proposed approach to the
regulation of tissues. The bottom line
is the most important. It's a tiered,
risk-based approach.
[Slide.]
I
did not see the final agenda until last night, and I found out that I am being
followed by Mary Beth, who is going to be talking about this, so I will talk
even faster than I normally do.
These
are the key provisions of MDUFMA. I
will let you look at those in the handouts or read them, I will let Mary Beth
expand on it.
[Slide.]
These
are the bottom-line numbers for it.
What is interesting is in the recently enacted final budget for the
fiscal year we are now virtually halfway through, the appropriate amounts were
not provided, as well as in the President's budget for the next fiscal year,
not all the funds that were planned have been requested.
[Slide.]
The
first year fees range from $154,000 for a premarket application, to a little
under 2,200 for a 510(k). I will just highlight again the bottom line, which is
that without reauthorization, the allowable sunset in October of 2007.
[Slide.]
Finally,
when I started with FDA some 32 years ago as an investigator, I was told never
tell people you are from the FDA and you are here to help them.
I
like to say this because over the past years, that has changed. We do believe we are here to help you and
anytime we can, members of the committee, members of the public, these are the
quickest way to contact us.
Thank
you. Should I ask if there are any
questions, Linda? Okay. I always do what I am told to Dr. Smallwood. She has me well trained.
Are
there any questions I can answer real fast without going off schedule? Committee members?
Thank
you very much. It's a pleasure.
DR.
NELSON: Thank you.
Next
on the agenda is Dr. Mary Elizabeth Jacobs to talk about MDUFMA.
Medical Device User Fee and
Modernization Act of 2002 - Mary Elizabeth Jacobs, Ph.D.
DR.
JACOBS: Good morning. Last December, we had a rather thorough
discussion of the law including user fees, performance goals, and all the key
provisions, so today I am giving you a short update.
[Slide.]
I
would like to cover the guidances and the FR Notices which have come out, what
the current performance is, implementation, and planned reports.
[Slide.]
On
February 25th, we posted the guidance everyone was waiting for, which
differentiates among all the different types of supplements which have
different fees, so that guidance covers
the questions. It is interim guidance
waiting for feedback, but it is pretty detailed and so far we are not getting
too many questions on it.
Secondly,
it discusses small business qualifications.
[Slide.]
We
also have FR Notices. These are on our
web site, FDA.gov. Go to Devices, then,
to the User Fee page, it is quite easy.
These discuss all the user fee payment procedures, and of interest to a
lot of people, it announces the establishment of the public docket, which has
been since the December BPAC.
You
go to FDA.gov. Go to Dockets, to the
list of them. You will see it quite
easily. You can not only send in your
own comments electronically, but you can also read all the comments that other
people have sent in.
[Slide.]
Briefly,
I just want to remind you that for all three types of our submissions, PMAs,
510(k)s, and BLAs, we have cycle goals for all three of them, and for two of
them, we also have what are called "decision goals." These change from 05 through 07, but this
illustrates for you, for PMA goals, that we have cycle goals and then we have
total decisions goals.
[Slide.]
This
shows the same for 510(k)s. You will
see we have what people think of as quite a challenging goal for the total
decisions.
[Slide.]
BLAs,
just as a reminder, under MDUFMA, we now have for resubmissions a new kind of
resubmission, which is things that have shorter updates, and we have only two
months to review that.
[Slide.]
This
shows our receipts as of March the 3rd.
Our 510(k)s are running a bit higher than they ordinarily do. We are quite pleased because so far we have
met all of the FY 05 MDUFMA goals.
[Slide.]
Process
changes that have been involved in this include a new document courier service
among all of our different locations. We
have more CDRH scientists serving on our teams, which has helped to speed up
our alliance with them.
We
have had additional Least Burdensome training, and we are going toward a
practice of finishing the reviews by mid-cycle and then working very actively
with the companies to resolve the problems before the first letter goes out.
[Slide.]
Implementation. We now have more reference materials on the
web site. I encourage you to look at
those. The Office of Combination Products
has been established. Guidance is
continuing under rapid development.
We
have had a lot of outreach. This is
only one example, but all of our roundtables and liaison meetings have had
updates, and I want to particularly draw your attention to something that has
just been announced.
On
April 9th, there is going to be a live satellite broadcast and webcast with
staff from both CBER and CDRH answering your questions and explaining the law,
so if you are interested, you will see the instructions for this on your web
site.
[Slide.]
What
are the reports that are coming up that are related to CBER? I am not dealing with the ones that are GAO
reports on what the appropriations are, et cetera, or the reprocessed devices. We don't review any reprocessed devices at
CBER, so there are a number of reports on that, that are just pertinent to
CDRH.
But
at the end of April, the criteria for third-party inspections will be
published. In August, there will be
information on devices used just for pediatric patients. In October, we will
have accredited third parties using the criteria which are published in April.
[Slide.]
October
26th is the report which goes on review by Centers other than CDRH, and we have
gotten questions on this. Yes, we are
planning to report using the MDUFMA goals on that even though only one goal
kicks in this year officially.
Also,
there is a report on the Office of Combination Products. Finally, November 30th, there is a report on
FDA as a whole and what our performance has been in the first year.
[Slide.]
Again,
I am referring you to the web site, very easy to find under FDA.gov, either
directly from that or going through Devices.
[Slide.]
Finally,
I would be happy to talk with any of you about any of your concerns or any
problems that we need to address.
Thanks.
DR.
NELSON: Thank you.
Any
comments or questions? Yes.
DR.
STRONG: There was a rumor around the
hallways that one of the changes might include a change in submissions for
instrumentation relative to blood screening assays, that you were going to
require BLAs rather than 510(k)s. Could
you clarify that?
DR.
JACOBS: We are using the same
instrumentation rules and software rules that CDRH is using, and they are a
little bit complicated because it depends on what your first submission is, so
that if you have a platform which is already being used, and now you want to
put a new test on it, you then need to supply when you submit for that test,
the instrumentation and software that is specific to that test.
But
let's say you want to have your very first use of a platform with a test which
is a BLA test. Then, you need to supply
all of the instrumentation and the software when you supply that test.
We
would need to discuss with you what your plans are. It may be that the company would like to have a separate 510(k)
for that platform because they plan to have other tests on it. They may prefer to send it in with the BLA.
So,
the basic rule is if you are already using the platform, you only send in as
part of your test what is new and specific to that test, and we incorporate by
reference all the prior ones.
Now,
I think what you are zeroing in is the second case that I talked about, is if
you want to have a test for the very first time, which is a BLA test on a
platform, then, we would need to talk to you about whether you would want to
send it in with that or whether you would want to send it as part of a 510(k),
which has shorter time frames.
Anything
else? Thanks.
DR.
NELSON: Next, is Dr. Elliot Cowan, who
is going to talk about the CLIA Waiver for the OraQuick HIV Rapid Test.
CLIA Waiver for the OraQuick Rapid
HIV-1 Antibody Test - Elliot P. Cowan,
Ph.D.
DR.
COWAN: Thank you.
At
the last BPAC meeting, I had informed you about the approval of the OraQuick
Rapid HIV-1 Antibody Test. Since that
time, there has been a significant development that I just wanted to formally
inform you about.
[Slide.]
The
OraQuick PMA was approved on November 7, 2002 as a moderate complexity device
under CLIA.
[Slide.]
With
an intended use to detect antibodies to HIV-1 in fingerstick whole blood
specimens, as a point-of-care test to aid in the diagnosis of infection with
HIV-1, and a test that would be suitable for use in multi-test algorithms
designed for statistical validation of rapid HIV test results when other rapid
HIV tests become available and data is supplied to us to demonstrate that use.
[Slide.]
OraQuick
was approved as a restricted device with the following restrictions: Sale is restricted to clinical laboratories
that have an adequate quality assurance program including planned systematic
activities to provide adequate confidence that requirements for quality will be
met, and where there is assurance that operators will receive and use the
instructional materials.
[Slide.]
OraQuick
is approved for use only by an agent of a clinical laboratory.
[Slide.]
Test
subjects must receive the "Subject information" pamphlet prior to
specimen collection and appropriate information formally known as counseling
when test results are provided.
The
test is not approved for use to screen blood or tissue donors. Everyone who purchases the OraQuick test
will receive a letter which states the following: "By purchasing this device, you are doing so as an agent of
a clinical laboratory and agree that you or any of your consignees will abide
by the restrictions on the sale, distribution, and use of the device."
[Slide.]
To
remind you a bit about the performance of this test, the clinical trials
demonstrated a sensitivity of 99.6 percent with a 95 percent confidence
interval of 98.5 percent to 99.9 percent.
Specificity in the clinical trial was demonstrated to be 100 percent
with a 95 percent confidence interval of 99.7 to 100 percent.
[Slide.]
OraQuick
was granted a CLIA waiver on January 31, 2003, as announced by President Bush
during a press conference which followed the announcement of his HIV
initiative, obviously, a very high profile event.
The
waiver was granted on the basis of data that was submitted to FDA, and that
consisted of a study that was done at four sites, consisting of 100 lay users
who had no prior laboratory experience, who tested a panel of 6 masked
randomized specimens, 2 of which were negative, 2 of which were low positive,
and 2 of which were high positive.
The
bottom line from these studies was that there was no statistically significant
difference between the lay user results and the correct results.
[Slide.]
Where
we are now is that the package insert for the waived device is in preparation,
and that package insert will contain details on the waiver studies.
[Slide.]
A
critical piece of this is that the restrictions that were developed for the
device at the time of approval will remain in place for the waived device.
A
centerpiece to this, of course, is the quality assurance restriction.
[Slide.]
There
have been obviously many questions about what constitutes an adequate quality
assurance program. To address this, the
Centers for Disease Control and Prevention held a working group at the end of
January--actually, it turns out it was just days prior to the waiver
approval--to bring together national and international experts to discuss what
types of quality assurance issues should be addressed for the OraQuick Rapid
HIV-1 Antibody Test and rapid testing in general.
The
recommendations from that working group are now being collated and a document
is being prepared which will be posted on the CDC web site when appropriate for
use by their funded programs and also will be available for the public, so that
they will be aware of the types of quality assurance issues that should be
addressed when running a test like this.
I
would also like to let you know that for those who are interested, on Thursday,
April 24th, from 1:00 to 3 o'clock, there will be a satellite broadcast by CDC,
which is called an Update on Rapid Testing for HIV. It will be live along with some taped interviews to bring
together folks who are interested in this and for CDC to discuss issues related
to rapid HIV testing and especially related to quality assurance.
Thank
you very much.
DR.
NELSON: Thank you.
Paul.
DR.
SCHMIDT: You described the customer
letter. Is the customer the person who
wants to test himself, is that what you mean?
DR.
COWAN: No. This is not a self-test.
This is for purchase by laboratories, and one of the key restrictions is
the testing has to be performed by an agent of a clinical laboratory.
A
customer is defined as the person who is purchasing the test. The way that the system is set up, the only
people who can purchase the test are those who are registered with CLIA either
as a moderate complexity lab or, now, those who would possess a certificate of
waiver.
DR.
SCHMIDT: I understand. I was worried about the words "agent of
a clinical laboratory" and "consignee" as words that would go
over the average user. Thank you.
DR.
COWAN: Yes, thanks.
DR.
FALLAT: What is the purpose of the
testing of the lay user if it's not going to be used by the patient? What was
the meaning of that waiver?
DR.
COWAN: A lay user could be someone who
is not formally trained in laboratory science, for example, someone who has a
Certificate of Waiver would purchase this test and give it to his or her staff
to run. The staff may have a high
school education, for example, or may not even be a high school graduate.
One
of the key provisions of moderate complexity testing is that there are
educational requirements and training requirements. Those are not imposed with CLIA waiver. I need to emphasize it is not designed as a self-test and that is
covered by the agent of the clinical laboratory.
DR.
NELSON: Thank you.
The
final committee update is the TRANS-Net Pilot Program. Alan Williams.
TRANS-Net Pilot Program
Alan E. Williams, Ph.D.
DR.
WILLIAMS: Thanks, Ken.
[Slide.]
The
TRANS-Net program was first presented in concept to this committee I believe in
the fall of last year, and we have recently completed two months of pilot work
with the program involving 5 transfusion services and 4 blood centers, so it
seemed appropriate to give the results of this pilot and some of the progress
that has been made to date and looking forward into the future, what some of
the plans are with respect to this program and potentially integrating it with
the HHS Blood Supply Program and other studies that are out there in the field.
[Slide.]
Just
very briefly, I think it is quite clear to everyone that the blood supply and
ways to monitor the blood supply have been the focus of intense interest
particularly over the last year or two in part stimulated by the CJD deferral.
This
has reached the level of congressional subcommittees and the New York States
legislature and, as described here, the HHS Advisory Committee for Blood Safety
and Availability, which at its September meeting last year made some specific
recommendations that HHS should actively increase public awareness of the
ongoing need for routine blood donations by healthy individuals, the Department
should maintain and/or increase funded support for blood supply monitoring, and
should also support initiatives to improve management of blood inventories.
I
didn't want to go through all the details associated with these
recommendations, but, in fact, some were very specific about actions that
should be taken.
[Slide.]
At
this meeting and in several presentations subsequent, FDA has presented a
concept of three goals related to blood supply monitoring and areas that should
be covered, the first being to monitor the breadth, the impact, and the
duration of blood and reagent shortages.
There
are programs in place which do this either for various elements of the blood
supply system or collect the data, but it is not available in real time, or
collect it for blood centers and not transfusion services.
As
I will mention in a couple of slides, there is some room for improvement, and
this gave rise to the concept of developing TRANS-Net.
[Slide.]
The
second goal, I think a very important one, is to monitor the long-term blood
collection and utilization trends. This
was conducted as a biannual survey funded for a number of years by the National
Heart, Lung, and Blood Institute. Over
the past four or five years, this has been a biannual survey run by the
National Blood Data Resource Center, an affiliate of the American Association
of Blood Banks.
I
think the data from those studies have really been very unique and very
important to understanding trends in the blood collection and utilization.
[Slide.]
The
third goal is to specifically monitor blood center inventory either by
capturing inventories aggregated by the major blood collectors or from
individual blood centers, and should reserve depots be developed around the
country, obviously, one would want real-time inventory data for those depots.
So,
those are three major goals to establish a conceptual design for a monitoring
system.
[Slide.]
I
mentioned that there are some opportunities to improve monitoring specifically
monitoring related to shortage. I think
one area that particularly needs to be addressed is sensitivity to local
shortage.
While
there are data available, it appears that a program that could capture a very
broad-based data input from both transfusion services and blood centers would
potentially allow us to focus in at the regional level when shortage occurs as
opposed to reading it or hearing about it in the media as typically occurs
today.
A
system obviously should have national representation. Data ideally should be available in real time because if one
needs to make appeals or otherwise conduct interventions, obviously, real-time
data is more relevant for doing that.
We
feel that blood center information and transfusion information gives slightly
different perspectives on supply availability, and we feel that a sophisticated
system should be able to integrate those two sets of data and build a model for
current supply.
Ideally,
this model could then be used to predict situations in the future, such as the
holiday situation this past year. There
were two mid-week holidays. It
shouldn't have been a surprise.
Potentially,
had the situation occurred in the past, we would have been able to predict a
particularly difficult time in conducting blood drives, and not had the
sustained blood shortage that was seen particularly during the January and
early February period.
Any
system clearly needs fairly broad data access, ideally, even available to the
public, so that it can be used to help stimulate recruitment of donors, but
clearly the data needs to be available and can be utilized by government and
industry to create interventions as necessary to help sustain supply.
[Slide.]
Specific
to TRANS-Net, I want to acknowledge the team that has been working on the
program. The project manager for the
program is Captain Manette Niu assisted by Sharyn Orton, Amy El Naggar, Stan
Pawlowski, and myself at the FDA Office of Blood.
We
have also worked very closely with Voxiva Corporation, which I think has put a
lot of development work into the program, developing the platform and making
their proprietary software available for telephone data reporting.
I
would also like to thank numerous colleagues who have participated in the
program both in the early design phase as consultants, and then subsequently in
the pilot phase.
[Slide.]
Now,
what are some of the design characteristics? I should say that this design was
put together under active consultation with 12 hospital transfusion services
and blood centers largely done in the second half of last year.
One
goal is to keep the system very simple, to utilize data routine compiled by the
facility, so that there wouldn't have to be specific efforts made to determine
the data that are needed.
The
reporting would be daily, on a weekday, reporting with a simple
shortage/no-shortage option as the first field of reporting.
Denominators
for reporting should be available. We
would like a daily report from any site participating in the program, so that
if a shortage is reported, we know that the same site would be represented if a
no-shortage situation existed there.
We
did want to recognize some local variations in a shortage definition. I have a slide that goes into this a little
bit more, but while we recognize the need for a standardized datapoint, in
fact, institutions differ one from another, and we wanted to be able to allow
for that to define shortage in a way that it is a shortage to that particular
institution.
The
primary system is a web-based system, and I will show you some shots of the
actual web pages, but information indicates that about 40 percent of
transfusion services probably do not have regular web access available. This
could change within the next six months, but that is the figure we are working
with now.
Because
the intent is to make this a population-based system rather than a sample, we
wanted to give even the small and more remote transfusion services the
opportunity to report, so through Voxiva, there is an option for telephone
reporting of data for sites that don't have web access available.
This
is a voluntary system, there is no payment specific to the reporting
sites. A question frequently arises
what is the incentive for sites to report.
Well,
as I will show, the data from the entire system will be available through the
web in a real-time manner. The data
will be available rapidly, essentially as soon as it goes in and goes through
server, it will be available on the map.
Secondly,
there needs to be meaningful use of the collected data, not only distributed to
those who have a vested interest, but made available to inform policy
decisions, made available to inform government and industry, to, you know,
tailor appeals as needed, and even to shuttle the information in times of
emergency to the disaster task force, or FEMA, or other agencies that
specifically need to know where there is a blood situation around the country.
We
also feel that on the public side, that the data could perform some support
functions for recruitment efforts, such as making available to the local media
exactly what the blood supply situation is in a given area.
I
think it is fairly common knowledge that the public is willing to donate blood,
but you need to ask them, and if you make this a daily reminder of what the
blood situation is, it could well help recruitment.
[Slide.]
As
mentioned, the system is designed to be population based. Given the web information transfer, that is
easily accomplished. There are
5,000-plus transfusion services in the U.S., so that potentially, all blood
centers and transfusion services would have the capability to participate although
we would want them to register for participation, so that we could get
predictable responses and data submission.
A
brief registration form will be requested for participating sites. This will allow data verification, follow-up
tracking of the site for consistent participation so as to develop a
denominator, and obviously, would give us contact information if anything
specific arises that we would like to get more information about.
Data
entry is very simple. If a site forgets
to report on any given day, there will be an e-mail sent to that site at a time
chosen by them, reminding them to submit their report. That e-mail contains a URL, so it is simply
a double-click on the link, and if there is no shortage, a single-click to
report "no shortage," and they are done. This actually worked very well during the pilot. It seems to be a very simple and
trouble-free way of reporting.
[Slide.]
Now,
I mentioned that we are allowing a little bit of flexibility in shortage
definition, so it can be institution-specific, could change by hospital, by
blood center, by geographic region. It
could change over time, could be change based on differences in the supply
network.
Because
there have been some questions about this just recently, so I wanted to give a
couple of examples. These aren't necessarily inclusive of all possibilities,
but, for instance, transfusion services sometimes need to preserve their
Rh-negative units and would give Rh-positive units to male patients because
medically, although it is not an ideal situation, the likelihood of causing
future problems in terms of transfusion is there, but it is largely a
controllable situation.
Now,
this varies from institution to institution. Some institutions would never do
this unless they really did not have the Rh-negative units available. Some institutions do it fairly commonly, so
there is a difference in policy with respect to that.
A
second difference between institutions, we know even among the pilot sites,
that it lists at least one site maintains an emergency reserve. This happens to be a frozen red blood cell
reserve.
So,
although they may not meet a patient's support criteria, if they have to go
into that frozen reserve to get blood to meet that patient's needs, that would
certainly constitute a shortage situation for them. That simply wouldn't be an option for a site that didn't have
that reserve.
[Slide.]
In
terms of data management, the plan is that the electronic data will be received
and mapped at a central FDA monitoring site.
This potentially could also be a contractor, in fact, Voxiva is the
server host for the pilot.
Registration
of facilities allows verification and follow-up of selected reports.
Data
access will be through software that considers the geo-spatial density and
frequency of shortage reports, and I will show you what I mean by this language
in a moment. Basically, the shortages
can be color coded by geographic area on a given map.
Software
can also be set up, so that if any shortage criteria meet a certain cutoff, an
alarm can be sounded or an ALERT message at any predetermined threshold.
Clearly,
we need to get some experience with the study to determine what that threshold
would be, but that is a potential for the system.
[Slide.]
The
data would be available on an interactive public web site using the GIS
software and color-coded maps. These maps would have a drill-down capability,
so that although the first map would be, for instance, the U.S. map with
states, if you click on a certain state, you could potentially go down to the
county level or even less.
We
originally set up a rule being familiar with OMB requirements that you couldn't
drill down to less than 5 participants, and in the pilot, we also put that rule
into place. If we made an error, we
wanted to make it an error on the conservative side, that we wouldn't display
somebody's data that were identifiable and get into trouble for that.
In
fact, the pilot recipients' response, in fact, expressed the opinion that they
wanted to see their data, and they didn't really care if it was individually identifiable. So, I think there is a message there that
down the line perhaps we could set up a consent option there to override that
rule.
[Slide.]
This
is a screen shot for the data entry for TRANS-Net. This comes up a single click on the URL as the registration by
user ID and pin number.
[Slide.]
This
is the initial data screen. You can see
on the left side, there is simply a shortage/no-shortage option. If you click
"no shortage," you are done, the data is recorded and you exit the
system.
The
sites from here diverge a little bit for the blood centers and the transfusion
services.
[Slide.]
if
you click on "shortage," you get a drop-down list of characteristics
related to that shortage. The list here
is for transfusion services, includes criteria probably familiar to most of
you, delay of elective surgery, delay of emergency surgery, delay of medically
necessary transfusion, et cetera.
There
is a similar list with criteria specific to blood centers.
[Slide.]
This
is an example of the mapping, clearly, a state map with some color coding
related to different shortage situations.
Now, we could define shortage any way that was appropriate using the
data in the system. One way would be to
use the shortage designation by the site, but, in fact, if we wanted to tighten
up that criteria and produce a map showing where surgeries were being delayed
and in a very severe situation, that mapping function could be modified to do
that, as well.
[Slide.]
This
is a drill-down to the Washington area using the same software. These are not real data, these are
artificial data.
[Slide.]
Now,
just to conclude, I wanted to obviously give some information about the pilot
study. As all of you know, we
experienced a rather prolonged shortage following the holiday period.
This,
in part, was likely due to the fact that we two mid-week holidays, and there
was a blood shortage recognized fairly broadly by the 3rd of January, which
then resulted in appeals from the major blood organizations, as well as
Secretary Thompson.
The
TRANS-Net program, as well as the HHS program, ABC, Stoplight, et cetera,
showed that, in fact, there was a shortage, recovery was somewhat prolonged,
and then complicated by the blizzard several weeks ago.
Now,
the scheduled start of the TRANS-Net pilot was the 6th of January, and we fully
intended to use mock data for the pilot, the reason being, number one, we
didn't expect any problems to be going on and we wanted to see some variation
in the data, so we would have some things to work with; and, two, with mock
data, we felt that we could produce the maps and test the functionality of the
report maps, as well.
Well,
what happened when we recognized that the problem that existed following the
holiday, by the second day of the pilot, we asked the sites to then report real
data, and, by the way, could you go back to the beginning of the week and do
that. So, the pilot actually now
consists of real data.
[Slide.]
This
is just a graph of shortage reports shown separately for transfusion services and
blood centers, just a very rough cut of the data line graph. You can see the number of sites reporting
shortages on the y axis versus date on the x axis.
You
can see from the 6th of January through about the 20th or so, there were a
larger number of sites reporting shortage.
This underwent a gradual decline, so that by the 10th, at least the
transfusion services had more or less normalized, whereas, the blood centers
were still reporting some spot shortages.
Interestingly,
this again went up again near the end of February. I don't know a specific explanation for that, but we did start
getting increased reports at the end of February.
[Slide.]
This
is just a first attempt to deal with some of the criteria. What we did was weight some of these criteria,
for instance, delay of elective surgery, delay of medically necessary
transfusion were weighted more highly that some of the other factors.
You
can see a similar trend of the scale based on the weighted criteria, but I
think one thing that is evident is that the amplitude of the graph is
considerably higher, so it does give us a little more flexibility to assign
some importance to shortages that are reported.
[Slide.]
Just
a few bullet characteristics about the shortage. Of 191 transfusion service shortage days, 35 percent of the time
the transfusion services reported that they had a shortage. Just from a very limited number of sites, it
appeared that they recovered somewhat sooner than the blood centers, and again,
there was a slight rise at the end of February.
Prevalence
of shortage reports by blood center days was a little higher, 48 percent during
the period. There didn't appear to be a sustained return to a no-shortage
status.
So,
a hypothesis which I think makes sense, but it remains to be advanced by data,
is that blood centers provide a more sensitive and early monitor for shortage
whereas, transfusion services probably reflect more acute need related to that
shortage.
[Slide.]
Finally,
just some characteristics of the criteria that were reported. For transfusion services during that pilot
period, there were no delays of emergency or elective surgery. There were 11 transfusion service days where
strategic reserve was tapped. There
were no delays of elective surgery.
There was one instance of a delay of medically necessary treatment, and
24 transfusion service days reported support of Rh-negative patients with
Rh-positive blood.
[Slide.]
Finally,
for blood centers, the critical shortage media appeals were reported on 11
blood center days, use of strategic reserves 10 blood center days, and order
were cut, 31 of the 176 days.
[Slide.]
So,
where does the program go from here?
This was first phase, 9 sites pre-OMB review. I think the next step is there are plans to have a discussion
with the Department on potential integrating TRANS-Net with the HHS blood
supply monitoring system obviously to try to prevent dual reporting and
optimize the data collection.
Once
that plan is developed, we would like to conduct the second pilot with several
hundred participants which will allow testing of the telephone system with a
little more depth.
Monitoring
blood supplies is more at the regional level, and allow functional mapping of
the data, and then ultimately, at a time that we can't predict right now, the
third phase, we would like to roll it out to the full population of blood
centers and transfusion services.
We
feel there are some potential expansions to the program. It could be used as an information conduit
for things like blood center inventory, platelet supply, adverse event
reporting, reagent and supply shortage monitoring, and, for instance, using the
e-mail function, a transfusion service that was short on any particular blood
product could, in fact, issue a broadcast e-mail message to surrounding
participants and actually have those sites call into that service if they had
the product available rather than calling around from the site that needs the
product.
So,
that is the description of the current status of the program, and hopefully, we
can give you another update in a few months.
Thanks.
DR.
NELSON: Thank you, Dr. Williams.
Are
there questions? Jim.
DR.
ALLEN: A comment and a question. This is very nice and I am delighted to see
you moving forward on this. My concern,
as with surveillance for communicable diseases during critical periods of time,
i.e., you know, all of this fancy reporting that is postulated during a
terrorism event is likely to break down because when people are the busiest,
they don't bother to report.
The
same concern here, that we have an event where there really is a critical
shortage, you send out the reminding e-mails, people just aren't going to have
the time to respond, so that this system tends not to gather the data in real
time when it is most critically needed, until we can get to the point that we
can get some sort of automatic data mining or computer-based reporting from
existing data.
Second,
going back to an early slide, the HHS Advisory Committee on Blood Safety and
Availability, the point one that DHHS should promote increased public awareness
of the ongoing need for routine blood donations, if I remember correctly, and
perhaps Dr. Harvath can comment on this, NHLBI had such a program 10 or more
years ago. I think it was cut because of
absence of funds.
I
wondered, is HHS broadly, not necessarily just FDA, planning to reinstitute
such a program to work with and support blood centers in their call for blood
donations?
DR.
NELSON: Liana, any comments?
DR.
HARVATH: I can't comment on behalf of
NHLBI. I thought that the plan was, as
in the past, to approach all of DHHS and talk to the major players that are
interested rather than focus only on the Heart, Lung, and Blood Institute.
I
don't know, maybe you know more than I do about that, Alan.
DR.
WILLIAMS: Well, the Heart, Lung, and
Blood Institute program goes way back.
It was called NEBRP, National Educational Blood Resource Program. It had very limited NGLBI funding. I think it was staff support for the
program, but largely, they were attempting to run it on corporate funds.
But
specific to the recommendation, I think there has been activity at HHS. I believe that the major tissue recruitment
campaign has now, at least in a modest sense, begun to include blood donation
in some of its awareness campaign.
I
don't know of any specific funds to help blood centers with recruitment
campaigns, but I think there also is another initiative to spur on donation by
federal employees and specific campaigns related to that, so I think there is
movement at the HHS area.
DR.
SCHMIDT: Two comments, Alan. First of all, we certainly need such a
system and you are well on the way.
The
first comment is you haven't discussed a situation that exists in some part of
the country that I am sure most of the members of the committee are unaware of,
and you are talking about the blood center and the transfusion service, the
transfusion service being the traditional hospital blood bank where blood was
sent to be under the control of the pathologist who charged so much per
cross-match, and it worked very well.
But
there are some parts of the country, as I know you are aware, where there has
always been a different system, where it has been recognized that the blood
center has the medical experts who know how to take care of patient and
nowadays it is called transfusion medicine.
It
is certainly true of Florida Blood Services and Seattle, and I understand now
in Cincinnati, Pittsburgh, where I guess the system we have had for 60 years in
Tampa is that we rent space inside the hospital where we place our employees
working for the blood center, and the blood is sent there. They don't work for the hospital, but
everything is billed through the hospital, therefore, they have control from
vein into vein, as it is called.
So,
that really affects the situation here, so we have been doing it for 60
years. We see this picking up.
Pittsburgh is relatively new in the system, and Cincinnati, as I understand,
and it is maybe because the pathologist can no longer get his two bucks per
cross-match.
Also,
we have electronic cross-matching, as we call it. So, you have a system that differs from the traditional way of
doing things, and may be increasing, and I know it's built into your system, I
am sure, but I just wanted the people on the committee, who don't have that in
the traditional Red Cross system, and most of the non-Red Cross blood centers
in the country just don't have that, but it does exist and it may grow.
Do
you have any comments on how you would feed that in?
DR.
WILLIAMS: I guess the major comment is
we did consider that. In fact, we had
some of these combined services in the pilot program, as does the HHS program.
After consideration, we felt that it would be most relevant to consider those
services as reporting as transfusion services, that those would be the most
relevant data from those combined activities.
But,
yes, there certainly are a number of them out there, they tend to be large, and
they are increasing.
DR.
SCHMIDT: The other comment, I think I
made before, is this really FDA's job, and maybe someplace in HHS, you are
certainly doing it very well, but just drawing some parallel, when we talk
about quality, we talk about benefits to the patient, but we are not allowed to
talk about the cost-benefit ratio. FDA
doesn't consider costs, I mean it's just benefits.
Now,
we are talking about not quality, but quantity, and I know there must be
something in the FDA law that allows you to do it, but it would just seem that
you are spending a lot of good time in which people think you should be working
on quality, on working on this quantity problem, and I am sure that has been
considered and you may not want to comment on that, but to some of us, its
seems pretty strange that you are into this business.
DR.
WILLIAMS: I will comment briefly and
then I guess Jay wishes to, as well.
There
was a discussion for some time as to where a monitoring system should live
within the HHS. I would say it largely
resides at FDA because FDA had the interest to design a system and put it
forward as a potential way to conduct blood supply monitoring.
It
could live at FDA, it could live with a contractor, it could live
elsewhere. I think this system is not
necessarily FDA unique, but I would add that FDA in some sense does have a
vested interest in the data because so frequently now, policy decisions are a
careful balance between blood availability and preserving safety. That was fully manifest to the DCJD
decision, but almost every decision made now that emerges is a factor.
DR.
SCHMIDT: The comment on that would be I
don't think FDA has great experience in what goes on in hospitals. I mean the
things that happen there in relation to the blood supply and everything else, I
mean every hospital in time of disaster gets all the blood it can and squirrels
it away and all hospital technologists are part squirrel, I think.
So,
they have different interest in what goes on. They are really not interested in
the rest of the community with a national supply, and you kind of have to know
these people to work in this system.
They are trying to beat your system as well as they can.
DR.
NELSON: Jay.
DR.
EPSTEIN: I think Alan hit the nail on
the head here, which is that there is a close interplay between considerations
of safety and supply. This issue came
to the fore when we were considering deferrals for VCJD risk that might have a
large impact on supply, and we discussed with the Department, David Satcher at
the time, Surgeon General and Assistant Secretary for Health, took the position
that we needed to closely monitor the supply impact of those policy decisions.
Roughly
at the same time, the FDA proposed adding to the Blood Action Plan, which had
become a departmental initiative, an item on monitoring and increasing the blood
supply, which had a number of dimensions, which included optimizing strategies
for donor recruitment and retention, eliminating barriers to safe donation, and
also monitoring of blood availability.
We
have come to see shortages as safety concerns. We do have enough latitude in
our mandate to consider product distributions as a part of what we can examine,
but more broadly, we have simply come to see shortage concerns as safety
issues.
We
do have shortage officers in FDA dealing with a multiplicity of product areas
including blood, also reagents that are needed in blood manufacturing sometimes
in shortage situations. Do, for these
various reasons we get involved.
Now,
the question is whose responsibility is it in the end, and I think that we have
evolved to a point of view that it is a shared responsibility. We have pieces of it at FDA, piece of it at
the Department, pieces of it historically and some currently at NHLBI, and most
recently, as Alan suggested, the combined campaign for organ and tissue
donation with blood donation has given a piece of the responsibility to HRSA.
So,
there is a shared responsibility and, of course, the blood industry itself has
a paramount interest and concerns, and we have been developing better
coordination with the blood industry in the whole program of monitoring
increasing supply.
I
think it is simply an observation that the leadership has migrated from group
to group over time based on the interest, the availability of resources, and
where the problems seem to lie.
So,
my answer to you, Paul, is that it has simply been an evolving situation. I could not tell you that there is any one
place where the aggregate responsibility lies. That is not true today. Today, we have a shared responsibility which
is distributed to a large number of entities, and most of what we are trying to
do today under the broader aegis of the Department is integrate things.
We
are trying to integrate the monitoring that the blood industry is doing with
the monitoring that the government is doing, and to link it up with the
intervention strategies. We have moved
further down the pathway of coordinating appeals for blood.
In
other words, we now have mechanisms whereby high public officials become aware
of shortages, they have press statements ready to go and they can do it on a
dime, and they have done it. We didn't
used to have that situation.
We
are also working toward the possibility of having a better coordinated system
for prediction of shortage and addressing it through a more coherent system to
identify where blood surplus might be and move the blood.
So,
it is simply evolving and I think the name of the game today is coordinating
effort in a more rational way, as well as improving the systems themselves.
DR.
NELSON: Harvey.
DR.
KLEIN: Alan, I would like to commend
you. I think you are developing a very
helpful system for an early warning and early monitoring system. It looks like from your slides and
presentation that you are think about what the efferent arm, the mechanisms for
intervention might be.
What
I wonder, however, is anyone, to your knowledge, either within FDA or anywhere
else, look at the impact of shortages in a quantitative way, looking at
morbidity, mortality, the quantity of surgery that is delayed, for example, or
Rh-positive blood going to Rh-negative individuals, a number of the other
measures, so that we can really know what the impact of shortages has been on
the American public's health.
DR.
WILLIAMS: I think the attempts to do
that have been very limited. Those that
I am aware of, the NBDRC biannual survey asks participants how many days in the
course of the year there was delay of elective surgery, so that is one
parameter.
The
HHS program captures some of the same characteristics I presented here I
believe as write-ins. Also, there would be some data for the 26 participants in
that program. Then, there is the data
potential that TRANS-Net would have if this was implemented nationwide and we
captured the right variables, I think you would have that information.
Then,
there is one additional datapoint which I think is a very powerful one, the
American Hospital Association presented data at the advisory committee, and I
can't remember the exact numbers, but I remember it was over half, I believe,
of the hospitals had presented information indicating a shortage that had
impacted patient care at some level, and I think that was a very powerful
statement.
So,
I think it is important information. I
think there are various mechanisms that could capture it. I think this is one, but we don't have the
data readily available right now.
DR.
STRONG: Just a comment. As a participant in this program, one thing
I would encourage is that we try to reduce the number of data collection
points. We are involved in all three of
the ones that you have mentioned, and the HHS one is certainly the most time
consuming of those and probably has been the least productive in terms of
data. At least you are demonstrating
that there are differences from day to day in terms of supply.
Our
experience is that this one has been easier to work with and seems to be giving
us some data that is useful, but I would certainly encourage that there be some
cooperation to try to consolidate these efforts.
DR.
NELSON: How frequently do blood centers
and transfusion centers report, is this daily, weekly, or what?
DR.
WILLIAMS: This would be a daily report
at a set time of the day on weekdays, so it would be a daily reporting.
DR.
NELSON: So, five days a week, five
times a week.
DR.
WILLIAMS: Correct.
DR.
FITZPATRICK: Alan, I think it's an
elegant way of collecting data, and like we said before. One comment that I have had in the past, and
still have, is what is the plan as to what to do with the data now that we have
got the mechanism and the tool, and are collecting the data.
One
thing you have noticed already, that was a little intuitive, but you needed the
data to support it, was blood centers are more sensitive than transfusion
services possibly or it may turn out that way.
So,
it may turn out that getting 5,000 transfusion services to report may not give
you much more data than what you are already getting, depending on what you
plan to do with the data.
The
other thing is on the management piece.
Jay mentioned that, and I would support what Jay was saying, that the
cooperation, we are seeing more cooperation between agencies and discussion of
how to deal with this problem and how to deal with it and that shortage is a
safety issue, but both comments from you and from Jay on if we see an overage
in an area and a shortage in another area, moving that blood around is not a
mechanism available to HHS or FDA.
So, how is it, do you envision a way of doing
that, is that an anecdotal comment, or are you just suggesting that if the data
is available, people will see it and do something? I am not sure where that is going and that concerns me a little
bit.
DR.
WILLIAMS: Well, you are also referring
to I think the efferent arm that Harvey mentioned. There is no absence of thoughts as to how blood could be moved
around the country. The problem is that
is business practice, it is not under control of any single entity.
