SGDEPARTMENT
OF HEALTH AND HUMAN SERVICES
FOOD AND DRUG ADMINISTRATION
CENTER FOR FOOD SAFETY AND
APPLIED NUTRITION
CONTAMINANTS AND NATURAL TOXICANTS SUBCOMMITTEE
OF THE FOOD ADVISORY COMMITTEE
VOLUME I
Tuesday, March 18, 2003
8:00 a.m.
4700 River Road
Riverdale, Maryland
PARTICIPANTS
Francis
Fredrick Busta, Ph.D. Co-Chair
James E.
Heubi. M.D. Co-Chair
MEMBERS
Alex
D.W. Acholonu, Ph.D.
Lawrence
J. Fischer, Ph.D.
Marion
H. Fuller, D.V.M.
Lawrence
N. Kuzminski, Ph.D.
Ken Lee,
Ph.D.
TEMPORARY
VOTING MEMBERS
James
Anderson, Ph.D.
Robert
D. Baker, M.D., Ph.D.
Larry R.
Beuchat, Ph.D.
Henry M.
Blumberg, M.D., Ph.D.
Margaret
E. Briley, Ph.D., R.D.,L.D..
Laurie
J. Moyer-Mileur, Ph.D., R.D., C.D.
Marguerite
A. Neill, M.D.
Virginia
A. Stallings, M.D.
Phillip
Tarr, M.D.
Patti
Thureen, M.D.
NON-VOTING
INDUSTRY REPRESENTATIVE
Toby L. Simon, M.D.
TEMPORARY
VOTING MEMBERS
R. Bruce Tompkin, Ph.D.
C O N T E N T S
PAGE
Welcome,
Introduction and Charges 5
Christine J. Taylor, FDA
Administrative
Issues 16
Jeanne Latham, FDA
Remarks
by Chairpersons 21
Presentations by Guest Speakers
Current
Marketing and Use of Powdered
Infant Formula 27
Sue Ann Anderson, FDA
Tennessee
Investigation 48
Matthew Kuehnert, CDC [via speakerphone]
Other
Relevant Investigations 88
Karl Klontz, FDA
Clinical
Consequences of E. Sakazakii Infections 121
John Alexander, FDA
General
Microbiology - Ecology, Pathogenicity,
Subtyping, Etc. 171
Maria Nazarowec-White, Canada
Microbial
Detection--Clinical and Food 197
Don Burr, FDA
Resistance--Thermal
and Other 221
Robert Buchanan, FDA
FDA
Field Survey of Powdered Formula
Manufacturing 246
Don Zink, FDA
Public
Comments/Industry Speakers
Jon A. Vanderhoof 320
Les Smoot 329
Russell Merritt 347
Jatinder Bhatia, M.D. 351
Preliminary
Subcommittee Discussion on Clinical Presentations 388
P R
O C E E D I N G S
Welcome, Introduction and Charges
DR.
BUSTA: Good morning. We're here to
convene the Contaminants and Natural Toxicants Subcommittee Meeting on
Enterobacter sakazakii Contamination in Powdered Infant Formula.
The first
presentation this morning will be by Christine Taylor, who is the director of
this group. And without going any
farther, I will just turn this over directly to Christine from the start.
DR.
TAYLOR: Welcome and good morning. I am
Christine Taylor, Director of the Office of Nutritional Products, Labeling and
Dietary Supplements. Thank you for all for agreeing to participate in the
advisory subcommittee meeting. You are an illustrious group of experts and we
are looking forward to your discussions today and tomorrow.
My job
here today is to set the stage for the more in-depth presentations and white
papers you will soon hear and to set the stage for the regulatory context that
leads us to you ask you questions about Enterobacter sakazakii and powdered
infant formula. As I am sure most of you know, FDA's mission is ensure a safe
and adequate food supply. And recent findings have led us to ask questions
about E. sak and powdered infant formula.
I think
it's important to recognize that within the Center we view not only the
regulatory issue of powdered infant formula, but also it raises some
interesting microbial questions. So, in
pulling together this effort, many factors and groups within the Center were
involved.
I think
the first point I want to make is a kind of tried and true one, which is that
obviously one of the reasons we're here is that FDA has the mission of ensuring
a safe and adequate food supply, and certainly infant formula falls into
that. And the short summary--the one
sentence reason for being here--is that recent findings have raised questions
about Enterobacter sakazakii encountered in infant formula.
[Slide.]
Clearly,
in addressing this issue, FDA needed extensive scientific input. It needed to be of the microbiological kind,
the physiological and clinical kinds, particularly in the world of infants, and
the manufacturing and processing of powdered infant formula. This required a diverse set of expertise,
and the subcommittee that you see sitting amongst yourselves, before yourselves
and in front of yourselves really include quite a wide range of expertise,
including temporary voting members to round out expertise. And Ms. Latham later on will clarify various
members' roles.
Could I
have the next slide?
[Slide.]
As I've
mentioned before, our focus on infant formula.
Infant formula is a product
regulated by the Food and Drug Administration. And currently, in-place GMPs do not specify microbiological
testing for E. sak. Infant formula, as probably many if not all of you know,
comes in two forms: powdered and
liquid, and the powdered, which is the subject of this advisory committee
meeting, in terms of dollar sales, is about half of the infant formula sold.
Just a
little bit of regulatory context: there is an Infant Formula Act. It's part of the Food, Drug and Cosmetic
Act. And this particular set of
regulatory specifications does indicate that manufacturers must retain records
pertaining to the microbiological quality and purity of their product. I do want to point out in the next slide,
however, that Infant formula good manufacturing practices, while proposed, have
not yet been finalized. And, more
specifically, there are no current GMP regulations for powdered products. Many of you may know that liquid formula
comes under the Novak and Tennessee provisions, but powdered formula have no
such provisions.
FDA has
proposed GMPs to include practices to minimize opportunity for microbial
contamination.
Next
slide.
[Slide.]
You'll
note that despite listing a number of microbial issues, the agency did not
specify, nor has it specified, provisions for Enterobacter sakazakii
In short,
what I'll try to do in the next few moments is tell the story of Enterobacter
sakazakii in powdered infant formula, and then briefly go over the charges to
the committee; the questions that FDA is asking.
At that
point, I will turn the meeting over to your two co-chairs. The Committee is chaired by two very
prestigious individuals, and they will do a presentation of background
information--a series of white papers--and then proceed to an open comment
period, followed by a discussion set of recommendations.
The
staring point for the Enterobacter sakazakii was in April of 2001, an infant in
Tennessee died of meningitis with E. sak. It was a premature infant in a
neonatal intensive care unit or NICU. The Centers for Disease Control and
Prevention investigated the situation and determined a link between the
infant's death and E. sak in the powdered formula fed to the infant in the
NICU. Because infant formula
manufacturing is under the purview of FDA,
the CDC notified FDA. In response to
the CDC input, FDA undertook several activities. The Infant formula--what was
left of it on the market--was recalled.
And, in addition, FDA conducted
outreach to health care professionals. The agency began to examine products and
ingredients used to make this formula, and manufacturing, for the presence of
E. sak, and we . conducted some related research.
In the
area of outreach, we obviously focused on health care professionals in
hospitalized settings, because the incident at hand did occur in the NICU. We drafted and disseminated very widely a
letter to health professionals. We
created and posted an FDA "talk paper" on our website. And then in addition, CDC itself published
its findings in reported the Enterobacter sakazakii in the MMWR.
In our
letter to health professionals we emphasized certain things. Powdered infant formula was a potential
source of E. sak infections in infants, and we pointed out that the risk
increased with prematurity and any underlying medical conditions. We
indicated that low levels of E. sak can lead to infection and we also
highlighted the CDC findings in Tennessee regarding the fatal meningitis case. In addition, we recommended that powdered
infant formulas not be used in the NICU, unless there was no alternative
available. Oftentimes powdered formulas
are specialized formulas, and some physicians have indicated alternatives
wouldn't be available And we also
provided suggestions for reducing infection risks. These included the use of boiling water and certain feeding and
related practices in hospitals.
We did,
later on, revise this letter to health
professionals, and we focused largely on the question of boiling water
preparation because possible problems associated with the loss of
heat-sensitive nutrients, the physical changes to the formula, and perhaps even
inadequate E. sak eradication, as well as
injury to persons preparing the formula. And this was done in October of this year.
A second
part, in addition in addition to our letter and outreach to help health care
professionals, involved FDA's actually going into plants and testing infant
formula, as well as infant formula ingredients.. Our purpose then was to look for E. sak in powdered infant
formula products and the raw ingredients.
We sampled all domestic powdered
infant formula facilities as well as
all contract and manufacturing facilities. And, in addition, for
transparency and clarity, we also posted the analytical method we were using on
our website.
FDA, as a
result of this sampling, did find some positive low levels in both finished
product and in the raw ingredients.
It's been in positive finished products that we have identified that
have already been marketed, and there were two such products that were recalled
voluntarily by the manufacturers, and no known cases of E. sak infection
resulted from the consumption of these products is known.
In
addition, we did conduct some related research. As I've mentioned, we worked on the method for protecting from
Enterobacter sakazakii in powdered formula.
We also did various studies on heat [inaudible due to technical
difficulty]. And also we conducted some
studies to show effects of water temperature of nutrient levels in infant
formula, which appear
at this point to not have been finalized.
In
addition, there were related activities in response to the FDA outreach. Pediatric hospitals and laboratories also
[inaudible due to technical difficulty].
The
industry itself conducted analyses and investigations, and as probably many of
you know they worked hard not to distribute products that were found possibly
to contain this particular strain.
I'm here
largely to give you the story of why we're here, and what you'll find out after
the opening of this meeting is the specifics of much of what I've discussed,
ranging from the specifics on the Tennessee case, to the FDA efforts in product
sampling. And so, with your indulgence,
I'll simply give an overview for now to you that later on this morning there
will be a variety of white-papers presented, designed to give you the specifics
on available information once we get the final documents.
We'll
begin with the current marketing and release of powdered infant formula, fun
through the overview of the Tennessee investigation and other relevant
investigations, in Enterobacter sakazakii.
We'll provide for you information on the clinical consequences of the E.
sak investigation, as well as some of the general microbiology and detection
and [inaudible due to technical difficulty], and then end with the findings
from our previous survey in the infant formula industry.
Given all
of that, there are two basic charges to this particular subcommittee. The first charge is a request to
characterize the infants at risk, relative to Enterobacter sakazakii in
powdered infant formula; if there is a risk, to identify the populations of
infants at risk, with due consideration to whatever factors are relevant to
morbidity, other related areas, such as age, on immune status.
The second
charge to the subcommittee begins: "If there is a meaningful risk, how can
this risk be addressed? What
intervention strategy can be used in infant formula processing plans? Are there other intervention
strategies? We've taken a look at the
possibility of labeling and directions for use. Is it possible, based on available information, to specify
allowable lower levels of microbial detection of E. sak in infant formula, and
do allowable levels vary by risk characteristics of the infant. And then, finally, what are the critical
knowledge gaps and research priorities.
Those are
basically the two charges to the subcommittee.
There is
a final point relative to our efforts to finalize the infant formula final rule
on GMPs. The agency will be reopening the
comment period for this final rule. Any
issues that may come from the discussions today and tomorrow that would be
relevant to the final rule can be commented upon by interested parties at the
time of that comment period.
Again, I
want to mention that I'm so pleased and very proud of the fact that the agency
was able to convince the important people here at the table to come and discuss
the details of this very interesting issue, and also emphasize that I am
telling an initial story, and many of you might have questions about, in terms
of either the outbreak in Tennessee or the FDA's activities, will be both
addressed and answered by others following me.
And my
role--I'm setting the regulatory context about FDA's responsibility in the area
of Infant formula, and will end right there.
Thank you
all again. I'm happy to take questions.
DR.
BUSTA: Any questions or clarification from the committee?
[No
response.]
DR.
TAYLOR: I also, sitting at the table here, will answer questions that may come
up. Thank you.
DR.
BUSTA: The next item on the agenda is the administrative issues, and Jeanne
Latham will cover those.
Administrative Issues
MS.
LATHAM: Good morning. Is this on? I'm Jeanne Latham. I will be replacing Dr. Henry Kim as the Executive Secretary of
the FDA Contaminants and Natural Toxicants Subcommittee of the Food Advisory
Committee for the purposes of this meeting.
I want to
welcome everybody, and I'd like to read the conflict of interest statement for
the record.
By the
authority granted under the Food Advisory Committee Charter of July 202, the
following individuals have been appointed as temporary voting members by Joseph
A. Leavitt, Director, Center for Food Safety and Applied Nutrition: James
Anderson, Robert Baker, Larry Beuchat, Henry Blumberg, Margaret Briley, James
Heubi, Laurie Moyer-Mileur, Marguerite Neill, Virginia Stallings, and Patti
Thureen. Dr. Phillip Tarr is a special
government employee to FDA Center for Drug Evaluation and Research, and is
serving as a temporary voting member under the authority of Lender Ari
Skledeny, Associate Commissioner for External Relations.
The
issues to be discussed at this meeting are issues of broad applicability. Unlike issues in which a particular
sponsor's product is discussed, the matters at issue do not have a unique
impact on any particular product or manufacturer, but may have widespread
implications with respect to all infant formulas and their manufacturers. To determine if any conflicts of interest
exist, the committee participants have been screened for interest in companies
that make infant formula. As a result
of this review, in accordance with Title 19 of the U.S. Code, Section
208(b)(3), Dr. Virginia Stallings has been granted a particular matter of
general applicability waiver that permits her to participate fully in the
matters at issue. A copy of the waiver
statement may be obtained by submitting a written request to the agency's
Freedom of Information Office, Room 12A30 of the Parklawn Building.
We would
also like to note for the record that Dr. R. Bruce Tompkin is participating at
this meeting as the acting Industry Representative and a non-voting member of
the committee. In the event that the
discussions involve any other issues not already on the agenda for which FDA participants
have a financial interest, the participant's involvement and their exclusion
will be noted for the record.
With
respect to all other participants, we ask in the interest of fairness that they
address any current or previous financial involvement with any firm which makes
infant formula.
In
addition, I'd like to take a few moments to very briefly refresh everyone's
memory about Advisory Committee operations, particularly the roles and
responsibilities of the Food Advisory Committee members and FDA staff. The committee members have been provided
with a copy of a Committee Member Guide to FDA Advisory Committees, and there
are copies of the member guide available at the registration desk for anyone
who may be interested. The Member Guide
is in need of updating, but by and large it provides a good operational
overview.
As a
scientific regulatory agency, FDA needs access to highly qualified expert
external advisors who can provide scientific and technical advise. Thus, FDA uses advisory committees to
supplement the agency's internal expertise, and help the agency staff stay
current with state-of-the-art technology.
Committee members participate in an advisory capacity, and final
decisions are ultimately made by agency officials.
With
respect to the committee members, the chair of the committee--or, for purposes
of this committee, the co-chairs--preside at and conduct the meeting, and they
may ask the FDA staff for clarification at any time during the meeting. Each committee member contributes their
unique scientific and technical background, education and experience to the
committee process. The standing
committee members--and, in this case, that is the Contaminants and Natural
Toxicants Subcommittee--generally are voting members, including a consumer
representative who is voting, and an industry representative who is non-voting,
as specified in the charter.