I
think looking toward the future, I know DoD and others have spoken in favor of
some strategic supply depots which could be established around the country, so
that if you excess inventory either at the major blood centers distributed
around the country or at strategic supply depots, that could form a clear
source of supply that could be sent into a regional area that was hit hard at
any particular time. I mean that is a
future possibility, it is not one that occurs today.
I
think largely today, in fact, we have a situation where the best thing we can
do is make the data available to the entities that are capable of supplying the
blood, and that is in the private sector.
DR.
GOLDSMITH: I have two questions. One is do you have any plans to extend this
program to the noncellular components of blood and plasma derivatives. I know that some of the other agencies have
been interested in this.
Second,
you don't really have clear criteria for shortage and what does shortage mean,
and to look at some of the impacts of shortage. Is there any plan to develop these criteria in a more robust
fashion?
DR.
WILLIAMS: To answer the first question,
within this specific system, there is not an immediate plan to include
noncellular components. It is a little
different dynamic there, not that the system couldn't measure it, but this was
designed to meet a need in the whole blood and blood component area.
With
respect to the second component, I guess I don't fully understand the question
because I think we are capturing the criteria which define a shortage, and the
message I was trying to get across is we are not dealing with a dichotomous
variable as an endpoint here, shortage/no shortage.
We
are trying to characterize the shortage, weight it in a medically relevant
fashion, and then define the shortage according to those weighted
criteria. That is the goal.
DR.
NELSON: Jay.
DR.
EPSTEIN: I just wanted to comment that
FDA has already, for several years, been gathering monthly distribution data
for the major plasma derivatives, and that the PPTA does make public the
monthly report of aggregated data of availability of the derivatives, and we
think that that effort has served the patient community, as well, in helping
them understand the dynamics affecting product availability.
So,
we have been doing it, but it is not done on the daily basis that is I think
somewhat more needed, dealing with the transfusable components.
Open Public Hearing
DR.
NELSON: The next item is Open Public
Hearing if anybody wants to make any comments or ask questions about the
presentations to date. Are there any
comments?
[No
response.]
DR.
SMALLWOOD: For the record, I would like
to announce that Dr. Matthew Kinard from CDC has been screened to participate
as a guest of the committee for these proceedings.
DR.
NELSON: The next item on the agenda
will really take us the rest of the day, and that is discussion of the West
Nile situation.
The
first speaker is Dr. Hira Nakhasi, who will introduce the issue and give us an
update of current and previous activities of the FDA.
I.
West Nile Virus Donor Testing
A.
Introduction and Update of Previous Activities
Hira Nakhasi, Ph.D.
DR.
NAKHASI: While the projectionist is
putting the slides up, I would like to
start the session because this session is going to be, as Dr. Nelson
said, will be the whole day session. I
think by the time you finish this, I am sure you will be exhausted
The
topic of the discussion for today is the validation of nucleic acid and
serological tests to screen blood and plasma donors for acute infection with
West Nile virus.
[Slide.]
The
issues which we are going to be seeking advice from the committee today are the
following. First, is to seek advice on
the design of scientific studies which are needed to validate NAT, nucleic acid
test, as well as possibly IgM test that is the serological test for West Nile
as a blood donor screening test.
Second,
we would like to also seek advice on whether available data on the clearance of
viruses in the manufacture of plasma derivatives are sufficient basis to
obviate the screening of source plasma donations for West Nile virus.
The
third is obviously the implementation issue with regard to strategies to limit
West Nile screening to particular locations and time are appropriate.
[Slide.]
Before
we go to asking specific questions, I would like to give you some background
information and also what FDA has been doing for the last year since the
epidemic broke. The first slide shows
you basically what is West Nile virus because I am the first speaker, I would
like to give some brief background.
West
Nile virus is an envelope RNA virus. It
is a mosquito-borne flavivirus, infects birds primarily, but humans are
incidental hosts. The majority of the
human infections as asymptomatic, some 20 percent show flu-like illnesses, 1 in
150 infections result in meningitis or encephalitis.
As
I said last time, also, older age is by far the most significant risk for the
severe neurological disease. Viremic
period can occur 2 weeks prior to symptoms and last up to one month after the
initiation of infection based again also on the previous studies which were
publishes a long time ago.
[Slide.]
In
the 2002 U.S. outbreak of West Nile, besides mosquito transmission there were
other routes of transmission identified, for example, blood transmission,
transplantation, breast-feeding, occupational uses, as well.
The
magnitude of risk for West Nile from transfusion is at the moment unknown how
much it can be. Another interesting phenomena of this is that the titers of
this blood, you know, virus, is not as high as the other viruses like HIV and
HCV, and the important thing is viremia resolves rapidly after the
seroconversion to the IgM.
IgM
can persist for a long time period, in some cases up to one year, however, no
chronic state of West Nile infection has been reported so far.
[Slide.]
The
current status you will hear more about from Dr. Lyle Petersen from CDC, who is
going to be presenting this afternoon.
In the last year of 2002, there are 4,000 approximately cases reported
of West Nile cases, 263 deaths, and 2,741 cases of West Nile
meningoencephalitis.
Thirty-nine
states including D.C. is endemic for West Nile virus. The estimated risk for transfusion was calculated last year,
before the last year, in 1999, between 1.8 to 2.7, and that prediction came a
little bit higher in the last year endemic regions, which is highly endemic
regions, it was something like 16 per 10,000 with a mean of 6 to 8 in 10,000.
During
that epidemic period of August 28, as of January 3rd, now with the change as of
March 3rd, 61 possible transfusion-transmitted cases were reported, 21 were
confirmed from 14 blood donations, 19 were not transfusion related, and 21 were
incomplete because they could not get the follow-up samples.
[Slide.]
What
did FDA do during this time? FDA posted
a lot of alert notices basically August 17, and before the epidemic, you know,
when we knew about this, the epidemic had started, vigilance in excluding
symptomatic donors prior to actual reporting of transmission.
October
3rd, we issued a notice for facilitating development of donor screening and supplemental
tests.
We
had congressional hearings. We issued a
guidance document around October 2002, and we have been also consistently
talking to CDC, other State Public Health Departments, blood organizations, and
other components in HHS about the epidemiological case investigation of
transfusion-transmitted cases, providing advice on the deferral of donors and
withdrawal of products.
[Slide.]
Most
of you know that we had a West Nile workshop in 2002, where various issues were
discussed, basically, methodologies which will be suitable for donor screening,
proposed studies on prevalence in donors, and FDA interaction with how the
tests will be developed, strategies for inactivation of West Nile in plasma
derivatives, and implementation issues.
The
summary was presented last year, in December, of Blood Products Advisory
Committee meeting, so I don't want to go into detail.
[Slide.]
The
regulatory pathway, FDA's current thinking is that it is with the donor
screening and supplemental tests for West Nile will be reviewed as BLA, the IND
process approval, and software and instrument portion of the application will
be a 510(k) submission. Dr. Mary Beth
Jacobs explained earlier that depending upon what type of the instrument it is,
it will depend on whether you need a separate 510(k) or it is part of the BLA
application.
We
still believe that this is a major level of concern because it is used in the
donor screening.
[Slide.]
We
will also hear today the various regulatory pathways regarding the study design
and basically approval mechanism. Dr.
Robin Biswas will talk about the various issues related to approval
mechanism. Dr. Indira Hewlett will talk
about the clinical study design, basically, validation of clinical sensitivity
of NAT and IgM assays, clinical specificity, and validation in the absence of
reference assays, and reproducible studies.
[Slide.]
In
addition, she will also talk about unit and donor management, algorithms, and
how we need to follow up in those guidances.
[Slide.]
We
also hear our efforts in the development of reference panels for lot release
testing, as well as the qualification panels of well-characterized and pedigree
samples to establish relative sensitivity of NAT and IgM assays.
We
will also hear from Dr. Maria Rios about development of in-house TaqMan PCR and
IgM test assay because we would like to understand the distribution of this
virus n blood components, infectious dose, and the viral tropism.
[Slide.]
You
will hear from the industry folks today about their efforts about the nucleic
acid tests, as well as serological tests for whole blood, source plasma, and
supplemental tests.
[Slide.]
You
will also hear from Red Cross and CDC about their prevalence studies and to
answer some of these questions, because of the time constraints, I don't want
to go through the whole slide because you will hear from Sue and Lyle more
detail about their efforts in the prevalence studies.
[Slide.]
We
will also hear from the source plasma industry, as well as the testing of
source plasma donation, how it is to be done, as well as the viral inactivation
process. Dr. Mahmood Farshid will also
talk about the issues which are relevant to the viral clearance, basically,
whether we need to have viral clearance specific for West Nile or we do we need
an upfront screening of the source plasma as a source of added safety in the
NAT.
[Slide.]
We
will also hear from Dr. Alan Williams about the supply management and triggers
for West Nile testing in the event if the test is not available at the time of
epidemic or if the test is available, how this test will be implemented and
what will be the trigger, basically because, as we know, it is a seasonal
epidemic, it is not throughout the country all the time, and also the issues
with the minipool versus individual NAT testing and testing in the presence of
other related viruses.
[Slide.]
So,
finally, the questions which we will be posing to the committee are as follows:
First
of all, we will ask the committee to comment on FDA's proposed criteria for
validation of West Nile NAT and IgM assays for donor screening.
We
will also ask the committee's input whether they agree that the product and
process-specific clearance of West Nile agent, as opposed only to marker virus
should be demonstrated in order to adequately assure the safety of plasma
derivatives.
We
will also ask the committee do the committee members agree that screening of
all plasma for fractionation for West Nile would add a safety margin in the
manufacture of plasma derivatives.
We
will also the committee to comment on the scientific validity of possible
strategies to limit West Nile screening to particular locations or times
depending upon the epidemic surveillance information and test availability.
So,
I think with that introduction, I think I would like you to sit back, relax,
and listen to the whole presentations the whole day, and by the end of the day,
you will be ready to answer some of these questions.
Thank
you very much.
DR.
NELSON: Thank you, Dr. Nakhasi.
The
first series of presentations are some industry presentations on NAT
testing. First, is Dr. Jim Gallarda
from Roche.
B.
Industry Presentations
Update on NAT Testing
James L. Gallarda, Ph.D., Roche
DR.
GALLARDA: There are some great cartoons
on West Nile virus on the web site, so I pulled one down because it's relevant
to what we think is going to be happening in the upcoming mosquito season.
[Slide.]
This
is a cartoon where a mosquito is on a deli bar sitting next to a patron saying,
"I'm the mighty mosquito, carrier of the West Nile virus, spreader of
fear, humbler of men. What do you have
to say about that?"
[Slide.]
Our
intention is to provide a technology that will do exactly this for the upcoming
mosquito season.
[Slide.]
My
presentation is entitled West Nile in Style - Roche Update.
[Slide.]
I
would like to give a review of what we discussed last time in the November
workshop for West Nile virus and to compare what progress we have made since
that time.
[Slide.]
The
distillation of our talk last time for our concerns about implementation of a
nationwide IND for screening for West Nile by PCR.
No.
1, as it was true in November to this very date, we have been really pleased
with the collaboration amongst the stakeholders in trying to figure out how to
do this, what normally takes three years, to collapse it down into nine months.
The
capital, training, and support costs that we are encountering right now, as we
said, are bearing true that this is substantial. The time frame, of course, is unprecedented.
We
had, in November, several concerns about how to execute a multi-site IND
although these issues have been remedied in discussions with the agency, as
well as technology transfer from a research type application to a full
GMP-regulated in vitro device.
[Slide.]
From
that time in November until a critical milestone that Roche reached in the end
of January, I would like to just review what we have been doing.
[Slide.]
Initially,
we had several options to explore how best to provide a PCR system that would
provide evidence of safety and efficacy for screening for West Nile, and we are
looking at our current AmpliScreen technology very closely, which really has
definitely got some advantages and some disadvantages.
The
advantages are, of course, it has been FDA approved, the customer can do single
unit or minipool testing for AmpliScreen.
There is an installed customer base throughout North America.
We
looked at a couple of options in the semi-automated systems and compared that
to other options that we had available which were fully automated technologies.
The
advantages, of course, with fully automated technology for sample prep is that
we have the option to do total nucleic acid extraction, which allows us to
simultaneously extract, not only RNA viruses, but DNA viruses or nucleic acids
from infectious bacteria or other total nucleic acid targets, so this was
something that we felt was rather important.
We
were looking at the potential to implement our next generation platform, which
is the TaqMan chemistry kinetic PCR, which really is a much more advanced state
of PCR.
We
also were trying to look at automation features that would truly provide for
pos ID, that is, a human being does not have the opportunity to, by human
error, introduce a mismatch of a donor ID with a particular PCR result.
Of
course, we were very concerned about improving the amount of user intervention
in a full system technology. What is hidden on the bottom of the slide is that
we also are aware of the agency's request to provide what we call "full
process controls," which mean that a control in a kit would mimic the
virus, that is, from the lysis step through detection step, the control would
assess the system's ability to perform as intended.
[Slide.]
So,
we have decided on January 31st was to commit to the fully automated
system. We felt that it was important
to take that step now to move into the future, which really is a future for
full automation.
The
system that I will be talking about, which is really in its infancy right now,
but is what we intend to go forward with for screening for West Nile, consists
of the current Hamilton MicroLab AT/plus 2 Pipettor, which will create pools of
the plasma for extraction and PCR amplification, and a COBAS AmpliPrep, which
is the automated device for total nucleic acid extraction, coupled with a COBAS
TaqMan Analyze which does the kinetic PCR for West Nile virus.
In
this particular iteration, we show what we call a docking station, which allows
for samples and results out with no human intervention. Because of the timeline for implementation
of West Nile, we are initially not using this feature, so these two boxes will
be separate.
[Slide.]
High-level
overview of what we are calling now the TaqScreen West Nile Virus System is we
will use a pool size of six on, as I said, the Hamilton MicroLab AT/plus 2
Pipettor.
The
system features ready-to-use reagents.
There will be automated sample extraction on the COBAS AmpliPrep,
automated addition of the master mix to extracted pools by the COBAS AmpliPrep,
so the user does not have to add the master mix to the extract.
Because
we are leaving out that docking station which couples the two devices together,
there will be a roughly 10-second user intervention to transfer extracted
samples from the AmpliPrep into the COBAS TaqMan.
The
separation of the two instruments allows for flexibility in the instrument
layout. A laboratory can choose to put
all devices, the Hamilton Pipettor, the COBAS AmpliPrep, the TaqMan into one
room or to separate those into rooms depending upon the structure layout of the
individual laboratory.
The
other thing that this automation system does is it provides for a rapid
introduction of new analytes since there is a generic sample preparation that
is applicable to other infectious agents that may be on the horizon.
The
throughput of a module, which consists of a Hamilton Pipettor, an AmpliPrep,
and a TaqMan is about 1,200 samples in a pooled format per module in a
two-shift scenario. All of our clinical
trial sites have, at a minimum, two sites, and depending on the volume
encountered in the course of a year, we increase that number of modules to fit
their needs.
[Slide.]
This
shows a COBAS sample prep in the context of a human being. This is a relatively moderate size
instrument. It provides for full automation through a generic sample prep
method. It clearly reduces the
potential for human error in manual sample extraction procedures.
It
also allows for increased operator safety since the operator is not doing the
extraction themselves.
AMPLILINK
is the software that will be attached to a personal computer driving the
system. It will be a Windows NT
interface and it provides for positive identification of the sample with the
test results.
What
I would like to talk about are some of the features of the AmpliPrep.
[Slide.]
The
sample preparation kit comes with cassettes that contain the reagents necessary
for sample extraction. These are, as I mentioned, ready-to-use reagents, there
is no mixing of any type. The operator
simply takes a reagent rack, which has a unique barcode associated with that
rack, and each of the reagents has a barcode set of information that as the
rack is inserted, a barcode reader reads all of these, so there is a link
between the individual reagent components and the particular run.
There
are LED indicators up here which indicate the status of the run. If it's green, it is in operation; if it is
not in operation, it will flash orange.
The LEDs allow the operator to feed the instrument with a continuous
flow of reagents in an ongoing process.
The
reagents themselves have self-sealing caps for increased stability. This allows us to do a run if we need to
take the reagents off the instrument to put in the refrigerator for the next day,
we can do that.
[Slide.]
The
samples are pooled in the Hamilton Pipettor into these S-tubes. These are 1.5 mL, small, microtubes that
have a special cap on them. Once the
samples are loaded into the AmpliPrep, there is no human removal of the cap. This is done automatically by the robot
onboard.
Each
of the pools has a unique barcode clip associated with it. As the operator slides these racks into the
instrument, the barcode reader reads all the information associated with each
of the individual pool and is captured in a data management server.
[Slide.]
This
is where the action is for sample prep.
There is a device called a specimen preparation unit, or SPU. It consists of a plugged specimen tip, so to
prevent contamination by pipetting, this is a plugged tip. It comes packaged in this SPU.
I
mentioned the S-tubes or sample tubes are these 1.5 mL tubes that have a
special cap that is gripped by a robotic device within the AmpliPrep extractor
to open the tube for sample pipetting.
All
of the extraction process goes on in these specimen reaction chambers. Any waste that is generated in the process
is deposited in the central chamber.
There is a lot of engineering features designed in this SPU to mitigate
contamination control.
[Slide.]
On
the COBAS TaqMan side, we are going to be using a COBAS TaqMan 96. It's a real-time, closed tube, amplification
and detection device.
[Slide.]
The
COBAS TaqMan has four independent thermal cyclers here and four holding spots
for K-tubes, which are the tubes that have the extracted sample and the master
mix added.
[Slide.]
Another
view of the COBAS TaqMan. Here are what
we call K-carriers or the tube racks for the actual PCR event. Sample racks can be slid in up to four
slots, which contain the extracted sample and master mix, and the four thermal
cyclers onboard. A robotic arm is
responsible for moving these at the appropriate point to the correct thermal
cycler.
[Slide.]
This
simply shows the K-tube or the kinetic tube that is the device that is loaded
into the COBAS TaqMan for PCR.
[Slide.]
This
is a slide that simply shows the technology associated with kinetic PCR. It is called fluorescence energy transfer
mechanism. It simply is a mechanism
that shows that through ongoing cycles, there is a cleavage event of a reporter
molecule that results in fluorescence with each cycle, hence, it is called
kinetic or real-time PCR.
[Slide.]
If
we take the TaqScreen West Nile Virus looking at the CDC lysate, these are the
types of growth curves that are generated with each cycle ranging from zero to
60 cycles when you see a growth curve of any type, and that indicates that you
have a positive result.
We
have 100 percent detection at 2.8 x 10-7 dilution of the CDC lysate,
and we see at 107 dilution about a 62 percent detection rate.
[Slide.]
If
we take the TaqMan portion of the West Nile Virus system and we look at a BBI
panel ranging from 1,000 to 25 copies/mL, this is the type of growth
curves. A high-titered sample, 1,000 in
this case, is shown in red, and the 25 copy/mL is shown in aquamarine.
This
is our benchmark to which we are developing now and optimizing a full system
from the Hamilton pipetting event through sample extraction, through TaqMan
PCR.
What
I would like to show you now are some slides that really illustrate where we
are or where we have been in the total system development of this, and where we
expect to be by the time we go live in July.
[Slide.]
Our
early full automation dataset or benchmarking against 100 copies/mL BBI panel
member shows growth curves that range from this type of event to this type of
event. At this point, we are getting a
92 percent detection rate for this particular dataset, and a dataset is
consisting of a particular process on the COBAS sample prep and formulations
associated with PCR.
[Slide.]
We
are moving by changing our process and formulation, which is currently
ongoing. We are starting to move these
growth curves to a higher level of fluorescence. Here, we are getting a nice 6
percent detection rate at 100 copies/mL.
[Slide.]
Really,
the sensitivity in the hit rate will depend on how a negative population
behaves in the TaqMan system. Here is a
group of 120 samples, which have a very nice flat line growth curve that will
establish our baseline in the future for deriving a certain performance in the
system.
Interestingly,
of 120 samples, one of these had a profile classic with a West Nile virus
positive sample. Since this came from California in the wintertime, we think it
is unlikely that this is a West Nile virus infected person.
On
root cause analysis, we identified that a technician had used a pipetter that
had previously been used for controls in this event, and we believe this is a
contamination of it.
[Slide.]
Critical
milestones going forward. Our plan is
to file our IND in April with the agency.
We have what we are calling a Phase I program, which consists of
training, installation at three high prevalence sites in the U.S., and to
initiate Phase I studies in the March to May time frame.
These
will focus on unlinked studies that will concentrate on work flow and training
of the operators. We will conduct some
non-clinical performance studies with the system early on. Mainly Phase I allows us to refine the
system, so that for what we call Phase II, we will have a polished system.
Phase
II training and installation will include nine additional U.S. sites, five
Canadian sites. We will initiate
studies July 1, 2003. This will be a
linked study and will be used to demonstrate safety and effectiveness in
detecting West Nile.
[Slide.]
Current
issues. This slide should look
familiar. We continue to be grateful to the inner stakeholder collaboration
that we have had with the agency and the blood screening community.
We
have significant capital, training, and costs. Especially now, we are
implementing our rollout phase as we speak.
The
new thing here is that because of the accelerated timeline, we are in the
process of optimizing the entire system which we think really introduces the
next generation in PCR screening, which is to provide full automation.
[Slide.]
These
are our partners in Roche globally. We
have instrument labs in Switzerland and Germany, our manufacturing site in
Belleville, importantly, our affiliates in both Indianapolis and in Canada, and
an outstanding research group in Alameda and development group in Pleasanton.
Thank
you.
DR.
NELSON: Thank you very much, Dr.
Gallarda.
Any
questions or comments? Jay.
DR.
EPSTEIN: Thank you very much, Jim.
Could
you just clarify whether the sensitivity at 100 copies/mL is for a neat sample
or one diluted 1 to 6?
DR.
GALLARDA: Right now we define the 100
copy/mL sensitivity as an analytical sensitivity. If you have a sample, individual or otherwise, if there are 100
copies in 1 mL, the detection rate is at that level.
So,
at a 95 percent detection rate at 100 copies/mL, we would expect a sample
coming in at 1,000 copies/mL diluted 6-fold to be reliably detected.
DR.
NELSON: Is your HIV-HCV NAT, does that
also use a pool of 6?
DR.
GALLARDA: No, the currently licensed
system uses a pool size of 24.
DR.
NELSON: So, do you conceive of this,
then, as a multiplex assay or is it separate from the HIV-HCV testing?
DR.
GALLARDA: This is an entirely separate
technology from the licensed HCV and HIV, where we really want to migrate
towards is full automation and ultimately the single unit.
DR.
ALLEN: Given that it is a totally
separate system, blood centers that want to implement testing for that would
need to purchase or at least additional equipment, find laboratory space for
that, and integrate that into their routine flow?
DR.
GALLARDA: Right. The sites that we are working with, both in
U.S. and Canada, those sites are currently users of the AmpliScreen technology
and have production setups for HCV and HIV.
Additional
space is used for this system, and depending on the laboratory's structure, we
can place it in a large centralized room or in multiple separate rooms.
As
far as the capital costs, we predict it to be under cost recovery, and the cost
recovery would include all costs that would support a large-scale IND.
DR.
FITZPATRICK: The IND is for pools of 6
only?
DR.
GALLARDA: We will be doing pools of 6
and single unit PCR testing.
DR.
NAKHASI: In the Phase II, which you
said that there will be linked studies and there will be 9 sites, where will
these sites be located, Question 1, and Question 2, which I guess I asked you
also last time, in case the epidemic is out someplace else, what are you
planning to do, how are you going to get the data?
DR.
GALLARDA: That is a very good
question. We chose 3 high-prevalence
sites for the Phase I rollout, which was primarily designed to allow us to get
workflow established, operator training.
These
3 sites include a site in Florida, Indiana, and in Texas. The other 9 sites on top of those include
the geographic distribution we have for the current AmpliScreen user base,
which ranges from Puget Sound in the Northwest throughout 2 sites in
California. We have Kansas City, we
have Minneapolis, we have South Bend, Mississippi Valley, Houston, Texas, New
York, Orlando, Gainesville. It is a
broad distribution across the geographic U.S.
The
majority of those are from the Mississippi Valley to the East, which is where
historically, West Nile has had higher activity.
As
far as trying to implement a faster rollout, I think it boils down to
discussions with the agency on what kinds of activities might be acceptable to
put what is really marginally being produced into a GMP environment. This is our goal, if there is anything that
can be done that is mutually agreeable between ourselves and the agency on now
to accelerate that process.
DR.
NELSON: Thanks.
Next,
is Christina Giachetti from Gen-Probe.
Cristina Giachetti, Ph.D., Gen-Probe
DR.
GIACHETTI: Good morning.
[Slide.]
This
shows the objectives for the program and is to develop and manufacture a
TMA-based assay for detection of West Nile virus in blood, plasma, and organ
and tissue donor specimens.
The
program has two phases and will support a Phase I clinical study to determine
RNA reactivity in archived linked samples collected in areas of potentially
high West Nile virus incidence rate during 2002.
The
Phase II clinical protocol will allow for widespread donor screening, and it is
anticipated that this trial will be initiated in July 2003. Dr. Sue Stramer will talk more about the
status in her presentation.
[Slide.]
These
are our goals for assay development. We
have analytical sensitivity goal of at least 95 percent detection at 50
copies/mL, detection of genetic variants belonging to Lineage 1 and 2 with
similar sensitivity, a specificity higher than 99.5.
We
are going to add an internal control to validate each reaction, and the system
is going to be completely compatible with the Procleix eSAS (semi-automated)
and the TIGRIS (fully automated) instrument platforms.
The
Procleix eSAS system is our current licensed platform that is used for
screening for HIV and HCV.
[Slide.]
Here
is a schematic of the Procleix Semi-Automated System. We use TECAN pipetter to pipette calibrators and target
capture. The sample processing is done
in our target capture system, and then we have amplification and detection that
occurs in regular water bath, and finally, the readout is in the luminometer.
[Slide.]
This
shows our basic technology. I am not
going to go into too much detail because most of you are familiar with that,
but if you have questions, you can ask me.
For
specimen processing, we use target capture/magnetic microparticle
separation. This allows for viral
lysis, nucleic acid capture, or removal of unwanted specimens, so that we are
able to isolate the RNA which is ready for amplification.
[Slide.]
Amplification
is with transcription-mediated amplification, or TMA, which utilizes reverse
transcriptase and T7 polymerase.
[Slide.]
The
detection is with the hybridization protection assay that utilize acridinium
ester labeled probes.
[Slide.]
The
detection we do with the dual kinetics analysis to be able to differentiate
between our internal control signal and the target West Nile virus amplicon.
[Slide.]
I
will be talking the aspects of performance regarding the specificity in normal
blood donor specimens, specificity in the presence of other blood-borne viruses,
analytical sensitivity, and clinical sensitivity.
[Slide.]
Specificity
in normal donors was done testing for 175 frozen plasmas and 100 fresh
plasmas. The initial reactive rate was
zero percent and initial internal control proliferate was 0.69 percent with no
repeat internal control failures. This
was set for internal controls very similar to the ones that we observed for the
HIV assays.
[Slide.]
We
tested the specificity and other blood-borne pathogens, and this include
hepatitis A virus, cytomegalo, Epstein-Barr, hepatitis B, hepatitis C, HIV, and
HCLV-1 and 2. We tested several numbers
of specimens for each of them. In all cases, the assay was non-reactive, which
gave us a specificity of 100 percent in this type of populations.
Our
statistics regarding average single to cutoff for internal control and analyte
were very similar to what we observed in the normal population.
[Slide.]
We
determined analytical sensitivity using different sources of materials. We tested an in vitro transcript. The transcript was quantitative at optical
density. Then, serial dilutions of the
transcript were made from 300 down to 0.1 copies/mL.
We
tested 40 replicas at each copy level to determine our positivity rates at each
level, and then we used a probit analysis to determine our 95 percent and 50
percent detection limits.
The
95 percent detection limit is 8.2 copies/mL and the 50 percent detection limit,
2.9 copies/mL.
[Slide.]
This
shows similar study performed in this case with the CDC viral panel. This panel was provided to us by Dr. Robert
Lanciotti at the CDC. This panel is
quantitated in PFU/mL. We performed
serial dilutions of this panel going from 1.1 down to 0.0015, in plaque-forming
units/mL.
Again,
we test about 20 replicas for each copy level, and we used probit analysis to
determine again 95 percent and 50 percent detection limits with our assay.
The
95 percent detection limits was 0.008 plaque-forming units/mL, and the 50
percent detection limit is 0.001 PFU/mL.
[Slide.]
This
shows determination of analytical sensitivity using the West Nile Virus BBI
panel that belongs to Lineage 2. This
panel contains 13 members, which expands from 1,000 to 30 copies/mL and some
negative panels.
We
tested 4 replicas of each panel member and we were able to detect with 100
percent detection all copies levels including the one with 30 copies. The negative panels gave nonreactive
results.
[Slide.]
What
we did to be able to determine our detection limits with Lineage 2 panel, what
we did again was serial dilutions, tested different replicas of different
copies levels, and again determined 95 percent and 50 percent detection limits,
which are 9 and 0.45 respectively.
So,
all along, our analytical sensitivity results correlates well regardless which
is the source of materials we are using.
[Slide.]
This
showed clinical sensitivity data. What
we did here, what I am showing is reactive results obtained from the CDC case
investigations.
Dr.
Sue Stramer, as well as Lyle Petersen, will talk more about the study. Here, I would just like to focus on which is
the performance of our assay in the samples.
We
received 384 samples of blind specimen from Dr. Sue Stramer, and we did the
testing. Here are the different cases,
and our results in the Procleix West Nile Virus as duplicate samples or at 1 to
8 or 1 to 16.
Also,
we tested the TMA confirmatory assay that uses different primers and probes,
and our results are compared with the CDC TaqMan assay. For the first 9 cases, we have completed a
correlation of the results between our neat samples diluted 1 to 8, 1 to
16. Similar results were obtained with
our confirmatory assays and with the TaqMan CDC assay.
For
two specimens, what we obtained was positive results while the initial results
for the CDC TaqMan assays gave negative results, but was positive once used a
higher sample input.
Finally,
there was one specimen that was positive in our assay, but was negative the CDC
assay. When we tested the specimen
diluted, sometimes the results were positive and sometimes the results were
negative. This indicates that this
sample has a very low copy level.
[Slide.]
Here
is a summary of our results for the performance of our assay so far. We
have no cross-reactivity with other blood-borne viruses. The initial reactive rate in normal blood
donor population was zero percent for an n of 575, for 100 percent specificity.
[Slide.]
Analytical
sensitivity shows 95 percent detection between 7 and 12 copies/mL for Lineage 1
and Lineage 2 virus, and between 0.005 and 0.042 PFU/mL for the CDC virus
panel.
The
50 percent detection rate were between 2 and 4 copies/mL for Lineage 1, 3 and 6
for Lineage 2, and between 0.001 and 0.003 PFU/mL for CDC panel.
[Slide.]
Our
clinical sensitivity data shows that the Procleix West Nile Virus assay has a
clinical sensitivity equal or better than the CDC TaqMan Assay.
All
the reactive results obtained with the Procleix West Nile Virus Assay were also
confirmed with an in-house TMA assay using an alternative amplification region.
[Slide.]
So,
our general conclusions show that the performance to date meets design goals
for specificity and sensitivity, and that the results support feasibility of
pool testing.
[Slide.]
Acknowledgments. This project is funded in part with federal
funds from the National Heart, Lung, and Blood Institute.
Thank
you.
DR.
NELSON: Thank you very much.
Questions? Yes.
DR.
STRONG: What was your input volume for
your analytical sensitivity studies?
DR.
GIACHETTI: The input volume is the same
volume as is used in our current assay studies, 500 microliters.
DR.
NELSON: You didn't test other
flaviviruses, such as St. Louis or Dengue?
DR.
GIACHETTI: No, we haven't tested
them. We plan to test them. It is just a little hard to get the samples. There are a lot of regulations, but we are
trying to get them, yes.
DR.
FITZPATRICK: Where are you with plans
for automation of the method?
DR.
GIACHETTI: This is a semi-automatic
method and in parallel we are working with our fully automated TIGRIS platform
right now. We also have a second plan
for this TIGRIS instrument, and we are moving along in that instrument system,
as well.
DR.
FITZPATRICK: Do you have any plans for
an IND by July with TIGRIS?
DR.
GIACHETTI: No. This system will not be by July on TIGRIS.
DR.
LAAL: Are you considering getting more
samples from countries that have a much higher incidence like Israel or
somewhere to see if you will get more of that last patient that you had, which
you could detect by some assays, not by the others because your sample size is
pretty small.
DR.
GIACHETTI: Pardon me?
DR.
LAAL: Your size is pretty small, your
sample size. If you go to countries
where disease are endemic, you are likely to get a much wider range of viral
titers.
DR.
GIACHETTI: I think it's a good
idea. We don't have plans right now to
get a sample from other countries mostly due to the very short timeline for
this project, but this assay is going to be used for linked studies where we
are going to look at prevalence during the last year epidemic season in U.S.,
yes.
DR.
NELSON: Thank you.
Next,
is Dr. Bruce Phelps from Chiron.
Bruce Phelps, Ph.D., Chiron
DR.
PHELPS: Dr. Nelson, Dr. Smallwood,
members of the BPAC Committee.
Last
November, at the workshop, I provided an overview of the ongoing program at
Chiron to produce some reagents and materials that could be used in support of
development of West Nile virus assays.
Today,
I would like to update you on the status of the program at Chiron in particular
with respect to development of supplemental assays for West Nile virus and
providing other critical reagents in support of the Procleix West Nile virus
assay development ongoing at Gen-Probe.
[Slide.]
There
are basically four main areas of forming objectives for our program. We are, as I mentioned, developing a TaqMan
assay for West Nile virus application as an alternative NAT to support the TMA
assay under IND.
We
are also looking at propagation of West Nile virus in large quantities for use
as controls or calibrators to standardize the assays. In addition, we are looking at development of other assay
standards which can be used, not only for nucleic acid testing, but also for
developing serological tests. Indeed,
we are looking at reagents that may be useful for serological assay
development.
[Slide.]
As
I mentioned, we are developing a qualitative West Nile Virus TaqMan assay at
Chiron to support the Procleix West Nile Virus assay development program.
The
assay will be full validated by our current plan and may be used as a
supplemental alternative NAT during Phase II trials to be initiated at the
advent of the 2003 mosquito season in July.
[Slide.]
We
have used the capsid region of the West Nile virus genome as the target for
amplification in this assay. It is a 965 nucleic acid nucleotide RNA transcript
from the capsid region is also included as the internal control in the assay.
Currently,
the sensitivity is between 10 and 50 copies of purified RNA transcript from our
current data.
[Slide.]
This
is some data that we have generated using the CDC viral panel, viral lysate
panel that I think every one of the manufacturers who are currently optimizing
assays are using this panel, so it gives a fairly good comparison.
You
can see that down to 4 x 10-7 dilution we were able to detect 2 out
of 3 of the triplicates in that particular assay with our TaqMan assay. This is about 10-fold better in terms of its
sensitivity for dilutional panels relative to the CDC TaqMan assay, and is equivalent
to sensitivity at least within a half log sensitivity of where we are with the
current TMA assay development.
[Slide.]
We
are currently evaluating two methods for our target isolation, magnetic silica
absorption and magnetic beads for oligonucleotide capture. At this point, we haven't really locked in
on either one of these methodologies yet.
We are evaluating them both.
[Slide.]
In
terms of propagation of the West Nile virus, using the New York strain, the
385-99 West Nile virus strain that was originally isolated from New York, we are
propagating in Vero cells in BL3 facility in our laboratories.
We
have currently obtain viral titers of up to 107 plaque-forming
units/mL in several different viral cultures. I think we have about 6 or 7 so
far that we have completed in large quantity viral cultures.
[Slide.]
We
are also developing a quantitative TaqMan assay for use in-house that will be
used to estimate the viral genomic equivalents/mL and using supernatants of
these virally infected cells.
The
West Nile virus inactivation process that we are using is actually based on a
validated procedure that we have used internally for HCV virus. It's an heat-inactivation protocol, and we
have confirmed that it does inactivate West Nile virus by using Vero cell
culture and infectivity studies in Vero cells, and that is how we are providing
inactivated virus for use in controls and calibrators for potential use with
the TMA assay.
[Slide.]
At
this point, we have succeeded in producing viral suspensions of
heat-inactivated virus, which are the equivalent of 109
plaque-forming units of pre-inactivated virus, and these have been provided to
Gen-Probe for use in internal controls or potentially for other purposes in
developing the assay.
We
have also developed three RNA transcripts of different genomic regions of the
West Nile virus, and you can see the three areas that we have looked at here
pretty much spanning the entire genome, the 5-prime noncoded region, the core,
and the pre-membrane and membrane regions, the envelope and nonstructural regions
1 and 2A, and similarly, the nonstructural 5 and the 3-prime noncoded regions.
These
are the nucleotide sizes for each of these transcripts. These have been fully characterized and have
also been supplied to Gen-Probe for use in developing their assay.
[Slide.]
Our
ultimate goal is to provide a genetically engineered non-infectious full-length
West Nile virus genome, which could then be used as a standard for development
of West Nile virus assays and make this broadly available for anyone who would
want to use this as a standard.
We
are continuing to pursue this in our program in Chiron. Once it is fully characterized, as I said,
this would be made widely available for use as a standard in nucleic acid test
evaluation.
[Slide.]
In
terms of reagents for serological assay development, we are in the process of
cloning and providing expression in use of West Nile virus recombinant
proteins. We have four that are listed here on this slide, again, the
pre-membrane and the envelope regions, envelope protein, capsid protein, NS1
region, all from the New York strain.
Some
of these have been purified to the extent that we can do some pretty good
evaluations of them ongoing immediately, others are in the process of
essentially being scaled up and improving the cloning and expression in the U
cells.
[Slide.]
These
recombinant West Nile virus proteins can facilitate development of supplemental
or diagnostic tests to detect IgM or IgG antibodies in the blood and serum of
infected individuals, and we plan to evaluate these in multiple formats.
They
could also be used to generate monoclonal antibodies for antigen test
development if that should be something that we would want to proceed with in
the future.
[Slide.]
We
would like to acknowledge Dr. Laura Kramer and Alan Dupuis at the Arbovirus
Labs in Wadsworth Center, New York State Department of Health, for providing
the protocols and the much valued assistance to allow us to get culture and
propagation of West Nile virus underway at Chiron.