All
voting members of advisory committees are appointed by the Commissioner, and
must be cleared as special government employees under FDA's conflict of
interest regulations. Other advisory
committee participants may include consultants, experts and guest
speakers. At this meeting, the
consultants are temporary voting members with expertise infant formula and
pediatrics, who will participate in the discussions of the committee. The guest speakers will present pertinent
information to the committee, and will not participate in committee discussions
or vote.
Additionally,
the advisory committee process encourages public interaction with the agency in
arriving at decisions. Not only is
there a consumer representative--in this case, a voting member--appointed to
serve on the committee, but the public is invited to appear before the
committee during the open public comment period.
FDA
participates in a listening mode, and responds to questions or a need for
clarification by the committee as needed through the chairs.
Thank you
very much.
Remarks by Chairpersons
DR.
HEUBI: Good morning.
We would
like to have all of the committee members identify themselves and what their
skill set is that they bring to the table that helps them be uniquely qualified
to come today.
I'm Jim
Heubi. I'm a pediatric
gastrologist. I run a clinical research
center at the Children's Hospital, and I participate and actively pursue
nutrition-related research.
I think
if we'll just go around the table, from Dr. Tompkin.
DR.
TOMPKIN: I'm Bruce Tompkin, and I'm retired now from ConAgra Foods, where I was
Vice President of Product Safety. I am
a food-market biologist, and basically that's all I've done throughout the 39
years' experience I've had in the food industry.
DR.
BEUCHAT: I'm Larry Beuchat. I'm from
the Center for Food Safety, University of Georgia. My area of research is in food-borne pathogens-mostly. I do also work with yeasts and molds,
spoilage problems. I've worked with a
number of pathogens. I've worked, on
occasion, with infant foods, but not with E. sakazakii--rather bacillus serius
and, and never hemorrhagic before.
DR. TARR:
I'm Phil Tarr. I'm from Washington
University in St. Louis. I'm a pediatric gastroenterologist, and also have
appointment in microbiology. My
interest is in food-borne infections, particularly bacterial infections.
DR.
ACHOLONU: My name is Alex Acholonu. I'm
from Alcorn State University in Mississippi.
I'm a professor of biology. I'm
an epidemiologist in the area of microbiology and parasitology.
DR.
THUREEN: My name is Patti Thureen, and I'm a neonatologist at the University of
Colorado, and my particular research interests are in fetal and neonatal
nutrition and metabolism.
DR.
FULLER: I'm Marion Fuller. I'm with the
Florida Department of Agriculture and Consumer Services. I run the Division of Food Safety. I'm a veterinarian by training, with
certification in general toxicology.
DR. LEE:
I am Ken Lee. I chair the Food Science
Department at Ohio State University. My
work area is in the process-induced changes in nutrients.
DR.
MOYER-MILEUR: I'm Laurie Moyer-Mileur.
I'm from the University of Utah.
I'm a research associate professor in pediatrics and a registered
dietician with over 20 years of clinical experience in the NICU, monitoring
babies with lots of feeding intolerance.
DR.
NEILL: Good morning. I'm Dr. Peggy
Neill. I'm an adult infectious disease
specialist, currently at the Brown University Medical School in
Providence. I have background in public
health epidemiology and food safety, with a particular interest in E. coli 0157
infections.
DR.
BAKER: I'm Robert Baker. I'm professor
of pediatrics at University of Buffalo.
I'm a pediatric gastroenterologist at Children's Hospital of
Buffalo. My particular research
interest is in gut barrier function.
DR.
BLUMBERG: Good morning. My name is
Henry Blumberg. I'm in the Division of
Infections Diseases at Emory University in Atlanta. I'm also the hospital epidemiologist at Grady Memorial Hospital,
and my interests include hospital and molecular epidemiology and clinical
research training.
DR.
BRILEY: I'm Margaret Briley, profession of nutritional sciences at the
University of Texas at Austin, and my research is with children and pre-school
and nutrition issues.
DR.
FISCHER: I'm Larry Fischer, from Michigan State University. I direct the Institute for Environmental
Toxicology there. I'm a pharmacologist
and a toxicologist. My research often
deals with age-related changes in susceptibility to chemical toxins.
DR.
STALLINGS: I'm Virginia Stallings. I'm
from Children's Hospital in Philadelphia, and professor of pediatrics, and at
times have run a nutrition support service--a large part of that service is in
neonatal intensive care units.
Most of
my research is on children with chronic disease and in nutrition.
DR.
KUZMINSKI: My name is Larry Kuzminski.
I'm retired from the food processing industry. I was with Ocean Spray Cranberries, where I was a vice president
of research and development and operations.
And prior to that I was with the Kellogg Company, where I was vice
president of science and quality in Canada, and director of food and research
in the United States.
My
interests are in food safety, and in the application of HACCP principles in
manufacturing processes.
DR.
BUSTA: And I'm Frank Busta, and I'm a professor emeritus a the University of
Minnesota, food microbiologist and have worked my entire life, mostly, with the
bad guys. And I'm pleased to welcome
all of you. It looks like a tremendous
committee, and the entire group is covered.
DR.
HEUBI: Jeanne tells me that I should announce that Dr. Anderson is unable to
attend because of family-related problem today. And I do want to remind you to use the microphones whenever you
speak because, obviously, the audience can't hear unless we do, plus we can't
hear ourselves.
And I
want to refer to your package that includes materials in this blue folder--that
includes the handout slides from the presenters.
And we're
going to make every effort to stay on time today, so that we can give full time
and deliberations that they're asking us to make today.
Yes?
DR.
STALLINGS: Who is our consumer representative?
MS.
LATHAM: Dr. Margaret Briley.
DR.
STALLINGS: Okay. Good. I just wanted to know for sure. Thank you.
PRESENTATIONS BY GUEST SPEAKERS
DR.
BUSTA: So, our first presentation today is going to be on current marketing and
use of powdered infant formulas, by Sue Ann Anderson.
The
guidelines state that we're to have 20-minute presentations, with 10 minutes
for questions from the committee.
Current Marketing and Use of Powdered
Infant Formula
DR.
ANDERSON: Good morning. This morning
I'm going to talk about how powdered infant formula is currently marketed and
used in the United States.
If I
could have the first slide, please?
Next slide? There should be one
before that. Well, in any case then, my
talk will focus on three topics: the types and uses of formulas currently
marketed in the United States; preparation of powdered formula for feeding; and
feedings to infants in health-care settings.
My
remarks this morning will be brief and very general in nature. Greater detail can be found in the White
Paper that is part of the briefing materials for the Committee for this
meeting.
In order to identify what the products are and who the consumers
are, I should begin by defining "infant formula" and
"infants." Infant formulas
are products intended for use solely as a food for infants. They are simulate human milk and are
suitable as a complete or partial substitute for human milk. Next
slide, please.
[Slide]
Infants
are defined as persons not more than 12 months of age. This slide set is not my slide set. We'll go ahead and work with it, though.
With
those definitions in mind, I would like to provide a little information on the
introduction of formula for infant feeding.
Powdered infant formulas were introduced well over a hundred years ago,
in the late 1800s. Liquid forms were
not introduced until the mid 20th century, with liquid concentrates
coming onto the market in the early 1950s, and ready-to-feed products coming
onto the market about 1960.
It should
be noted that powdered infant formula are not sterile products, however the
liquid products are. After the liquid
forms were introduced, the proportion of infant formula sold in powdered form
decreased. The lowest proportion of
sales of powdered infant formula occurred from about 1970 to 1980. Around 1980, technical advances in the
processing of powdered infant formula resulted in products that were more
readily dispersed in
water, making it easier to reconstitute the powders
for feeding. Since 1980, the proportion of powdered infant formula sales has
risen and in 1999, sales of powdered infant formula accounted for about half of
the dollar sales of infant formula products in the United States. This estimate
was based on data collected from supermarket, drug store, and mass merchandiser
scanners by
Information Resources, Inc. I should also note that the proportion of
infant formulas sold in powdered form in the United States differs from that of
other countries. In most other countries, almost all infant formula is sold in
the powdered form.
I should also note at this point that the
proportion of infant formula sold in powdered form in the United States is
quite different from that in other countries.
In most other countries, almost all infant formula is sold in powdered
form.
We'll see
what the next slide is.
[Slide]
There is
also a difference in the cost of feeding of the infant formulas, with the
powdered infant formula being the least expensive, and concentrate
intermediate, and ready-to-feed the most expensive product.
Most
infant formulas are formulated to meet the nutritional needs of infants with
generally good health status.
And, if
you will bear with us for just a couple of minutes, we're going to see if we
can find the right slides here.
[Pause.]
MS.
LATHAM: This is Jeanne Latham. I just
want to apologize for our technical difficulties. This is what happens when you try to perfect a presentation and
you provide many, many different versions of it. So, hopefully, we're going to find the correct one.
[Pause.]
DR.
ANDERSON: Okay,.why don't you advance and I'll tell you where we are. One back.
Most
infant formulas are formulated to meet the nutritional needs of infants with
generally good health status. Included in this group are term and preterm
infants, with term infants being those
born at or later than 37 weeks gestational age, and preterms born before 37
weeks gestational age. These products are available in both powder and liquid
form.
Could I
have the next slide, please?
[Slide]
Other infant formulas are designed to meet
the needs of infants with special medical conditions such as genetic disorders
of metabolism (for example, PKU) or other system disorders, for example
gastrointestinal diseases. With the exception of protein hydrolysate formulas,
this category is available only in powdered form.
Could I
have the next slide, please?
[Slide]
Okay. Because the infant population is at risk for
nutrition insult and infection, proper preparation, storage, delivery, and
disposal of their feedings are critical for prevention of food-borne
infections. In order to provide
consumers with accurate information for preparation and use of infant formulas,
there are specific Federal regulations for infant formula labels. Preparation
and use instructions required on infant formula labels include 1) product
storage, 2) sterilization of water, bottle, and nipples, when necessary, and 3)
dilution for powder and liquid concentrates showing major steps for
preparation, including a required pictogram for illustration.
Could I
have the next slide?
[Slide]
This next
slide shows the pictogram from the Code of Federal Regulations, depicting
instructions for formula dilution. The wording on the slide is difficult to
read, but it does say "Sterilization is recommended. Your physician will
decide if it is not required."
Next
slide.
[Slide]
Other
information is also required on infant formula labels, and it's required to caution against improper preparation or
use. Cautionary statements such as the following are required:"The health
of your infant depends on carefully following the directions for preparation
and use;" "Use as directed by a physician." And, also, a "Use by" date
required on the infant formula. The
"use by" date is a date
selected by the manufacturer on the basis of tests or other information showing
that the formula, until that date, under conditions of handling, storage,
preparation, and use prescribed by label directions, will contain not less than
the quantity of each nutrient as specified on the label and will otherwise be
of acceptable quality.
Next
slide.
[Slide]
Each
manufacturer designs their product labels to conform to these regulations and
the wording and pictograms on labels will vary for each manufacturer. For
example, directors for preparation of water for reconstituting infant formulas
generally fall into two categories. On
this slide, you see the first category, which
includes some direction to boil the water
and to cool it before adding to the infant formula.
Next
slide.
[Slide]
This may be
hard to see, too, but this is a sample from an infant formula label where it
does say to boil the water. It says to
boil the water for a minute, then cool;
pour the cooled water into a bottle;
add the powder and cap bottle, shake well and feed immediately.
The
second category ---next slide.
[Slide]
The
second category of instructions for water preparation includes an instruction
to consult with the doctor about the
need to boil water.
Next
slide.
[Slide]
And a
sample pictogram from a label with this sort of information says, "Your
baby's health depends on carefully following these easy directions. Ask your baby's doctor if you need to boil
sterilized water for formula and bottle preparations, pour the desired amount
of warm water into a bottle, add the powder, cap, shake very well and it does
include a statement "After feeding, throw away any formula remaining in
the bottle."
Next
slide.
[Slide]
Instructions
for feeding and storage direct the consumer to feed immediately or cover, refrigerate, and use, either within 48 hours--next slide--
[Slide]
or, on
some formulas, in 24 hours.
Some labels--next slide--
[Slide]
--also
include instructions for disposing of the unused formula, such as "Discard
unused formula after feeding," or "Throw away prepared formula left
in the feeding bottle, or cap within one hour after feeding begins.".
Examples of such statements are shown on the next slide.
[Slide]
Use of
commercially sterile infant formula products is not always feasible in health
care settings and, in some situations, formulas must be prepared from
nonsterile, powdered products for hospitalized infants. The American Dietetics
Association (ADA) has developed guidelines for preparation of infant formulas
in health care facilities. These guidelines include detailed recommendations
intended to decrease the risk for microbial contamination and growth during
infant formula preparation, storage, and use in health care facilities. The guidelines have been updated recently,
and are available on line at the URL shown on the screen.
I will
not go into detail about the recommendations for preparation of formulas, but I
should say that the ADA guidelines for delivery and disposal of infant formulas
for nipple-fed and tube-fed infants ---there are guidelines for these groups in
health care settings.
Next
slide.
[Slide]
For
nipple-fed infants, the instructions are given to feed within 4 hours of
preparation, cover and refrigerated for up to 24 hours. If the formula is warmed, it should be
warmed quickly, in less than 15 minutes; and to discard any product remaining
in the bottle one hour after feeding begins.
Next
slide.
[Slide]
For
tube-fed infants given intermittent feedings, the formula should be, again, fed
within four hours of preparation, covered and refrigerated for up to 24 hours
if not used immediately. For
intermittent feeding, it should be packaged in amounts for one feeding or for a
4-hour period and for continuous feedings, it should be given in a way so that
the hang time would not exceed four hours.
This has
been a brief and very general overview of the current marketing and use of
infant formulas. What I would like for you to remember--if I can have the last
slide--
[Slide]
--what I
would like for you to remember as we proceed through today's agenda are
the following points: About half of the
market share--the dollar sales in the United States, is for powdered
products. This is based on 1999 dollar
sales. Powders are not sterile
products. There is a small percentage of infant formulas that
are not available in commercially sterile, liquid forms, and methods given for reconstitution and delivery are
variable.
Thank
you.
DR.
BUSTA: Thank you.
Are there
any questions?
Robert.
DR.
BAKER: I'm Baker. I have a clarification point.
The
labeling is required, is that right?
But the exact wording, and sometimes even the content of the label is
decided by the manufacturers.
DR.
ANDERSON: There are certain requirements--there are certain elements that are
required on the label, and there are certain statements that are required.
Linda, if
you would go back to--about six or seven slides? Keep going.
Okay. This one.
[Slide]
The
statements that are in quotation marks--the first two statements--those
statements are required.
If you go
back one more slide. One more. One more.
[Slide]
Information
related to these topics is required, but there is no wording that is specified
or directed by FDA. The manufacturers
is given latitude in that regard.
DR.
HEUBI: I have a question--Jim Heubi.
Do we
know, as an example--first--I have a two-part question. One, do the powdered formula labels include
a statement that they're not sterile?