Thank
you.
DR.
NELSON: Thanks, Bruce.
Comments,
questions? Jay.
DR.
EPSTEIN: Thank you very much, Bruce,
for the presentation.
One
question here. You mentioned that you
have the goal to develop the NAT assay as a supplemental NAT, but you linked it
to support of the Procleix assay development. Of course, we are aware that you
have a special collaboration with Gen-Probe, but I guess the question in the
long term is do you seek potential approval of a supplemental NAT as a
free-standing test, and if so, have you thought about the possibility of making
your assay available to other candidate developers of NAT screens.
DR.
PHELPS: At the present time, we have no
plan to seek approval of this test for a more broad use. We are currently developing only to support
the TMA assay under IND.
DR.
NELSON: Mike.
DR.
BUSCH: Bruce, all three presentations I
think looked very good in terms of progress.
What I am interested in is the data, now that we have it, molecular
transcripts, and especially the comparative endpoint titration of the CDC
plaque-forming unit standard versus these more quantitative viral load type
assays.
Do
we have insights into the relationship between plaque-forming units and
copies? Some of the data I think
Christina shows suggested that there may be about 1,000 plaque-forming units
per genome if you just compare the 50 percent hit rates on the PFU dilution.
I
am wondering, with your data, or your sense of what that relationship is, and
then also whether perhaps others may comment on the infectious dose that might
be evident in animal models, how many plaque-forming units are required to
transmit if that is known in animal systems.
DR.
PHELPS: Currently, Mike, we are doing
the studies to determine exactly what the plaque-forming units versus copy
number would be detect in the TaqMan assay.
It is in the same ballpark as what Christina was showing for TMA, around
3- to 500 plaque-forming units, and we are continuing to refine that as we
complete the studies.
As
far as the infectivity level, we don't have any data at this point to respond
to that. Perhaps someone else could
answer that.
DR.
NELSON: Other comments?
DR.
SMALLWOOD: Would those speakers
following the break please check with the audio-visual gentleman there to make
sure that your presentations are loaded and they are the proper order.
DR.
NELSON: I have got 25 after, so let's
come back at maybe of 11:00. We will
take a half-hour.
[Break.]
DR.
NELSON: The next presentation is Dr.
Andrew Conrad from NGI.
Andrew Conrad, Ph.D., National Genetics
Institute
[Slide.]
I
am going to highlight a few points that have been made many, many times, but
they are relevant because they become interesting and part of what we did here.
The
most important thing here is that the West Nile virus is obviously an envelope
virus. The incubation period is rather
short and I will present some data that supports that concept.
[Slide.]
The
literature talks about the IgM to IgG conversion. When we encountered original patients, when the literature
encountered original patients, 37 percent of the patients only IgM positive,
but they converted to IgM-IgG within 4 to 5 days.
The
IgM, and again this will be some data that is relevant for your later thoughts
about the role of IgM in this testing, the IgM was stable with 50 percent of
the patients having IgM positivity after several months.
[Slide.]
At
National Genetics, we have developed both a quantitative and qualitative test,
and I won't go into massive detail.
Instead, I will give you some data instead of showing you what the test
looks like. I will give you a brief
description, but then I am going to give you data from serologically positive
individuals, and these are important.
These
were individuals that came into our Clinical Divisions of the laboratory where
doctors suspected them of having West Nile virus. They were febrile or demonstrated other sequelae of
infection. Then, I will finish this
talk with the results from preliminary screening, and I am going to use the
data from preliminary screening of 40,000 plasma donors for West Nile
virus. This will hopefully give you
some ideas of the proportions and the magnitudes of these issues.
[Slide.]
Again,
in order to do this, we looked at all sorts of different regions of the virus,
and West Nile virus fortunately is conducive for making tests, and I think from
the previous speaks in the whole blood arena, that fortunately, West Nile virus
lends itself to some testing.
But
I thought the most important part about is in our validation studies, we found
that we could get tremendous concentration of the virus through
ultracentrifugation and in dilutive medium, which meant that we could increase
sensitivity almost at will by increasing the sample size. That may be important because there has been
a lot of talk about West Nile virus being in fairly low concentrations, and I
think I will show you some data which supports that.
[Slide.]
The
next two slides we can skip through quickly. They are just what we used the
same poor snowy owl liver that the whole world has used to generate strain
385-99, made serial dilutions, quantitated those serial dilutions, determined
that the amount that we had in our culture supernatant was 1.2 x 109
copies/mL.
This
sample was diluted in negative human plasma to create positive quantitative
standards. Again, these are instead of
transcripts, which we also have, we thought that we were going to use these
because it better controlled for whole viral extraction.
[Slide.]
This
is just what the assay was looking like, and you can skip through these. These are the readouts that the computer
sees from the Southern blots.
The
next one, as well, you can go ahead.
That's the quantitative sort of 40-cycle version. Go ahead, skip through.
[Slide.]
We
were then blessed with the hard work from the CDC about also having the panel,
which was these 9 members. Unfortunately, when we tested all 9 members, they
came up positive 100 percent of the time, all dilutions, so we had to make
further dilutions down another 1,000-fold, I guess, and if you look at the next
slide, we will see some numbers that we translated.
[Slide.]
In
the 100 microliter reactions that we used to do this, we had 95 percent
detection in the smaller reagent reaction volume of 0.01392 PFU/reaction, which
turns out in our 1 mL assay to mean that we have a 95 percent detection of 6.63
copies.
We
all say these numbers, of course, tongue in cheek, and I wouldn't bet if
someone said 6.63 and someone else's was 8 that there is really any difference.
These are still based on relatively small numbers. We did triplicates, there different lots of the same Lanciotti
dilution and then in replicates that you can see. So, any of these numbers are loose, but we are getting some idea.
Under
our calculations, we ranged here from anywhere from 500 copies/PFU to 1,013 if
you use those two spreads of those numbers of copies/PFU. I knew that that was a question previously
asked, and that's somewhere in the infectious unit in the tissue culture models. Again, I think that may be dependent on the
conditions of your tissue culture.
So,
this in hand, we thought, okay, well, we have an assay that is fairly
sensitive, let's move on to the clinical samples, which is the next slide.
[Slide.]
We
were given 140 clinical samples that came in to our clinical laboratory
environments; 38 of those samples turned out not to be West Nile virus, and
they developed no sequelae of the disease, they turned out to be
hypochondriacs.
The
other 102 were people who obviously weren't and what is interesting, and I
think is most important here, is that of the 102 samples that we were able to
detect IgM or IgG in, only 10 of them were viremic. This again supports the concept that it looks like there is a
rapid--upon the appearance of IgM or IgG--there is a rapid clearance of the
virus.
With
the average viral load in these 10 samples being 45 copies, and it ranged from
20 to 100 copies, and I think that that is again important, that these were
extremely low level viremic samples that 10 out of the 102 people who truly
were West Nile virus candidates.
So,
I think that that is again an important thing to keep in mind.
With
this data in hand, we thought it was now time to move forward into a true
asymptomatic population, at which point we began to investigate plasma donors
from a very limited source that were participating in sending us samples in
through our FDA-approved mechanisms, test mechanisms where we have those pools
that I am sure many of you have heard of far too frequently.
[Slide.]
Again,
we used those standard algorithms. The
magic cube that we have allows easy analysis of samples the size of 512 or 64,
since both are simultaneously generated on the TECANS, and our licenses are for
pools small than 512, so we figured we would take those shots at it. We could do pools of 512, 64, or 8, all
without changing anything. We picked
these two because we figured we would rather use higher volume test analyses
than smaller pools. It makes the amount of work less, and that is what we
specialize in, being lazy.
[Slide.]
The
first effort, we were able to locate 7,107 samples that were blinded, however,
we did some geographic information based on how they came in. Of those 7,107 samples, 5,366 were from very
high incidence areas.
So,
when I begin, I don't want you to start making calculations of incidence and
prevalence when you know that this was front weighted to cherry-pick into areas
and the time period when there was likely to be higher infections.
The
remainder of the samples, the 2,000 or so different samples came randomly
throughout our catchment. National
Genetics probably sees an awful lot of plasma, but this was a limited supplier
who catches, happens to be fairly geographically dispersed.
[Slide.]
In
that first week, we were able to locate one West Nile virus NAT-positive sample
out of the 7,107, and statisticians would be impressed by the power of this
calculation, thus, determining 1 out of 7,107 is the incidence.
The
titer of the individual sample was determined to be 2 x 105, which
was interesting to us because this was higher than what people traditionally
thought the viral loads for West Nile virus in humans would be.
We
went on to fortunately grab that sample blinded. I can't identify the donor, but we could identify the sample, and
found it to be negative for IgG and IgM, and that is important. That was the first true window period
donation that we were able to locate.
So,
thrilled with our performance, we decided to roll onward and look to see if we
could expand that while rallying against people's hassles of having to do all
this stuff. In the next slide, we were
able to get 33,000 blinded samples from the same systems.
[Slide.]
We
screened 64, in pools of 64. These
samples, however, were collected in
late October 2002. In this case, the
samples were distributed more widely throughout the U.S. and did have samples
that did not represent the high prevalence areas, and the data follows.
[Slide.]
Eleven
additional samples tested West Nile virus positive from these 33,000, 3,300,
for an incidence of 1 in 3,300 is extremely important to note that in the
plasma industry, these could represent the same donor repeatedly donating in
this catchment period. It is not like
whole blood where the donor times would be spread out.
So,
what I am talking here is donations, not donors, and it may be that these
incidence numbers are dramatically skewed by the fact that a single donor
donated three times, four times, whatever number of times in this, because they
can donate as frequently as twice a week, and the average that we have gotten
in our system typically is more than seven times in a three-month period.
So,
5 of these samples were detectable in pools of 512 or greater, so the other 6
were not, they were only detectable in the pools of 64. The viral loads of these samples were very
low with none of the viral loads being above 13,000 copies/mL.
[Slide.]
The
range was anywhere from 250,000, which was the high sample that we spoke about
earlier, down to 100 copies/mL, which were the range of these samples. The serologic status, I am getting today on
some of them, but I will tell you that we haven't gotten IgG, that of the low
samples, the ones that were below 1,000, all of them have been shown to be IgM positive,
I do not know the IgG yet, so that is important to know.
[Slide.]
In
addition, quantitative tests have been shown to demonstrate the efficacy of
inactivation methods against West Nile virus, and I think you will hear more
about this afternoon. We are continuing
to work with plasma suppliers to continue to perform these pilot studies, INDs,
whatever you want to call them. We
might go forward if appropriate.
It
appears that West Nile viremic samples are pretty easily detectable in pools of
64 and definitely detectable sometimes in pools of 512, which surprised us
because it would probably be efficacy in which pooling can be performed and the
virus precipitated.
[Slide.]
The
final slide I have is that in our small study, and it looks like these things
have come true, that IgG and IgM-positive, very low titer samples are one class
of samples that we are going to get.
So, in other words, the appearance of IgM and definitely IgG causes a
precipitous drop in viral loads.
There
is a second class of samples which are IgG-negative and IgM-negative, and those
I am going to call mid-titer samples because they are certainly not as robust
of infections as we have seen in some of the other viruses, parvo or HCV. So far we have not produced what we would call
a very high titer viral sample, and we will continue to look.
The
pace at which we continue to look, we are fully operational to look in any
scale we need to because it is just added right onto our already FDA-approved
systems. There is no modifications that
need to be made other than the primer pairs.
So,
as the industry deems necessary to get the inactivation data, we will look if
we have to. That's all.
DR.
NELSON: Thank you, Dr. Conrad.
Questions
or comments? Jay.
DR.
EPSTEIN: Thank you very much, Andy.
Can
I get you just to clarify, since you used the concentration procedure, what
would you say is the sensitivity for detection of the neat sample when placed
in the configuration of a pool of 64?
Is it 6 copies/mL or is 6 x 64, or something in between?
DR.
CONRAD: I will tell you that remember
that because of constraints in the amount of material we had in certain panels,
that it gets confusing, and I apologize for this. Sometimes we were only afforded enough material to test 100
microliters, and therefore, we would have to extrapolate sensitivities to the
mL amount by timesing that sensitivity or actually dividing it by 10 to get a
lower sensitivity if you had a whole mL.
That
is based on the efficiency of the precipitation, so as you know in our FDA
licensed assays, we have licensed both the 100 microliter 1 mL, 2 mL, and you
can see that the sensitivities ratchet downward depending on--we always report
in sensitivity/mL, but right now the assay that we are representing here is a 1
mL assay, which we are claiming has 6.6 copies/mL in the neat sample.
If
we did a 2 mL, it would be something, 8 or 9 times more sensitive, or 3 mL, so it depends on what becomes
relevant in the examination, like all the clinical samples we used, we only had
100 microliters on in order to spare those samples. So, we were able to detect 12, and that is I think part of the
problem.
We
try to normalize against because we believe that in this case, with the proper
diluent, that the ultracentrifugation really precipitates this virus better
than it does with other ones, and we haven't seen floating that we saw with
other flaviviruses like HCV because we took the top and the bottom pellets off,
and we haven't seen that as long as you put the appropriate diluent in the
plasma before spinning.
DR.
GALLARDA: Jim Gallarda from Roche.
Andy,
you describe your copy sensitivity in the terms of Poisson statistics. Does that give you an actual copy number of
a detectable unit?
DR.
CONRAD: Well, obviously, we do it two
ways, Jim. I mean we like to use the
final thing of the extractable unit, and you are right, because is it a true
copy, but we also try to compare that against transcript, which we assume has
100 percent efficiency although there are some RT issues in there.
I
didn't mean to imply that we just used
Poisson. We have always gone two ways.
We have always taken transcript against Poisson, trying to use the
Poisson to control for extraction functionality and the transcript, so that is
an amalgam when we get those numbers, and we assume the unity that you need for
Poisson distribution is a derivative of having both the transcript data and
that extraction data.
DR.
LEW: I just wanted to clarify that
there was absolutely no processing prior to detection of these pooled samples.
DR.
CONRAD: I am sorry. "Processing," what do you mean?
DR.
LEW: Processing, at this stage, it is
just pooled, you know, sterilely for testing.
DR.
CONRAD: It is pooled according to our
standard FDA algorithms. We take the
plasma, we combine it in an algorithm where an automatic robotic device
combines the plasma into these pools of 64 or 512, is then lifted off those
machines, extracted with standard methodologies that I think many of my
predecessors have described in better detail than I, and then amplified in our
case using PCR, and that is what happens to it. But by "processing," this is not ready for
fractionation if you mean any processing of that kind, none of that, this is
just raw plasma in a pool.
DR.
NAKHASI: Hira Nakhasi from CBER-FDA.
Andy,
I am just a little bit curious to know, you mentioned that the late summer
samples of 2002, you had an incidence in the pool of 64, the problems was 1 in
7,000, whereas, you go to the late October 2002, again, at 64 dilution, you had
1 in 3,300. It seems to be much higher.
Summer
will be much higher problems than later October.
DR.
CONRAD: The problem is remember as I
catch more, this is a relatively small supplier of plasma, so as I catch more
time, remember I took one week to get those 7,107, and I got 1 guy. That is why I really want to make that
clear, that the incidence of 33,000 could have been 1 guy going four, five, six
times.
DR.
NAKHASI: I see.
DR.
CONRAD: So, the problem until we move
into IND and unblind this is that I can only give you donational prevalence and
incidence, but that in no way infers that there was an increase--there is an
increase in the amount of the donations, but that doesn't mean that there is
more donors who are succumbing to the virus, which would be a traditional way
to look at an outbreak of an epidemic.
It
may and most like has to do with the longer duration of this relatively small
group who I am now following to garner 33,000 samples. You can see appreciably longer than I
followed to take a snapshot of one week.
Does
that make sense, because I don't want to engage in some paranoia here that it
is going up in October, it is probably going down, especially because how low
those viral titers were. These weren't
all new incident infections. They
looked much more--with the IgM presence--like an ongoing infection.
DR.
NELSON: Thank you.
Next
is John Callaghan from Tetracore.
John Callaghan, MS, Tetracore
MR.
CALLAGHAN: Good morning.
[Slide.]
As
we saw at our last meeting, we have a real problem with West Nile, we need a
test, we need it soon, and this should be in a NAT format and also we need an
instrument, platform, and software that is FDA approved.
What
we have from Tetracore, we have a NAT assay format, but it is not on a platform
that is compatible yet. It could be made compatible, but we have some unique
applications for our test that might be interesting to this audience.
[Slide.]
What
we have is a rapid vitrified real-time RT-PCR for West Nile. This target a 3-prime UTR region and what I
mean by "vitrified" is we take the reagents and we dry them and use
the test, so it could be stored in a dried format.
The
potential uses for this test could be for supplemental or confirmatory test of
positive or indeterminate samples identified by the NAT screening test, and another
possible use would be for organ and tissue donor screening, and we have data
that I am going to show you here today that supports our ability to identify
West Nile in heart, lung, kidney, spleen, brain, and other tissues from
infected birds, so this is not human data, but we are actively seeking
possibilities of using this test on human organs and tissues.
[Slide.]
What
we were looking at in the design of this test was we wanted a broad specificity
among West Nile and Kunjin viruses, and I will show you why we decided Kunjin
was important in just a moment, but we wanted a test that had no
cross-reactivity with other flaviviruses or clinically similar viral diseases
such as the other encephalitides.
Also,
we tested some human sera from parenterally transmissible diseases that the
blood bank would routinely screen for, and I will show you that data in a bit
here. Also, we wanted to have maximal sensitivity. We wanted to make the test quantitative, and we wanted it to be
easy to use and have excellent test indices.
[Slide.]
So,
what we did in the original design is we took a look at the phylogeny of West
Nile virus. You can see that it is
broadly divided into two groups, and I have heard them referred them to as New
World and Old World, and the isolates that we are seeing here in the U.S. are
the New World group, and then the isolates from Uganda and Africa, other parts
of Africa are classified into Old World group II.
Also,
you will notice down here that all of this group here are Kunjin viruses, so if
you want to have an assay that is going to be able to detect all of the members
of both of those groups, we decided to pick a design with primers and probes
that were 100 percent homologous or nearly 100 percent with every member of Gen
Bank representing all of the groups here of Kunjin and West Nile, both groups I
and II.
[Slide.]
This
just shows some of the data that we have got from one of the platforms was an
ABI-7700 instrument showing the range of linearity across several logs. On this particular instrument, we are able
to get down to between 2 and 20 copies/volume tested.
[Slide.]
The
other approaches we have been using, the Cepheid Smart cycler, which is a
portable instrument that has 16 individual wells. These can be linked together and you can get up to six
instruments running off of one computer at one time.
So,
this is nowhere near the kind of volume that you are going to need to screen
blood units, but if you were in a confirmatory situation, you may not have that
many positives to analyze, and this particular format might be useful. These instruments are relatively inexpensive
compared to some of the other instrumentation, so there would be an advantage
to having a system like this.
[Slide.]
What
we have done with our reagents is we take the primers, the probes, the enzymes,
all the perishable materials and we dry them down in the presence of trehalose.
Trehalose is a glass-forming solute, so it gives you the appearance of a
glass-like sliver kind of on the edge of a slide.
So,
this is Cepheid Smart Cycler tube, so we have had a lot of experience in our
company drying down RT-PCR assays for RNA viruses for a number of different
agents using this particular format.
[Slide.]
The
way that you process the sample, we are using the Qiagen extraction kit, but
basically, you would add your sample to the viral lysis buffer. Our input volume is 140 microliters. The virus is neutralized and the RNA is
stabilized, and you go through the RNA extraction process.
For
the test itself, we have the dried stabilized mixture containing all the
components in the tube, and we have a universal rehydration buffer that we
add. We bring the volume up to 25
microliter reaction, you know, put in 22.5 microliters of the rehydration
buffer and 2.5 microliters of your template, then add it to the machine and
test, and the results are out in about 35 minutes.
[Slide.]
So,
the test features are it is a single tube method. It is a reduced potential for contamination, a simplified
handling of reagents because we have it in a dried form. In the vitrified form, it breaks the chain
of refrigeration, so the handling of reagents is further simplified.
We
have done shelf life studies on other assays showing that we get up to two
years in accelerated shelf life studies, so, you know, leaving the test at room
temp and then applying it to other accelerated temperatures.
These
results can be transmitted electronically through the Internet, and there is a
possibility, I mean even though we haven't approached this, but it is
conceivable it could be linked up to a BIS system.
Of
course, these types of tests generate much less waste than the standard PCR
methods.
[Slide.]
We
would package it something like this.
This is another assay that we have done for veterinary application, but
basically, the tubes would be packaged with the desiccant and could be stored
at room temperature.
[Slide.]
These
will be produced in a GMP facility. We
are a company located here in Gaithersburg.
[Slide.]
Now,
this is a panel of specificity that we did on some of these agents. As you can see, we have tested a number of
different flaviviruses, and there is no cross-reactivity. We tested several isolates from group I, the
New York crow isolate.
We
were working with the National Zoo here in D.C., and we got a number of
different isolates from multiple species, multiple sample types, so we are able
to detect at least several members of group I, and then from group II, this is
representative of the BBI panel, the first one that they had available.
We
also got an isolate from Romania that we tested, and then a Kunjin isolate from
Australia, so these were all positive by our test. Then, we tested some human sera that we got from the University
of Maryland, representing HIV-1 positives, HTLV-1, hepatitis C, hepatitis B and
A, and then also at the last meeting, we had heard that some of the serological
assays cross-reacted potentially with syphilis-positive samples, so we tested
that, as well, and there was no cross-reactivity among those.
[Slide.]
This
is just showing some of the data on the ABI-7700. It has been shown that we can get both groups of West Nile and
Kunjin.
[Slide.]
Also,
for specificity, we decided to take nucleic acid extracts from a number of cell
lines that are commonly used for the propagation of viruses, and there was no
cross-reactivity with our assay with any of these nucleic acid extracts. I would like to point out it is a very
complex mixture of sequences, so it is good that there was no cross-reactivity.
[Slide.]
Some
of the veterinary samples that we had worked with, we were working with Cornell
University, and we had a number of different animal species here and a number
of different tissues. One real
interesting finding was that we were able to work with cloacal swabs from birds
and fecal samples, as well, and detect West Nile in these, but also notably, we
had heart, brain, kidney, spleen, lung, liver, and CNS from a horse, and we
found that with the assay that Cornell uses, they use a nested RT-PCR, and we
had 100 percent correlation between our real-time PCR and theirs on the sample
panel tested.
[Slide.]
Also,
these are samples that we got from the National Zoo here in D.C., and of all
these different species, we identified West Nile in 12 different species.
[Slide.]
Also,
multiple samples, so the plasma that we used from animals, there were
represented multiple species, and then also the tissues, there were multiple
tissue types. Often we would get like a homogenate, a blend of tissues to test,
but we made a lot of positive identifications throughout this past summer with
the veterinary samples.
[Slide.]
Now,
the technical challenge was the quantitation. What we decided to do was take a
look at the Armored RNA. Now, you can get this from Ambion in Austin, Texas,
but the problem is that you would need an NS5 assay to amplify this and ours is
a 3-prime UTR, so we needed a bridge between the two assays to quantitate the
positives that we were using, so we developed a second assay that targets the
NS5 region.
Then,
on that, we tested the Armored RNA, which is a known quantitative standard that
you can add. It goes through the whole
process of extraction, so it's a known standard that you can put into your test
system. You know what goes in, you know
what comes out.
Then,
along with that, you could compare like, for example, the sample from an
infected sparrow, you could compare it against your standard curve and
quantitate with that, then, run endpoint dilution in parallel on the 3-prime
UTR.
[Slide.]
So,
we ran those in parallel on the Smart Cycler.
[Slide.]
Then,
also on the ABI-7700 to compare.
[Slide.]
That
quantitation showed that the sparrow sample we had came out at 2250
copies/volume tested, was 2.5 microliters.
[Slide.]
So,
if we run an endpoint dilution, this is just data from the Smart Cycler showing
down at log 4. That would be equivalent
to about 2.25 copies/volume tested. So,
if you converted that back, it would be in the range of 7.5 to 75
copies/mL. We still need a lot more
data to show the reliability of these figures, but at least that is a good
start, I think.
[Slide.]
The
specificity so far has been 100 percent among all the panels that we have
tested. There has been no
cross-reactivity with other members of infectious disease panels that we have
selected. There have been no false
positive in samples collected where the disease is absent, and the sensitivity
is between 2 and 20 copies/volume tested.
We
have just recently received the panel from the Red Cross just a few days ago to
start testing human plasma, but since we just got it, there just wasn't enough
time to finish the work before today's presentation, but that work is ongoing
right now. Also, we would like to
identify partners to collaborate with for testing human tissues and organs for
West Nile virus.
[Slide.]
Some
of the advantages that we have right now is excellent specificity, a broad
coverage that detects both West Nile groups I and II and Kunjin viruses, but
doesn't amplify other flaviviral near neighbors, and it so far looks sensitive,
that we could get down to less than 20 copies/volume tested, and we can make
this qualitative or quantitative. Then,
we could supply these reagents in wet or dry form, so that if there were other
instrument platforms, they could be used on there.
We
have detected virus in multiple tissues and fecal material and plasma from
infected birds, and it is very fast turnaround time. On this particular instrument, if you just had a handful of
samples, you could do the extraction and testing, and have results in under two
hours.
So,
basically, that's it from Tetracore.
DR.
NELSON: Thank you.
Questions? Yes, Judy.
DR.
LEW: Actually, I have some concerns
about the sensitivity and maybe you can explain that to me. You are talking about 20 copies/volume
tested, but the standard test only requires 2.5 microliters, and if we know
that humans tend to have low viral load, you try to do that extrapolations,
because with the birds, they have very high viral load, so it would be much
easier to detect.
How
sensitive is it going to be if a person who has just in the thousands copies/mL
or maybe 10,000 copies/mL?
MR.
CALLAGHAN: Well, I think we would have
a much better feel for that as soon as we are done with the panel that we have
received, because those are plasma from human donors that some of them are
infected.
So,
we have just begun that testing, so we are going to have to do quantitation on
those, as well, to get a better feel, and also do many more replicates of the
extracted RNA to get more confidence in our real level.
The
work is ongoing, so we are not completely finished with that yet, but this is
just some preliminary data showing that we have got down to those levels on
those types of samples.
DR.
NELSON: Thank you.
The
next speaker is George Dawson from Abbott.
We
are just now moving into serological tests from NAT testing.
George Dawson, Ph.D., Abbott
Laboratories
[Slide.]
DR.
DAWSON: I am going to update you today
on some of the work we have done on West Nile virus IgM testing.
[Slide.]
We
talked last time in early November about some of what our goals were, and it
was really to try to understand in a little more depth what is the marker
profile of an infected person, what is the overlap between RNA detection,
antigen detection, and IgM detection, what are the markers associated with
symptomatic acute infection.
So,
we have been working on developing an IgM class antibody test for detection of
antibodies to West Nile virus. We have
obtained two different recombinant proteins from two different laboratories.
We
took a lot of time to get the Material Transfer Agreements signed, but we have
been working in earnest on that. One of
them was signed in early November, the other not completed until early January,
so we don't have a ton of data to present today, but I am focusing mainly on
data today.
So,
we evaluated these two proteins for ability to detect IgM. We also wanted to have capability to do PCR
in our lab, so we looked at different primer pairs and their ability to detect
RNA in a panel of samples provided by BBI.
Our
major goal is to look at the utility of IgM testing in three different
settings. One is in blood screening,
one is in reinstatement of RNA positive donors, and a third is in diagnosis of
acute infection. We are relatively sure
that the best test around for acute infection will be IgM.
[Slide.]
Here
is a depiction of the viral genome. I
will call your attention to the different sets of primers that we have
developed and looked at their ability to detect RNA. We looked at different sets of primers and we selected, in bolded
red there, degenerate primers that were built specifically for West Nile virus
detection.
We
have been using RT-PCR test and using nested PCR, running agarose gels to
detect the amplicons.
[Slide.]
I
mentioned we obtained two different recombinant proteins. Protein 1 was a truncated envelope protein
expressed in eukaryotic cells. The
second protein was a pre-membrane/membrane/envelope gene expressed in
eukaryotic cells and actually secreted into the supernatant fluid as vesicles
believed to be secreted as viral-like particles and to allow the envelope
protein to be folded in its correct conformation that would more simulate the
native virus.
This
protein 2 came from CDC. We actually
received from CDC the protein itself, as well as the cell line that produces
this protein.
[Slide.]
We
looked at two different assay formats with these two proteins. With protein 1, we use an indirect assay
format. This assay entails coating a
solid phase directly with antigen, reacting the antigen-coated solid phase with
sample. IgM, if present, would bind to
the antigen-coated solid phase, and would be detected in the second step with a
conjugate directed against IgM or against IgG because we also wanted to look
at, at the same time we are looking at IgM detection, we wanted to look at IgG
detection.
[Slide.]
For
protein 2, we used an IgM capture EIA.
This is a three-step assay, uses a solid phase coated with antibodies to
heavy chain of human IgM, so in the first step, IgM is specifically captured on
the solid phase. After a washing step,
antigen is added.
After
an incubation step, the antigen antibody complexes will be bound to the solid
phase, unreacted antigen would be washed away, and we then subject the
complexed solid phase with a conjugate specifically directed against West Nile
virus envelope protein.
[Slide.]
We
have done more testing than I am showing today, but here is what I will focus
on for today's presentation. We tested volunteer donors from areas that were
not endemic at the time that the blood donations were taken.
We
looked at some panels that were categorized as IgM only or IgM and IgG only by
a CDC assay run at a state health laboratory.
We also obtained paired human samples from acute West Nile virus
infection.
We
received the CDC proficiency panel and we are in progress with an experimental
infection in Rhesus monkeys, and this study is one that I proposed last time
that we would get involved in. We have
made good on that, and we are in the midst of doing this testing. I will show you just a little bit of data
from that study as I conclude this presentation.
[Slide.]
With
protein 1 for both IgM and IgG, we found a lot of bounce in the population, and
we found reactives IgG and IgM with it, with this assay format, indirect assay
with protein 1. These were
RNA-negative. The IgM-positive was IgG-negative, the IgG-positive was
IgM-negative, so there was no co-detection of antibodies.
With
the second protein, which we felt is more specific, these samples were
negative. In any event, we had to cut a
cutoff somewhere and what we did is we just took a bottom line of looking at
about 7 standard deviations from the population mean, set the cutoff there, and
then proceeded with our studies.
[Slide.]
With
protein 2, with the three-step IgM capture assay, we tested 241 volunteer
donors from a non-endemic area, and found that none of the 241 samples were
repeatedly reactive. We set a cutoff
which nicely kept the samples that we assumed to be negative as running
negative.
[Slide.]
We
then began to look at some of the samples that came to us, categorized as being
IgM-positive and IgG-positive, and we found that if you looked at this, a
dotted line running across the graph here, and we have sample number on the x
axis, S/N values on the y axis, and you can see that all 8 are positive with
protein 2, only 5 of the 8 are positive with protein 1, so we are beginning to
have some concerns about protein 1's ability to detect IgM.
[Slide.]
Then,
we looked at samples that were IgM-only, and here there are 9 specimens that
are IgM-only. Protein 2 detected all of
these. Again, you see the dotted line
running across is where the cutoff would be set. All 9 positive with protein 2, and only 3 specimens were positive
with protein 1. So, we are seeing some
very clear differences between these two proteins and ability to detect IgM.
[Slide.]
A
second set of pedigreed samples we got were 12 pairs of specimens that were
diagnosed as IgM-positive for West Nile virus, and we see here the 12 patients
aligned on the x axis, S/N values on the y axis, comparing this time it is not
protein 1 versus protein 2, it is protein 1 alone here, and the number at the
top of the bar indicates the days post onset that this sample was tested.
You
can see that a couple of them are very nicely positive, patient 2 and patient
9, patient 7, the first bleed positive, et cetera, but a lot of these were
negative, again another concern that protein 1 may be missing true acute West
Nile virus-infected individuals.
[Slide.]
With
protein 2, however, we found that all 12 of the first bleed specimens were
positive for IgM, and 11 of the 12 of the second bleed specimens were positive
for IgM, so again we are finding that the second protein that we are
evaluating, the CDC protein works very nicely in detecting the acute phase
infection.
You
will note that in some cases, patient 1, the IgM value goes up between day 11
and 21, other ones, as in patient 2, remains the same. Patient 3, the IgM values goes down, so we
see a lot of different types of responses.
For those experienced in serologic tests, this is what you see. There is
not a monolithic type of response. You
will see different things with different individuals.
[Slide.]
Both
assays here are using protein 2. We
received a 20-member panel from CDC, and we tested these. We have the panel member on the x axis, on
the axis, the S/N value obtained.
We
have, in the lighter blue, the CDC values that we received from Fort
Collins. On the dark blue is the Abbott
IgM S/N values, the point being that we detect correctly all the samples that
were sent, and, in fact, our sample to negative values are substantially higher
in our hands. It may be because we have
a lot of experience developing solid phase tests, and we are actually in this
assay that I am talking about today, we are using actually an IgM-coated solid
phase that we manufacture for other IgM tests, so we have taken all the bugs
out of how to coat that solid phase, how to keep the background very low, and
how to make the solid phase consistent and reproducible and able to detect IgM.
[Slide.]
I
mentioned that we have an ongoing study.
This is just a schematic, so I can address what we found, in general, is
that we are seeing RNA anywhere between 5 and 10 days, from day 2 through day 5
in some animals RNA positive, and other
animals, it goes out as late as day 10 post-inoculation, but there were no RNA
positives beyond day 10.
IgM
in the five different animals we look at came up in every individual, ranging
between day 9 and day 11 post-inoculation.
IgG follows about 4 or 5 days later.
It seems to be coming up around day 14.
So,
we are seeing some overlap of RNA detection and IgM detection. One of the animals we clearly saw
IgM-positive, RNA-positive bleeds.
Just
go back there for one more second.
We
are working with collaborators at another site, and we are doing the IgM and
IgG testing. We are going to have
hemagglutination inhibition data, plaque reduction neutralization tests. We have RT-PCR in my lab, and we also have
infectivity being done. So, we will
have a pretty nice profile of available tests on this experimental model.
It
doesn't depict it on the x axis, but actually, these animals were bled every
day for the first 14 days post-inoculation.
[Slide.]
So,
we have looked at two different recombinant proteins and we have developed IgM
tests. Protein 2 seemed to do a much
better job in detecting antibodies of IgM class than protein 1.
I
don't mention here, there is just not enough time, but we did do also some IgG
evaluation. Protein 1 works very nicely
for IgG, and protein 2 also works for IgG, so we don't see a big difference in
performance with protein 1 versus 2 for IgG detection.
So,
we are continuing to look at viremia and the relationship with IgM
detection. We have a cell line in-house
that we have obtained from the CDC, that we are generating internally. We are producing antigen. This antigen is showing to work similarly to
what has been working with the CDC antigen.
We
are looking at prototype assays first on polystyrene beads, has probably a more
rapid assay development cycle, and also microparticle-based assays.
We
are also expressing additional proteins internally from envelope, I mean 1 in 3
of envelope protein. We are looking at NS3.
We will be looking at core.
[Slide.]
We
have studies planned or in progress.
Here, we are continuing to optimize the assay. We are looking, how are you going to confirm a positive, and this
something you can spend quite a bit of time talking about because probably the
best confirmation is plaque reduction neutralization tests, but it is very
tedious, time consuming, and it requires very specialized training and equipment.
RT-PCR
will work probably on early acute phase samples, but it is probably not going
to help you out on most of the clinical samples coming in, in an
asymptomatic, in a diagnostic lab.
Another
confirmatory strategy is to look at IgG detection with protein 2 or to look at
both IgM and IgG on alternate proteins.
I mentioned NS3, core, and other envelope proteins we are looking at and
expressed in different host cells.
External
studies, we are not really embarking on anything right now, but we are looking
forward to participate in the study on quarantined units to find out what we
see with IgM and RNA and IgG, and possibly to participate in the donor
reinstatement studies, and we will be looking to align our tests with the
testing done at different state health labs.
Thank
you for your attention.
DR.
NELSON: Thank you.
Questions
or comments?
DR.
LAAL: How many animals do you have in
your macaque study?
DR.
DAWSON: There is five animals.
DR.
LAAL: And they are all infected with
the same dose?
DR.
DAWSON: They were infected with the New
York strain, intradermal inoculation of 105 PFUs/mL.
DR.
LAAL: And in all of them, you see this
fall in titers?
DR.
DAWSON: That is a schematic, it doesn't
represent any reality. Some did fall,
some did not fall, so it is hard to generalize.
DR.
STRONG: Are you using a blocking
reagent for IgM assays? I would be a
little concerned about specificity.
DR.
DAWSON: With protein 1, in an indirect
assay, we used different, typical types
of blockers, animal sera, detergents, and, as well, we are using goat antihuman
IgG or whatever it is, antihuman IgG, so we can make sure that we are detecting
the IgM and it is not being masked by IgG.
In
the second assay format, the IgM capture, we used some of the diluents that we
use for hepatitis A and hepatitis B, which have different types of protein
blockers to optimize specificity, so I think we are in pretty good shape for
that.
DR.
STRONG: And you are using sort of the
classical format. Have you looked at
chemiluminescence, as well?
DR.
DAWSON: No, we have not, but we
will. Like I said, we didn't get
materials that were going to be of any use to us until mid-January. Some of these assays require using
monoclonal antibodies, especially that IgM capture, you are going to have
monoclonal antibodies that you purify the antibodies and label with your
chemiluminescent tag or whatever enzyme you are going to have, so we are
working along that path.
DR.
EPSTEIN: Thank you, George.
You
mentioned interest in the potential development of IgM as a donor screen. I am just wondering whether you contemplate
having any products available for IND use in the 2003 summer months.
DR.
DAWSON: I think that is a difficult
question for me to answer because, you know, it would require a pretty large
volume of tests, and we would really need to meet with CBER to talk over how we
would implement that plan. We think for
this year, we could probably participate in some external studies to look at is
there value here.
What
I am seeing so far is that I would love to get the 20 or 21 samples that were
linked to transmission to find out how many of these are IgM-positive. What we have heard, what I have heard at
other meetings, with IgM testing, is that most or all of these samples were negative
for IgM.
So,
we are here if, you know, the community believes there is a need for an IgM
test for screening, but I think current consensus--and please correct me if I
am mistaken--is that NAT will be done.