And then, secondly, do we have any knowledge about how informed
neonatologists are, or providers for preterm infants are in terms of
understanding the non-sterile nature of powdered formulas that might be used in
that venue?
DR.
ANDERSON: To answer your first
question, it's not required that there be a statement on the label that says
that the powders are not sterile products--commercially sterile products. And I'm trying to think quickly--and I don't
remember seeing a statement on a powdered product to that effect.
With
regard to knowledge of neonatologists about the nature of these products, I'm
not aware of any surveys that have been done to collect that information.
DR.
HEUBI: Is it the general sense that they understand this, or not. I guess that's a question that I have in the
back of my mind right now.
DR.
ANDERSON: My inclination would be to say they have a better understanding of it
now than they did before the E. sakazakii incidents occurred, but I don't
really have a basis for saying how much they do or do not know.
DR.
HEUBI: Okay.
DR.
THUREEN: I polled our neonatologists at several hospitals in town, and one of
about 25 had any clue how to prepare the infant formulas. It's usually done by a milk lab, and they
really--you know, it was totally out of their realm of knowledge. It just wasn't anything they thought about.
DR.
BEUCHAT: A question concerning the tube feeding--
DR.
HEUBI: Please identify yourself.
DR.
BEUCHAT: Larry Beuchat.
A
question concerning tube feeding and perhaps also relates to some of the other
recommendations: are there recommendations with regard to the tubes themselves,
or any other vessels or containers into which the actual formula comes in
contact before the baby consumes it?
The re-use, the hygiene and so on?
DR.
ANDERSON: Those would be products that would be packaged for delivery of the
formula, and those would have to meet guidelines from one of our sister
centers. I'm trying to figure whether
it would be drugs or devices--probably devices--that they would have
specifications for delivery systems--for those types of products.
DR. TARR:
Is it possible for us to get a list of--
DR.
HEUBI: Please identify yourself.
DR. TARR:
Phil Tarr, from St. Louis.
Is it
possible for us to get a list of the formulas that are not available as
liquids?
DR.
ANDERSON: As specific products, or as types?
DR. TARR:
Probably both.
DR.
ANDERSON: We could certainly do it as types, and I could see if we could get
the specifics for you.
DR.
MOYER-MILEUR: Moyer-Mileur.
In regard
to delivery systems, as far as tubing for continuous drip feedings, while the
products themselves may be sterile when they're initially used, if they are
reused then they're no longer that. And
that would all be dependent on each individual NICU and their policies. So it would be really hard to--for us to
answer that or even to control that, I would think.
Hopefully
at a level-three--in a level-three nursery the delivery systems would not be
re-used, and that would not be their standard of practice. The worry would be if you had a level-two,
or say even a level-one nursery where they're keeping babies who are 32, 33
weeks and feeding, and maybe not quite as knowledgeable about these products.
DR.
HEUBI: Heubi.
Just as a
point of clarification, do we have a general idea about how long NICUs utilize
this equipment that is used for feeding?
Is it 24 hours, 48 hours, 72 hours?
Is there any sense about this?
DR.
ANDERSON: I'm not able to comment on that.
I think--I can say it varies among institutions, but further than that,
I couldn't comment.
DR.
MOYER-MILEUR: You know, I can't either, because I think if you're dropping in a
continuous drip-feeding tube, you leave it as long as possible.
DR.
HEUBI: I guess I was more thinking about the bag and the tubing that leads to
the tube--
DR.
MOYER-MILEUR: They should be changed every four hours.
DR.
HEUBI: The tubing should be as well?
DR.
MOYER-MILEUR: The connecting tube, not
the actual--
DR.
STALLINGS: Stallings.
I would
echo what Laurie was saying. I think in
most level-three nurseries, the feeding system would be treated more like an IV
feeding system, but with the--you know, when you change the set you change the
whole set.
For a
nasogastric feeding, of course the tube in the infant would not be changed four
times a day and, in general, it would be left in I think it's about three days
before it would be changed electively.
So, you
really do, in the NICU, when you're talking about these products, you're
generally going to be thinking about feeding tubs. And then as the infants age and approach term, they'll go to
nipple-feeding and more to the devices that we think of as usual infant
feeding.
But,
you're right, we have to think about the plastic bag--the formula being mixed,
storage and all of that, but then the plastic back, the feeding tube, the
connection to the feeding tube that's in the infant. So there are a number of different components.
DR.
HEUBI: Are there any additional questions?
Thank you.
Oh, I'm
sorry--Dr. Beuchat.
DR.
BEUCHAT: Beuchat.
Just one
question--for Virginia. What is,
then--what could be the length of time that the tube would be in the infant
without removing it, and still feeding it?
DR.
STALLINGS: The nasogastric? It probably
varies per center, but in the stressed neonate, I think it's about three days
that because of the stress of moving a tube in and out of the baby, that you
wouldn't be changing it every 24-hours unless it occluded, or there was some
other reason.
I really
can't--somehow three days sounds about right, but I can't remember
exactly. But every nursery should have
a protocol about electively changing that.
But, again, as Laurie was saying, I think some of our concerns--there's
so much movement now to keeping more stable babies in second--the different
level nurseries that there's probably more nasogastric feeding that's being
done in those settings than used to be done.
So there is a site issue and an education issue here.
DR.
HEUBI: Heubi.
And you
have to realize that some of these babies have naso-duodenal tubes placed and
that's the context. There's not a big
move to replace them periodically. So
they might be in for longer periods of time.
Any
additional discussion or questions?
[No
response.]
All
right. I think we have to move on to
our multi-media presentation. Dr.
Kuehnert is not available to present
personally, but he's going to be available by telephone. He's involved with the investigation for the
current respiratory outbreak in Asia, and we're pleased to have him available
to present today for us. And as soon as
we make our connection, we'll be ready to go.
Tennessee Investigation
DR.
KUEHNERT: [Via speakerphone] Can you
hear me okay?
Thanks
for inviting me to speak. I'm sorry
that I couldn't be there in person, but an emergent investigation precluded me
coming. But I appreciate the
opportunity to present by phone.
What I
wanted to do today is discuss primarily the case in Tennessee in 2001 that
resulted in Enterobacter sakazakii meningitis and death in an infant who was
hospitalized.
MS.
LATHAM: Dr. Kuehnert?
DR.
KUEHNERT: Yes.
MS.
LATHAM: This is Jeanne Latham. You are
obviously not able to hear the mikes at the table very well, and I just wanted
to remind you that we will be recording the presentation, and we will have to
try to figure out how we're going to ask you questions, because Dr. Heubi was
trying to get your attention, and we weren't able to, but we will work that
out.
So, go
ahead and do your presentation. Thank
you very much.
DR.
KUEHNERT: Okay. Okay.
And, to
discuss the case, discuss the resulting MMWR and interim recommendations, and
also offer some discussion concerning the implications and possible activities
going forward.
Could I
have the next slide, please.
[Slide]
So this
should say "Background."
Enterobacter sakazakii is a gram negative rod that was classified as a
yellow-pigmented variant of E. cloacae, until it was designated as separate
species in 1980. And the reservoir is
unknown, as far as the original source of the organism.
Next
slide, please.
[Slide]
Clinical
characteristics. This is a pathogenic
organism with a particular affinity for the nervous system. Complications are commonly quite serious,
including necrotizing enterocolitis, sepsis, meningitis. And in addition to meningitis, cerebral
abscesses, cysts, infarctions, fairly extensive cerebral damage. And outcome is poor. In most of these infections, there is an
impaired neurological outcome. In fact,
that's expected in the vast majority of cases that occur, and fatality rates
has ranged, in the literature, between 40 and 80 percent.
Next
slide, please.
[Slide.]
Potential
sources of the organism--as I said, the reservoir is unknown, but powdered
infant formula has been associated in the past, before the case in 2001, with
outbreaks of E. sakazakii infections in neonates. The organism has bene traced to a number of steps in the powdered formula preparation
process. It's been found in freshly
prepared or refrigerated powdered formula, in utensils and equipment that's
used in formula preparation, such as blenders and other tools used to make
formula, in the unreconstituted product, and also in unopened product.
Powdered
formula products, as I said, have been associated with these outbreaks,
including meningitis, sepsis and necrotizing enterocolitis. Other studies have looked at powdered
formulas for contamination by bacteria and there was one particular study that
looked at Enterobacteriaceae in general, and found contamination at low levels,
including over half of products from a number of different countries, including
the United States, and specifically 14 percent of powdered formula samples were
contaminated with E. sakazakii, and most of you are probably familiar with this
literature. However, the concentrations
of the organisms were quite low.
Next
slide, please.
[Slide.]
This is
outline of presentation. Let's skip
over this.
Moving on
to case description--the next slide.
So, on to
the 2001 case. A male patient was
admitted to the neonatal intensive care unit in April 2001. Gestational age was 33-1/2 weeks, delivered
by C-section. APGAR scores were 4 and 7
at one and five minutes, and the birth weight was 1,270 grams.
Patient
was started on enteric feeding with powdered formula and breast milk, and then
on day 11 of life, developed sepsis and neurologic symptoms.
Lumbar
puncture was performed which was consistent with meningitis. White cells and red cells were present,
although at actually low numbers, but the protein was high, and the glucose was
remarkably low.
Cerebral
spinal fluid culture grew Enterobacter sakazakii. The patient was treated with ampicillin and Cefoxitime, but did
not do well despite full support, and became pulseless and was resuscitated
with pressors.
As the
clinical course progressed, it was clear that the neurologic was severe, and
that there was no significant brain activity.
And so support was withdrawn, and the patient expired at day 20.
Next
slide.
[Slide]
The
facility in which this occurred with the University of Tennessee at
Knoxville. It's a regional referral and
tertiary care center; fairly large facility--360 beds. It has a level-three NICU with 55 beds
that's composed of two parts: and intensive care nursery with 27 beds, and an
intermediate care portion with 28 beds.
They
looked back--because this was an unusual organism, the hospital epidemiologist
looked back to see whether they had seen E. sakazakii in specimens in the
recent past. In fact, they looked in
the last three years and didn't find anything from 1998 to 2000. However, they did find two isolates detected
in March 2001, in addition to this case.
So this prompted a further investigation.
Next
slide.
[Slide]
So the
study objectives were to ascertain whether there were additional cases of E.
sakazakii infection or colonization that they had missed clinically to
determine the source of the organism and to develop measures to prevent further
infection.
Next
slide, please, that says "Case Finding."
[Slide.]
A
cross-sectional prevalence survey was performed, and what this was was
basically looking at all patients in the NICU during the case patient with
meningitis was ill, and that was defined as April 10th to 20th
2001. I'll refer to that as the study
period.
They
assessed all these patients for stool colonization. In addition, they looked for clinical reports from the
microbiology laboratory for E. sakazakii and did a review to see what symptoms,
if any, patients had. And a "case
patient" was defined as any NICU patient with the E. sakazakii-positive
culture during the study period, either from the stool or other sources.
Next
slide.
[Slide]
Case
finding: 49 patients were hospitalized during the study period. There were nine case patients, and of these
nine--this slide shows the site of infection or colonization--six E. sakazakii
from the stool, two from tracheal aspirates, one from the urine, and one from
cerebrospinal fluid, which was the index case.
I would
want to add here the footnote that you have 10 sites here, and that's because
one patient had E. sakazakii in the stool and the urine.
Next slide, please.
[Slide.]
Next, a
cohort study was performed. These are
the variables that we looked at, combining the literature concerning infant
formula and also just nosocomial infection variables that are commonly looked
at.
Just to
high the highlights, we looked at, obviously, modes of nutrition: split formula
into powdered versus liquid versus ready-to-feed; also looked at whether the
formula was given by continuous feed or by bolus. And that's pretty much what I wanted to highlight there.
Next
slide, please.
[Slide.]
These
were the results of the cohort study.
The only variable found to be significant was powdered formula use. So, just to walk you through this table, of
the 30 patients that were exposed during the study period, nine were ill and
were case patients. I'm sorry--of the
49 in the cohort, 30 were exposed to powdered infant formula and, of those,
nine had E. sakazakii and were cases.
Of the remaining in the cohort, 19 did not have powdered formula, and
none of those patients had been given powdered formula.
There were
some other things that I can't really say approached the statistical
significance but were, I think, of some note, is that the continuous had a
P-value of .16, and the absence of breast milk use was also--had a P-value of
.16.
Next
slide, please.
[Slide.]
Next,
observational laboratory studies were performed in the facility; looked at
formula preparation, storage and administration. I'll just briefly gloss over these; cultured the environment and
materials for formula preparation in patient care, including the preparation
area in the NICU, and cultured lots that were in use during the study period,
including powdered formula from open containers.
Next
slide, please.
[Slide.]
So this
should say "Laboratory Studies."
Laboratory studies were performed by CDC and included confirmation of
isolates from the cohort study, cultures of open cans of formula. They were also able to obtain unopened cans
of formula of an identical lot number that was supplied by the manufacturer,
and we cultured that as well. Culture
method was done according to a modified protocol of inisol, and all study
isolates and selected historical isolates were compared by pulse-field gel
electrophoresis.
Next
slide, please.
[Slide.]
Environmental
and formula cultures--these are the results--showed that none of the on-site
cultures that were performed, either in a preparation area or in the NICU, grew
E. sakazakii. CDC cultures grew E.
sakazakii from a single lot of powdered formula, and this was from both an
unopened can and an open can. The PFGE
patterns were indistinguishable between isolates of either the cerebrospinal
fluid of the case patient that died from meningitis, and also the opened and
unopened containers of powdered formula.
And the
other interesting sort of aspect of this is the PFGE results also suggested
pattern diversity among other isolates from the cohort study compared with
previously collected strains, and this is shown graphically in the next slide,
which says "PFGE Results.
[Slide]
And what
you see here is a gel, and the PFGE results, and you see lanes two through six
showing a CSF isolate, a respiratory isolate, stool isolate, urine isolate and
the two formula isolates being identical.
However, there were other patterns that are different, and these include
some stool isolates from the cohort study, as well as historical isolates from
isolates that were pulled from the freezer, from other previous investigations.
The
implications of this, I guess we can get into in the discussion.
Next
slide.
[Slide]
Observational
studies. The hospital detected no
breaches in infection control. The
formula was prepared according to manufacturer's instructions on the label. It was mixed with sterile water,
refrigerated for less than 24 hours.
And their guidelines at the time were to use the mixed product within
eight hours, and the hang time, although I'm not sure of the exact time, they
said it was around six hours.
Next
slide.
[Slide]
Intervention. The facility, at the time, prescribed a fair
amount of powdered formula in the facility, to about 50 percent neonates in the
NICU. As a result of this event, they
changed the formula preparation site from the NICU to the pharmacy. The principal formula used was switched to
liquid ready-to-feed, although they still had to use some powdered formula
selectively for special formulations.
They changed their allowable hang time for mixed feeds from eight to
four hours. And, to date, they haven't
seen any further E. sakazakii or clinical isolates.
So, in
conclusion, concerning this case the source of the case of Enterobacter
sakazakii infection was traced to receipt of powdered infant formula. The only significant--it was the only
significant risk factor in epidemiologic study, and there were matching isolate
patterns on PFGE concerning--from the powdered formula and from the patient.