IgM could be there for donor reinstatement, or if there was a turn in
thinking that IgM is required, then, we need to discuss that.
DR.
NELSON: it was my understanding that
the transition early, in the first few days, that IgM appears, that there still
may be PCR-positive infectious virus, so that theoretically, you might pick up
low-level viremia using an IgM.
I
just wondered when IgG appears, in other words, when IgG appears, does that
mean that the unit would not be infectious or that the person would not be
infectious at this point. Is there any
data on that?
DR.
DAWSON: I don't have data on that. I would like to ask a couple things of
everybody here. When you find a
NAT-positive, when anybody finds a NAT-positive, I think they should be
withheld in some manner, made available for IgM and IgG testing, and for virus
isolation, because there are labs that do this routinely.
Let's
get to the bottom of this thing and find out.
You know, there has been all these positives identified in those 33,000
samples, let's have some cooperation and see, you know, maybe make those
available to people that test for IgM, IgG, to experts in the field, send them under code, and won't have to
second guess.
DR.
NELSON: Thank you.
Charles
Tackney from Ortho. He is substitution
for Steven Alexander.
Charles Tackney, Ph.D., Ortho
Diagnostics
DR.
TACKNEY: Thank you. Steven is off on another jaunt.
[Slide.]
I
am going to show you today some early data on a prototype assay that we put
together a few months ago after the November meeting to try to see what sort of
tools we could mobilize to answer some of these questions that were coming up.
If
you noticed, I use the word "user-friendly" here, and I hope when we
get done, maybe I will leave you with the thought that the assay is rather
facile to do, it has certain benefits of performance, and even though it is a
very complex subject, potentially, it's even deployable.
[Slide.]
I
would like to just reiterate what George said because we seem to be bringing up
the rear on immunoassays. It is a very complicated topic, and we do have a
research tool here, it is a very good tool, but it is a very complicated
medical question that we are trying to answer.
I
think what we want to do is say that we have put together some things, we have
some observations and some new tools of reagents that might help answer some of
the critical epidemiologic and immunologic questions about this virus which are
really not clear, I think, today.
It
is not your typical flavivirus using the HCV paradigm. So, take this as a contribution towards the
knowledge really and be aware that we are perfectly amenable to discussing any
scenario around these tests because we really don't know where to go with it.
The
test uses a 10 microliter sample, it's a microplate format, and lab people prefer
to call this right now a hand-cranked assay.
It does not take advantage of any of the process and the excellence that
we can put into a full-blown assay, but I think you will see there is enough
data to justify that it is working.
It
is a homogeneous reaction in solution that involves a biotinylated anti-mu
capture reagent that is mounted on a SAC well, either a microplate or a
cup. It has a less than 2-hour time to
result. That time can be pushed down
below an hour when it is brought onto one of our automated platforms, and it
uses the chemiluminescence readout typical of the ECI platform.
[Slide.]
Very
simply, the patient sample is diluted down in liquid to the anti-mu capture
reagent. The reaction there takes place
almost instantaneously. It is incubated
in a SACwell for 30 minutes in this format on the SACwell, it is washed. A conjugate is added, and the term
"conjugate" here we are going to put in quotations. I will show you what that means, a
"conjugate" is added, substrate and chemiluminescent readout, and
time to result 30 minutes.
In
the typical predicate assay that this comes from, which is our HAV or HB core
IgM assay, these incubation steps are 15 minutes each, so that is why I say a
hand-cranked assay.
[Slide.]
What
you have is a conjugate which, in this case, you should keep in mind is either
a monoclonal antibody to a ligand, and that ligand can be a recombinant
antigen, a peptide, or a viral lysate, or it can be a directly labeled analyte,
an HRP-labeled material.
In
the assay I am going to show you, we are going to use an envelope recombinant,
and we have a very substantial effort here in cloning. We have basically cloned all of the relevant
proteins, the structural and some of the non-structural proteins, in both
bacteria and, where applicable, particularly for the envelope protein in
mammalian systems, and we have a monoclonal in this case to that envelope
protein. That is what I will be showing
you here.
The
biotinylated anti-mu capture monoclonal in solution captures the human IgM,
captures down onto the SACwell in a rather seamless manner, and the assay
basically is pre-formatted, so there is no coating, if you will, here.
[Slide.]
We
have not invested a lot--and we will--we have not invested a lot of energy, and
this was put together for the purpose of this meeting, in getting the kind of
numbers of patient samples and things that would be statistically
relevant. We have very, very few actual
West Nile patient samples available to us, and that is a big problem, and we have
only looked at a small cluster of normal patients that we just pulled out of
the archive.
So,
the concept of cutoff here, I want you to give me the benefit of the doubt that
I am not declaring a cutoff. This is a
proof of principle assay, does the reagent format, as it is put together,
detect things that we can monitor against the CDC format assays right now, and
then we would move on from there.
The
assay would be generally very, very quiet in terms of light unit output had it
not been for one outlier, so I have not tried to manipulate the experiment to
choose my best. Out of 41 samples, this
is what the spread of ALU units looks like when we read it.
I
am going to leave that number in there and declare a 225 ALU average
approximately, just give myself 2 standard deviations, which would not be
typical of one of our assays, and say that the cutoff is going to be 400 ALUs.
[Slide.]
If
you do that, you will see that there is a nice bundling down here towards the
left except for this one outlier, but I am going to stay with the 400 ALU for
now.
[Slide.]
These
are samples that were provided to us very generously by Dr. Meyers at the
Maryland Health Lab, and I have to say--I don't think he is here--he has been
donating a lot of counsel time to us, helping us to understand the medicine and
get reagents and recruit reagents, which is not trivial, and echoing what
George said, it is really difficult for us to get things. I know everybody is trying their best, but we
are not getting everything that we are really seeking in a really timely
fashion, especially these new challenge samples that we believe are coming
through.
If
you look at Bob's data here, they have all been PRNT-positive confirmed. They were sent to us in the blind with the
following commentary as per his interpretation of their lab data. This one was told to us to be a West Nile IG
equivocable with a Mac ELISA of 3.7, which would put it right on the cusp of
positive at the Mac ELISA.
This
is an IG-positive who is negative in the Mac ELISA and then a series of IgMs
including one that scored negative in that ELISA, the Mac format, but is
PRNT-positive.
This
is one generation of our assay using that 400 cutoff and clearly, we are going
to call this one positive right here, assuming it's above 1, and most likely it
is a positive.
This
one statistically would become a negative sample very easily if I were to spend
the time formatting this out properly, and all the say down here, you see we
have a very, very nice signal range of these positives including this fellow
here, who is clearly positive.
We
have done this several times, and we have had these samples assayed several
times here at the Health Lab, and we are seeking more samples like that
especially those infectious window samples if we could get them.
[Slide.]
This
is just a very, very minor piece of data just showing that the assay is highly
dilutable, it has got a robust dilutional capability for what that's
worth. It's alive, if you will, and it
would help to get some quantification of these results, which is not really
trivial with the current assay.
[Slide.]
We
identified a patient down in Texas, we just call it the Texas patient, that was
scored positive in the CDC assay. It
had hemagglutination titer, specific titer over here. We put it through our assay, and we drove it down. We stopped at that endpoint
unfortunately. You could see here again
we have got a nice dynamic range picking up that patient very clearly.
[Slide.]
We
do have in our archives a whole array of exotic all sorts of agents. We popped out a bunch of dengues for which
we don't have a lot of background pedigree information, but we do know their
basic serotypes. You could see here
these are really quite hot samples and may form the basis for some sort of a calibrated
panel for us, and also reinforcing the fact that I shouldn't be calling this a
West Nile assay, this is a flavi assay, and that could be a good thing or a bad
thing depending on where we want to go with this assay as a clinical tool.
[Slide.]
What
are we going to do in the future? Well,
the answer is really we are not sure.
We want to participate, we want to help. We really enjoy the subject.
The staff is pretty excited about it.
You know, you get tired of the old hepatitides. We certainly plan to transition this assay
onto the vitreous format if possible, pick up some of the advantages of that
platform, make it more portable.
We
need to optimize assay performance. We
have no doubt that the small amount of time we have spent we can quiet that
down to make it typical of some of our other assays where these ALUs get down
below 100 reproducibly.
We
can get the validation down around the reps, the precision, and so on. We want to do lots of studies with
experimental samples, challenge samples, interference samples. We are doing that now. We are seeking those from other people, and
we really want to be involved in someone's trial, looking at these interesting
retrospective studies and seeing what it means today given that data that we
could accumulate with this assay.
So,
we are here to help, but we really don't have a decision on where we will be
next summer, as I think George eloquently put it, I think all the organizations
want to do something.
Thank
you.
DR.
NELSON: Thank you.
Any
questions? Yes.
DR.
FITZPATRICK: On the IgG-negative,
IgM-positive sample, do you have nucleic acid test results on that?
DR.
TACKNEY: No, we do not, and we have
requested that. We do not have that in
our hands although it may very well exist in the laboratory, and we have
requested that. We are also waiting on
the CDC proficiency panel, the panel that has been described, as well as we are
also out in the field surveying our contacts particularly in the American
south, doctors who send us patients, send us interesting things that we can
look at, and talking to several principals here currently about what
opportunities might exist to get that information.
DR.
NELSON: Jay.
DR.
EPSTEIN: Do you care to comment on the
recombinant and antigen, on its origin?
DR.
TACKNEY: Sure. This is a mammalian-based recombinant
antigen. It is similar in performance
to the mammalian antigens that have been discussed earlier at other
meetings. I will tell you that our
feeling is that it is a confirmational structure. Working out a bacteria is not suitable for this antigen, we can
assure you of that.
We
have a cell culture system which is a suspension system, a mammalian-based
system similar to a myeloma, if you will, that is putting out this
antigen. We also have the very, very
earliest--and I won't be presenting any information on it--hints that we have a
fledgling antigen assay, have no idea what that means in terms of the
progression of the disease, it is purely Ph.D.-speak.
We
have NS1 cloned, NS3 cloned, capsid cloned.
We have an array of peptides because we had sort of a naive dream that
we would just pop out a West Nile-specific assay right out of the gate. That's not dead in the water yet, but is
proving very, very difficult, and actually, maybe we don't want that if you are
going to screen.
The
antigen is critical, as George said, not all antigens are made alike regardless
of their sequence.
DR.
NELSON: Thanks.
Next,
is Wallace Narajowski from Focus Technologies.
Wallace Narajowski, Focus Technologies
MR.
NARAJOWSKI: Good morning. I am Wally Narajowski with Focus
Technologies. For those of you that are
not familiar with Focus Technologies, we are a small company, a reference
laboratory, and a diagnostic product company.
We have been dedicated to infectious diseases for over 25 years, and our
headquarters are in Herndon and our laboratory is in Cyprus, California.
Some
of you may know us formerly as MRL, Microbiology Reference Lab. We are now called Focus.
I
am here to give you an update on our West Nile IgG and IgM ELISA product. I have to thank Dr. Hogerfe and Dr. Prince,
who are really the technical people behind this. They are back at the shop
making sure we can get product out the door actually starting within a couple
weeks.
My
purpose here today is to give you an update on our products for West Nile
virus. Currently, our goal is to work
with the FDA to have an FDA-cleared clinical diagnostic product available this
season.
Starting
next week, we will actually be supplying the West Nile antigen for the Public
Health System. We are manufacturing
that for them and we will be starting to ship that next week. We are targeted to have an ASR for both IgG
and IgM available starting in April.
[Slide.]
The
key reason we can do this is last year, in our reference laboratory, we
performed over 36,000 serologic assays for West Nile. As you can see, this chart, this is the distribution of when
these were actually run, and our goal is to have the FDA-cleared product with
the FDA by August.
[Slide.]
Currently,
we are naming this product Flavivirus West Nile, and the reason we are doing
this is because we got the recombinant from the CDC, and this recombinant is
targeted for West Nile, but it also does cross-react with other flaviviruses.
Our
IgM assay is about a 2 1/2 to 3 hour microtiter standard ELISA format for a
capture type, 96-well, microtiter format, and our IgG is about an hour and a
half assay, very similar type of format that I think everybody in here is
familiar with.
[Slide.]
The
actual protein that we received from the CDC, and that is going to be part of
this, is the recombinant protein, and it is derived from the pre-membrane and
envelope genes of the West Nile. CDC
last year supplied this recombinant to all the Public Health System. This year, we are going to supply it to both
CDC and Public Health.
As
I mentioned, the protein retains both specific epitopes and cross-reacts with
other flaviviruses.
[Slide.]
We
utilized samples from our 2002 season to establish a cutoff, and this is really
the basis for establishing our cutoff for this assay. We started obviously with the 36,000 serologic specimens.
We
took 700 of those and used that as the basis for the cutoff, 150 of the 700 had
Public Health results, and 100 of the 150 had plaque reduction results
associated with them, as well, and that is how we established our cutoff. What I am going to show you next are the
results of the assay that we are going to release in April utilizing this cutoff.
[Slide.]
This
is for our IgG. We first did a Southern
California where we did normals, and we did a mini-seroprevalence, and it was
very similar to what we found with dengue many years ago, 95 percent negative,
and there was an underlying IgG positivity rate.
Specificity
compared to our reference lab was 95 percent.
The four outliers were in the equivocal range. Sensitivity was 97 percent and again 3 of the 4 in the equivocal
range.
[Slide.]
Our
IgM assay, we have 100 percent concordance for negative sera versus our
reference lab, and 96 percent concordance on positive, 24 of the 24 Public
Health IgM, plaque reduction were positive with our IgM assay, and 118 of the
samples from Public Health, either ELISA or plaque reduction were 100 percent
concordance, as well.
In
summary, our goal is to introduce an ASR format with the products that you see
here, probably starting the end of April, and we hope to have an FDA-cleared
product in time for August. We did meet
with the FDA actually Tuesday of this week, and they are onboard, if we give
them good clinical results, they say they can make it happen.
Thank
you.
DR.
NELSON: Thank you.
Questions?
DR.
GALLARDA: Jim Gallarda from Roche.
Are
you providing the antigen only or are you providing all reagents for the test
methodology?
MR.
NARAJOWSKI: We are making available
both to Public Health, the antigen, as well as the ASR reagents, but most of
them are going to probably perform their own assay like their protocol last
year, but they do have the option to purchase the reagents from us, as well.
DR.
GALLARDA: I guess my question is
related to the quality control from a manufacturer providing uniformity in
manufacturability as compared to distribution of the components to multiple
labs, how that might affect the reliability of the assay in the setting.
MR.
NARAJOWSKI: The way that it is
configured is there is a reagent pack, as we call it, a convenient pack that
has the plate and the conjugate and the cutoff as part of that convenience
pack. Then, all the other reagents,
they can purchase from our catalog, and that should take care of any of the
uniformity issues.
Does
that answer your question? Okay. Any others?
Okay. Thank you.
DR.
NELSON: Thank you.
Next,
is Dr. Lyle Petersen, who is going to give us an update on the transfusion-transmitted
cases.
C.
CDC Update on Investigations of West Nile
Virus Transfusion-Transmitted Cases
Lyle Petersen, M.D., CDC
DR.
PETERSEN: Good morning.
What
I am going to present is work from a number of different topics.
[Slide.]
What
I will first do is talk about the transfusion transmission investigations,
first talk about the characteristics of the recipients, characteristics of the
donors, and talk about comparison of symptoms of implicated and non-implicated
donors.
Then,
I will go on to discuss the estimates of risk of transfusion transmission
during the 2002 season, talk a little bit about a viremia study which I think
Sue Stramer will follow up on that after me, and then talk about some data we
have from our experiences from IgM antibody testing in our laboratory.
[Slide.]
To
go on to talk about the transfusion transmission cases, we wanted to determine
the number and type of components transfused to recipients in the four weeks
before illness onset. Basically, what
happened was is that through our national surveillance system for West Nile
virus, possible transfusion-transmitted cases became known, and these were
people who had received transfusions in the four weeks before developing West
Nile virus infection.
So,
we tried to retrieve what we called initial donation samples, which are
retention segments and then transfused components that were there from the
donor at the time of donation. Then,
for all of the donors, we then followed up them with a follow-up questionnaire
and got a serum sample for IgM antibody testing.
These
were all from donors of components received by the index recipient, and we also
went back to try and find co-component recipients of donors who we identified
with transfusion transmission related West Nile virus infection.
[Slide.]
So,
the criteria we used to define a transfusion-transmitted case was actually
quite strict, and we required either a donor to be PCR-positive and/or
culture-positive from an initial donation sample, and IgM-positive at
follow-up, or to be PCR-positive and/or culture-positive and have a
co-component recipient that was IgM-positive.
The
third category we considered as a definite transfusion-transmitted case was a
PCR equivocal donor who was IgM-positive at follow-up and also had a co-component
recipient that was IgM-positive.
The
last category of a person we considered was a co-component recipient of a
person proven to be, by the above case, by the above criteria, and that
co-component recipient had to be IgM-positive, as well.
[Slide.]
So,
we received reports of 61 possible transfusion cases as of March 1st,
2003. We determined that 19 were
definitely not transfusion related, 8 didn't have West Nile virus infections, 3
had their transfusions after symptom onset, 2 had symptom onsets more than 28
days before the transfusion, and 6 had West Nile virus infection, but all
donors were IgM-negative.
Now,
that left 21 additional cases that we called inconclusive due to incomplete
donor follow-up, and we have pretty good evidence that many of these 21 cases
could have been transfusion related, but we just didn't have the data to
categorize them according to the scheme that I mentioned on the last
slide. That left 21 confirmed cases of
transfusion transmission of West Nile virus, and this will be our final tally,
I think, for the last season.
[Slide.]
In
looking at the recipients, this slide depicts the underlying medical conditions
or reasons for hospitalization, and you can see that these were a pretty sick
bunch of people, to begin with, 6 had stem cell or organ transplants. Nobody got the infection from the organ
transplants in this series, but these were post-organ transplant patients who
were on immunosuppressive drugs.
Four
had cancers, mainly hematological malignancies, 8 had obstetrical or surgical
procedures requiring transfusion, and 3 had other medical problems.
[Slide.]
Of
these confirmed cases, 12 of them had West Nile virus meningoencephalitis, and
1 had West Nile fever, and the rest were asymptomatic.
Of
the 13 symptomatic people, one of the curious things was the incubation periods
among these people were rather long, and they had incubation periods from 2 to
21 days, the median 11 after the implicated transfusion, and this was an
unexpected finding.
These
people received blood from a median of 36 donations ranging from 2 to 274, and
of the outcome, 6, or 30 percent, died, 1 due to West Nile virus
meningoencephalitis. Among these
people, it was very difficult to really determine if their underlying condition
killed them or the West Nile virus killed them, or both, to this outcome data
is a little bit unclear.
[Slide.]
So,
all three types of the major blood components were all implicated, red cells,
platelets, and fresh frozen plasma, and the maximum interval between the date
of donation and the date of transfusion was 33 days for red cells, 5 days for
platelets, and 44 days for fresh frozen plasma.
[Slide.]
In
looking at the donors of these 21 people, we had a total of 726 donors returned
for follow-up testing, and 92 percent completed an interview.
Out
of the 21 people, there were 14 donors implicated. A couple of the donors had transmitted to more than one
recipient, so that is why there aren't 21 donors, there is only 14. All had evidence of viremia and importantly,
all were IgM-negative on their initial donation samples, and all were linked to
at least one recipient with West Nile virus infection.
Seven
of the 14 donors were linked to more than one recipient with West Nile virus
infection, and actually, one recipient had received transfusions from 2 viremic
donors, and it was unclear which of the viremic donors had transmitted to the
recipients.
[Slide.]
So,
this slide summarizes the 14 donor labeled A through N. You can see that their dates of donation
corresponded to the peak periods of the epidemic last year, in late August and
September mostly.
One
thing that is very interesting is the symptom onset and to the time of
transfusion. The ones with no data are
the asymptomatic donors who had not symptoms at all, and there were 5 of those,
and the minus figures are people who had symptoms before their donation, and
plus are people who had symptoms after their donation.
In
some cases, there is a range where the donor couldn't really remember.
Now,
I would like to express some extreme caution in trying to interpret these data
because you have to remember that these donors were asked to recall a very mild
illness that had occurred months before, so the timing of their illness onset
relative to their donation was a bit unclear, and a couple of them, like donor
A, had some kind of sinus infection before their West Nile virus infection, and
it was really impossible to exactly determine when her symptoms began.
The
last two columns are the PCR test results, both for retention segments and for
plasma. The retention segments turned
out to be very difficult to test and somewhat unreliable. We both had false positives and false
negatives associated with testing the segments, but the plasma samples were
held under good conditions, and these are the results in plaque-forming
units/mL. As you can see, all of the
viral levels were quite low.
[Slide.]
So,
the next thing we wanted to look at was symptomatic versus asymptomatic
donors. What we found is the
symptomatic versus the asymptomatic donors had a median of 44 and 45 years
respectively. The mean viremia levels
in plasma were higher for the symptomatic donors compared to the asymptomatic
donors.
The
viremia levels seemed to decrease as the time from illness to donation
increases.
So,
if you look at the chart there in yellow, going from donors who reported
symptoms before donation to donors who reported symptoms after donation, you
can see that the donors who reported symptoms onset right about the time of
donation had the highest levels of viremia, and this was statistically
significant.
[Slide.]
So,
we looked at the implicated donors versus non-implicated donors, and as I
mentioned before, we had 14 implicated donors, and we had 654 other donors who
were IgM-negative, who we used as a comparison group. Three of the symptoms of these donors were independent predictors
of West Nile virus infection, and those were fever, eye pain, and new rash.
Now,
looking at the fever column here, you can see that 9 out of 14 of the
implicated donors reported fever compared to 3.7 percent of the non-implicated
donors. These 9 donors were all of the
symptomatic donors. The other five
people were completely asymptomatic and had no symptoms at all.
[Slide.]
So,
we tried to look at what kind of symptoms could discriminate the implicated
donors from the non-implicated donors, and as I mentioned earlier, fever caught
all of the symptomatic implicated donors, but 3.7 percent of them, of the
non-implicated donors, reported fever, so that, by itself, would not be a very
good screening tool, so we tried to look at combinations of symptoms.
You
can see that fever and eye pain, or fever and new rash seemed to be pretty good
predictors of acute West Nile virus infection.
[Slide.]
So,
what I tried to do then is look to see what the sensitivity and specificity in
predictive value positive of these combinations of symptoms would be. If you look at the first column there, I
have incidence per 10,000 donors, and I just came up with a number. There is some rationale for this number,
which I will explain in a minute.
But
looking at, for example, fever, if you assume that the incidence is 10 per
10,000 donors, if you assume the sensitivity of fever as 64 percent, the
specificity as 0.963, the predictive value would be 0.174, or put in another
way, for every West Nile virus viremic donor, you would exclude 57 normal
donors.
If
you look down at these combinations of fever and eye pain, and fever and rash,
the predictive value positive is actually quite good.
[Slide.]
Now,
I would like to go on to the next topic and talk about the estimated risk of
transfusion transmission in the United States during the 2002 period. These results are based on a model that we
published in Transfusion, in the August issue I believe it was, last year, so I
won't go into the methodology.
But
what we found was that the average risk in the United States last year was 0.36
per 10,000 donors with a maximum risk of 1.55 during the peak of the
epidemic. Now, I have also presented on
this slide, state specific estimates of risk, and you can see that among these
higher incidence states, the risk was considerably higher than the national
average, with a high in Michigan of about 4 per 10,000 donors overall during
the epidemic.
[Slide.]
In
this slide, I just showed the same data showing the actual curve of the
estimated risks for the same states, and as you can see, during the peak of the
epidemic, in Michigan, we estimated that around September 1st, that the risk
was about 10 per 10,000 donors.
In
some high-risk cities, such as Detroit, the risks actually were up to like 20
per 10,000 donors.
[Slide.]
So,
then, we decided to try and validate this model with the Red Cross and Sue
Stramer, and did a West Nile viremia study in which we took samples from
Cleveland and Detroit during the first three weeks of September 2002. According
to our model, we estimated that the risk would be about 8.2 per 10,000 in that
population.
We
tested all of these samples by TaqMan PCR in our lab, and the very preliminary
results--I want to emphasize very preliminary--of the first 5,761 tested to
date, the point prevalence of viremia was 6.9 per 10,000, and that was based on
4 positives. But what I learned this
morning, walking in the room, there was actually 6 positives, so the point
prevalence is actually higher than that, and it is probably going to come very
close to our estimated point prevalence of viremia.
So,
I think our model for transfusion risk is probably going to be pretty close.
[Slide.]
Now,
one of the things I am always asked is what are the trigger points in trying to
predict future epidemics particularly in this season for making decisions about
blood donor screening.
We
have a national system called ArboNet, which is a national surveillance system,
which we look at birds, mosquitoes, horses, and humans, and West Nile virus
infection in those. It is a real-time,
web-based reporting system.
The
first measure I call epizootic measures, which is dead birds. What we found is that dead birds are
extremely sensitive to detecting West Nile virus epizootic activity in an area,
but not very specific for human disease, so as a discriminator to look for
future West Nile hot spots, it is probably not very good.
What
people have tried to do is quantitate dead bird surveillance to get a better
measure of West Nile virus risk. What
we found is that in certain places it does correlate with human risk.
If
you look at indexes like dead birds per square mile, you find in places with
higher measures of epizootic activity, it does appear to be correlate with
future human risk, but this is very difficult to evaluate because the national
surveillance is not completely standardized across the whole country.
So,
certain states will test a few dead birds in a county and quit, others will
test hundreds, so it is very difficult to try and translate these data into a
national or a local estimate on a national level of risk.
The
other things we look at is mosquitoes which are not timely and not universally
done. It is very hard to do mosquito
surveillance. Horses, it is the same
problem with birds except horses are less evenly distributed. Dead horses aren't particularly useful in
Manhattan, for example.
So,
if we look at all these epizootic measures, probably none of them are terribly
useful for determining trigger points
for making decisions about blood donor screening.
[Slide.]
The
thing that is probably more useful is human illness surveillance. The one thing we do not encourage or
standardize is surveillance for West Nile virus fever because there are
probably 500,000 cases of West Nile fever last year in the United States, so it
is just impossible to do surveillance for something of that frequency, so we
really try and do West Nile virus meningoencephalitis, which is universal
across the country, and from those data, we actually can estimate transfusion
risk, and those data were used to give those models the data that I presented
earlier, and we have come up with a simple formula that people can use to
estimate transfusion risk in their area.
We
can get a good idea of the size of the future epidemic from early season human
cases. So, for example, if an area has
got human cases in June, we know that there is going to be a huge problem later
on.
The
one problem is that there is a short lead time between human cases and
transfusion transmission, and among these counties where we found
transfusion-related cases, the donors of these cases often donated only a
couple of weeks after we had recognized the first cases in those counties.
So,
human illness surveillance is probably a good trigger point, but the lead time
is rather short.
[Slide.]
I
would just like to end with a little bit of information about experience in our
lab with IgM antibody testing and in the sera submitted to CDC from 1999 to
2002, IgM was detectable in serum and CSF on the date of onset in 99 percent
with 6 exceptions in serum of 800 tested and in 10 exceptions of CSF in 800
tested.
I
would like to put one caveat to that, is that when somebody submits samples to
us and writes the date of onset, we don't actually go back and try and verify
that that is the case, so the date of onset may not exactly have been correct.
The
other thing is most of these people that got submitted samples were from people
with meningoencephalitis, and not West Nile virus fever, but the bottom line is
that at least in people with meningoencephalitis coming in your lab on the date
of onset, most people are already IgM-positive.
Almost
everybody is IgG-positive by day 7 post-onset, we also found the P to N ratio
was 3 to 5 times higher to West Nile virus than SLE.
The
other thing is IgM persists for a long time.
[Slide.]
This
is the slide that shows P to N ratios in days post-onset up to 400 days, and
you can see that with cutoff of 3, that a large number of people are still
IgM-positive 4- and 500 days out. This
data was published this month in the Emerging Infectious Disease Journal, which
has an updated chart to 500 days out.
[Slide.]
I
would just like to say that all of this work, it was really a tremendous amount
of work, and there were a lot of people involved in collecting it all, and I
would particularly like to thank Sue Stramer and the others from the Red Cross
and the other blood collection agencies, and FDA, for helping out with all of
these investigations.
Thank
you.
DR.
NELSON: Thanks.
When
the donors, the 600 or whatever, were questioned, and you looked at those that
were implicated and those that weren't by its symptoms, did either the
interviewers or the donors know that they were linked to the transfusion case
at the time of the interview or was this blinded?
DR.
PETERSEN: They all knew that there was
a possibility that they were linked to a transfusion case. Each of these cases had an average of 36
donors. Nobody knew which one it was
until they got questioned and then the samples went to the lab and they were
tested.
We
had no clue which one it was at the time of the questioning.
DR.
NELSON: A few of them had a fever at the
day of donation. My understanding of
the way that the transfusion services work is that usually, temperatures are
taken. Actually, when I have donated,
the temperature is taken prior to collecting the unit. Did that occur just when they went home or something,
after donating, or why were some febrile at the day of donation?
DR.
PETERSEN: I think there is two things
to consider. One is we didn't ask them
specifically what time of day, we just asked them what day, so we don't know if
they were febrile before or after. The other thing is keep in mind the big
caveat here. You are asking people
about a mild illness two months earlier, so the reliability of this data are a
big questionable.
DR.
WILLIAMS: I just wanted to probe a
little bit on the morbidity and mortality of the recipient cases.
Of
the 12 meningoencephalitis cases, one case died. Of the remaining 11, can you say anything about long-term medical
impact of that? A second question. Six of the recipients died. I gather from what you said earlier you
couldn't either rule in or rule out West Nile involvement. Could you just say
anything more about that potentially?
DR.
PETERSEN: All I can say is it is really
hard to determine what people die from, because we have looked through these
charts just in incredible detail, and a lot of these people were extremely sick
to begin with, and particularly where it got difficult is with somebody with an
unknown febrile illness, some of these people were septic before they got their
implicated transfusion, so it was just really difficult to figure out killed
them for one thing or if they even had West Nile virus illness.
Some
people it was very clear-cut where they just got the transfusion and developed
the meningoencephalitis. Others,
particularly if they had milder symptoms, it was very hard to sort out and some
of these people we could never sort out to any kind of degree of certainty.
The
other thing I might like to add is that there was a certain selection bias of
how we got these people, because the ones who had, let's say, the index cases,
the ones who were identified with possible transfusion-related West Nile virus
infection, by definition, had something that alerted the doctor that they may
have had West Nile, so most of those people had meningoencephalitis, but if you
looked at the co-component recipients that we went back and identified, most of
those people were either asymptomatic or had mild illness that we couldn't
separate out from their other underlying illness.
DR.
ALLEN: I think that is an important
point. Obviously, the volume of infectious blood that one gets from the
mosquito is very different from what one gets in a transfusion, so that is
interesting that they still could have gotten that as a co-component recipient
and still had very mild illness. That
says a lot I guess about the host response.
DR.
PETERSEN: Right.
DR.
ALLEN: The other major question that I
am struggling with is this whole issue of viremic states continuing despite the
presence of IgM antibody. It would nice
to be able to parse it out along those lines if you could in terms of accepting
or rejecting donors.
From
this study, I just want to reiterate, as far as you were able to tell based on
the segment analysis of the donors, there were no donors who were IgM-positive
at the time that they donated that blood, is that correct?
DR.
PETERSEN: That's correct. We did have one case where there was
somebody that was IgM-positive and viremic, had a very low level of viremia at
the same time. This was a person who was actually a blood donor who
subsequently got West Nile virus infection and called the blood collection
agency. The blood collection agency
pulled the plasma. That person was both
at the time of donation.
Unfortunately,
the recipient of that person's blood died, was very sick and died before we
could go back and investigate the person, so we actually do not know if that
person transmitted infection to the recipient or not, but everybody else was
IgM antibody negative.
DR.
DAWSON: George Dawson, Abbott
Laboratories.
I
had two questions. One is from some of
the meetings I have been at, it has been stated that only about 1 percent of
individuals infected developed CNS involvement, meningoencephalitis, and we
have only seen 20 cases of transfusion associated and 13 of those were
encephalitis or meningitis.
What
is the total number of transfusion-associated transmissions do you think
occurred? That is one question.
The
second question is--and it will be a difficult one to answer, but it certainly
wasn't 20, it might have been some number with a thousand, you know, with a
comma--I have it heard it discussed also that people or birds or animals, et
cetera, may be negative in their blood for viral RNA, but the virus may be
harbored in tissues.
I
don't know if you have any evidence on, you know, maybe some of the individuals
who have developed encephalitis or meningoencephalitis, did they become RNA
negative in serum and positive in their tissues, either brain, CNS, or other.
Does
that pose a matter of concern for this committee in looking at using RNA only
as a criteria for reinstating donors because if you are non-viremic, but it's
at 1 virus/mL and you can't detect 1 virus/mL, could it be harbored at low
level and periodically come out in serum.
DR.
PETERSEN: I think your second question
is of real interest particularly for the organ transplant community. There are data that do show that you can
find live virus in organs of people when they are not viremic, and the best
data, there is a 1954 paper by Severne and Gore [ph] where they experimentally
infected people as part of some experimental cancer treatment. These were not normal people, these were
people with cancers.
There
is a table in that where you can actually see that some of the people are not
viremic at least by the measures you could determine in 1954, but they could
isolate virus from various organs. So,
that is of concern.
I
think as far as the issues for blood donor community, I think you just have to
go with your data, and the data show that of all the known transmitters that we
have, all were IgM antibody negative, and it is probably the best data you are
going to ever find.
As
far as the risk goes, there is probably hundreds or maybe, I don't know how
many, I haven't done the calculations, but based on my model, you can actually
do the math and figure out how many transfusion transmissions probably
occurred.
Our
system was probably extremely insensitive for determining transfusion
transmission because we were first focused on trying to find the sickest
people, to begin with, and second of all, all these cases came to light way
late in the season, after the initial organ donation cases occurred in early
September, so we really didn't have good surveillance for this all through the
season, we were just mainly picking up people towards the end.
DR.
DAWSON: Thank you.
MR.
CAVANAUGH: Dave Cavanaugh from the
Committee of Ten Thousand.
I
wanted to follow up. Dr. Williams asked
you a two-part question and one part didn't get answered, which was the
long-term effects, and since there was something in the press from Canada in
late February about 80 percent having chronic neurological damage of those
hospitalized, I was wondering if you could comment on that story and then on
the findings that you found.
DR.
PETERSEN: As far as the long-term
effects of West Nile virus infection, there are no good long-term studies
looking at the long-term effects of West Nile virus infection. We are in the process of doing those right
now.
Some
of the best data come from the New York City, people identified in the 1999 New
York City outbreak. These people were followed up a year later. About 50 percent of them reported, and this
is self-report, some kind of residual neurological problem, but well done, long-term
studies are lacking and we are in the process of doing them right now.
DR.
STRONG: I was interested in your
opinion of the progression of this epidemic.
We have seen mostly cumulative data.
What is happening on the back side of this, everything is moving west,
but what is happening, say, in New York with the surveillance data?
DR.
PETERSEN: That is actually a good
question. What has happened is that in
places where there has been West Nile virus infection in humans, that has
persisted in the same places. The story
has kind of got buried because there was a huge epidemic in the Midwest this
year, but if you looked at New York State, New York State, who has now had four
years of experience of the virus, had more cases last year than they have ever
had.
So,
where it has been, it appears to stay, and despite the huge epidemic last year,
in which my estimates are about 500,000 people became infected, that is only
about 2 percent of the population in that area, so there is stili a huge
susceptible group of people.
DR.
STRONG: And the bird population, are
the numbers there about the same, as well?
DR.
PETERSEN: The bird population, we don't
exactly know which species are the primary reservoir species, but we suspect
house sparrows are one major reservoir species, and what we found last year in
Louisiana was that about 30 percent of the house sparrows got actually infected
in that area where they have the huge epidemic and epizootic.
The
problem with these birds like house sparrows is they turn over every year and
so you have a whole new fresh susceptible crop the following year, so it is a
little bit hard to generalize from levels of birds what is going to happen in
people.
DR.
SCHMIDT: It has been said that as far
as testing blood donors, it won't be worthwhile in a few years because
everybody will have been exposed by mosquitoes, but what you just said, that is
not true.
DR.
PETERSEN: I can't predict what will
happen. If you look at what has happened with St. Louis encephalitis, which is
a related flavivirus, very closely related flavivirus, what you see with St.
Louis encephalitis outbreaks is that is focal epidemics involving hundreds of
people, there are occasional regional epidemics involving thousands of people
like what occurred with West Nile last year, and in some years there is nothing
that happens, there is a handful of cases, and trying to predict where and when
all these epidemics will happen is very, very difficult to determine.
Now,
is West Nile virus like St. Louis encephalitis? In some ways, it is, and, in fact, there was a very large
epidemic of St. Louis encephalitis in the Midwest in 1975, that actually,
interestingly enough, affected the same states, the same cities, and, in fact,
the same neighborhoods as the West Nile virus epidemic did last year.
So,
we think St. Louis encephalitis may be a good model for West Nile. The caveat is that West Nile virus produces
viremias in birds that are hundreds and millions of times higher than St. Louis
encephalitis virus.
We
think its epidemic potential has been much, much greater than for St. Louis
encephalitis virus.
DR.
NELSON: It also kills birds, which St.
Louis is unlikely to or has not been reported to do.
DR.
PETERSEN: Yes, West Nile kills birds
and St. Louis encephalitis doesn't, but that is actually another interesting
story, but the bottom line behind that story, which is quite fascinating, is
that it appears that it is not all strains of West Nile virus that kills birds,
it is very particular to the strain that is circulating here and the identical
strain that is circulating in Israel.
As
far as its killing birds, the species that are probably the most important for
maintaining the virus like house sparrows, which get huge viremias and there
are lots of those kinds of birds, the mortality rate is rather low, and it
kills 99 percent of American crows, but there are relatively few crows, so it
is probably not a species that maintains the virus.
DR.
NELSON: I think we need to move
on. Thanks, Lyle.
Sue
Stramer from the American Red Cross.
D.
Donor Serologic Studies of WNV 2002 Outbreak
Susan Stramer, Ph.D., ARC
DR.
STRAMER: Thank you.
First
of all, I would like to thank all the collaborators who I couldn't fit on the
slide, both from the Red Cross and outside of the Red Cross, who provided
information and data for this presentation.