So we
concluded that powdered formula, which is a non-sterile product can be
contaminated with E. sak and can cause fatal meningitis, and that use of
powdered formula should be carefully considered in the neonatal health care
setting.
Next
slide.
[Slide]
After
this, Mead Johnson voluntarily recalled Portogen powder.
Next
slide.
[Slide]
And an
MMWR was published which included summary interim recommendations for the
NICU. This included to select formula
products based on nutritional needs; that trained personnel should prepare
products using aseptic techniques; to follow manufacturers recommendations for
preparation of formula; that the administration of hang time should be less
than four hours; and that there should be written hospital guidelines,
including notification, reporting and follow-up available in the event of a
product recall. And there are some
aspects of that last bullet that I think are difficult for hospitals to follow,
and I'll get into that a little bit in the discussion.
Next
slide.
[Slide]
We also
asked that cases be reported, particularly concerning invasive infections in
infants younger than 12 months to the state health departments, CDC and, of
course, FDA Medwatch.
Next
slide.
[Slide]
So, just
to conclude here, the questions I think that sort of come up with this case is
when this case first emerged, well, the question was is whether this is an
emerging pathogen. And in trying to
answer that, there are a subset of questions that come up. Again, what is the reservoir of the
organism? Is this--has it changed? Is there something new as far as the
reservoir? Has this been around all
along? And what is the endemic rate of
the E. sakazakii colonization or infection due to powdered infant formula? Since we don't have surveillance for it, we
don't know, you know, how many cases there have been.
Just as
an aside, we have a nosocomial infection surveillance system, and we looked
back at isolates--at infections that have been reported due to E. sakazakii,
and we found one where there was an isolate from the CSF in 10 years of
surveillance. And this is approximately
300 hospitals. So this is a very
unusual organism for a hospital to find, and I don't think that this Tennessee
facility's experience in not seeing an isolate for three years from NICU
patients is unusual.
And,
next, what's the role of specific methods for preparation and use to promote
growth and reach the threshold in clinical significance. So if it exists in formula, what are the
factors that make it matter in causing clinical disease. And, as an adjunct to that, what are the
predisposing risk factors for infection?
Next
slide.
[Slide]
And so I
just sort of split sort of the separate issues, which I think encompass the
spectrum of issues that cross agencies and different both clinical, laboratory
and manufacturing arenas concerning the manufacture of the formula: screening
during manufacture, issues about preparation and storage and use; treatment of
infection if there--you know, there's very little in the literature about
treating E. sakazakii in general; and, finally, issues about case reporting and
surveillance of infection, which I've already touched on.
Next
slide.
[Slide]
So,
concerning manufacturers specifically, I'm sure what's going to be discussed
today is going to be concerning processing and implementation of screening,
which I understand has already begun to some extent. Concerning preparation, storage and use, development of guidelines
or recommendations. The American
Dietetic Association had had some recommendations previously that had been
disseminated but was out of print at the time of this event, and I understand
that they're developing revised guidelines.
And,
finally, case reporting and surveillance--this was the issue I touched on
before, which was about hospital record-keeping. They usually don't record whether--they record, of course, in the
orders whether formula has been given, but not necessarily whether it's liquid
or powdered, and certainly not what lot number has been given. So, in trying to track--do a trace-back
investigation of the case, it's very, very difficult, because oftentimes the
hospital doesn't now whether the patient even got powdered formula, or whether
liquid ready-to-feed. And so this
provides a particular challenge in the way formula documentation is currently
set up.
Next
slide.
[Slide]
So, as
far as future plans, what we've currently been involved with FDA has been
sporadic case investigations, and sort of case finding. I think a case series description would be
very useful, and there are a couple people working on that. As far as policy on formula preparation, I
mentioned the American Dietetics Association.
They also have done surveys of preparation and use. I think that's important. And also, obviously, revising the
guidelines.
Concerning
laboratory research, we've been involved with some, and I know FDA has been as
well, concerning the growth characteristics of E. sakazakii, and also, specifically,
the effect of two things: one, competitive microbial flora. I mean, we've done studies looking at E.
sakazakii growth, but one of the questions is how does it grow differently when
it's in sort of the milieu with different organisms, as it is with infant
formula, and also about the effect of heat inactivation on growth.
Next
slide.
[Slide]
I'd like
to acknowledge many people who have given input over the last, actually, couple
of years and, particularly, the hospital that originally detected the case in
Tennessee.
Next
slide.
[Slide]
That's
the end of my presentation. I'd be
happy to answer questions.
DR.
HEUBI: In order to answer questions, either we'll have to--
DR.
KUEHNERT: Oh, yes, I'm having a lot of trouble hearing you.
DR. HEUBI:
I think what we ought to do is each of us, maybe, should go and pose the
question with the microphone to him, and then he can respond. That way we can all hear what he says. Do you want to do that? There's a microphone over there.
So, if
you'll queue up, that would be great.
Now,
please identify yourself.
DR.
STALLINGS: Hi. It's Virginia Stallings,
from Philadelphia. Thank you for being
with us.
My
question is: how hard is this bug to grow and identify in a busy microbiology
clinical lab? Because that will give us
some framework to consider the data we have at hand.
DR.
KUEHNERT: Yes. Yes. I heard the question.
There are
two aspects to that. One is whether E.
sakazakii can be easily grown in a clinical specimen. I think that most laboratories are pretty well suited to
that. We have had situations where they
weren't sure whether it was run of the mill E. cloacae or E. sakazakii, because
sometimes the yellow pigment isn't that strong. But it appears that from what I understand--I'm not a microbiologist--but
from my understanding, is that the methods used in most clinical microbiology
laboratory should pick up most of them.
Now, the
issue concerning finding it in infant formula is different. You know, some hospitals that have suspected
that they might have E. sakazakii in their formula have tried, you know, a
number of different methods which really don't work, like putting the powdered
formula just directly on media, or even trying to do--trying to culture it,
just using standard methods. And I'm
sure FDA will speak to this, but there are--I mentioned a modified protocol,
and it is a fairly intensive, somewhat complicated protocol that takes a number
of days to grow it out of formula. And
the biggest problem--I don't want to belabor this too much, because I'm sure
it's going to be discussed later--I mean, the big problem is that you've got a
lot of microbial flora in the formula that competes against the E. sakazakii,
and so you have to pick--it's sort of a needle in a haystack to pick it out. And that's some of the difficulty in trying
to isolate it from formula.
So, the
short answer to the question is: yes, I think microbiology laboratories should
pick it up in clinical specimen, but no, I think they're going to have
difficulty from infant formula.
DR.
NEILL: Peggy Neill, from Brown University Medical School. I have two questions, and they're unrelated.
The first
one is that in your slides, it would probably be approximately "9,"
for "facility characteristics," at the bottom of the slide it
indicates that there are two isolates of E. sakazakii detected in March 2001,
but I'm trying to discern from the text here that they appear not to have been
either a clinical isolate at all? Or
they were certainly not a clinical isolate from the NICU.
DR.
KUEHNERT: Right. Yes. Let me address that and try to remember what
the significance of those isolates.
If I
remember right, one of those patients was included in the cohort study--well,
let me back up. The two isolates, I
believe, were respiratory isolates.
They were not thought to be causative for infection. One of the patients was included in the
cohort study and, I believe, had a positive stool but did not have a clinical
isolate during the study period.
The other
patient, I think, had already been discharged, and their illness was due to
another issue, and they did not have the isolate for comparison--they did not
have either respiratory isolate for comparison at the time of the study. They had been discarded at the time that
they noted the problem.
DR.
NEILL: Okay. Thanks.
The
second question pertains to your PFGE profiling?
DR.
KUEHNERT: Yes.
DR.