[Slide.]
I
will cover two basic areas in this presentation, preclinical studies really
taking off where Lyle and Christina Giachetti left off just previously, cover
the case investigations that Lyle outlined, and prevalence studies that Lyle
called the viremia study.
The
two types of viremia are prevalence studies that I will talk about. One, the CDC study that Lyle has already
mentioned; and two, a Gen-Probe study or the Phase I study that will be part of
the IND for Gen-Probe.
Then,
I want to talk about our Phase II studies, which is really systemwide IND
implementation of West Nile virus testing, and some of my comments I believe
will be very helpful to this afternoon's discussion as the FDA goes through
guidelines for implementation.
Lastly,
I will talk about contingency planning, what happens, although we say we are
going to be testing by July 1st, what if the mosquitoes aren't quite as
compliant and we have West Nile human activity prior to that.
[Slide.]
The
first study involved the case investigations from CDC. Those were the possible transfusion
transmission cases during the last calendar year. We were involved in the investigation of 32 cases, and from those
32 cases we were able to obtain co-component frozen plasma from which the other
products from the donation were transfused and possibly associated with a West
Nile-positive recipient.
So,
many of these cases were described in the 21 confirmed cases by Lyle and the
other 21 potentially linked cases. In
those cases, as was described, red cell segments were tested from implicated
donations and follow-up samples from implicated donors.
We
also were able to obtain 383 frozen plasma unit samples from these 32
cases. They were tested by CDC TaqMan
RT-PCR assay, donor follow-up was tested by the IgM ELISA.
CDC
has two protocols for testing. One is
the standard input assay which was done on all of these samples, however, if a
donor was found to be IgM-positive and negative on the standard input assay, a
second assay was used, which is the high input TaqMan assay, which has about a
10-fold increase in sensitivity.
One
other comment I would like to make about these 383 plasma, they were in a panel, so I will not only
show CDC data, but I will also show panel results from Gen-Probe. We have also
sent the panel to National Genetics Institute, and we will have those data
soon. They have also been sent to
Tetracore as has been referenced, and any manufacturer who is interested in
testing these samples may do so.
[Slide.]
So,
the 383, and many of these were already described, but you will see their
context a little bit differently, we had 11 total RNA positives from either the
retrieved frozen plasma unit or the stored red blood cell segment.
Ten
were plasma reactive and 2 of these 10 were also reactive in the segment. Two of the 10 were nonreactive by the
standard PCR assay and required the high input PCR assay for reactivity.
The
11th, or one additional, was plasma equivocal, but segment was reactive, and
this result possibly was false positive.
When we look through the data, you will see that.
Eight
of the 11 had follow-up samples collected and in all 8, IgM was demonstrated
upon follow-up.
[Slide.]
Now,
these are the Gen-Probe results for the same 383 specimens. There were 15 initially reactive and 2
within a 10 percent negative gray zone.
So, those were all retested.
Three, the initially reactive, were IR, that is, initially reactive only
the reactivity did not repeat.
The
2 gray zone samples also did not repeat.
Of the 12 that were repeat reactive, 11 of 12 had very high
signal-to-cutoff ratios and had reactivity in replicates at 1 to 8, and 1 to 16
dilutions, also with very high S to CO values.
One
of the 12, and this is the case Lyle ended with discussing, with the equivocal
IgM results, had a moderate S to CO value with plus/minus reactivity at both
the 1 to 8 and 1 to 16 dilutions.
Eleven
of the 12 were the ones I mentioned that were reactive at CDC, so in comparison
with Gen-Probe, 100 percent of the CDC reactives confirmed, and here we had 9
of 12 donors with follow-up samples available.
All 9 demonstrated IgM positivity on follow-up.
[Slide.]
This
is a line listing and I will show three slides of this, of increasing
complexity. So, we will start with the
simplest first.
These
are the cases. These are the
CDC-positive results. Here you can see
2 that were positive in the red cell segment and the plasma. These are very interesting samples that have
the strongest reactivity, as you will see in the next slide, and represented
the points closest from collection to transfusion that Lyle showed in the
previous presentation.
These
were the two samples that were initially negative, but then were positive
following high input PCR. these
represent means of either duplicate or triplicate testing in the Gen-Probe
assay. Each color is a different
dilution. This shows you the IgM data
on the last column.
These
three were not considered confirmed cases according to the CDC definition
because they did not have IgM seroconversion in the donor, and this is the IgM
equivocal case.
[Slide.]
So,
how what I have added are the CDC plaque-forming units/mL or way to get an idea
of how much virus was present in all of these samples. The PFU was derived from a standard curve of
previously calculated viral PFU/mL, and the standard is run with each TaqMan
assay.
So,
here are the two samples that were positive in both red cell segments and
plasma. You can see here they have the
highest viral loads at least by PFU/mL, and the red cell segment, as Lyle
pointed out, was not as reliable, and there are lower viral loads from the red
cell segment.
Here
is the sample I referred to that is potentially a false positive since the
plasma result only gave us an equivocal result.
[Slide.]
This
was the center column that were color coded. These are the replicate
signal-to-cutoff ratio in the Gen-Probe test.
I show you this for the reason that these samples that were linked to
transfusion transmission, and were the most important, these were the
IgM-negative samples also had great dilutional capacity and that they were
detected, except for the one sample which we will get back to, by all
dilutions, and you don't see the dilutional strength decreasing at a 1 to 16
dilution. If I just show you plus or
minus results, you don't know from the plus value how strong that signal is and
the confidence of that positive result.
Here,
you see the one weakly reactive RNA-positive sample, replicates were reactive,
but then as you go into further dilutions, we did have discordant results.
[Slide.]
Moving
into the next study, we have created at the Red Cross, a repository of 89,000
samples, and these were collected from 6 high-risk regions during the West Nile
virus epidemic last year. These have
been stored frozen, and the samples have been saved for West Nile virus study
with IRB approval using a waiver of informed consent for these studies.
Samples
have been retained in a database with basic demographic information. 8,000 are in the process of being tested by
CDC using the high input PCR assay, and this is the viremia study that Lyle
mentioned.
They
are composed of the first three weeks of September from Cleveland and two weeks
from Detroit. We selected these by
metropolitan zip codes, and the preliminary results to date have already been
discussed. We have six positive of
5,761 and many more with equivocal results or marginal reactivity seen at long
cycle times, so we will have to do additional testing to determine how many of
these equivocal and other borderline reactives are true positives.
[Slide.]
So,
what we are doing with the bolus of these samples from this repository is we
will do a Phase I preclinical study.
The test samples will be tested individually and we are targeting about
35,000 to 50,000. The purpose of the study will be to define the West Nile
virus RNA prevalence and risk during the 2002 U.S. epidemic, determine the
efficacy of pooling. From positives, we
can determine the viral loads, we can perform 1 to 16 dilutions or 1 to 8
dilutions, and be able to see their reactivity.
This
will also serve to begin clinical sensitivity validations for the Gen-Probe
assay. This will be a linked study, so
we will be able to go back to donors and identify IgM seroconversion to confirm
the test results, that is, the RNA test results, and we hope to go back to the
consignees, be able to identify the recipients and determine the infectivity of
transfused products.
[Slide.]
These
two slides just show the samples as we collected them by week beginning with
the first week of September. This is
when I requested samples be retained, and they really kicked in at about
September 5th. So, these are samples
that we collected from St. Louis, Detroit, and Cleveland.
[Slide.]
Memphis,
Gulf Coast. These represent our
Mississippi collections. We have fewer
collections in the Chicago area, but whatever they are will be tested.
[Slide.]
The
next two slides are older slides of Lyle's, but it was the information we had
at the time that we created the repository and served as the basis for some of
the site selection. So, these are five
of the cities that we have selected and at that point in time, what their estimated
average risk was. Mississippi is not on
here, but we know the epidemic occurred a little bit earlier and Lyle showed
about 2 per 10,000 is the average risk.
[Slide.]
At
the time, this has been more fine-tuned in the graph that Lyle showed, but it
just shows the relative risk at the time we pooled the repository samples for
the various areas that appear on the repository.
[Slide.]
How
will we do the study? The testing was
slated to occur in March. Now, we have
slipped to April. Our FDA inspection
team in the Southern California area helped us move the study out a little bit
by doing an inspection of the laboratory, but they are now gone fortunately.
Anyway,
so we are targeting now to begin the study at least instrument validation and
training mid-April and towards the end of April we will start testing the first
samples.
Study
duration will be a minimum of nine weeks.
It will involve staff from all of our NAT labs and that will facilitate
their early training on the assay. The
testing will occur by week starting with samples from the first week in
September and continue until two weeks of NAT-negative results are obtained.
If
you remember, the previous slide I showed you at the beginning of September,
the peak was the highest and we believe with the first couple of weeks of
testing, it will rapidly reach two weeks of NAT-negative results because the
curve declined very rapidly.
We
will use IND reagents and a draft package insert. Final assay software, we will not have available. I mentioned this is a linked study, so
positive donors and recipients will be contacted for samples. We will use a similar questionnaire to that,
that was used for the CDC case investigations, and the data will be submitted
to FDA in support of pools of 16 and the use of the Gen-Probe reagents.
[Slide.]
Then,
our implementation for Phase II will be our full scale, as I just said,
implementation, which has been approved through management at Red Cross. We are targeting, as the rest of the
industry is, an implementation date of July 1st.
We
will use the same NAT labs within the Red Cross that do HIV and HCV testing,
using the same pool of 16, but using a Gen-Probe West Nile assay in addition to
the HIV and HCV assay. We will have a
"companion" IND with Gen-Probe, and the study has already been
submitted to our IRB for review.
During
the first three months of testing, we will have to report results from the
testing labs to the regions using a manual reporting process that is fairly
comparable to what we used when we launched HIV and HCV NAT in 1999.
[Slide.]
Our
protocol for testing will be again pools of 16. If a pool of 16 is reactive, they will be resolved to the
individual NAT reactive donation. All
nonreactive pools, the donations from those will be released and nonreactive
individual donations from reactive pools will also be released.
I
want to mention, because FDA will discuss this, this afternoon, NAT negative
donations in NAT reactive unresolved pools, that is, if a pool is reactive, but
all 16 donations test negative, will be released based on the efficacy of the
model we have for HIV and HCV NAT, and that model efficacy was based on the
fact that reactivity of NAT reactive unresolved pools could never be
reproduced, and many studies were done trying to reproduce unresolved NAT pool
reactivity.
Also,
at the Red Cross, we have done extensive follow-up studies demonstrating no NAT
reactivity or seroconversion upon subsequent donation from donors in NAT
reactive pools, and this included 15,689 donors.
We
all know and recognize that the undiluted sample represents the gold standard,
and this is a quote from the FDA document.
"The test result on the individual donation is considered to
indicate the infectious status of the donor."
So,
we believe that those donations from NAT reactive pools that don't resolve are
acceptable for use.
[Slide.]
We
will confirm NAT reactivity by an alternate NAT method, most likely we will do
PCR once we have NGI sensitivity, that is likely the procedure we will
use. We will confirm NAT in both the
source tube and the retrieved plasma unit, which we will try to collect from
each region as we do for HIV and HCV.
We
will also do investigational IgM testing assuming we find an IgM test that will
be available in the July time frame.
Otherwise, we will have to use CDC or a reference lab. We will have to plan contingencies for that,
but that will also be done in the source tube and the retrieved plasma unit.
Our
policy for retrieval and lookback under the IND period will be donations for
the prior 28 days from NAT reactive donors.
[Slide.]
NAT
reactive donors will be temporarily deferred until follow-up testing has been
performed, and this is the criteria of our follow-up test results. If the donor demonstrates West Nile RNA
clearance by individual NAT, which is how we will do the follow-up testing, and
they produce IgM, the donor can be reinstated as long as 28 days from index has
elapsed.
If
the donor is RNA positive, another follow-up sample is required until they
demonstrate clearance of virus.
If
the donor is RNA negative, and IgM negative upon follow-up, we will yet request
another follow-up if the donor has been confirmed in index by alternate NAT,
meaning we know that this is a true positive sample. The reason we want another follow-up sample is we want to eliminate
the possibility of intermittent viremia, which we don't believe occurs with
West Nile, but that has never been proven.
Lastly,
if the donation is RNA negative, IgM negative, and has not been confirmed by
alternate NAT, no further follow-up of the donor is necessary because that
would be classified as a false positive on the test of record screening.
At
the end of the study period, donors not enrolling in follow-up will be
automatically reinstated and we will also use a questionnaire for positive donors.
[Slide.]
Why
follow West Nile virus NAT-reactive donors?
Well,
under an IND, and actually in a complex operational setting, a single protocol
leads to the fewest errors. Also, the
additional data provides donor the complete information on the meaning of their
reactive investigational test results.
Another
reason is we cannot mandate when a donor will provide a follow-up sample, and
we need to wait for all of the follow-up results prior to reinstatement, so I
just give you a little example to ponder.
If
the donor provides a follow-up sample on day 27, and then if we are allowed to
reverse deferral, the donor could donate on day 28, the unit could be
transfused, the components could be transfused.
We
then subsequently, based on the follow-up sample, could get NAT-positive
results and then be in a very sticky situation.
Really,
the major reasons have to do with the performance of a test under IND, in which
the performance characteristics of the investigational test and viral dynamics
in West Nile virus human infection is not well established. Without the follow-up data including all the
IgM data, we will never be able to prove this.
True
confirmation of infection is IgM seroconversion.
[Slide.]
What
will we do for contingency planning?
Well, this is currently what we have in the mill. We are trying to create a three-month target
of FFP, however, production and storage is logistically difficult.
We
also, as I mentioned, in the prevalence study, we will have one lab up and
testing, so in the event that there is a small outbreak or there is a small
number of donations that need to be tested, we will at least have one lab up
with limited capacity.
Lastly,
we will create a systemwide 30-day inventory of NAT tubes and perhaps
associated pools. We have to work out
the logistics for testing once we bring the test up systemwide under IND.
These
30-day inventory tubes can be used as needed, depending on reports of human
West Nile cases for the entire system or just, as I said, as needed, if there
is specific focal points for testing.
Thank
you.
DR.
NELSON: Thank you.
Questions
or comments?
DR.
STRONG: You have got Phase II. This is not Phase IIA, right?
DR.
STRAMER: This is Phase II, yes,
correct.
DR.
STRONG: So, you will be doing
pre-release testing.
DR.
STRAMER: Yes. What Mike is referring to, in the beginning of NAT, there were
multiple phases of the rollout of NAT, and many blood centers including the Red
Cross only released frozen products based on the results of NAT, and fresh
products were released, labile products, even before we had the NAT results
because of turnaround time issues, et cetera.
When
this test is released, I believe the entire industry will do real-time testing,
and no product will be released without the benefit of a NAT-negative West Nile
result.
DR.
STRONG: Do you have any information on
the stability of the sample? You have
got segments that are not showing as a high a viral load as the unit. Is that because of degradation or is that
just because it's a small sample, do you know?
DR.
STRAMER: The red cell segments are
highly diluted, so it could be a combination of instability to the red cell
degradation and then viral degradation, or it can be purely a dilutional effect
of the way we have the red cell segments saved.
DR.
STRONG: What about plasma versus
serum? Are your archived samples--
DR.
STRAMER: Yes, all of our archived
samples are EDTA plasma stored in Becton Dickinson plasma preparation
tubes. Stability studies, unlike for
HIV and HCV NAT, where we were very aggressive in participating in the
stability studies, we are not doing any stability studies. We are leaving those for the manufacturer,
but our assumption is we are already processing NAT under worst case storage
conditions, because HCV is very labile, and I would anticipate that West Nile
is not going to differ from HCV.
DR.
DAWSON: I have two questions. One, in your initial screen of the 5,700
samples, you found I think 6 positives.
Were any of those tested for IgM?
DR.
STRAMER: Not yet, no.
DR.
DAWSON: That will be interesting to see
that.
The
second thing is you mentioned using IgM testing in your testing algorithm and
that you are going to five or six sites.
How many tests are you planning on that, approximately, over the season
to need, and how many would be ideal to have for IgM? Just ballpark.
DR.
STRAMER: It depends how many reactives
we get. I mean that is really
unknown. If we have no prevalence, we
will not use a single IgM test.
DR.
DAWSON: So, when you said IgM as
needed, what you meant was that if there were NAT-positive, you would be
testing those donors and follow-ups for IgM.
DR.
STRAMER: That's right, and it is part
of our confirmatory.
DR.
DAWSON: You weren't saying that a
co-screening with IgM and RNA?
DR.
STRAMER: That is not our plan, and I
also want to say the plan I presented is really what we can do, our system is at saturation. If we hope to be doing anything by July 1st,
we have to make the systems as analogous to our current systems as possible,
otherwise, we don't have rapid implementation.
DR.
DAWSON: Would it be possible for you to
send us under code those six samples that you found positive, so we can test
them for IgM, IgG, whatever else we have and any excess you have of the 21
transfusion-associated cases, I would like to try our hand again, send them
under code, let's see what we find serologically, so that I can know with a
clear conscience and data that we are not shunting any IgM because we do want
to participate as needed in IgM testing, and screening included.
DR.
STRAMER: For samples we have units
from, such as the 32 case investigations with 383 frozen plasma units, you
know, everyone is interested in having volume of those. We certainly will be
willing to share some of that volume for IgM testing.
Now,
regarding the repository samples, the ones that were first tested by CDC, those
will be tested also by the Gen-Probe assay first, and then positives will also
go on to NGI for confirmation and quantitation. We also want to perform dilutional studies on those, and we would
like to, in addition to that, perform IgM.
Just how far can you stretch a 2 mL PPT will be the question.
DR.
DAWSON: Okay. I am still interested, so keep us in mind.
DR.
STRAMER: All right. I know your number.
DR.
KLEIN: Sue, do I understand that the
first dozen specimens from donors that you showed, that 3 specimens tested
positive for RNA in two separate laboratories by two different technologies,
but negative for IgM, and are considered false positives, is that correct?
DR.
STRAMER: No. The 12 samples that I showed, 11 of which were positive at CDC,
12 were all positive at Gen-Probe; 9 of the 12 were positive for IgM in donor
follow-up, but negative for IgM at index.
The
one index sample that was equivocal, it had a variable set of test results at
CDC, but when all was said and done, I have like several e-mails that say, in
all capital letters, this was not IgM positive in index.
That
sample demonstrated IgM at index, demonstrated IgM positivity on follow-up, but
let's say very weak RNA reactivity at index and very weak reactivity of IgM at
index. So, it was kind of a cross-over
sample, you know, so you can imagine RNA coming down and IgM going up.
DR.
BUSCH: I just wanted to clarify. The one-month inventory that you talked
about at the end, is that going to be something that you are going to
systematically do, henceforth, you are going freeze down and retain both the
pool and the individuals for all your NAT screening, or is this just a contingency
that will be put in place transiently to potentially need to test, and would
that testing be done only in hot sites or if testing were delayed, you don't
intend to back-test the entire inventory for a month once testing is licensed
or IND available?
DR.
STRAMER: What we are doing now, if we
don't plan our contingencies now, they are not going to happen, especially in a
system like ours that is complex.
So,
in order for us to be ready for any event, we can store probably 30--what we
talked about is a 30-day inventory.
Those PPDs would be frozen down, and in order to even do this, you know,
freezers have to be obtained, they have to be temperature mapped, all the
validation, so it is not an easy undertaking.
We
would store those samples in the event that there are pockets of West Nile
human activity, and then we would have to decide within Red Cross, within the
blood industry, do we just test those pockets that had West Nile activity, or
do we test the entire United States, I mean our entire inventory.
I
think we don't know that as of yet, but if we don't have the samples, we won't
have the ability to make that decision.
DR.
EPSTEIN: Sue, I just want to focus
again on this issue of low-titer samples and possible correlation with
IgM. It seems to me that we have two
pieces of data that are pertinent.
Lyle
has showed us that in the transfusion transmission studies, all of the index
samples in case of transmission were RNA positive. On the other hand, Andy Conrad showed us that if you take
community-based surveillance samples, it is not at all uncommon to find
low-titer viremic samples that have a positive IgM.
What
we don't know is whether samples of the latter sort (a) transmit, and (b)
whether they would be picked up or missed in a minipool screening assay. So, I think that it becomes very important
for us to design the Phase II trial based on the result of the Phase I trial.
In
other words, when you run through the first 35 to 50,000, you presumably will
find some positives. If there are false
negatives at 1 to 8, or 1 to 16, I think we then have an open question whether
we need to look for IgM when we move to Phase II.
It
would be very informative, of course, whether those samples that are false
negative at 1 to 8, or 1 to 16, have positive IgM. If they don't, we don't expect to find anything in Phase II, but
if they do, then, they are more like the samples that Andy Conrad showed us,
and I would think that even though we haven't proven that such a configuration
is associated with transmission, we wouldn't want to overlook that possibility
when we do the screening studies prospectively.
So,
I am just a little bit uncomfortable that we know what to do in Phase I until
we see what we get in Phase II--I am sorry, the other way around--
DR.
STRAMER: Right, the other way around.
DR.
EPSTEIN: --what to do in Phase II until
we see what we get in Phase I.
DR.
STRAMER: Phase I, all the samples will
be tested neat, so we will be able to look at dilutional consequences, and we
will be able to look at IgM in those samples.
DR.
EPSTEIN: A second point. I was a little bit unclear, when you talked
about further testing of the NAT-reactive donors at 28 days. Not all donors will be retested. I mean I agree fully if you get a reactive
test on follow-up, you want to not re-enter the donor until they have ended up
retested or negative, so they remain deferred pending further outcome.
But
what about the donors that don't present for their follow-up, is the plan to
hold them indefinitely in abeyance?
What you said is that they would be re-entered at the end of the study,
and I guess the question is whether the non-returns could be re-entered at day
28 automatically or day 56 automatically.
DR.
STRAMER: We have had many discussions
internally about this, and that is why I went through that slide
specifically. It is very difficult to
design a study operationally that allows automatic reinstatement and follow-up
to occur at the same time, because errors will result and we have become very
error-averse.
So,
the easiest way for us to do the study and to guarantee that we have IgM data
is to require the follow-up sample, and if the follow-up sample clears virus,
demonstrates IgM seroconversion, the donor is automatically reinstated.
Now,
what happens with those other donors, really, the way we have the study set up
now, it depends on the length of the study.
If the IND period goes one year, those donors then would have to be
temporarily deferred for that period of time.
If
we learn more about the study as we are progressing through the study, we can
change policies prior to that, but at this point, we don't know the performance
characteristics of the test. The only
way to prove the performance characteristics of the test is to make sure we get
the IgM data and demonstrate seroconversion.
DR.
PHELPS: What is very important is that
the way that Sue designed the study, is that she will be looking at the
individual sample, because we believe the comparable sensitivity would have
caught all the viremic samples that I presented in those clinical cases, and
those clinical cases were people who again reported that they were febrile.
So,
I think that the role of IgM will be much more clearly sorted out under Sue's
paradigm where since she is testing an individual, if they remain viremic, wait
a period of time, then, become non-viremic, the importance of IgM will sort
itself out under her algorithm, because we really chose a different paradigm,
in a sense, she will be testing individually, at least we will track that data.
Now,
when you jump up to pools of 16, that may become relevant, but I think we will
learn that data in Sue's study.
DR.
GOLDSMITH: Did you address the issue of
the turnaround time, that you will be able to test the more labile cellular
components?
DR.
STRAMER: I didn't show all of those
logistics. When the five laboratories
were polled as far as increased workload and delay in turnaround time, so we
know what to communicate to our hospitals, there will be a one- to two-hour
delay in turnaround time due to testing, but that will be absorbed in our
normal process, so at the end of the day, there really won't be an ultimate
delay in turnaround time. At least
that's what they tell me. We will track
that and let you know or you will let us know.
DR.
NELSON: Thanks.
We
are going to have to break for lunch now.
[Whereupon,
at 12:30 p.m., the proceedings were recessed, to be resumed at 2:30 p.m.]
AFTERNOON PROCEEDINGS
[2:30 p.m.]
DR.
SMALLWOOD: We are a bit behind, but
hopefully, we can make up some time.
The
next topic is Regulatory Pathway for West Nile Virus Testing, FDA Update. The first speaker is Martin Ruta, talking
about Guidance for Industry.
E.
Regulatory Pathway for WNV Testing (FDA Update)
Guidance for Industry
Martin Ruta, J.D., Ph.D.
DR.
RUTA: Thank you, Dr. Nelson.
I
want to thank everyone for participating today particularly Lyle from CDC, who
interrupted his vacation, and device manufacturers, who came and presented a
lot of interesting data this morning.
What
you are going to hear now are a series of talks, first, starting off with mine,
about the guidance document that we issued last fall, last October, plus some
industry action that was voluntarily taken after that.
Then,
you are going to hear some additional talks from folks at FDA about our
thinking about managing donors and units in the face of testing and raising
some questions and options that we have as we enter into the next season that
Dr. Williams will present.
[Slide.]
What
I want to do is go through a guidance document that we issued on October 25th,
2002, but as part of background, last August, we were dealing with West Nile
virus as a theoretical risk to the blood supply and very quickly it became a
real concern in organ transplantation and soon thereafter in blood transfusion.
[Slide.]
Since
August, blood transmission by West Nile virus was confirmed, and that is the
first time it was confirmed in this outbreak, so this is probably the most
studied investigation of West Nile and West Nile epidemic that there has ever
been, so we are learning a lot of new things.
Part
of the assumptions that we made in coming up with our original guidance, you
know, we have to look back and see are still valid. So, in order to assure the safety of the blood supply in the face
of the risk of West Nile virus, we took two initiatives.
One
is guidance on the management of donors and blood components, and the second is
to facilitate rapid development of donor screening tests.
[Slide.]
So,
there were some assumptions going into the guidance and some that came up
afterwards. One of the assumptions is
that human infections were expected to occur seasonally during periods of
mosquito activity, and we weren't sure if this would continue to occur
year-round in some parts of the country.
We thought in the southern states there might be ongoing infection, but
my understanding is that there hasn't been a case reported since last December.
About
80 percent of infected persons are asymptomatic, and many of the 20 percent of
individuals will have mild symptoms that are nonspecific, or flu-like illness,
and again, as we have heard, 1 in 150 develops more severe symptoms.
The
ratio of the viremia can be up to 29 days. This is based on a 1950 study that
we have heard about of injecting cancer patients with the virus.
Continuing
with the assumptions, viremia resolves rapidly after seroconversion to IgM, and
we have seen that IgM positivity can persist for going on two years.
We
know of no chronic carrier state for this virus, and the last bullet is
something we didn't know about prior to the guidance, that we have learned
about since, and you have heard this morning, is that there can be donors who
exhibit symptoms prior to donation, are apparently healthy on the day of
donation, and can transmit West Nile virus.
[Slide.]
What
I want to do is walk you through the guidance document that we issue last
October, and then you will hear further thoughts later on from Dr. Williams.
So,
we issued a guidance on Recommendations for the Assessment of Donor Suitability
and Blood and Blood Product Safety in Cases of Known or Suspected West Nile
Virus Infection.
[Slide.]
The
recommendations included that donors or potential donors with the medical
diagnosis of West Nile virus infection be deferred until 14 days after the
condition is considered to be resolved and at least 28 days from onset of
symptoms or diagnosis, whichever is the later date. At the time, our current thinking was that in the absence of
current or recent symptoms, an IgM positive antibody test result alone should
not be grounds for deferral.
[Slide.]
Donors
who report an otherwise unexplained post-donation febrile illness suggestive of
West Nile virus infection in the setting of West Nile virus transmission in the
community. Again, the trigger here that
we told people for last fall was human cases in the community, those donors
should be deferred for 28 days from the onset of illness or 14 days after the
condition is considered to be resolved, whichever is the later date.
Donors
whose blood or blood components were received by a patient with a possible case
of transfusion- related West Nile virus should be deferred for 28 days from the
date of potential transmission. Bear in
mind that there was a very intensive epidemiologic investigation that was going
on last fall, in which CDC was doing a lot of work to investigate whether
transmission was actually occurring through blood and, in collaboration with
the blood organizations, were identifying possible donors who may have been
implicated in transmission.
[Slide.]
FDA
recommended that blood establishments actively encourage donors to report
post-donation illnesses that might potentially be associated with West Nile
virus, that is, flu-like symptoms that include a fever, occurring within two
weeks of blood donation in the setting of active West Nile virus transmission
in the community. These are
post-donation reports.
[Slide.]
In
terms of managing the units from the donors, we recommended that in-date
components from current, prior, and subsequent collections be quarantined and
retrieved if a donor later reports a medical diagnosis of West Nile virus.
The
product quarantine and retrieval should cover a time period dating back to 14
days prior to the onset of illness and 28 days subsequent to the onset of
illness. Again, our thinking at the
time is that in the absence of symptoms, an IgM-positive antibody test result
should be grounds for product quarantine and retrieval.
Medical
directors should exercise judgment when a donor reports an otherwise
unexplained post-donation febrile illness in the setting of active West Nile
virus transmission in the community.
[Slide.]
Again
reminding everyone that there was a very intense epidemiologic investigation
and that donors were considered to be potentially associated with transmission
of West Nile virus if the infected recipient received the donor's blood
components within 28 days before the onset of symptoms in the recipient.
For
each associated donor, product quarantine and retrieval should occur for the
in-date components that were collected in a 28-day window period prior to, and
subsequent to, the suspect donation that might have been implicated in that
transmission.
[Slide.]
We
also recommended that when a blood establishment received information that a
donor has a medical diagnosis of West Nile virus, the blood establishment
should notify transfusion services to permit lookback recipient tracing and
notification.
If
the post-donation illness was not diagnosed as West Nile virus infection,
actions to identify prior recipients were not appropriate.
In
the context of the epidemiological investigation, if there was a suggestion
that a specific donor was a likely source, you know, again, there were cases
where many donors were being investigated, but if there was one there that
might have been a likely source of transmission of West Nile virus to a
transfusion recipient, the blood establishment should consider lookback
notification of other recipients.
[Slide.]
That
is the end of the guidance document that we issued. There were a number of other events that occurred after that, and
I just want to highlight one of them.
That
was an action taken by industry, and this was a voluntary action to withdraw
products from the market. On December
12th of 2002, the AABB, ABC, ARC, and DoD put in place a voluntary market
withdrawal of selected frozen transfusable in-date products collected in areas
that experienced West Nile virus during 2002, as a preventive measure, to
mitigate against the risk of transmission of West Nile virus through blood
transfusion.
States
were identified by the CDC and time periods identified as to when there were
peaks in transmission, and the risk period was defined as seven days prior to
the onset of symptoms of the first encephalitis case, and seven days after the
onset of symptoms of the last encephalitis case in the respective state.
That
is the end of my presentation and it kind of brings us up to date where we were
as of last fall in our thinking that led to the guidance. Again, we are dealing with a very intensive
investigation, the most intensive investigation that has ever occurred for this
virus.
You
are going to hear from Dr. Hewlett some thoughts on donor and unit management
in the context of testing under IND, and from Dr. Williams about some of our
current thoughts or possible options for donor and unit management in case
testing might not be on line for the whole country if there is a West Nile
transmission again this season.
DR.
NELSON: Thank you.
Are
there questions? Okay.
Next,
is Dr. Robin Biswas, who is going to talk about the approval criteria for West
Nile virus testing.
Approval Criteria - Robin Biswas, M.D.
DR.
BISWAS: Good afternoon.
The
next three presentations are closely related and I will go through my slides
quickly to save time and also because a lot of what I will say will be gone
into in more detail by Indira Hewlett and Maria Rios, the following speakers.
[Slide.]
In
regard to the approval mechanisms for assays used to test donors for West Nile
virus, what you should know is that tests used in the manufacture of blood and
blood components are reviewed by FDA as biologic products under the PHS Act.
This
means that a manufacturer needs to present to FDA, an Investigational New Drug
application, which is sort of a trial plan for the device, and after FDA
approval of the IND, i.e., that plan, the manufacturer goes out, gathers the
data, and then presents the data to FDA as a Biological License Application.
[Slide.]
This
is just the list of the things to consider.
[Slide.]
In
regard to clinical sensitivity, what we mean by that is the reactivity of the
assay in individuals with known West Nile virus infection. So, you can test samples from persons with
clinical West Nile virus infection, and another way to do that is to test
samples in epidemic West Nile virus regions.
[Slide.]
In
regard to analytical sensitivity, we mean by that the reactivity of the assay
in samples containing the analyte of interest, for example, West Nile virus
RNA.
To
do that, to get a handle on that, you can test serial dilutions of samples with
the RNA or test serial bleeds from individuals with the disease; if possible,
samples collected closely together very early in the infection. Of course, for this to be useful, you need
standard reagents with defined units, and Maria Rios will be going into that in
more detail.
[Slide.]
Now,
early estimations, we were told that the viral load in samples associated with
the transmissions, as Lyle Petersen has described this morning, that the viral
load varied from about 3,000 to 5,000 copies/mL, so our current thinking is
that the sensitivity of the NAT should be targeted at least 100 copies/mL in
the individual donation to ensure 100 percent detection of 1,000 copies/mL in
the individual donation.
Of
course, as data is gathered, and it is being gathered at a great rate, and as
technology improves, this target might change.
[Slide.]
Clinical
sensitivity refers to the reactivity of an assay in individuals without West
Nile virus infection, and you would like that to be low in the blood bank
setting, of course. Otherwise, you are
not going to use many suitable units.
You can study this by testing healthy individuals, such as blood donors,
persons in winter or in non-epidemic regions.
One needs also to test individuals with other diseases.
I
have included up there the other related viruses. We said earlier that we would encourage manufacturers to design
their tests, so that they could pick up these, as well, but, of course, there
is a problem in getting samples. I have
sample scarcity there. It seems to be
more a problem of getting ahold of those samples, they may be available elsewhere
in other parts of the world.
[Slide.]
In
regard to clinical specificity, the assay should be tested in the end-user
setting with a U.S. donor population, in geographically separated donor sites,
at least three lots of the assay, and confirmatory additional and supplemental
testing needs also to be addressed, and Indira will go into that in more detail
in a moment.
[Slide.]
In
regard to analytical specificity, this is the reactivity of the assays with
samples not containing the analyte of interest, and obviously, it overlaps with
clinical specificity.
You
can test samples with other disease markers, and you can test samples with
potentially interfering substances to assure that the assay has very good
specificity.
[Slide.]
This
slide deals with controls during the actual manufacture of the kit. Controls and calibrations during the kit
manufacture are needed to assure lot-to-lot consistency of assay reagents and
the finished kits.
The
control reagents need to have varying degrees of reactivity, and the endpoint
titration curves from testing the assembled kit with panels of samples should
have slopes and midpoints that fall within validated acceptable limits.
[Slide.]
In
regard to assay stability, studies on at least three lots are needed to
demonstrate stability claims for whatever time periods or under what conditions
that the company claims.
[Slide.]
In
regard to reproducibility and operator proficiency, a panel of plasma and sera,
composed of positive, negative, and weakly reactive sera, should be tested in
at least three sites with different operators with at least three lots of the
assay.
Each
study site should demonstrate operator proficiency with the panel before
screening donors.
[Slide.]
in
regard to instrument and software, the instrument and software portion of the
application should be included in the BLA, in the Biological License
Application.
If
it has been approved or cleared for another use--that mean another assay--you
need to submit data demonstrating that it meets the new intended use of the
assay that you want to now get approved.
There
is a CDRH guidance for this, and I have it up there.
[Slide.]
Additional
guidance documents, I have listed here.
There is a guidance in regard to the HIV NAT tests that came out in
1999, which is very helpful, and considerations in this document are applicable
to other gene-based tests.
[Slide.]
Another
guidance document, a rather old one, but still very relevant, is this one that
I have here from August 8th, 1989, about anti-HIV test development.
[Slide.]
The
way forward. We will continue to work
with other components of the Department, with NIH and CDC, and the
manufacturers and blood organizations to facilitate assay development.
If
necessary, FDA would allow widespread study of appropriate tests under
IND. I think Indira will be going into
that in more detail.
Sponsors
are asked to seek FDA's guidance and to submit a pre-proposal before initiating
studies to support an IND.
Thank
you very much.
DR.
NELSON: Thank you, Robin.
Questions
or comments? Okay.
Next,
is Dr. Indira Hewlett, who is going to talk about clinical study design, unit
and donor management.
Clinical Study Design, Unit and Donor
Management
Indira Hewlett, Ph.D.
[Slide.]
DR.
HEWLETT: I am going to be discussing
FDA's current thinking in regard to clinical trial design for validation of West
Nile assays and some proposed approaches to unit and donor management based on
test results.
[Slide.]
By
way of background, all reported transmissions by blood transfusion has occurred
in the acute, viremic phase, indicating that this phase of infection would be
the ideal target for intervention.
Therefore,
NAT may be the most appropriate strategy to interdict infectious donations.
Mostly
likely, NAT on pooled donations will be implemented first due to practical
considerations and since platforms for HIV and HCV NAT on pooled plasma have
already been licensed.
But
there are concerns about the impact of pooling on the sensitivity of NAT assays
and the ability to detect specimens with low levels of virus.
[Slide.]
Also,
it is known that viremia and detectable IgM co-exist in the acute, late phase
of infection--there has been a lot of discussion about it this morning--but
lack of data on West Nile virus transmission by donations, that are minipool
NAT negative and IgM positive, suggests that it may be useful to explore
whether a strategy involving a combination of NAT and IgM testing would need to
be considered in IND studies.
Finally,
although there is lack of evidence of West Nile virus transmission by plasma
derivatives, screening of source plasma collections may increase the margin of
safety achieved by virus removal and inactivation methods.
[Slide.]
FDA
recognizes the need to implement testing in a timely manner and would therefore
allow large-scale studies and widespread use of tests under the Investigational
New Drug Application if necessary, similar to HIV and HCV NAT implementation.
This
approach would facilitate introduction of testing in blood and plasma centers
while evaluating test performance.
FDA
would recommend routine use of licensed screening tests to detect acute donor
infections with West Nile virus when tests are available.
[Slide.]
As
part of clinical validation, FDA has traditionally required that a test be
evaluated for clinical specificity, clinical sensitivity, and reproducibility.
I
will be discussing clinical specificity and sensitivity. Reproducibility has already been discussed
by the previous speaker, Robin Biswas.
[Slide.]
For
validation of the clinical specificity of a NAT or IgM assay for West Nile
virus, the test would have to be evaluated in a low-risk population using
specimens from low-prevalence areas.
In
general, FDA considers a sample size of 10,000 tests, pooled or individual, to
be adequate for clinical specificity determinations. Data on clinical specificity could be obtained by identification
of negative cases during prospective studies conducted at various clinical
sites under IND and retrospective studies of repository specimens identified in
clinical and donor settings during previous epidemics.
[Slide.]
For
investigational NAT assays, clinical specificity of NAT false positive or
reactive specimens could be established by additional testing with an alternate
NAT, that is, a NAT assay which uses primers from a different region of the
viral genome, IgM, and follow-up testing.
Investigational
IgM test results could be confirmed using a NAT and a second IgM test and
discordant IgM results resolved by plaque reduction neutralization assays.
At
this time, testing of all donations by both NAT and IgM during clinical trials
is something that may need to be considered since testing of minipool and IgM
assays are not well established.
[Slide.]
For
clinical sensitivity determinations, limited numbers of samples exist,
therefore, multiple approaches may need to be considered.
Clinical
sensitivity for both NAT and IgM could be determined using repository specimens
including transfusion and West Nile virus illness related, community-based
specimens from donor and community settings.
In
addition, identification of positive cases during prospective IND studies
conducted in blood bank and community settings, and testing of seroconversion
panels, if available, could provide useful sensitivity data.
[Slide.]
For
sensitivity determinations, reactive screening NAT and IgM results could be
confirmed by an alternate NAT and IgM assays including follow-up testing with
NAT and IgM.
For
a diagnostic indication, specimens from cases of West Nile virus illness in
community and clinical settings could be tested. This intended use, of course, is regulated by the Center for
Devices.
[Slide.]
If
the investigational test is intended for detection of various strains of
flavivirus or JE serocomplex or the West Nile virus strains, the ability to
detect these strains would have to be demonstrated.
These
studies could be conducted with repository specimens from previous epidemics
and using well-characterized panels.
[Slide.]
There
are no existing licensed or well-standardized assays for NAT or IgM
testing. Therefore, FDA is considering
some additional approaches for test validation.
FDA
has initiated efforts to assemble an in-house qualification panel composed of
pedigreed specimens as an additional tool for clinical sensitivity evaluation.
[Slide.]
FDA
believes that testing of a common set of pedigreed specimens by all candidate
investigational tests may be a useful additional approach to determining
whether assays have equivalent sensitivity.
Also,
testing of all reactive samples identified in clinical studies by all manufacturers
seeking licensure of West Nile virus tests would help further establish the
relative sensitivity of candidate assays.
[Slide.]
This
combination of approaches should provide adequate data to evaluate clinical
sensitivity for donor screening.
For
this purpose, however, collaboration between blood, plasma, and clinical
centers, test kit manufacturers, and governmental organizations is necessary in
sharing of samples which will further facilitate test evaluation and expedite
licensure.
[Slide.]
I
will now move on to discussing supplemental assays which are intended to
confirm reactive screening NAT or IgM results obtained with the individual
donation.
Supplemental
NAT would confirm results for screening NAT, and a supplemental IgM assay, of
course, would confirm either screening NAT or an IgM test result.
[Slide.]
Clinical
specificity for supplemental NAT assays could be obtained by testing a minimum
of 500 repository or prospectively collected specimens from low risk and low
prevalence areas.
Clinical
specificity could be confirmed by testing with an alternate NAT and IgM assays.
[Slide.]
Clinical
sensitivity for supplemental NAT assays could be obtained by testing samples
with reactive NAT results on the individual donation, and clinical sensitivity
could be further confirmed by follow-up testing with screening and supplemental
NAT and IgM tests.
[Slide.]
Similarly,
clinical specificity for supplemental IgM assays could be obtained by testing a
minimum of 500 repository and prospectively collected specimens from low risk
and low prevalence areas, as with supplemental NAT assays.
Reactive
IgM results could be confirmed by a second IgM and an alternate NAT. Discordant IgM results could be resolved
using plaque reduction neutralization tests.
[Slide.]
Clinical
sensitivity of supplemental IgM assays could be obtained by testing samples
with a reactive NAT or IgM result on the individual donation.
Reactive
results of the screening NAT and supplemental IgM test could be confirmed by a
second IgM assay and follow-up testing with an investigational and the second
IgM test.
Discordant
results again could be resolved using a plaque reduction neutralization assay.
[Slide.]
I
think this has already been stated by Dr. Biswas, but I will just say it again,
that FDA's current analytical sensitivity standard for West Nile NAT assays is
100 copies/mL for the individual donation, and that this standard may be
revised as tests become more sensitive or as additional data obtained on the
levels of viremia and infectivity in future studies.
[Slide.]
I
will now switch to the topic of unit and donor management on the basis of test
results.
[Slide.]
West
Nile virus detection systems will most likely be minipool NAT based, similar to
existing licensed platforms for HIV and HCV.
Consensus algorithms for unit and donor management based on test results
may be needed across testing platforms.
During
the clinical trial, FDA will consider strategies for unit and donor management
in order to gather data regarding which of these strategies would provide
maximum assurance of safety against West Nile virus transmission by blood and
plasma.
[Slide.]
This
approach may be necessary since the performance characteristics of
investigational tests and viral dynamics in West Nile virus infection of humans
are not well established. However, at
this time, FDA is considering some interim approaches for unit and donor
management, and we may revisit these strategies, if necessary, when IND studies
are completed.
[Slide.]
Similar
to algorithms currently in place for HCV and HIV NAT, reactive NAT results on a
master pool could be confirmed by testing individual donations with the same
NAT.
Non-reactive
units could be released, reactive units would be quarantined and destroyed or
labeled.
Donors
with reactive NAT results would be deferred for 28 days. This is a temporary deferral consistent with
the longest known duration of viremia that has been described so far in the
literature.
Reactive
results in the individual donation could be confirmed using an alternate NAT
and IgM test.
[Slide.]
During
clinical trials, reactive investigational NAT results could be confirmed by
follow-up testing. Donor may be
retested prior to 28 days. Follow-up
testing would include the investigational NAT, alternate NAT, and IgM testing.
If
the follow-up sample is NAT reactive by the investigational NAT or alternate
NAT, the donor remains deferred for an additional 28 days.
The
donor may be eligible for reinstatement if follow-up sample prior to the 28
days is negative by the investigational NAT, alternate NAT, and IgM positive.
However,
the donor would continue to remain deferred for the 28-day deferral period.
[Slide.]
This
slide is just a schematic of what I just said to you, so we will skip it and
move on to the next slide.
[Slide.]
If
individual donation NAT is used, non-reactive units may be released. Reactive units could be retested in
duplicate by the investigational NAT, and reactive results confirmed by an
alternate NAT.
Non-reactive
units may be released, reactive units
destroyed or labeled appropriately, the donor is deferred again
temporarily for 28 days, notified and counseled.
The
sample could be tested by alternate NAT, IgM, and/or plaque reduction
neutralization test to confirm investigational NAT results.
Follow-up
testing during the 28-day period would include investigational NAT, alternate
NAT, IgM. Again, if a NAT is reactive,
donor is deferred for an additional 28 days as with minipool NAT testing.
[Slide.]
Next
slide, please.
[Slide.]
We
will skip this one, too.
[Slide.]
Reactive
IgM results could be confirmed using a second IgM and NAT. If reactive on NAT and IgM, the donor is
deferred for 28 days, notified and counseled.
Non-reactive
NAT results again could be confirmed using an alternate NAT.
If
the sample is NAT negative and IgM positive, the donor may be reinstated.
If
testing was not performed during the 28-day deferral period, the donor could be
reinstated after this time.
[Slide.]
In
summary, I have tried to outline FDA's current thinking regarding approaches to
validation of West Nile virus NAT and IgM screening and supplemental tests, and
some considerations for unit and donor management.
Clinical
validation could include a combination of retrospective and prospective studies
to increase sample size in clinical trials.
Of course, this would involve the collaboration between industry and
government organizations to facilitate timely introduction and licensure of
West Nile tests for blood safety.
I
will close with that and thank you.
DR.
NELSON: Thank you, Indira.
Questions
or comments?
DR.
SOLDANA: John Soldana from CBS.
Indira,
I am slightly confused about the detection limit for NAT. I think you said it was 100 copies/mL for
the analytical sensitivity, and I think the previous speaker said 100
copies/mL, so that 1,000 could be picked up every time.
I
think that makes the 100 about 50 percent detection limit, so are we talking
about a 50 percent detection limit or a 95 percent?
DR.
HEWLETT: As I understand it, we are
talking about a 95 percent detection rate at the 100 copies/mL, such that the
1,000 copies can be detected in the original donation.
DR.
SOLDANA: Thanks.
DR.
GALLARDA: Indira, I have two
questions. One is regarding validation
of IgM assays with regards to clinical specificity. You touched on clinical sensitivity for both NAT and IgM, but you
didn't or at least I missed maybe the clinical specificity requirements to
validate an IgM assay. That's the first
question.
DR.
HEWLETT: I actually did have it on one
of my slides. I can go back to it, but
it is basically confirming with a second IgM test and resolving discordant
results using a plaque reduction neutralization test.
DR.
GALLARDA: Thank you. The second question I had, had to do with
you mentioned that if you have an initial NAT reactive on individual testing,
you could go and then repeat it in duplicate by NAT or do alternate NAT or IgM.
If
the unit is tested reproducibly at two times after initial positive result, and
those two replicate test points are both negative or one is positive, what does
that mean with respect to donor management and unit management?
DR.
HEWLETT: If you have got a discordant
repeat test, obviously, you would want to run an alternate NAT. The IgM is really more as an additional
tool. I think we are more focused on
confirming your NAT test result with another NAT assay, so that is how the unit
would be handled.
Then,
of course, you would reflex into follow-up testing in the event you have got
two NATs that are positive, you would want to do follow-up testing to confirm
the NAT result including seroconversion to IgM.
That
is how you would deal with the unit.
The donor obviously is going to have to be notified and counseled. There will be deferral triggered by the NAT
reactive test result that you obtain on the second test, on the duplicate test
where you have either one or both as being positive. That would trigger the deferral.
The
alternate NAT, of course, is to further confirm and to validate the test result
of the original NAT test.
DR.
GALLARDA: If the initial NAT screening
test result is positive, and the two replicates for the same screening NAT
assay are negative, what would you do with that unit?
DR.
HEWLETT: You would still want to test
using an alternate NAT at least during the IND phase, so we can collect that
data to be able to say, well, you are done once you have a negative result on
your duplicates, so it sort of back-validates the assay.
DR.
GALLARDA: And that donor would be
deferred until the 28-day follow-up period.
DR.
HEWLETT: Right.
DR.
PETERSEN: Just one question. If I understood you correctly, to look at
the specificity of the IgM antibody assay, you were contemplating testing with
a second IgM antibody assay?
DR.
HEWLETT: Do you have other
suggestions? There are no reference
assays, there are no really reference materials for these assays, so you have
to work with the tools you have, and that would essentially be testing it with
another test, another test for the same marker.
DR.
PETERSEN: I think probably you want to
do a neutralization test.
DR.
HEWLETT: Yes, that is actually part of
the algorithm.
DR.
PETERSEN: But not another IgM test,
because the problem is that because of the cross-reactivity of the
flaviviruses, you are going to get yourself into real trouble because even a
low prevalence area, you may have somebody that may have had yellow fever
vaccination, dengue, et cetera, previously, and you are going to end up with
some problems sorting it all out without a new test.
DR.
HEWLETT: I think the plaque reduction
obviously is going to have to play a role in sorting out results of the IgM
tests, there is no question that that is going to have to be invoked and have
to play a role. Thank you.
DR.
LEW: I think the question was asked a
little earlier, but slightly different, but I am still concerned about the 100
copies/mL, because someone had also presented, and I think it was American Red
Cross, saying that they are going to look at pools of 16 versus I think the way
the design was, if you have a 1 to 6, or pools of 6, then, clearly, the level
of 100 should detect that 1,000, you are quite comfortable.
But
when you start moving on up, and you are getting larger and larger pools, is that
something you all are comfortable with?
DR.
HEWLETT: Yes, you are right, I think
that is a very, very important point because obviously, we are looking at a
disease where virus levels are not as high as what you would see in HCV or in
HIV infections.
The
more sensitive the test, obviously, it is more desirable. So, the 100 copies is just to set that limit
as being the most desirable or the minimum level that we will accept at this
point based on what is achievable by assays that are being developed at the
moment.
I
don't know if that answers your question.
DR.
LEW: I guess I was just concerned if I
was correct that the American Red Cross are looking at pools of 16, I honestly
can't remember that presentation.
DR.
HEWLETT: Yes, I think that is what we
heard this morning, but Sue is going to get up and clarify it for us.
DR.
STRAMER: There are multiple factors
that you have to look at for sensitivity.
Pool size is only one of those factors.
Certainly, the sensitivity of the test, if the test is ultra-sensitive,
it gives you more flexibility to do a pool of up to 16 donations.
So,
the data that Gen-Probe showed at 50 percent hit rate was 2.9 copies/mL, which
is considerably south of 100 copies/mL, so we feel, with that sensitivity, it
will allow pools of 16. From the case
investigations that I showed, the Gen-Probe test, even in a pool of 16 and
replicates of 3, had S to CO values greater than 30 in all the CDC-confirmed
samples.
So,
we have some feasibility data at least that shows pools of 1 to 16 work on
clinically relevant samples.
DR.
HEWLETT: One does need to bear in mind
that although these are diluted samples, there are ways to concentrate the
virus and that is what manufacturers are doing. They are trying to find ways to concentrate what is in the
diluted samples, so they can get as much of it bound to the plate and to
enhance sensitivity of detection.
DR.
NELSON: Dr. Maria Rios is going to talk
about panel development and in-house testing.
Panel Development and In-House Testing
Maria Rios, Ph.D.
DR.
RIOS: I will be talking about the
panels for regulatory use and standardization of West Nile assay.
[Slide.]
The
need for panels for West Nile virus is quite obvious from what we heard this
morning, there are multiple newly developed assays for blood screening and it
is the concern of transmission of West Nile by blood transfusion.
However,
there is a lack of reference reagents for the assay standardization in terms of
viral quantification and sensitivity evaluation, and what was what we just
heard about when Indira was questioned about how many copies and how to detect
that.
There
is also an acknowledged scarcity of human clinical specimens. The availability of animal specimens was
made by CDC, and the most available was the flamingo strain from NY99, but the
human isolates for evaluating these assays in standardization is still a need.
[Slide.]
There
is a lack of consensus in the viral load titer, as you have heard several times
here, that the viral titer has been referred as plaque forming unit. Just to clarify what plaque forming unit is,
it is now many plaques you get in one plate that is covered with the cell in
the plates, are corresponding to viral activity lies in the cells and making
empty holes in the plate.
So,
we don't really know how many virus are required for forming a single unit or
single plate. So, it is estimated that
the viral particle in a PFU can be as broad as one infective particle to even
1,000 if they are localized in a single spot.
Therefore,
there is a need for correlation between RNA copies, actual copies in the PFU
unit. Because the non-infectious
particles that is known to be present in flavivirus, and specifically in West
Nile, those defective particles are not detected by PFU, but they are detected
by PCR. So, the viral RNA is present,
it is detectable, but it cannot be seen or identified in plaque forming unit.
Copy
number determination is necessary for us to proceed with a definition of
analytical sensitivity and therefore fulfill the regulatory requirements as we
have been discussing about what is the real sensitivity, what is a PFU, what is
a plaque number.
FDA
and CDC have been working jointly towards the development of standards, and
initially, CDC have made available the flamingo NY99 isolated in New York from
a flamingo in 1999, which was titrated based on PFU units.
The
lysate of flamingo 99 was made available to the companies that were developing
assays. One has to bear in mind that,
first of all, the units in PFU, when you do dilutions to try to tell your assay
sensitivity, you are not really talking how many virion is there, how many RNA
copies are there, but how many infective detection can be seen, therefore, you
don't know exactly your RNA copy number.
[Slide.]
So,
as I said, CDC provided the isolate and then provided FDA with the NY99, the
viral seed, so that we could grow, do titration, identify plaque units number,
and try to correlate to RNA.
The
CDC also provided to the FDA the first FFP unit that was implicated in the West
Nile transmission by transfusion from which the co-component, the red cell, was
proven to be the transmitter of West Nile infection. The virus was isolated and propagated, expended for manufacturing
of panels.
[Slide.]
These
panels would be used with the purpose of lot release and for the licensure and
post-market surveillance for NAT. We
also plan on using panels for IgM tests.
The
qualification panels for evaluation of relative sensitivities of
investigational NAT and IgM assays are necessary for the post-market
surveillance.
[Slide.]
Both
NY99 provided by CDC and the FDA-Hu2002 isolates are in the process of being
characterized at molecular level. We
are doing viral infectivity determination by performing PFU assays both at the
FDA and in collaboration with the New York Department of Health.
We
are further doing cytopathic effect and titration of the virus to know how many
infective virus are there. In parallel
to that, the same expended virus will be used to determine the RNA
measurements.
By
doing that, we would know the actual RNA copy numbers by measurements of the
RNA by fluorescence and optical density determination with TaqMan compared to
in vitro transcription RNA of known concentration. Then, we can calculate based on the size of the virus how many
RNAs and therefore, how many viral copies are there.
Once
this is done, we can correlate fairly accurately the PFU unit with the RNA copy
numbers.
The
final panel specification will be only established after we have done this and
we have done these two procedures in collaboration with the companies, and they
are performing these assays in other laboratories that have some excuse in
doing that, and we will break down, correlate the results, and come to a common
ground of correlation PFU and RNA titer.
[Slide.]
For
the IgM panel, we would like, FDA would like to have clinical specimens, and we
urge here that you please share your specimens with us, between yourself and
with us. We need clinical specimens that will contain varying titers of antibodies
for West Nile and some members that are also NAT positive, as we heard
here. Some are IgM and NAT
positive. That will be needed for the
validation.
The
panel will be evaluated in collaborative studies, as well, using various
candidate IgM assays.
Specifications
for NAT and IgM panels has to be established based on results of collaborative
studies.
[Slide.]
Our
plan is to have at least 100 pedigreed clinical specimens, and these 100
samples might include RNA positive only, IgM positive only, and dual RNA and
IgM positive, and please help us to make this happen.
FDA
also recommends that all reactive specimens identified in the IND clinical
trials be made available to all manufacturers through sharing of samples, so
that the assays can be cross-compared.
[Slide.]
The
sensitivity qualification panel, animal strain has been used, as I mentioned to
you, the NY99 was available during early test development, but the desirable
source material that it is the same for that one that we are testing, the human
panel.
The
fact that CDC provide FDA with the sample and the isolate will allow us to use
human isolates for developing these panels.
FDA
plans to formulate a panel for sensitivity evaluation or qualification of the
assays using both the animal, because it was the original strain that was made
available for the people developing the assay, as well as human strain.
The
panel will include members with very high or high copy numbers, a broad range
and very low, so that we now, if we are not quite clear about how high one
viremic stage can be, we don't miss anything.
[Slide.]
In
summary, FDA is preparing lot release and qualification panels. Panels will be made available when
adequately standardized, and FDA recommends testing of qualification panels
when the investigational INDs are performed for all applicants.
Thank
you.
DR.
NELSON: Thank you, Maria.
Questions? Yes, Judy.
DR.
LEW: I was just going to make a comment
and maybe a question, as well.
When
you are talking about comparing the PFUs to actual viral load, are you going to
try to do that concurrently? Just
because of the experience with HIV, we know freeze/thaw can really affect your
RNA copies, so if you send it off separately, that may not work.
DR.
RIOS: No, what has been done is that we
expanded our most wanted mail off the viral isolate, so the same unit or the
same volume of 500--actually, it was 750 mL--was pulled together and was
shipped simultaneously in dry ice from the same stock.
I
am working, at the FDA, the group is working with the same, as well as the New
York Department of Health, was provided to NGI, Chiron, Gen-Probe--not Chiron,
I have to send it to Chiron--Gen-Probe, Roche, and all the players in the game,
so that we would have, each one of us have one type of measurement, and try to
come to a common ground.
Then,
with the PFU, we establish both FDA and New York State Department of Health, we
can compare it, but it is the actual same isolate, it is the same bulk
material.
DR.
LEW: It may be the same bulk material,
but I am just saying I know how things are shipped around, and if you do your
PFU at FDA, and then you have to freeze it down and send it to various places--
DR.
RIOS: No, it was frozen before. I asked them to report to me whether or not
the sample came there in good condition, otherwise, don't use it. The only place that is performing PFU is the
New York State Department of Health that has been doing this consistently, and
we want at least two laboratories performing and correlating data.
DR.
GALLARDA: A comment and a question,
Maria. The copy number determination, in working with the NIBSE and W. Cho, on
making international standards, I think John Soldana can attest to the
difficulty of saying this is a copy because technologies have different ways of
defining copy. So, it sounds like it is
a work in progress, how to do this, is that correct?
DR.
RIOS: Uh-huh.
DR.
GALLARDA: On how you establish a
commonly defined term copy number. I
think you have already started the ball rolling, right, by sending out your
initial panel to testing laboratories.
DR.
RIOS: I can give you just what our
approach has been there. Like I was
explaining, the same bulk material was extracted, making sure that you only use
RNA as carrier, and then all the measurements were taken in fluorescence
measurement using fluorescence RiboGreen from molecular probe, and they were
correlated.
Then,
the concentration of RNA was translated into molecules per volume using a
number based on the size of the virion, and try to correlate back. It is very hard to define it, to say what is
one virion, but it is the best that we can come close to what we can call a
unit.
That
is why I would like to have from all of us.
I am planning on talking to Soldana.
DR.
GALLARDA: The question I had was
OD-260s would be influenced by TRNA, which has aromatic compounds.
DR.
RIOS: I am not using TRNA.
DR.
GALLARDA: Oh, I thought you said there
was--
DR.
RIOS: No, a void.
DR. GALLARDA:
Thanks.
DR.
NELSON: Mike.
DR.
BUSCH: Maria, just a couple comments on
the PFU versus copies. What you are
doing is using a tissue culture expanded strain. You know, with HIV, there has been a long-term controversy over
the relationship between copies and TCID versus infectious units.
DR.
RIOS: Absolutely.
DR.
BUSCH: I think the same kind of work
you are doing could be done as well with actual plasma from primary material
rather than expanded tissue culture.
DR.
RIOS: The difficulty, Mike, is that we
don't have even a small volume to do.
Imagine to have for the same people to get a handle on, I don't know, a
couple of units or something to do that.
Also,
when you have to put in the counter, your plasma, you are counting with a lot
of factors. Not every plasma, even
though it is positive, you can get a good isolate, and when you grow and you
have a standard, then, you can work more homogeneously and getting normal
material like natural.
DR.
BUSCH: I think in the units that Sue
has identified and that may be identified from some of the other studies, there
should be large volumes of viremic units that could be subjected to parallel
studies.
DR.
RIOS: Sure, but this is ongoing, it is
almost done. I needed to have the unit
a month or two ago.
DR.
BUSCH: The other comment, it is
actually more important perhaps than copies per PFU, is copies per human
infectious unit, and this is where I think the kind of studies we heard from
Abbott in appropriate relevant animal models where we could do titrational
endpoint inocula and correlate that with titers will give us the answer whether
we really need ID-NAT.
DR.
RIOS: Absolutely, but when you run a
PCR and you say that it was, I don't know, 104 or 103 or
106, you are not knowing whether or not you are measuring defective
particles in there, that may give you a higher signal, and then that is when
the infectivity will play a role.
Yes,
Jay?
DR.
EPSTEIN: I just want to comment that,
you know, the big picture here is to establish a well-characterized reference
material that can then be provided to a wide number of candidate manufacturers
to enable meaningful comparisons, whether the copy to PFU ratio is or is not
characteristic of a clinical sample is perhaps less important than having a
single, well-characterized material.
DR.
FITZPATRICK: I am just trying to
correlate Indira's talk to yours. In
the clinical specificity, she talked about investigational IgM test results
could be confirmed using NAT and a second IgM, and then discordant IgM results
evaluated using the plaque reduction assay.
From
your talk and the discussion, I am a little concerned about the evaluation of
the plaque reduction as--are you evaluating discordant result to find out if
you have an infectious donor, or are you evaluating discordant result to see if
they were infected with West Nile, and it doesn't sound like a plaque reduction
or a plaque forming unit would tell us that.
DR.
RIOS: I think you are looking at
both. First, you are looking at the
infectious unit when you do the plaque reduction or if the antibody was really
specific or if you are getting some cross-reactivity.
I
don't know, we have never looked at flu.
I am sure that in the middle of next epidemic, when everybody is going
crazy, somebody that has headache and fever, we will think they have West Nile. If we don't have a sensitivity that is such
that we know that flu is not cross-reactive, somebody that goes somewhere, we
know the world is a global village, so we need to use both parameters.
What
I was referring mostly regarding constructing the panels, is to make sure that
when we say we would like an analytical sensitivity or a qualification assay or
validation assay, we can give a panel and say you test this and you give it to
us, and we know where we are standing.
IgM
is okay, it is confirmation whether it is specific or not, but again, we can't
rule out a previous infection a year ago if we cannot identify positive by NAT
test, and so on, and so forth.
I
forgot to mention that we are developing house tests to cover most of the whole
viral genome because most of the assays won't use the same viral genome, and we
want to make sure that the panel we are generating we will be reliably
detecting any of the genome segment of the virus.
DR.
FITZPATRICK: I am not sure the PFU is
going to help you evaluate a discordant IgM result, though.
DR.
RIOS: Not a PFU, no.
DR.
EPSTEIN: I think the confusing that is
being engendered here is whether we are using a culture for virus titer or we
are using a neutralization titer of antibody. What we are saying here is that if
you have an investigational IgM assay and you get a positive result, how do we
know it is a true positive.
The
most definitive available test is the plaque reduction assay to confirm that is
a West Nile specific IgM, so we see that as the ultimate test. The question is how available will that
assay be, and short of that, what we are saying is that if you have a positive
IgM result of an investigational test, corroboration that it is a true positive
could be obtained by other IgM assays unrelated to the particular test that was
used to screen and/or if there is a concordant NAT positivity because the odds
of having, you know, a true positive if you have also a positive NAT, we would
think are very low.
But
we did hear the comment from Dr. Petersen earlier that we should be wary of
attempting to confirm an IgM test with an IgM test, but the reason that we have
put that forward, that's statistical confirmation rather than analytical
confirmation, but in the back of our mind is that if there are several thousand
putative positive IgMs that come up in the various studies, we may not be able
to do plaque reduction assays on all of those samples, so what else can we do.
Is
that clarifying for you?
DR.
FITZPATRICK: That is helping.
DR.
RIOS: Are you trying to comment about
what Mike mentioned about using actual sample to do PFU for validation, because
I think it is two different--one is a tool that you can use a reference to
quantitate that unit. You don't have to do the PFU in the unit if you have a
viral count or that you have standardized and you have a big volume and you
know one PFU correspond to X, Y, or Z.
Of course, we are going to talk about log, but it won't be 1 to 1,000.
DR.
FITZPATRICK: That helps. Thanks, Maria.
DR.
DAWSON: Just one more comment on the
IgM alternate testing. I think that if
you saw the data that Sue Stramer presented this morning, that perhaps 1 in
1,000 donations during peak season may be positive, and there is about a
million donations per month, that would mean you would possibly get 1,000 RNA
positives in September-October time frame, doing plaque reduction
neutralization tests would be very, very difficult. In fact, my understanding of the epidemic last year, that CDC
stopped doing plaque reduction neutralization tests because there were so many
tests to run, they couldn't do it.
So,
it is very time consuming, so I think that second IgM test will be of value
especially if you go into a second host cell, which will not have the same type
of cross-reactivities, and you would have to validate that second IgM assay
with plaque reduction or some other suitable supplemental test.
DR.
NELSON: Thanks. According to the schedule, next is
lunch. So, presumably everybody has had
lunch.
I
think we will move to Alan Williams, who will talk about blood supply
management and triggers for West Nile virus testing.
F.
Blood Supply Management and Triggers for
West Nile Virus Testing - Alan
Williams, Ph.D.
DR.
WILLIAMS: Thank you again.
[Slide.]
If
tests with adequate sensitivity and high levels of predictive accuracy are
available nationwide before the first human West Nile case occurs, we will be
in pretty good shape in terms of preventing transfusion transmission of the
agent, but if not, and, in fact, the first anniversary of the first 2002 human
case comes in 87 days from now, we potentially need to think about contingency
plans.
The
focus of this talk will be to discuss interventions, both some different levels
of testing, as well as other potential interventions, and triggers largely
obtain from the surveillance data available through CDC, that might serve as
start and stop triggers for other potential interventions.
I
think it is important to mention that this is a discussion simply to put some
of the options and some of the pros and cons on the table for discussion. While you can expect FDA to focus its
thinking quite soon in order to give time for ramp-up for any potential
interventions, this talk shouldn't be interpreted as FDA current thinking. I am simply putting items on the table for
discussion.
[Slide.]
First,
I actually want to compliment CDC for their effort on addressing this
epidemic. Through the ArboNet system,
which involves both human and animal surveillance, as well as the intense
post-transfusion study presented by Dr. Petersen earlier today, not to mention
Lyle's uncanny timing in publishing a paper Modeling Post-Transfusion
Transmission of West Nile that published a matter of months before the first
case actually occurred, I think they have done a tremendous job.
In
fact, we can expect that in the coming year, mosquito and animal data will be
available, as well as human data regarding West Nile epidemic. However, as described earlier, the epizootic
data is probably going to be of somewhat limited use for various reasons which
Lyle detailed, and it is more likely that the human epidemic data will serve as
a more useful trigger for some of the potential options.
Now,
the West Nile virus meningoencephalitis data reported to CDC, our understanding
is that is quite comprehensive and complete nationwide. West Nile fever data, on the other hand,
does vary from community to community.
Some areas of the country work up fever cases, get the testing done, and
then they become categorized as West Nile fever cases, other areas of the
country do not.
One
consideration for defining a human trigger would be to use the combination of
these two levels of reports and allow the fact that surveillance might be a
little tighter in one area of the country versus another.
Now,
when the voluntary market withdrawal was instituted for fresh frozen plasma and
other frozen products last year, it utilized human West Nile case data at the
state level. In fact, data is available
at the county level, and potentially allows a little more fine tuning of use of
some of the surveillance data. One
could use individual counties or county plus a surrounding county to define a
region.
As
reported earlier, the transfusion transmission studies, the actual cases were
recognized by CDC last year, and a close investigation of the known 14 cases
has led to, albeit it limited data, on the viremia and the symptoms of known
implicated donors.
[Slide.]
Just
an observation that West Nile is the first mosquito-borne TTD which is
indigenous to the U.S., arguably, the first vector-borne if one doesn't
consider babesiosis.
It
has epidemic clustering both in time and geography, and I think in considering
interventions, one needs to keep this in mind, it differs from some of the
other types of post-transfusion infections that we have been dealing with, and
it raises new issues regarding donor screening and blood testing.
NAT
technology clearly paved the way for direct rapid testing of donors. The flexibility involved in NAT raised the
potential for developing a test within a nine-month period, which is really
quite incredible.
[Slide.]
What
are some of the unique challenges and questions related to West Nile? First, is related to testing, the biggie,
geographic area and timing of the 2003 epizootic and epidemic is totally
unknown at this point.
CDC
feels I think that some of the early data will help to be predictive of the
likely extent of the epidemic, but even the early data are not yet available.
Secondly,
some aspects of West Nile virus NAT assay performance are unproven at this
early stage. These involve not only
sensitivity and specificity of the assays, but also scale-up capabilities, you
know, will, in fact, all the equipment or software, et cetera, be available
when it is needed even if the actual reagents are available.
Location
of test sites, INDs are currently under development, will they be adequate to
cover any sites having epidemic West Nile virus if that site happens to be
covered by a different IND and there is problem with test availability or
something.
So,
all these things need to be considered to be reactive to cases as they occur.
I
think it is probably a reasonable approach that implementation plans should be
compatible with a worst case 2003 epidemic, and then we can really readjust as
data tend to prove that otherwise.
There
are considerations of minipool NAT sensitivity, which leads to the question are
other additional protections needed to prevent transfusion transmission or
minimize it.
[Slide.]
With
respect to donor screening, we heard this morning some limited data on the
pre/post-donation donor symptoms, 3 of 14 implicated donors reported
pre-donation fever plus or minus other symptoms.
The
background prevalence of fever independently is 3.7 percent, but combined with some
of the other symptoms, comes down a little bit to a little less than 2 percent,
and potentially, some combination of symptoms might have good predictive value
without undue loss of the normal donor population.
Rash
and eye pain were mentioned this morning.
In fact, I think we need to look at the data a little harder to see
whether those additional symptoms were before the donation event or after the
donation event, and exactly how they would fit in.
We
need more precise donor loss estimates.
This was from a controlled sample built into the design of the
post-transfusion case investigations, but it would be very helpful if industry
could, in fact, conduct some surveys of these symptoms for various time
periods, one week plus or minus the donation event, two weeks or three weeks,
et cetera, because the current data relate to three weeks, and are at a limited
number of samples.
A
large point is that there are persistent blood supply problems, and these
increasingly point toward a quite fragile donor base, so whether we are talking
about specificity of an assay or a screening of a donor, all of these will
impact the current blood supply.
We
do need to keep in mind the donor loss in this circumstance would be
"temporary," probably related to 28 days post-initial recognition,
but even temporary loss of a donor has its own inherent problems because once a
donor is deferred, sometimes they tend not to come back as readily, so that has
its own implications.
[Slide.]
Post-donation
information reported in 5 of 14 of the implicated donors you heard this morning
are reported post-donation. Again, the
background prevalence would be about the same, but we know that post-donation
reporting tends to be inefficient and the products may well have been
transfused by that point.
[Slide.]
Another
potential intervention--and this is one that I think needs to be considered
very cautiously--would be real-time suspension of blood collection or blackout
in effect of blood collection in a geographic area experiencing human West Nile
cases.
Now,
this was retrospectively sort of the approach taken in 2002, when there was
voluntary market withdrawal of fresh frozen plasma and other frozen products in
parallel with human West Nile virus cases defined at the state level plus or minus
a week, I believe.
The
potential for transmission would arguably be reduced by this approach because
the epidemic tends to have microfoci, so that if one simply stopped collecting
blood in an area that appeared to define the epidemic, one could quite possibly
intervene in potential transmission cases.
However,
a couple big areas of concern with the blackout of collection, first, is that
if you establish that as a precedent, it may, in fact, not be sustainable if
the epidemic worsens, and you may, in fact, have to change standards midstream
to maintain an adequate blood supply.
Even
if done at a local level, there is a potential for severe strain on supply.
[Slide.]
So,
those are some of the potential interventions. What are some of the potential
triggers? Some of the obvious ones,
West Nile cases in animals, the epizootic, West Nile cases in humans, the
epidemic, are these going to be defined by geographic area, and available would
be state, county, and some combination of county data.
One
could also perhaps envision a situation where a large county, for instance,
Cook County in Illinois, shouldn't be treated the same as a very small county
in another state. One could potentially
tie a case rate per 100,000 population to a trigger.
The
actual scope of the regional and national epidemic also would be a factor in
defining the state of the epidemic.
Another
factor, potential trigger, would be a defined end of human cases and a defined
end of the epizootic.
[Slide.]
Additional
triggers. Test availability and
effectiveness. Is the test available in
very limited supply or is it available and able to be implemented nationwide,
and what is the effectiveness, the sensitivity of the test.
Is
there evidence of local or regional blood shortage? Clearly, it is always a balance between implementing measures to
protect from a safety threat versus introducing potential compromised safety by
having inadequate blood supply, so that needs to be a trigger, as well.
In
fact, changes in the NAT testing prevalence itself could be of value
particularly in signaling the end of epidemic in a certain area.
[Slide.]
So,
what I have done in the balance of the talk is simply combine some of these
factors and again just for discussion purposes, put out some potential interventions
and their triggers.
To
begin with, considering NAT testing, what would be the start triggers for NAT
testing? First of all, say that NAT
testing, high-quality NAT tests are available under IND in unlimited
availability. One would consider that
the most appropriate response would be U.S. testing, universal testing as soon
as possible.
Now,
say the test is available only in limited availability. The potential trigger would be the
epizootic. I know Lyle mentioned this is of limited usefulness, but I think one
thing it does is it signals the potential for human exposure and is available
for a period of time, roughly a month, as I recall, prior to the first human
cases being developed. So, it gives
some sign to put something in place before a human case actually occurs.
Say
a test has very limited availability, only a single lab has a test, and one
needs to institute some form of protection, then, potential of the human cases
could serve as a trigger, but by then you already have transmission in place
and a little late in the game.
[Slide.]
What
would be the triggers for stopping NAT testing? In an unlimited availability situation, I think this is something
that needs further discussion. One can
always make the argument that if you have a geographically or temporally
focused infection going on, that even if it is not going on in a certain area
of the country, a donor can always get on the plane from somewhere else in the
country and attempt to donate in that area.
As
I said, this needs discussion, but perhaps the West Nile NAT prevalence itself
could be a driver as to whether NAT testing should be continued once the season
is defined as being over.
In
a situation where the NAT test has only limited availability, a potential
trigger for that would be a defined end to the epizootic, and this could be
defined at the county or similar level, and under very limited availability,
similarly, defined end to the human cases.
[Slide.]
What
are some other potential intervention triggers? Donor screening for West Nile symptoms and product quarantine and
retrieval based on post-donation information.
There
would be a couple way to potentially implement this. One would be the typical direct screening of donors, which in
many centers now is done by self-administration of the donor form, would
involve modifications to the form, which takes some time.
It
could also be done by adequate upfront information to potential donors and
inclusion in an information sheet. In
fact, most of the donor deferral occurs before the donor ever comes to the
blood center, so I think there should be consideration of that, as well, if
this is planned.
What
would be a trigger to start donor screening? I think here would be another
situation where a start of a defined epizootic in an area would be appropriate,
but in the case of supply concerns, potentially, one would consider the human
cases. In either situation, one would
need to carefully monitor supply.
Donor
screening, when would be best for it to stop?
Similarly, West Nile epidemic or potentially human cases, probably the
epizootic would be somewhat more protective.
[Slide.]
Cessation
of blood collection in West Nile endemic areas, the blackout concept. One consideration would be that such a
strategy might only be appropriate to start if an epidemic is highly localized
and there is no test available. That
would be one situation where it might be arguably an appropriate response.
One
would obviously need to carefully monitor supply and the start of such an
intervention could be defined by human cases at a carefully defined geographic
level. Stop of that approach would be
end of the human cases at again the same geographic level or availability of a
West Nile sensitive test.
[Slide.]
In
summary, the uncertainty of the timing and the scope of the 2003 West Nile
epidemic, if there is one, requires planning for several simultaneous
interventions to maintain a safe blood supply.
I
am actually hoping that there will be adequate mosquito control, so that we
will see a lot less than we had last year, but again that is another variable
which is unproven at this point.
Pre-definition
of intervention triggers will assist the planning process, and we recognize
that blood collection facilities need time to put in place whatever is recommended.
Planning
will develop more focus as the 2003 surveillance information becomes available,
and FDA very much seeks comments from the committee regarding the interventions
and the trigger concepts presented.
Thanks.
DR.
NELSON: Thanks, Alan.
Comments? Paul.
DR.
SCHMIDT: We heard before that the
highest levels of HHS had interest in maintaining the blood supply. It would
seem that before the media starts in, that there really should be proactive
action.
The
public believes you, they believe the CDC, but this might perhaps have to be
kicked upstairs to the Assistant Secretary of Health, if there is one, or the
Surgeon General, and to lay it on the line and say what we know, what we don't
know, what might happen, say it in advance.
It
is sort of like--I hope this doesn't kick down the idea--but it is sort of like
Code Orange, if it doesn't happen, then, you take credit for it, but the point
is you lay it out in advance, before the panic starts.
DR.
KLEIN: I really think that this is
really very creative thinking and it's the right time to do it, and I really
applaud your doing that. I do want to
make a comment, though, about donor history.
It
is a comment that relates to some of the past donor questions that have been
imposed upon the blood collectors. We
spend a lot of time, a lot of money, and a lot of science validating assays, as
we heard today.
We
spend no time, no money, and no science validating donor history questions,
many of which have almost no sensitivity and even less specificity. They do a great deal of damage.
So,
while I think the idea is actually a good one, before anyone comes up with a
question that is then published and imposed upon the blood collectors, I think
we ought to do some kind of validation of the donor history, so that we can be
sure it does something to enhance blood safety, and not something to detract
from blood supply.
DR.
NELSON: Part of the problem, of course,
is that we don't currently have a West Nile epidemic, so we could tell how many
false positives there were, but we couldn't measure the true positives.
DR.
KLEIN: I am well aware of that. We don't have a variant CJD epidemic
either. One could argue, I suppose,
that having imposed a series of questions, we haven't seen a single case, so it
must be working.
I
don't mean to be facetious, I really would caution us, because we have made
mistakes in the past that I think have been costly, not so much in terms of
dollars, but in terms of donors, and I think we need to think very carefully so
that we don't make such errors again.
DR.
ALLEN: I agree with Dr. Klein. This is a very intriguing presentation, and
I thank you and the FDA for it. It
raises, in my mind, some real serious considerations that have to be looked at,
and those aren't our questions to be considered this afternoon, so I will be
brief, but let me just mention a few.
This
issue of clinical illness and symptoms, I think that does need to be looked at
very carefully. My own personal
standard before I donate is I am totally healthy for at least a week. Even if I have had a respiratory infection
or something, and I get called, you know, two or three times in a row, please
come in and donate, I don't do it.
One
of the questions I routinely get asked is do you feel well today, and I think
that is an appropriate question. It
might be extended somewhat. I think
something could be done here without changing things too dramatically, that
might help increase deferral for relatively minor symptoms either in the pre-
or post-donation period. I think that
definitely needs some consideration.
The
issue of geographic suspension of collection, I am really concerned about that
one. I think it is going to be
difficult to explain to the donor population.
I think that potentially that is one that when you try to reinstitute it
in an area, you are going to find that donors may not be coming back in, having
been turned away for something that they ill understand.
I
think it is going to play havoc with the local collection systems, you know,
who may have to shut down perhaps, depending on the criteria, for weeks at a
time. Given what we heard, that even in the high incidence areas, what was it,
2 percent of the population may have been infected in a given season, I think
this is something that we have to look at very, very carefully before taking
any recommendation on.
Also,
the issue of starting and stopping testing. I, because of cost and a whole host
of other considerations, don't want to see any more testing done than
absolutely essential. It increases the
probability of errors, it increases costs, time, the necessity to purchase and
maintain expensive equipment and all the rest of it.
Nonetheless,
we have long established in our country that blood is collected under a single
standard, and that was obviously extremely important during the HIV
epidemic. We didn't establish local
standards here and there. We didn't
establish a minimum set of standards.
We established a national standard.
I
think that there is a lot of reasons why that concept needs to be maintained,
or if it is going to be changed, needs to be looked at very carefully.
I
would like to hear from Roger Dodd, I don't know if Sally Caglioti is in the
audience, but what is the impact in a laboratory of starting and stopping
testing at certain periods of time in the year rather than having here is a new
standard and we are doing it on every unit that goes through and is going to be
verified as acceptable for release.
What
about maintenance of equipment that isn't used for a period of time, you know,
is left sitting idle and then may need to be cranked up on short notice if
something happens?
I
think these are issues that are worthy of exploration, but I think we have to
look very carefully at all of the implications before making any decisions.
DR.
STRONG: I would just like to amplify
those comments since I have some operational responsibilities at a blood
center. Starting and stopping anything,
whether it be a questionnaire or donor testing, is a major undertaking. We have to write SOPs, we have to go through
validation exercises, we have to train staff.
All
of those things take time, and it could easily be that it would take you the
period of time that you were stopping to stop, and as long to start again, that
you wouldn't have accomplished anything.
So,
from a very practical point of view, the starting and stopping really doesn't
seem even feasible.
In
terms of stopping collections, if we are actually able to hit this timeline and
starting in July, of course, we are starting in the summertime. The summertime is notoriously one of our
most difficult time with keeping up with collections.
I
just can't even imagine that anyplace in the country could stop collections and
make up the deficits no matter how small.
We struggle every summer to get through that. I also commend you for coming up with some optional scenarios.
These don't seem very practical and it is going to be very difficult for
us to implement either of those two options.
DR.
FITZPATRICK: It's great that we brought
these up and I echo Harvey's comments, but one thing is unfortunate that on the
agenda, you have asked for discussion, but there isn't time for discussion.
Second,
there won't be another BPAC until the end of June, and by then it will be too
late to have your mechanisms in place, so you are going to have to do this
without much discussion.
You
have already established a trigger and that is 1 July. You have informed the industry and the
testing, the manufacturers, that by 1 July we are supposed to be testing the
entire blood supply. So, I think you
have already established a trigger to start testing and set a date. So, I am a little confused by that.
You
have said that it is 82 days until the first reported case from last year, and
we don't have a strategy, and I am concerned about that. I am concerned about the fact of there is
two impacts to stopping collection in a geographic region. One is the obvious impact of the blood
supply in that region, how do you maintain it, how do you get donors, what is
the impact on the donors.
The
other is a personal impact, what do you do with that staff that has been idled,
and how long are they to be idled, and what is that collection center to do
with their idled staff? Do they pay
them? With what? Is there any mechanism available to them to
keep that staff on for an unknown period of time? I think that is a very unworkable idea.
Starting
and stopping testing, Dr. Strong I think addressed that very eloquently. One thing that I don't see that was discussed
previously is that when this started, we identified a set of patients who were
at risk. There were a couple of
comments on others about labeling the blood as tested or untested, or as West
Nile positive or West Nile negative, and using clinical judgment to provide the
blood to the appropriate patient. I
think that is a possible strategy that could be used in those areas where there
is a geographic solution or problem.
If
there is a trigger point of human cases being reported and you have a
geographic region where you think the blood supply is at risk, and we don't
have enough tests, then, yes, I think you should probably divert tests to that
region to test, but we have those problems that Dr. Strong mentioned, if they
aren't testing already, then, they have to be trained, they have to be
validated, they have to be assessed, and that all takes time, and quite
frankly, between now and July 1st is probably not enough time to even do what
we are being asked to do by July 1st.
So,
unfortunately, there aren't a lot of solutions, we have some conundrums
here. The impact is unknown, the risk
is known, and the interventions are few. So, I think it might be best to maybe
have some special session to look at what can be done, because the public is
aware from last year that there is a problem, and we are aware that patients
will be at risk, so I think some interventions need to be come up with, and to
me, besides those you have mentioned, the one of identifying the at-risk
population and trying to protect them may be the most quickly and easily put in
place even though it may not be the most effective, it will at least be
partially effective.
DR.
SCHMIDT: Unlike you all, Florida has
had a very warm, wet spring, at least south of Gainesville, and the talk is
already on that things are going to start early this year.
DR.
NELSON: On that optimistic note, I
would like to maybe move on and we will have some more discussion in a little
bit.
The
next topic is testing of source plasma and the clearance of West Nile virus in
plasma-derived products. First, is
Mahmood Farshid from FDA.
G.
Testing Source Plasma Donations and Clearance
of West Nile Virus in Plasma-Derived
Products
Mahmood Farshid, Ph.D.
[Slide.]
DR.
FARSHID: My brief presentation is with
two questions or two issues with regard to West Nile and plasma-derived
products, which they are noted here.
The
first one is whether viral validation data on model flaviviruses are sufficient
to demonstrate West Nile virus clearance.
Basically,
the question is the current validation data, which is done on model viruses, is
sufficient or the specific validation needs to be done.
The
second is whether the clearance steps in the manufacture of plasma derivatives
obviate the need to screen donations of source plasma.
I
will address the first question first and try to present some pros and cons.
[Slide.]
The
question which is frequently asked, are current viral clearance steps effective
against West Nile virus, and the clearance level predicted for West Nile is
comparable to those obtained with model flaviviruses based upon taxonomical and
physicochemical similarities between the viruses, as we heard in presentation
today, it is an envelope virus, and it is a flavivirus.
There
are some data presented in the last workshop, and we will hear more today which
indicate that these predictions are confirmed more or less based on this data
which presented publicly.
Also,
it needs to be noted that no transmission of West Nile by plasma-derived
product has been reported.
[Slide.]
However,
to provide further assurance on the robustness and reproducibility of West Nile
clearance in various plasma-derived products, specific viral validation studies
should be conducted using West Nile virus.
[Slide.]
The
rationale for further validation studies are indicated here. The clearance results vary depending upon
the virus and the product being tested.
Basically, for plasma derivative, viral validation studies must be
product and process-specific.
That
is to say that generic validation is not applicable to human-derived products
or products that may be contaminated with human pathogens.
So,
the key word here is the specificity of the validation and its relevance of a
particular process and product.
[Slide.]
This
is simply as example to underscore the need for the specificity in terms of
validation studies, which shows the different manufacturing conditions could
substantially influence the clearance capacity of a given inactivation step.
Here
is 6 different manufacturers and activated hepatitis A and albumin by
Pasteurization. As we can see, there
are huge differences in the level of the clearance as low as 2.9 for the
concentration of 4 or 5, and as high as 6.6, the same thing for the concentration
of 20 to 25 percent, as low as 2.5 and 6.6 log being reported.
[Slide.]
This
slide basically here, the intention is not to outline what is required for
validation, but again to underscore the importance of having a specific
validation for a given process or product.
We put some of the parameter which is required for validating the small
or laboratory scale model, and to establish the relevance to the actual
manufacturing process.
One
need to include all the critical process parameters within the clearance steps
performed at the fullest scale, and adopt actual production methods as far as
possible. Assess the impact of
unavoidable differences, and maintain relative values, such as volume and
geometries, and also absolute values, such as time and temperature.
Accurately
transfer the process from laboratory scale to full scale.
[Slide.]
The
question of what type of virus need to be used to perform these validation
studies, whether model viruses would be acceptable or the relevant virus should
be included.
The
current approach is that the actual virus of concern should be used in viral
validation studies if technically feasible.
Specific
model viruses can be used in validation studies if technical/experimental
limitations do not allow use of the relevant virus.
Basically,
viral validation studies, since it is being performed in a small scale,
inherently has a number of variables, and one needs to avoid introducing new
ones, and for this reason, using the virus of concern in doing validation
study, it goes a long way to produce reliable data which could be extrapolated
to the actual manufacturing process.
[Slide.]
For
example, this is the panel of viruses which are being used to validate for
plasma-derived products, and overall for human-derived product which the
relevant viruses are HIV and HAV. These
two viruses, there is tissue culture available for them, and they could be
used.
Specific
model viruses are used, such as BVDV and Sindbis for HCV, because no tissue
culture is available, however, it need to be noted that initially to establish
the effectiveness of a given inactivation steps, animal studies were performed
using actual HVC, and the acceptance of the model virus, such as BVDV, is based
on a large number of data which has been generated over the years.
PPV
and CPV also are used as specific model viruses to basically model for human
B-19.
[Slide.]
The
conclusion is that studies on specific--and the cue here is the
specific--studies on specific viral inactivation and removal steps in the
manufacture of plasma derivatives should be conducted with West Nile virus to
confirm the reliability of current clearance results, which are based upon
validation studies using model viruses.
This
basically addresses the first question or first issue.
[Slide.]
The
next question is whether the clearance steps in the manufacture of plasma
derivatives obviate the need to screen donations of source plasma.
[Slide.]
Current
approach for risk reduction of transfusion-transmitted viruses essentially is a
complementary approach which includes donor selection and donor testing, and
inclusion of inactivation and removal step in the manufacturing process.
This
complementary approach would provide sufficient margin of safety, and neither
of these steps alone would be sufficient to achieve that.
[Slide.]
In
case of West Nile, although there is still discussion regarding some of these
points, but overall one can say that because of the pathogenicity and
transmissibility by blood, prevalence in the donor population at least during
the peak season and in high prevalence area, and availability of screening
assay if it becomes available, and one cannot ignore the public demand assuming
that the assay is available and the virus is pathogenic and it can be
transmitted by blood.
[Slide.]
Doing
testing of source plasma will result in reduction of viral load in the
manufacturing pool, and by that, increases the clearance capacity of the
manufacturing process.
The
viral load, as we heard this morning, overall is low, however, considering the
peak season and high prevalent area, inclusion of the testing will reduce that
load and the overall burden on the inactivation and removal steps.
Testing
will be additional layer of safety as it is applicable to other relevant and
pathogenic viruses that currently we are dealing with.
Thank
you.
DR.
NELSON: Thank you very much, Dr.
Farshid.
Comments,
questions? Okay. Thank you.
Dominique
Pifat from Bayer on behalf of the PPTA.
PPTA Presentation
Dominique Pifat, Ph.D.
DR.
PIFAT: Good afternoon. I would like to thank you for the
opportunity to talk. As was just
mentioned, I will be making this presentation on behalf of all member companies
of the Plasma Protein Therapeutic Association.
[Slide.]
Next
slide, please.
[Slide.]
As
Dr. Farshid just mentioned, these are three of the questions that were posed to
the panel, and these are the three questions that I will be addressing.
Does
the experience with West Nile virus support the effectiveness of model virus
concept? Donor deferral criteria and
robust clearance data obviate the need for West Nile virus screening of plasma
for fractionation? And product and
process-specific clearance of West Nile virus assures the safety of plasma
derivatives?
Again,
I want to emphasize that we are dealing here with plasma derivatives
manufactured products.
[Slide.]
The
use of model viruses is extensive in the industry. The best example is that hepatitis C virus is not itself tested
in the clearance provided by the manufacturing processes. Instead, most companies in the industry use
a bovine viral diarrhea virus as a model, and I think this has been successful
if one bases that on the safety of the products.
Hepatitis
C belongs to one genus of the Flaviviridae family, BVDV belongs to another, and
West Nile virus, which is also an envelope virus, a member of the Flaviviridae
family, belongs to a third.
[Slide.]
So,
model viruses, we feel are an effective surrogate for closely related viruses
specifically when it comes to envelope viruses. Different envelope viruses are very susceptible to a number of
different inactivation methods, and I am going to show you data to support
this.
Envelope
viruses, with a broad range of physical characteristics, have been shown to be
similarly susceptible to a number of inactivation methods, and I will show you
data to support that, as well.
[Slide.]
So,
the model virus approach is adequate, we feel, to predict behavior of viruses
with similar characteristics, and we feel that there should be a risk-based
approach to decide whether full validations with a new virus should be
required. Not all viruses present the
same risk profile, and viruses with differentiating characteristics should be
considered whether it is necessary, in the terms of whether it is necessary to
conduct full viral validations.
In
addition, the industry is committed to performing studies to verify the
validity of model virus concepts when a virus of concern appears, such as West
Nile virus. So, the question here is to
do full validations or just verification studies.
[Slide.]
I
am going to switch gears here and talk about plasma screening, the second
issue. Plasma screening for West Nile
virus, we feel would provide no significant increment in the safety of plasma
because of the pathophysiologic features of the virus.
As
we have heard today, West Nile virus titers are relatively low. The viremia appears to resolve relatively
rapidly, and there is no documented chronic stage for West Nile virus
infections.
In
addition, we have donor deferral requirements, inventory hold that are
currently in place, and as I will show you, we have robust viral inactivation
and removal throughout our manufacturing processes.
[Slide.]
I
am going to now address in greater detail, inactivation and removal of West
Nile virus through our manufacturing processes. The safety of plasma derivatives from West Nile virus has been
adequately assured through virus inactivation and removal studies.
As
we had committed to doing at the workshop in November, PPTA member companies
have now performed virus inactivation and removal studies using West Nile virus
itself, not in a model, and we will show you in a minute that existing methods
show very robust clearance across processes and products.
[Slide.]
I
have grouped these data from all the different companies based in inactivation
procedures, so I am going to deal with pasteurization first, so that is heat
treatments in aqueous solutions at 60 degrees Celsius, with or without
stabilizers demonstrate a very effective inactivation.
[Slide.]
In
this particular slide, we are dealing with a factor VIII product. I will show the comparison of inactivation
between BVDV and West Nile virus, West Nile virus being the purple line here,
and you can see that pasteurization results in very rapid and effective
inactivation of West Nile virus to the limit of detection in this product.
[Slide.]
This
is another factor VIII preparation, different manufacturing process, again
extremely rapid inactivation of West Nile virus. It looks like 6.5 logs of inactivation for West Nile virus, again
very similar to what we had demonstrated with BVDV.
[Slide.]
This
is an IG preparation, again very similar data.
West Nile virus is inactivated to the limit of detection, as is bovine
viral diarrhea virus, and other viruses.
As you can see here, yellow fever virus, which is another Flaviviridae.
[Slide.]
This
another immunoglobulin preparation, very similar data again. So, the inactivation by pasteurization does
not seem to be affected by the product stream.
All of these different products behave very similarly when they
pasteurized in terms of the inactivation of West Nile virus.
[Slide.]
This
is a human plasma protein solution, and again, pasteurization results in
extremely rapid inactivation of West Nile virus, in this case greater than 7
logs inactivation, and as a point of comparison, BVDV is inactivated rapidly,
as well, and to the limit of detection, as well.
[Slide.]
This
is a completely different product, prolastin, and again, West Nile virus is
inactivated very rapidly to the limit of detection, as is BVDV. So, I have shown you very different products
where pasteurization works equally well.
[Slide.]
Pasteurization
of human serum albumin from a third company, again, very similar data. West Nile virus and BVDV are both
inactivated to the limit of detection.
[Slide.]
This
is another human albumin preparation.
The protein concentrations in this product are very different from the
slide I just showed you, and again, that doesn't seem to have a significant
effect on the ability of pasteurization to kill West Nile virus.
This
slide shows other flaviviruses, BVDV, tick-borne encephalitis virus, as well as
togavirus, Sindbis virus.
[Slide.]
I
am going to switch now to solvent detergent. Now, the different member companies
used different cocktails of solvent detergent.
Some companies used TNBP cholate, others used TNBP tween, others TNBP
triton, and what I would like to show you is in all different products that we
have looked at, with all these different combinations of solvent detergent,
there is very effective inactivation of West Nile virus.
[Slide.]
This
is plasma-derived factor VIII. Again,
West Nile virus is inactivated very rapidly, greater than 6 logs reduction,
BVDV gives similar results. In this
experiment, we have used half the concentration of TNBP tween that is used in
manufacturing, so even at half the concentration of standard TNBP tween, we
still get complete inactivation of West Nile virus to the limit of detection.
[Slide.]
This
is an immunoglobulin preparation, again very rapid inactivation of West Nile
virus to the limit of detection even at half concentration of TNBP
cholate. This is a different solvent
detergent.
[Slide.]
Again,
another company's product, solvent detergent.
This is TNBP triton, again, we see very similar results, inactivation of
West Nile virus to the limit of detection.
Now, in this particular case, they have used only 5 percent of the
solvent detergent concentration that is actually used in manufacturing. So, this shows that there is a tremendous
margin of safety in terms of the ability of solvent detergent to inactivate
West Nile virus.
[Slide.]
We
have looked at some other methods of inactivation, as well as removal.
[Slide.]
This
is vapor heat treatment. Again, West
Nile virus is inactivated very rapidly here.
The kinetics of inactivation are similar to BVDV, but we start out here
with a much higher concentration of virus, but nevertheless, the West Nile
virus is inactivated to the limit of detection again.
[Slide.]
This
is a removal technology, nanofiltration, and we can see here that you can get
greater than 9 logs removal using the Asahi 15 and nanofilters, so again, very
good removal here of West Nile virus within this process.
The
data that I have shown you are one manufacturing step at a time. Of course, most of our manufacturing
processes include a number of these viral inactivation steps, and as long as
these viral inactivation or removal steps operate by different mechanisms,
then, we can add the removal that they provide throughout the process, and that
is how we obtained the overall viral clearance for the processes.
[Slide.]
This
is again to show you, this is just a subset of the data that are available, but
if you look at a whole variety of envelope viruses, these are not only
flaviviruses now, these are different viruses, such as herpes, pox, rhabdo,
retroviruses, and a number of others. You can see that for those viral
inactivation methods that actually target the envelope, such as pasteurization,
solvent detergent, caprylate, you get inactivation of all of these viruses to
the limit of detection.
So,
there is a body of data that show that actually these viruses, these envelope
viruses behave very similarly when subjected to pasteurization, solvent detergent,
and other technologies.
[Slide.]
One
question that might come up is, is it possible to overwhelm the system, is it
possible to overwhelm the reduction that is afforded by the manufacturing
processes. I would like to point out
that all these data show greater or equal to a certain number of clearance,
which means that the clearance is to the limit of detection, and if we had
virus stocks available that were higher titers, we could show yet greater
clearance.
As
I mentioned, multiple steps are incorporated in the manufacturing processes,
and based on the data that we have heard today, in terms of the potential
viremias in humans, and also the ratio between infectious particles and copies
or particles, and the overall clearance that is afforded by our processes for
these envelope viruses, it is very difficult to envision a situation where you
would overwhelm the capacity of these manufacturing processes to clear these
viruses.
[Slide.]
In
conclusion, we feel that product and process-specific clearance of West Nile
virus do assure the safety of these plasma derivatives, and based on these
clearance data, we feel that West Nile virus screening of plasma for
fractionation is not warranted because of the robustness of the clearance data.
Again,
the experience we have with West Nile virus demonstrates the utility of model
virus concept in that West Nile virus turned out to be inactivated very
similarly to BVDV and other model viruses.
Thank
you.
DR.
NELSON: Thank you, Dr. Pifat.
Questions,
comments?
DR.
STRONG: You didn't comment on the
possible effect of having immune plasmas in your pool, whether or not that
would affect the activation process or make it more susceptible.
DR.
PIFAT: You are talking about antibodies
to West Nile virus in pools?
DR.
STRONG: Yes.
DR.
PIFAT: Well, I think for immunoglobulin
products, for instance, that would provide additional safety. For the products that don't contain any
immunoglobulins, that would have very little effect.
DR.
LEW: Just out of curiosity, has anyone
looked at the patients who received the plasma products to see if they have a
higher rate of West Nile IgG to show past infection now that we have gone
through one cycle? I realize it's all
new, but remember the high-risk groups have been burned, they may be very
interested in seeing if they were at risk this past summer.
DR.
PIFAT: I am not sure I understand your
question. You are talking about these
patients having immunoglobulins to West Nile virus based on passive transfer?
DR.
LEW: Right. I mean the idea was during the summer, there was some little
mini-outbreaks throughout the nation, and I am sure there were patients there
who received plasma products using this, and that might be a very easy way
quickly to see at least during the epidemic time in these areas, there wasn't a
higher incidence of patients who received these products and then showing
evidence of infection.
DR.
PIFAT: That has certainly not been
looked at. There has been, of course,
as has been reported earlier, no transmission from plasm-derived products. But the other thing to take into account is
that the plasma collected during the epidemic season, there is a delay in the
time it takes to manufacture the product.
So, during the epidemic, we would not have seen that.
At
this point, this plasma has been processed.
You know, most of the plasma that would have been collected in the
summer would have been processed by now, but I am not sure we have the
information to answer your question.
DR.
GOLDSMITH: I guess this is kind of a
comment and a little bit of a question.
I think the use of model viruses is very excellent when the pathogen is
not easily cultured in the laboratory.
In this case, with the West Nile virus, it can be cultured in the
laboratory and apparently at very high titer, at least according to some of the
manufacturers, but not all.
The
products that are used by people who use plasma derivatives include IGIV, and
the people who use that are primary immune-deficient patients, which are a
corollary group I think to those who have been infected by West Nile virus in terms of transfusion of cellular
components and have resulted in death.
So,
I guess from the perspective of that group of knowledge, there should be
assurances given to the patient communities that West Nile virus has been
tested for and has been eliminated from the product, if there is a proposal to
not reduce the viral load on the front end.
I see some real differences manufacturer to manufacturer in terms of the
starting inocula, and it may therefore impact on the end results that are shown
here.
If
there really is an epidemic, you could include potentially, I don't know, 100,
1,000 people carrying 1 x 105, which would mean at the end of the
day that you might overwhelm the system because you haven't tested it to a high
enough level.
So,
I would just, again, by way of comment and question, ask if additional data
could be obtained to get further assurances with larger inocula if there is a
plan to not test on the front end to reduce the viral load.
DR.
PIFAT: We are very aware of the
patients that are potentially receiving immunoglobulin products, for
instance. One important point to
remember is that when you talk about 10, 100, 1,000 patients being incorporated
into a manufacturing pool, you are talking about an arithmetic scale. The clearance that we get for these
processes are on the order of 1012 logs, so even if those patients
were incorporated in a manufacturing pool, if you do the calculations, there is
no reason to believe that the clearance of the manufacturing processes could be
overwhelmed.
DR.
GOLDSMITH: Let me just follow up. Are there really two steps then in the
process that you can show independently that removed 106 particles?
DR.
PIFAT: For most products, that is correct,
yes.
DR.
GOLDSMITH: Is that true for all
products?
DR.
PIFAT: For all of our products, that is
true.
Open Public Hearing
DR.
NELSON: I think we are now at the open
public hearing, I think Dr. Dodd wanted to make a comment for AABB, ABC, and ARC.
DR.
DODD: Thank you very much, Dr. Nelson.
I
am Roger Dodd. I am appearing here as
president of the American Association of Blood Banks, and I am speaking on
behalf of the AABB, America's Blood Centers, and the American Red Cross. My comments relate to West Nile virus
testing.
Actually,
many of the comments that are incorporated in this presentation have already
been made by the committee itself.
The
U.S. blood banking organizations have taken significant steps in response to
the 2002 mosquito-borne West Nile virus epidemic. The organizations have worked with CDC and with FDA to conduct
the case investigations that established that West Nile virus was
transfusion-transmitted.
As
this new scientific knowledge was acquired, the organizations communicated to
both their membership and to the general public that West Nile virus could be
transmitted by transfusion. The
communications indicated that the risk was small but real and that the benefits
of necessary transfusions outweighed the small risks due to West Nile virus.
In
2002, several actions were taken to minimize the risk of
transfusion-transmitted West Nile virus infection. An increased emphasis was put on the need to obtain post-donation
information from donors and to retrieve and quarantine units that were donated
by persons with a high index of suspicion of West Nile virus infection.
At
the end of the 2002 mosquito transmission season, a voluntary market withdrawal
of frozen plasma products intended for transfusion was initiated for components
derived from collections during the West Nile virus transmission season in
states with human cases of West Nile virus.
This
action resulted in the withdrawal potentially of over 150,000 such
components. At the November 6th FDA
West Nile workshop, the blood organizations indicated their full support for
the development of West Nile virus screening tests that would be put into place
for the 2003 transmission season.
Currently,
the organizations are planning for the introduction of such testing by the
target date of July 2003, and you have heard some of these plans. The planning includes participation in
preparing and submitting INDs to FDA, modification of laboratory space for
installation and validation of additional equipment where needed, staff training,
and revisions of blood center computer systems. The blood organizations are fully committed to implementing such
testing as soon as reagents and systems are available, even ahead of July 1st,
if necessary.
Although
several other actions are under consideration by FDA and others to minimize
West Nile virus transfusion risk in 2003, the blood organizations believe that
blood donor screening by minipool NAT will be the most significant intervention
to decrease West Nile virus transfusion risk.
We
have heard today some discussion about the potential of using IgM tests as a
screening measure, and this concerns us because our general experience is that
IgM tests tend not to be particularly specific although the data we have seen
today are quiet encouraging, but we believe in, and encourage, the performance
of assessments of the need or otherwise for IgM testing in such studies as Dr.
Stramer indicated as Phase I studies rather than as a broad-scale
implementation unless we know that this is really going to be needed.
A
review of initial data from West Nile virus transfusion-transmission cases
revealed that, in retrospect, a few
donors reported symptoms compatible with West Nile virus infection days to
weeks prior to their transmitting transfusion or donation.
But
as you saw from Lyle, the most current data indicated that only 3 of the 14
implicated donors for whom we have data reported pre-donation symptoms and in
at least one case, these symptoms were much earlier than the donation,
suggesting that these nonspecific symptoms might have been coincidental with
another condition and not to West Nile virus infection.
The
CDC data also indicate that while fever was reported as the most consistent
symptom for implicated donors, 3.7 percent of non-implicated donors reported
fever three weeks before to three weeks after the implicated donation.
Based
on the available data, it appears that the sensitivity and specificity of
additional pre-donation donor questioning would be low. It would be expected to be even lower once
West Nile virus minipool NAT is in place.
Furthermore, it is anticipated that questioning about fever or other
symptoms in the 7 to 14 days before donation will be nonspecific and may result
in deferral of large numbers of donors.
The
committee is reminded that this was found to be the case when a new question
concerning symptoms of diarrheal illness to prevent Yersinia enterocolitica
infection was piloted a decade ago.
Actions
that decrease the available blood supply should be taken with extreme caution
since blood shortages now unfortunately are frequent. Thus, the blood organizations are concerned about the potential
impact of adding an additional donor-screening question.
We
recommend that appropriate information about the impact on blood availability
be obtained before making a decision on this measure. The Red Cross, and I learned today the ABC, are in the processes
of planning joint studies to try and define the impact of such donation
questions to aid in decision-making.
With
regard to post-donation information, the blood organizations agree with the
need to emphasize that donors report such information promptly to blood
centers.
The
current FDA guidance on this issue should remain unchanged since the guidance
is consistent with procedures currently used by blood centers for other
post-donation information and is based upon medical director evaluation of the
information and a decision on appropriate action.
Blood
centers will continue to conduct investigations of possible transfusion-transmitted
West Nile virus cases, as this will be important to evaluate the efficacy of
donor screening. The blood
organizations will work with the Centers for Disease Control and Prevention,
and with their local state health departments to coordinate these
investigations.
As
you heard, blood centers are actively stockpiling FFP and other frozen plasma
products in anticipation of the onset of West Nile virus clinical cases this
year. In accordance with opinions
expressed by CDC scientists, the current plan is to stockpile such components
in a specific geographic area until the first human West Nile virus case is
reported in that area.
These
components would be available for use throughout the country and would be used
preferentially in place of frozen plasma products collected in regions with
West Nile virus clinical cases prior to the onset of screening. This frozen stockpile of units should be
considered safe for transfusion and should not require retrospective West Nile
virus testing after such screening is introduced.
While
it is possible to put such a program in place for frozen components, it is not
possible to duplicate this program for liquid components with limited shelf
life. If mosquito-borne West Nile virus
human cases occur in a geographic region prior to implementation of West Nile
virus donor screening, it would not be possible to cease blood collections in
that region.
The
blood supply is too fragile to implement such a strategy. Lack of available blood would be a greater
risk than the very small risk of West Nile virus transfusion-transmission that
would exist at the onset of the mosquito transmission season, when very few
mosquito-borne cases are expected.
This
point was made repeatedly and eloquently by FDA during last year's epidemic. The blood organizations will encourage their
members to work with local and state health departments to ascertain whether a
significant early epidemic is occurring in a specific county or region.
The
blood organizations are committed to minimizing the risk of West Nile virus
transfusion transmission in 2003 while maintaining an adequate blood supply.
We
believe that both tasks can be accomplished if testing is introduced at or near
the July 1 target date. If, however,
such testing is significantly delayed and if there is an unexpectedly large
early season West Nile virus outbreak, the blood organizations will work with
the Food and Drug Administration to consider appropriate policies.
Thank
you.
DR.
NELSON: Thank you, Roger.
Is
there anybody else who wanted to make any statements during the open public
hearing? Mike. Briefly, hopefully.
DR.
BUSCH: Very brief. I just wanted to show two slides to
summarize the studies that NHLBI decided to fund within the context of the REDS
group.
[Slide.]
The
first relates to the recall activities that occurred last year. The ABC sites around the country actually
recalled something in the range of 63,000 units, which I understood the value
of those was over $2 million, as a result of the discussions about concern over
potential transmission from these frozen products.
We
made an effort with the ABC program, Celso Bianco, to survey all the sites and
identify the units that were collected from the highest risk regions, highest
risk time periods, and ended up, at this point, had 1,500 and anticipate 2,000
of the highest risk units will have been identified and are going to be
sub-aliquotted and made available to all the companies.
They
will be tested by both Gen-Probe and Roche NAT, as well as the IgM and IgG
assays from the two major manufacturers, Ortho and Abbott, the goal being to
both assess the true safety impact of that large recall, which was kind of done
very quickly, and assess whether there truly were a substantial number of
viremic units, and then also to identify the viremic and seropositive units,
which there will be large volumes that can be made available to all the
manufacturers and to FDA.
The
second study is to collaborate again with the serology manufacturers, and both
Ortho and Abbott have been in close dialogue on this, to apply these serologic
assays to the serial samples that we have collected over the last three years
in the highest risk RADAR-REDDS group regions, which would include the Detroit,
Pittsburgh, and Chesapeake Baltimore regions, to look at the evolving
seroconversion rates through the courses of these three summers, and model,
therefore, the incidence of the infection during these three years.
That
will allow us to understand incidence over time and combining that with the
understanding of the early viremia, develop an incidence window period model
that is a little more direct. Lyle's
strategy involves extrapolating from clinical encephalitis cases to estimate
the prevalence and incidence in the donor pool. So, this will be a way to actually measure incidence in the donor
pool during these epidemic years.
[Slide.]
The
other activity is to actually, with these Ortho and Abbott assays established
for purposes of these retrospective studies, we are then planning to also make
these available to the NAT IND programs to the extent that this works out and
that we could actually make these validated assays available for donor
follow-up testing, and to collaboratively also, with all the programs, as we
have done with HIV and HCV, the goal will be to also monitor the yield around
the country in an ongoing context.
The
final piece, which I think may be of interest to the discussion, is another
strategy that we have discussed or sort of developing protocols related to,
would actually trigger the retention of stored specimens, the individual
samples, as we implement minipool NAT this coming summer.
In
regions where there is observed minipool NAT yield or active CDC case
reporting, the individual samples from those focused regions could be stored,
and as Sue described, they could be stored, if it's routine, for a month in the
Red Cross, but in other regions, I think there is no plans to routinely store
samples.
So,
the idea would be if there is minipool NAT yield in a particular donor region,
the individual samples could be saved, and then those samples could
subsequently be batch tested perhaps in an unlinked context to determine how
many samples that had low level viremia detectable only by ID NAT were missed
within those regions, and also one could test serologically all those
individual donation samples to see were there IgM positives and again
understand the incidence in a region in which minipool NAT yield is being
observed, which would then validate this incidence window period model.
Thank
you.
DR.
NELSON: Thanks.
Harvey.
DR.
KLEIN: I just wanted to comment. Since I am not an employee of the National
Heart, Lung, and Blood Institute, this is not a conflict of interest.
I
think this is just, for the record, very important to note, that studies such
as the REDDS study and the RADAR studies, which are prospective studies, are
very expensive, but incredibly important in the retention of those
repositories, have a value that is difficult to estimate at the time when they
are being done, but we can see from these kinds of studies, carry on for long
periods of time.
DR.
NELSON: I think we have learned a lot
from repositories, and we will continue, not only with West Nile, but other
agents.
Why
don't we take five minutes.
[Break.]
Questions for the Committee, Discussion
and Recommendations
DR.
NAKHASI: Let's get this session rolling
again. I guess by now you should be
knowing what the questions were and what were the answers, so it should not
take more time to answer these questions.
The
questions for the committee are, first of all,
Question No. 1. Please comment
on FDA's proposed criteria for validation of West Nile NAT and IgM assays for
donor screening.
Maybe
we should take one question at a time.
DR.
NELSON: I think so. Let me see if I understand what the criteria
were. In terms of specificity of the
NAT, you were talking about looking at 10,000 specimens in an area with
specimens that should be negative in the absence of an epidemic, et cetera, and
negative subjects.
In
terms of the sensitivity, you wanted a sensitivity of 100 copies/mL in either
an individual or a minipool.
DR.
HEWLETT: That would be the analytical
sensitivity of the test regardless of the pool size.
DR.
NELSON: Right. You have both the NAT and IgM assays. Let's discuss the NAT first. Does anybody have any questions or comments
about the criteria for sensitivity or specificity of the NAT?
DR.
ALLEN: My brain doesn't function too
rapidly sometimes. I assume that in
looking at the calculations of the ability to detect 100 copies/mL, that you
have looked at the sample size and the probability that there will be at least
one in there and that that has all been taken into consideration.
With
pooled samples, I know that we are talking about very minute sample volumes in
there, and I assume that that has all been taken into account to assure there
is a reasonable statistical probability that there will be a minimum number.
DR.
HEWLETT: Yes, as well as what is known
about the highest titers or at least what we know about circulating titers,
which is I think somewhere in the range of 105. We have heard some numbers and somewhere in
that range, so we would take that into account, as well, in trying to set a
limit of detection, as well as what is actually achievable in assays that are
being developed today.
I
mean that is another consideration obviously, if, you know, assays of today
don't meet that sensitivity, then, we would have to think about it, but what we
have learned is that most of the companies have assays that are well below that
limit, so it is something that is achievable for licensure.
DR.
NELSON: Are there any other comments?
Do
we need to vote on this, I mean we are happy with it or we are not happy with
it? It is not a yes/no question.
Well,
it seems reasonable from what we know about it--go ahead.
DR.
FALLAT: I would just say I have been
very impressed with hearing all of the various companies and how far along they
already are, and I take it, as you just said, the criteria you have set up, it
a realistic goal that July 1st, we will have something in place with those
criteria. Is that the bottom line?
DR.
HEWLETT: Yes, to some extent, because
the other thing we were trying to accommodate was the scarcity of samples, you
know, there are very few samples actually that are available for assay
validation, so that is why we have taken the approach that we would accept
testing of repository specimens, that we would accept perhaps testing of a
pedigreed panel, which we are trying to put together, and that we would also
accept some type of cross-comparison of the different assays that are being
developed using specimens that are identified as reactive in clinical
trials. So, a couple of different
options that we have laid out for manufacturers to pursue in trying to validate
tests.
DR.
NELSON: Not a part of your question is
the issue of whether it's a multiplex pool including testing for HIV, hepatitis
C, or if it's just a pool that is a separate sample tested for West Nile. Presumably, the sensitivity should be
similar in either of these formats, but maybe not.
DR.
HEWLETT: Obviously, it would be better
if they are of equivalent sensitivity for the different strains of West
Nile. Now, if they are combining it
with other viruses, again, obviously, ideally, it should not be at the expense
of compromising sensitivity of the other virus. I think Jay wants to say something.
DR.
NELSON: If a manufacturer included data
on the sensitivity, just testing for West Nile, but then included West Nile in
a multiplex assay where other viruses were tested at the same time, would that
satisfy FDA's criteria for adequate sensitivity?
DR.
HEWLETT: If I understand correctly,
Ken, you are saying that if a test for West Nile is developed, not independently,
but in combination with the other tests, they would have to validate the entire
multiplex, so that is an added issue.
DR.
NELSON: They would have to show the
sensitivity however it were going to be used.
DR.
HEWLETT: Right.
DR.
EPSTEIN: I think at the moment, that is
quite theoretical because you have heard that the assays that are being
developed are stand-alone.
DR.
HEWLETT: Basically stand-alone, yes.
DR.
EPSTEIN: I wanted to comment on the
earlier point about tests being ready by July 1. FDA didn't say we want tests by July 1. We want tests as soon as possible in the next warm months. In 02, the earliest cases were reported in
May. You know, from May to July is a
long time if we are talking about blood safety, and I think that we shouldn't
overstate the prospect of having tests available.
This
is why we have had, you know, the scientific workshop in November. It is why we have been revisiting the issue
periodically with the BPAC. We are
doing everything we can, and I think you heard the efforts that the companies
are making to move test development as quickly as possible, but I don't think
we should overstate certainty here.
You
know as well as we do what there is in the mill at the moment and how the
progress sounds, so we would all like to be optimistic, but I think it's
guarded optimism, and that is why you have heard us also talking about the
doomsday scenarios, you know, what if we don't have a test or what if we don't
have enough tests for enough regions.
So,
we just want to be very careful not to overstate where we are with development,
cautiously optimistic, and I wish it were sooner than July.
DR.
NELSON: Go ahead, Harvey.
DR.
KLEIN: I would just like to make the
comment that the tests so far all look very good, they look very sensitive, but
if perchance it should turn out that they are a little less sensitive than we
would like, I would hope that we wouldn't let the perfect be the enemy of the
good, because we never have with other assays, and we would eliminate most of
West Nile even if we let a few cases through, and I am sure you have thought
about that already.
DR.
HEWLETT: Yes, we have, and, yes, it is
better to have something in place rather than have nothing, so, yes, we agree.
DR.
NELSON: Judy.
DR.
LEW: I was just going to ask if FDA had
any guidance on how you would choose the other confirmatory NAT or IgM assay
because you kind of left it open that if you have a positive, and you want some
sort of confirmatory test, but there is obviously no gold standard.
So,
what would you recommend to industry or whoever, how do you choose, and who is
going to pay for those studies? I mean
how are you going to work that all out?
DR.
HEWLETT: For the NAT test, obviously,
an alternate NAT would be a good supplemental test to pursue. These are not
necessarily our positions. These are
just scientifically valid approaches.
An IgM assay to confirm the NAT test would also be another very viable
approach, and it is only by doing these studies that we are really going to
understand which of these, or would one need a combination of these approaches
to assure safety.
So,
that is why we are proposing these different approaches to be pursued under IND
during clinical trials and we will be able to have a better sense of exactly
what kinds of strategies would work best at the time of licensure.
DR.
LEW: I guess what I am trying to
address is like within a company, they make certain primers, and that is their
NAT test, and then within the same company, they choose totally different areas
of the virus and say, okay, that will be our confirmatory, or are they
obligated to go to a different company, or go to CDC, or some other place that
does TaqMan or a different type of test, a nucleic acid test.
DR.
HEWLETT: They can pursue different
options, develop their own supplemental test.
They can work with other manufacturers who are developing tests, and you
have heard about some of them today, that are focusing more on the supplemental
side of testing than the upfront screen.
So,
I think there are different options here.
DR.
NAKHASI: And if I may add, that is why
we would like to have the qualification panels, the pedigree panels which would
be then tested against, so that samples from different tests would be tested
against each other.
In
the absence of a gold standard, that would be the easiest way to do it.
DR.
NELSON: Jay.
DR.
EPSTEIN: I think that there is a
distinction that we make. You have
heard us use two terms, supplemental tests and alternate NAT, and what we are distinguishing
here is that in the context of the trial, other research methodologies may be
available to help validate or confirm the investigational result.
Those
other technologies we call alternate technologies because we are not implying
that they themselves are then also being validated as free-standing, approved
tests. So, when we use the term
"supplemental" tests, what we are saying is that someone goes through
the exercise to validate a free-standing test, which is intended to be used for
further testing in clinical use.
What
it would take to validate a supplemental test, I think it is implied that it
should work regardless of which screen you use. So, more work is needed to show that it is appropriate in
combination with other screens.
So,
for example, one would not be happy if it, say, had the same primers as the
screen. You would want it to have
different primers, but if it is to be a free-standing supplemental test, it
would have to have different primers than all the other screens or a different
basis of technology, something that makes it a truly "orthogonal"
test. So, there is more flexibility
when we talk about alternate assays.
DR.
NELSON: The second part of this
question relates to the IgM assays, and I didn't get the sense that there would
be screening, that there would be even adequate validation or assays, or
whatever, to also screen donors for IgM by July 1st or perhaps even July 1st of
2004, but I think obviously, the issues about criteria for validation are
different in these two assays.
Does
anybody want to make any comments on this? Apparently, there are
cross-reactions with other flaviviruses in the IgM assay, and they are
certainly quite different issues.
DR.
GOLDSMITH: I think we heard data that
showed that the IgM assay is not predictive, that the people who were infected
last summer were infected by IgM-negative/NAT-positive sample. So, as a screening test, it seems to me it
doesn't play a very predominant role.
DR.
NELSON: Right, only if it picks up a
small percentage who have a very low viral load that is not picked up on the
NAT, but then you have got the problem of the specificity of the IgM in the
face of other flaviviruses and the long persistence of IgM, perhaps persistence
from one season to another, that makes it difficult as a donor screening assay
I think.
Does
anybody want to add to that or disagree with it?
DR.
STRONG: I think that's the role for
that particular assay assuming that it qualifies. It is more of a confirmatory test to show seroconversion.
The
only other comment I might have about the supplemental assays here would be the
issue of the plaque reduction neutralization assays. I think the comment was made from the audience that that might be
impractical particularly if we have specificity problems, then we have to try
to demonstrate specificity using NAT.
That is certainly not available in very many labs.
DR.
HEWLETT: Right. I think that is why we proposed the second
IgM with the discordance being analyzed in some other way. The only other tool we can think of at this
point, it has been in use. I agree that
it is not widely available, but it's a tool that is known to be sensitive and
more specific. So, it is a tool that
could be explored during IND studies.
DR.
NELSON: Certainly, on follow-up sample,
the IgM, as far as we have known, is fairly sensitive in somebody who actually
has a West Nile infection, so that would be a very good use, to confirm in a
subsequent sample, whether or not a person actually was infected.
But
as far as an overall donor screening assay, it is perhaps quite a bit less
useful.
Jay.
DR.
EPSTEIN: Yes. I just want to clarify the FDA's current thinking about the
IgM. None of us really thinks that you
should screen with IgM and then defer all IgM-positive donors.
The
thinking is that if minipool testing proves to be insensitive, then, there
might be a value to screen with IgM, but the implication is that the
IgM-positive sample would then be tested on a single unit NAT, and we don't
know whether that need exists or not.
We
aren't going to know until repository samples, as has been proposed by Red
Cross, are tested with individual NAT and then retested at dilutions, and then
we will know.
DR.
NELSON: That study could give us quite
a bit of important information.
DR.
EPSTEIN: Dr. Busch mentioned the
possibility to retest samples from regions having positive minipools by single
unit NAT when we are in the clinical phase.
I don't know whether there is the intention also to test those samples
by IgM, but I think that would be another opportunity.
In
other words, if you have a region of human outbreak, if you are getting NAT
positives in donor screening, if you go back to that cohort of samples, you
have a second chance to look for samples that might be single unit NAT
positive/IgM positive, which would once again tell you whether you might have
picked those up with IgM.
I
think the concept is not to screen the whole donor base and defer the donor and
pitch the unit based on IgM, nobody really thinks that.
DR.
ALLEN: I think that is good. I think there certainly are lots of
additional studies that need to be done.
I applaud the approach of working collaboratively both with the test
manufacturers, as well as with the blood collection centers, to make sure that
we have got a variety of clinical specimens for testing and for analysis.
I
don't believe we have quite enough data yet to say that under no circumstances
could a NAT-positive, IgM-positive test or unit of blood be considered
non-infectious. I think we have heard enough data to suggest that it is not
likely to be infectious, but we have got a lot more to learn.
In
the absence of data suggesting that that is a real problem, I don't see a
reason to consider using IgM as a screening test for blood collection unless,
as Jay just indicated, subsequent studies indicate that there is a very real
benefit to doing so.
I
have got my own suspicions in terms of how that is going to turn out, but I
think it needs to be subject to study.
I think that the general information, going back to the specific
Question 1 that was asked, I think that the proposals that have been laid out
to validate the interpretation of the IgM assays, the sensitivity and
specificity, and so on, are quite adequate, and I commend you for the work you
have done and move forward on it.
DR.
NELSON: Let's move to Question 2.
Question
2 is I guess a yes/no question.
DR.
NAKHASI: Yes. Question 2 is: Do the
committee members agree that product and process-specific clearance of West
Nile agent (as opposed only to marker viruses)--that's the model virus--should
be demonstrated in order to adequately assure the safety of plasma derivatives?
You
heard the data from both, and the PPTA, as well as the presentation from
Mahmood Farshid.
DR.
STRONG: Just a question about the
question.
Is
the question directed at consistently using the agent to demonstrate
inactivation or is it that once validation of a marker agent could be
demonstrated, which of those two are we talking about here?
DR.
NAKHASI: It is I think whether we need
the specific clearance every time for the West Nile virus. Is that right, Mahmood?
DR.
FARSHID: Validation study to
demonstrate the clearance capacity is done only once for the given process and
product, so it is done at the smallest scale in the model outside the
manufacturer side.
DR.
STRONG: I understand that part, but you
can also validate the marker virus, which is a different validation.
DR.
FARSHID: The marker virus has already
been validated. The current manufacturing
process and current product has been validated by marker viruses basically to
show the clearance of HCV because they are being used as a marker for HCV.
The
question is whether this current data would be sufficient to demonstrate the
clearance of West Nile, or should West Nile be used in this validation study as
a relevant virus despite that will be done to do the clearance studies will be
West Nile or can we rely on the current data, which is done on the marker
viruses.
DR.
FALLAT: Didn't we see data on the West
Nile virus, however?
DR.
NELSON: Yes, but that was one
experiment, I guess.
DR.
FALLAT: Multiple experiments with
multiple different stages in the fractionation product, so that the 6-log
reduction occurs at at least two or more steps in many of the products. Wasn't that the data that we heard?
DR.
NELSON: I guess the question is what
more, in addition to the data that we have seen where there was some data where
West Nile was included with the marker, but what else would be normally needed
to validate this process.
DR.
FARSHID: As it was indicated, the
validation, which is done, should be product and processes specific, and the
validation package will be submitted to the FDA for evaluation. The thing that we saw here is basically what
we saw in public. We have not evaluated
the validation data, so basically, we have to take this result at face value.
If
the validation package is submitted to the FDA, then, we can evaluate it, and
based on that, maybe make a final decision on it.
DR.
FALLAT: I think what we saw was not
multiple steps for a single product. It
was a single step for multiple products.
We didn't see consecutive inactivation steps, and it was not a formal
validation study, as has been said by FDA, and I think that we have an
identified pathogen that causes deaths, we have a vulnerable population who
would be adversely affected, and I think that the manufacturers have an
obligation to test this virus in their system, and to do a formal validation
study that would meet FDA criteria.
DR.
FARSHID: The point can be made, what I
saw here is basically some of the result done on the clearance steps, which
they are robust. There are some
clearance steps which are validated for manufacturing process, for example,
precipitation, or removal steps, which they are not robust, and there is the
problem with reproducibility. We have
not seen result for those particular steps.
Currently,
we require that each manufacturing process to contain at least two major
removal and activation steps for each of them, and the cumulative log reduction
will determine the overall clearance capacity of the process.
So,
what we saw here is more or less fragmented and basically more emphasis on the
robust steps of evaluation. This is not to minimize the result, I think
they are very encouraging, but we like to see the validation package to
basically make the final evaluation.
DR.
NELSON: Do you want us to vote on this?
DR.
KLEIN: Could I ask a question?
DR.
NELSON: Yes.
DR.
KLEIN: It is both a comment and a
question. The comment is I guess if it's possible to do this kind of study, and
by that I mean if you get--we didn't see 6-log reduction, we saw more than
6-log reduction. We saw a 6-log
reduction in some instances in an hour where the period that you are going to
pasteurize is 10 hours, so you may not be able to get a titer high enough to
even test two separate steps in pathogen reduction here with this agent.
So,
I would say if it's possible to do that, I would like to see it done with the
agent. If it is not possible to do
that, I don't think you can hold up the companies because the science isn't
there. That is just a comment.
The
question is what does this mean in practice, do this mean in practice that in
the product labeling, you are now going to say it reduces West Nile virus, and
if they don't do the steps, they will still have everything exactly as it is
today, but it won't say in the package insert "reduces West Nile
virus?"
DR.
FARSHID: First, let me comment on your
first question, that when the steps are
validated, they are validated separately, so basically, if you use several logs
spike, it will be done for two separate logs.
They are not consecutively.
DR.
KLEIN: I understand that, but you might
be able to get a titer high enough to even show. We don't know what the reduction is in some of these steps. It might be 15 logs for all we know. I doubt it, but it's conceivable because we
saw very rapid disappearance below the level of detection for the virus in an
hour in some cases.
DR.
FARSHID: Basically, to comment on the
second question, in order to have a high degree of assurance that the
manufacturing process can clear a virus, we need to have the validation data
for it. Otherwise, if we want to rely
on extrapolation, and you think that is sufficient, that's a different story.
However,
for us to say that the process, with a high degree of assurance, can clear West
Nile virus without seeing the validation, I think may not be appropriate, at
least at that stage.
We
can say it by extrapolation and we can say with reasonable degree of assurance
that it would do that. This is
basically, as I mentioned, this is the approach which is being followed
regarding all the viruses, which they are relevant virus and also the other
viruses that we require validation for.
DR.
ALLEN: I am going to take a little bit
of a devil's advocate position on it and say that if there were any evidence
from epidemiological or clinical studies that there had been transmission of
West Nile virus through plasma-derived products, if there was any evidence that
this virus was particularly hardy or difficult to eradicate or to disrupt, I
would certainly concur with those steps.
I
have seen enough data today to suggest that this is not a significant problem
in these products and I would argue that absent that, I have difficulty asking
for additional regulatory requirements just so that we can be absolutely
certain under whatever.
There
are lots of other viruses out there. We
have picked up the things that we know about.
You know, I think there is a lot of things yet that we don't know about,
and I guess I have seen enough evidence today to suggest that this is not a
significant problem in this group of products.
I
would be interested as--I don't remember whether it was Dr. Lew of somebody--I
would like to see us perhaps take a group of patients who have been receiving
some of these products on a regular basis over the last 12 months, and do West
Nile virus testing, antibody testing on them to see if there is any evidence of
increased risk of infection.
That
is a fairly easy study that can be done, and absent that, I don't see that
there is any clinical evidence whatsoever that there has been transmission or
that there is a significant risk.
DR.
GOLDSMITH: The premise that you built
this on is that people have been exposed to plasma that was collected last
summer, and unfortunately, they haven't been. Plasma generally goes through a
long period of hold and observation.
You can talk to the manufacturers about how long that period of time
really is, but for it to be six or nine months is not unusual. So, it hasn't entered the manufacturing
stream or maybe just now is entering the manufacturing stream.
So,
we haven't really had a chance to see if this material transmits, and I don't
think we should think take that chance in all honesty.
DR.
LEW: If I could just add to that, I
will agree and disagree with you in the sense that I don't think the studies
have been done to show the safety, the epidemiologic studies, and because of
that, I don't think it is worth the risk, and as you just heard, obviously, it
is going to take a while for even people to be exposed.
If
we heard correctly, then, it may be that the epidemic is just going to grow
over the next few years as more mosquitoes are infected, and there is always
this population that is susceptible, so maybe even if we test it from the last
epidemic, we may not see it, but we test for the next epidemic, and then we
wait the whole cycle for those who are at risk to get those products, we might
see it.
It
is going to take so long to do this epidemiologic studies, why not just
validate it.
DR.
GOLDSMITH: I don't think the validation
is something that is done over and over again.
It is a set of experiments that are done one time on a model basis to
prove that the process is robust, and once you prove it, you get a claim in
your label, and I think that is the end of it.
I don't think anybody is asking the manufacturers to do this with each
lot and repeat and repeat and repeat.
We
are just asking them if it would be possible to do this one time and prove it
in the proper experimental theater.
DR.
KLEIN: What I was trying to get at is I
think if it is possible to do this with the agent, I think it should be
done. I wouldn't withdraw anything that
hasn't been tested for this until you can do the validation. I think that that is simply not going to be
labeled that it's safe for West Nile virus even though I think most of us
sitting around the table believe it really is.
But
absent the proof, I agree, I think if you have got the agent and you can do the
studies, I personally wouldn't rely on model viruses because I look at that as
sort of Stage I in your validation of any pathogen reduction system, and not
end stage. If you don't have the virus,
as with hepatitis C, then, you have nothing else to use.
DR.
NELSON: I think the concept of the
model virus came from hep-C and other viruses that can't grow and you don't
have a challenge.
Go
ahead.
DR.
FALLAT: I will just make a comment to
agree with what Dr. Goldsmith and Dr. Klein just said. Particularly when you look at Question 3, it
seems to me that if we say yes on this question, and that the answer comes back
that, in fact, there is adequate reduction from those one-time studies, I think
we can answer no to Question 3. So, I
think it all makes sense.
DR.
NELSON: The question is does the
committee agree that product and process-specific clearance should be
demonstrated in order to adequately assure--a yes answer is that West Nile
should be tested, validated, and a no answer is that the data from marker
viruses are sufficient.
DR.
SMALLWOOD: There are 11 members that
may vote on this question.
Dr.
Allen.
DR.
ALLEN: Yes.
DR.
SMALLWOOD: Dr. Davis.
DR.
DAVIS: Yes.
DR.
SMALLWOOD: Dr. Fitzpatrick.
[No
response.]
DR.
SMALLWOOD: He has gone. Okay.
Dr.
Goldsmith.
DR.
GOLDSMITH: Yes.
DR.
SMALLWOOD: Dr. Klein.
DR.
KLEIN: Yes.
DR.
SMALLWOOD: Dr. Laal.
DR.
LAAL: Yes.
DR.
SMALLWOOD: Dr. Lew.
DR.
LEW: Yes.
DR.
SMALLWOOD: Dr. Schmidt.
DR.
SCHMIDT: Yes.
DR.
SMALLWOOD: Dr. Harvath.
DR.
HARVATH: Yes.
DR.
SMALLWOOD: Dr. Fallat.
DR.
FALLAT: Yes.
DR.
SMALLWOOD: Dr. Nelson.
DR.
NELSON: Yes.
DR.
SMALLWOOD: Dr. Strong, how would you
have voted?
DR.
STRONG: Yes.
DR.
SMALLWOOD: The result of voting is a
unanimous yes. Dr. Fitzpatrick is
absent, and Dr. Strong, the non-voting industry representative agreed with the
yes vote.
DR.
NELSON: Okay. Now we are down to Question 3.
DR.
NAKHASI: Question 3. Do the committee members agree that
screening of all plasma for fractionation
for West Nile virus would add a safety margin in the manufacture of
plasma derivatives?
That
is the screening of the plasma to have an added value to the safety as is the
case of HIV and HCV.
DR.
NELSON: It's a little bit of a tricky
question.
DR.
FALLAT: The comment has been made and I
think I would agree with it.
DR.
NELSON: I guess the issue is yes, but
is the safety margin necessary or important.
Maybe we could just add the words either "necessary or
important," a necessary or important safety margin, so that if one voted
yes, one would feel that the source plasma donors should be screened because of
this necessary or important safety margin.
If
one voted no, you could still say that there was a safety margin, but it
wasn't, what, clinically or didn't have relevance to the safety of the
transfusion recipient. Is that fair? Jay.
DR.
EPSTEIN: Could I suggest that you want
to add the word "useful," without a useful safety margin.
DR.
NELSON: Okay.
DR.
FALLAT: It would seem to me a logical
way to state it would be to say that if, in fact, a plasma derivative product,
by doing Question 2, was proven not to be adequately reduced and made safe,
then, that product and any plasma obtained for that product should have the
pre-screening done.
In
other words, if you find a product that, for whatever reason, it only goes
through one step or something, and it is then used, I don't know what product that
would be, then, that would be reasonable to have pre-screening for that
product.
DR.
NELSON: That changes the question even
more.
DR.
FALLAT: Yes, I know it does, but it's
the logical thing to do, it would seem to be.
DR.
NELSON: Yes, because we could add
something, assuming that the product or process-specific clearance steps have
been validated. I don't think the
committee would want to vote yes, that they didn't need to do it, and then find
out that there was a product that West Nile wasn't cleared from, and that would
fall under that. I don't know.
DR.
STRONG: This is a question for the
FDA. Are you assuming that the
inactivation steps are going to work, and you would do this anyway?
DR.
FARSHID: Yes. The initial assumption was that the clearance step would work,
and the reason being, as I mentioned, these are considered to be complementary
steps, which together would provide the sufficient margin of safety, and that
is basically the current approach with regard to risk reduction strategy.
DR.
NELSON: It certainly is for HIV and
hepatitis C even though these viruses are probably inactivated.
DR.
GOLDEN: I just want to underscore what
Dr. Farshid was saying. I think that
for the viral clearance removal steps, we have the concept that you need
multiple steps because there can be failures, there can be manufacturing
failures for whatever reason, the right temperature, right incubation, right
concentrations of solvent detergent are not in the step, so that is one level.
The
other level is that the starting virus, I mean we have seen a lot of data,
which probably is reliable at this point, that the highest starting virus would
be 105 per collection, but you may have much higher titers in some
situations which we don't know about.
So,
having this screening test in place would definitely add, to my mind, another
level of safety, which is relevant and has been relevant for other virus
situations.
DR.
STRONG: On the other hand, you are also
going to have an increasing number of immune donors who are contributing to
that pool, which should neutralize the virus.
I think we have had examples in the past where we have removed
immunoglobulin-positive donors, and that has turned out to be a bad move.
DR.
ALLEN: I assume this would be NAT
testing only though.
DR.
GOLDEN: Yes.
DR.
NELSON: That is a good point, if the
screening were NAT testing, then, you would still have antibody-positive donors
in the plasma pool, and that probably would not be very much affected by the
screening of donors.
Jay.
DR.
EPSTEIN: I just want to comment that
part of what has been motivating Questions 2 and 3 is that FDA has developed an
historic approach to dealing with viral safety of derivatives, you know, learn
through hard lessons both with HIV and hepatitis C, and that we have this
combined approach of screening, which limits the titer of virus in a
fractionation pool, and inactivation, which provides assurance of virus
clearance, and the combination of the two provides a safety margin, in other
words, you cap the virus in the pool and then you have some assured level of
clearance.
Part
of what is going on here is what we are asking is, is there a good reason to
deviate from these well-established paradigms, because if the committee advises
us that we should not pursue rigorous viral clearance, we should not pursue
upfront screening, then, we want to know why this situation is different, and
what precedent is that establishing for other agents, because if the vote were
to be, you know, don't bother, then, we would want to know how to apply that
precedent should we be confronted with some other emerging agent.
So,
I think that is why we have phrased these as calling for the safety
intervention and seeing if you concur, because we are really asking you whether
you concur with our standard paradigm.
DR.
NELSON: Yes, and there are examples of
hep-C and transmission, IVIG, which underwent these removal processes that
failed. So, it's not a pipedream.
DR.
HARVATH: A question for FDA. I just wanted to say what we have heard here
today is an assumption that there will be tests available sometime in the
summer, but if we were to agree with this, given that there may be limited
testing available, I would think it would be more important to test your blood
components and to worry about this later after you have assurance that you have
a test that, first of all, you will have sufficient sources of the test,
because I see this as far less of a concern than the whole blood components
that we have heard about.
I
would just like to raise that point, and I am sure that that is part of your
strategy that you would put in place.
DR.
FARSHID: I think this has been
indicated in one of the slides, that the assumption is availability of the
test.
DR.
GOLDSMITH: I guess I would like to
support that stepwise concept that we deal with the validation first. If this testing is not done, then, we may
have a dichotomy of the plasma that is actually used to make products like
IGIV, because some of it will come from recovered plasma, it will come from
whole blood donations that will undergo NAT testing, and some will come to
source plasma that will not undergo, and then we will have two different plasma
streams, one of which may be more rigorously screened than the other, and I
think that sets up a precedent that probably is not something that we want to
put in place.
DR.
LEW: To me, the bottom line is, too,
that we are having to answer 3 without the data. I think this committee had dealt with that before. I didn't see any data that this is safe not
to screen plasma. I mean I think the
data suggests it is probably okay because of the low viral load, but we don't
even know what the infectious particle is.
It
may be much more infectious than HCV, I have no idea. So, we can't really say that it is safe without screening. So, when we don't have data, I think we have
to err on the side of safety in this particular case.
DR.
NELSON: Harvey.
DR.
KLEIN: I also tried to think of what
the down side would be in doing screening, and it seems to me that it's only
cost. I say "only," that's a
big thing for some people. But I think
unlike some of the other tests we do where we get donors out of the donating
pool and we are never able to get them back in.
Here,
after a month, literally anyone can come back in to donate. So, it seems to me that we don't have a lot
of the issues with the donor in screening that we have with some of the other
agents where there are false positives.
So,
it seems to me that it is hard to argue against there being a belt and
suspenders aspect. There is a safety
margin. Whether it is useful or not, I
think is a question, but it seems to me the down side is the cost of the test,
and that is probably a relatively small down side.
DR.
STRONG: Just to comment on that,
remember that there are quite different screening scenarios between the plasma
manufacturers and the whole blood screening people. The plasma people use very large pools, which are likely not
going to be effective in picking up these low titer viruses. So, they would have to set up a completely
different system in order to accomplish that.
DR.
NELSON: I guess the pool size is 512 or
higher, so it would have to be 107 or 106.
DR.
KLEIN: Again, you are not worried about
the low titer here, because you have two inactivation steps. You are worried about the ton of virus that
is here, so you are not worried about allowing a few people with low virus into
your pool.
DR.
NELSON: Plus you have antibody, I
guess, hopefully.
DR.
NAKHASI: I just wanted to comment. But we heard data today that in a pool of
512 and 64, you could pick up the sample and the prevalence was the same as
what was predicted, and the ways one can concentrate the samples and other
things, so one can overcome that problem.
DR.
NELSON: Right. We had data on it.
Should
we put "useful" in there or
leave it off?
DR.
NAKHASI: Do the committee members agree
that screening of all plasma for fractionation for West Nile would add a useful
safety margin in the manufacture of plasma derivatives?
DR.
NELSON: Do you agree with that
rewording? Okay.
DR.
LEW: Even changing the wording, I guess
my concern is we don't have the data to say that it would add a useful--what I
am saying is that if I was to err on the side of safety, yes, of course, we
should go ahead and screen. It costs
money, but it would be safer for immunocompromised patients, but we really
don't have the data really to know if it is truly going to be useful or not.
DR.
NELSON: Let's leave "useful"
off then, you are right. I mean we
don't have the data to vote on it, but
we are supposed to anyway.
A
yes would mean that it would add a safety margin, and a no vote would mean that
you don't think it would.
DR.
GOLDSMITH: Could we change
"would" to "could?"
DR.
ALLEN: I have got a question. Are we, in fact, saying if the committee
were to vote yes on this question, is that tantamount to telling the FDA that
we would be strongly supportive of their implementing a screening mandate?
If
we waffle on it in terms of could, and so on, I agree with Dr. Lew, I don't
think we have been presented with sufficient data especially if we add
"useful," although I like the question, and I agree with Dr. Harvath,
I think our primary concern right now is with blood components that are going
to be tranfused.
I
think this is an issue that needs to be addressed in terms of a recommendation
to the FDA a little bit further down the line once we get a little more data,
the tests have been licensed. I don't
think this is a high priority issue. I
think we gave a recommendation in terms of Question 2.
If
Question 2 is answered satisfactorily by appropriate tests, I think we have
made a significant step in that area.
Given that, I would be perfectly willing to defer a recommendation on
Question 3 until the tests have been licensed, we get a little bit more data,
and we know that all blood components are being screened.
DR.
NELSON: You could abstain.
DR.
ALLEN: I very well may, Mr. Chairman.
DR.
NELSON: That is a choice, I guess.
Let's
vote.
DR.
SMALLWOOD: Let me state the question
that we are actually voting on for clarity.
Do
the committee members agree that screening of all plasma for fractionation for
West Nile virus would add a safety margin in the manufacture of plasma
derivatives?
Roll
call.
Dr.
Allen.
DR.
ALLEN: Without repeat my statement, I
abstain at this time.
DR.
SMALLWOOD: Dr. Davis.
DR.
DAVIS: Yes.
DR.
SMALLWOOD: Dr. Goldsmith.
DR.
GOLDSMITH: Yes.
DR.
SMALLWOOD: Dr. Klein.
DR.
KLEIN: Yes.
DR.
SMALLWOOD: Dr. Laal.
DR.
LAAL: Yes.
DR.
SMALLWOOD: Dr. Lew.
DR.
LEW: Yes.
DR.
SMALLWOOD: Dr. Schmidt.
DR.
SCHMIDT: No.
DR.
SMALLWOOD: Dr. Harvath.
DR.
HARVATH: Yes.
DR.
SMALLWOOD: Dr. Fallat.
DR.
FALLAT: No.
DR.
SMALLWOOD: Dr. Nelson.
DR.
NELSON: Yes.
DR.
SMALLWOOD: The results of voting: 1 abstention, 2 no votes, and 7 yes votes.
Dr.
Strong, how would you have voted?
DR.
STRONG: No.
DR.
SMALLWOOD: The non-voting industry
representative agrees with the no vote.
DR.
NELSON: The final question is again an
essay question. Please comment on the
scientific validity of possible strategies to limit West Nile screening to
particular locations and times depending on epidemic surveillance information
and test availability.
As
I recall, I think that Dr. Williams' presentation was that the hope was that
there would be sufficient reagents and that the assay would be available for
uniform screening, but this I guess was in the also maybe perhaps likelihood
that they wouldn't be available by July, so this is an alternate scenario.
There
were also questions raised by the people who were actually working in the blood
collection and transfusion feel that it is not possible to turn this on and off
at different times of the year, and I was a little surprised at that because I
thought it might be possible given the fact that we have pretty solid
transmission times of the year and others where it is not, and I thought that
this might be a feasible strategy.
Discussion
on this?
DR.
STRONG: I might just comment that one
organization not to be named has still not been able to drop one test although
it has been allowable, so it can take more than a year to stop and start these
programs.
DR.
FALLAT: I think we heard a lot of good
comments after Dr. Williams' doomsday
talk, and I just wondered if one of those doomsday scenarios develops, how will
you really deal with it. What process
does one go through to really implement one of the major kinds of decisions
that you were talking about there of withholding or stopping?
DR.
NELSON: If you are going to have
elective surgery, move to California or something like this.
DR.
SCHMIDT: I think blood has to be a
uniform product. It moves all over the
country. The blood from New York goes
all over the country and that is true of other places, and people travel. I know you are just talking about testing
donors in an area you are worried about, but I think we have to keep the
product uniform.
DR.
KLEIN: I think if it's the situation
where you don't have enough assays, I think there are scientific data to say
that it may not be perfect, but it is probably better to do your screening in
an area where it's epidemic, and by labeling blood as being tested for West
Nile virus, you also give the physician the option, as Mike Fitzpatrick
suggested, of using that in particularly susceptible individuals.
I
look at that as an interim measure, as a gap measure until you have enough
assays. I quite agree that it is very
difficult to turn tests off and on. I
mean you have to revalidate your information systems for one thing, so that
once you did have enough of a test that was certainly acceptable for IND and
later license, you would use it uniformly, but if we should get in the
situation where we do have some tests, we just don't have enough, I think you
could use the kinds of strategies that Alan Williams suggested to best protect
the public health.
DR.
NELSON: There is an example of
screening for CMV, which is done selectively and units are transfused
selectively into selected patients, so those are patients here. We might talk about a time period or a
geographic area where, you know, very high risk, we would screen.
It
seems to me that that would be useful if we couldn't implement uniform
screening.
DR.
ALLEN: I think I spoke as to why I am
opposed to this kind of a scheme once we have the test licensed and readily
available. In a critical, you know, a
hyperepidemic period in a selected geographic area, I agree with that Dr. Klein
said, that it may be the better part of valor to try to do selective screening,
and so on.
I
think we have to think through, however, how we are going to implement this
sort of situation, and it may be that we alternate or, you know, that we move
apart from routine operating procedure, that we may need to designate a few
regional or national laboratory that is capable of handling that screening
wherever it is necessary to implement during the period of test shortage, and
that samples would then be sent by express mail to that one laboratory.
I
think if you are looking at blood collection centers around the country that
are doing their own laboratory testing, you are not talking about a practical
situation in terms of trying to get them geared up suddenly because there has
been, you know, the start of an epidemic of West Nile virus in that geographic
area.
It
is a stop gap measure. It is one that
if it looks as though we are not going to be able to roll the text out
coincident with the beginning of West Nile virus transmission in this country
this summer or late spring, we do perhaps need to look at alternatives, but I
think we have to look at the application of it very, very carefully to assure
that it is, in fact, going to work successfully.
DR.
SCHMIDT: Don't you have to change the
question to say if there is not enough to go around, because that is what you
are talking about. We have some precedent. As I remember the first HIV test was
made available in San Francisco before it was made available in other places.
But
we also have the situation of certain blood collection centers or agencies who
do more testing than is required, and word kind of gets around - our blood is
better than your blood because we are doing things we don't have to, so I think
we want to stay out of that.
You
have to change the question in order to accommodate what you are talking about
now.
DR.
NELSON: I don't know, Alan can answer
this, but it may have been that it wasn't only in the issue or shortage, it was
also in the issue of this being strikingly a seasonal epidemic, and therefore
do you want to screen for during the six-month period, all donors where you
know there is no risk. The only risk is
a false positive. It's a different situation
than HIV.
DR.
WILLIAMS: I think we are requesting
comments more specific to what to do in a test shortage situation. I think in the course of the talk I sort of
presented this scenario that if there were adequate testings, we could
reconsider whether full geographic testing year-round would be appropriate
after things are in place and we have test data available, so I think you are
correct in your assumption.
DR.
SCHMIDT: You will have to change the
question.
DR.
WILLIAMS: It's a request for a comment
rather than a specific question with a vote.
DR.
NELSON: We have filled up a blue book
with our essay.
Does
anybody else have any comments?
DR.
STRONG: Just that really the company
strategies I think are addressing this because at least what we heard this
morning, the rollout is to be to those areas which have had cases in the past,
and the ones that don't are the last to go up.
DR.
LAAL: Would it be feasible to have
designated centralized laboratories where it is possible to validate the test
in the next few weeks, months, and have samples from any area, geographic area
that looks like it is particularly badly hit be sent to that laboratory,
because you obviously can't do it all over the country, not knowing where it is
going to hit. Would that be feasible?
DR.
NELSON: There are central labs now
doing the NAT testing for HIV and hepatitis C.
I don't know, somebody who is a blood banker needs to answer that.
DR.
STRONG: The difficulty is that we all
have different information systems and different barcodes, and to validate that
would be a huge undertaking, so it is not really, not within the time frame we
are talking about.
DR.
NELSON: Actually, the development of
this assay has been remarkably rapid, and it looks like it's fairly well on
track. It is hard to know what will
happen. We may have it and there will be no epidemic, but we scared the
epidemic away, we scared that mosquito.
DR.
STRONG: I think that is a
demonstration, I think the FDA had it right, it's a demonstration of the power
of nucleic acid testing. You can see
from the antibody manufacturers how much longer it is going to take them to get
a validated assay in place, whereas, with nucleic acid testing once we have a
sequence, we can go with it although even having said that, to go with a whole
new platform and have it up and running in six months is a major undertaking.
DR.
NELSON: Any other comments? That is the benediction. We will see you again tomorrow at 8:30.
[Whereupon,
at 6:15 p.m., the proceedings were recessed, to be resumed on Friday, March 14,
2003, at 8:30 a.m.]