NEILL: And I suspect you know where I'm going with this. You alluded to the fact that lanes two
through six are clinical isolates that are identical, and they are from this
particular outbreak investigation. But
if I understood you correctly, you have other isolates farther to the right in
that gel, but I don't know which lane, and that those have different
pulsotypes?
DR.
KUEHNERT: Right. Yes. Let me try--I was going to discuss this, and
in the interest of time I did not. But
I'm glad you brought it up.
Okay. There are two groups of isolates here. Unfortunately, I don't have them labeled, so
I can't tell you which is which, but there are some--there are stool isolates
there from the cohort study that had different PFGE patterns from the index
case and the isolate from formula. And
then two or three of the other ones are from historical isolates from the CDC
freezer.
Now,
concerning the stool isolates, I mean there are a couple of possibilities for
explaining this. One is that the
patients just happened to be colonized with E. sakazakii from the stool, which
is possible--although we don't usually look for it in clinical microbiology labs. So it might be there, but I think that's
probably unlikely, because people do do screening for gram negatives in stool,
and have not frequently found this.
The other
explanation--which I think is more likely--is that there were multiple--there
may have been multiple--this is speculation on my part--but there may be
multiple isolates of E. sakazakii from that--either that implicated lot, or
other lots is possible, and that this represents isolates that were below our
level of detection for the lot that we sampled.
But, I
mean, there's obviously got to be some explanation as to where these came from,
and we don't have an answer for it.
DR.
HEUBI: Hi. Jim Heubi.
After
this initial case series was identified in the hospital, was there an effort
made then to check any of the formulas given to these infants, later during the
follow-up or new additional cases to know whether actually those formulas [off
mike].
DR.
KUEHNERT: Could you repeat that? I'm
not sure I fully understood that?
DR.
HEUBI: Well, what you reported was that there were no additional cases after
this series, including the nine that were colonized with one case. And then you made the comment about the fact
that there's been no additional cases since that time.
DR.
KUEHNERT: Mm-hmm.
DR.
HEUBI: The question I had was: was there any effort made to try to make sure
that the formula was or was not contaminated in any of those additional infants
might have been exposed to after the initial reports?
DR.
KUEHNERT: Well, my understanding at the hospital is I don't think they've done
any further stool colonization studies, nor have they done any cultures of the
formula. I think that they've done--I
mean, I don't know--a somewhat active surveillance, certainly in looking for E.
sakazakii on clinical specimens. But as
far as stool colonization, or directly culturing formula, I don't think that
they've done that.
Again, I
don't think they had the resources to do, you know, the modified protocol in
the first place to culture formula. And
so I don't think they've been doing that on an ongoing basis, either.
Does that
answer your question?
DR.
HEUBI: Yes. I guess as the devil's
advocate what I'm saying is that the changes they implemented may or may not
have had any impact on whether they have any future cases.
DR.
KUEHNERT: Yes. I think that's a valid
statement.
DR.
HEUBI: Thank you.
DR.
BEUCHAT: Larry Beuchat, University of Georgia.
I have
two questions. One is a follow-up to
Peggy's. Were the two March 2001
isolates, you know the PFGE patterns determined in this film--were they similar
or not from isolates that are shown on the gel?
DR.
KUEHNERT: Sorry, this is the March 2001 isolates?
DR.
BEUCHAT: Yes.
DR.
KUEHNERT: Yes, those were discarded before they knew there was a problem.
DR.
BEUCHAT: My second question is dealing with the four-hour hang time. What criteria--what science-based
information was used to determine and recommend that a four-hour hang time
should be used?
DR.
KUEHNERT: That's a very good question.
At the time that we made these recommendations, I think it was an
educated guess. We weren't sure even
that the hang time had a role in this.
I think the previous question, you know, spoke to that, that they made
these changes and didn't see any more isolates, and that doesn't mean that the
changes they made resulted in fixing the problem. But also the flip side of that is we don't know whether the
hang-time played role in it being a problem.
But we knew that it made sense, you know, concerning microbial growth to
shorten the time to as short as feasible.
And I think four hours got to the limit of what we thought was feasible
in a NICU.
Since
that time--I'm sorry I'm not there, because I would have brought some extra
slides, which I'm looking at here--our lab has done some studies on E.
sakazakii growth in the implicated formula, Portogen with Iron. And what you see is a curve that shows very
little growth until you get to about--beyond six to eight hours, in which case,
between eight hours and 24 hours you see a jump in growth from a log of 4 to
about a log of 9. And also we've done
studies looking at not only that formula, but also other manufacturers formulas
that have shown almost--well, actually, identical, statistically significant
differences in that growth.
DR.
BEUCHAT: On that growth curve that you were just describing, what was the
initial number of E. sakazakii in the formula?
DR.
KUEHNERT: At t-0 hours, 3 logs--so, 1,000 organisms were inoculated. And then you see about a log of 3 at t-2,
t-4--it goes to about a log of 4 at t-6 and t-8, and then it goes up
to--actually, it's log 8 at about 24 hours.
I want to
explain how this done. It was done
under clinical conditions, so the formula was mixed with sterile water, and E.
sakazakii was put in at that time. Then
it was refrigerated for 24 hours, and then hung. And that's t-0 was when it was hung. And so what I just described was under those conditions.
DR.
BEUCHAT: So the temperature would have been, perhaps, 70 Fahrenheit? The room temperature?
DR.
KUEHNERT: Yes--I don't have the breakdown to the exact temperature, but it was
ambient temperature, which I'd have to look at what actually she did. I think it was a little warmer than that, I
think.
DR.
ACHOLONU: Doctor, my name is Alex Acholonu.
I'm from Alcorn State University in Mississippi.
During
your delivery, talking about the case findings, you talked about examining
stool samples, tracheal aspirates, urine and CSF. May I know why blood is not checked as one of the fluids in the
body?
Number
two question: we have been told that this disease is most prevalent in
neonates, which mean children less than one month old. Did you, in order to rule out cases of
prenatal infection, check the mothers of the babies?
DR.
KUEHNERT: That's a good question.
We did
not do cultures of the mothers or of siblings, or household contact at that
time. Subsequent to this, there was
another investigation in another hospital in Tennessee, where we did do those
cultures--vaginal cultures and other--rectal cultures--of the mother, and did
not find it. But that's a very good
point, and that was not done in this investigation--to my knowledge.
The first
question, I didn't quite get. It was
something about checking some site?
What site was it?
DR.
ACHOLONU: Yes--checking the blood. We
are told that one of the effects of the disease is sepsis.
DR.
KUEHNERT: Right.
DR.
ACHOLONU: And I take it to be septicemia.
May I know why blood samples were not checked.
DR.
KUEHNERT: Yes. Okay.
That's
sort of a complicated issue. I guess--I
mean, at the time it was sort of dealing with, certainly, an infection-control
issue and an investigation. I'm not
sure--I wasn't the hospital epidemiologist who had to make this decision. Inga
Himmelreit was. But I mean, if I were
her, I might be uncomfortable doing blood cultures where it's not clinically
indicated. So I think that, you know,
all these patients were clinically stable, and so I think that probably was the
decision why they didn't do blood cultures.
Although blood culture is obviously a low-risk procedure, it's not a
no-risk procedure. And so I think that,
you know, that may have been why it wasn't done. Also, the yield probably would have been pretty low.
DR.
ACHOLONU: My last question: since the infection is more in neonates in the
intensive care unit, is it possible that some of the infections are nosocomial?
DR.
KUEHNERT: Do you mean that some of this--
DR.
ACHOLONU: Picked up from the hospital, rather than coming from the baby
formula?
DR.
KUEHNERT: Right. Right.
Yes, I
think that's a good point. I mean, as
far as the stool--I mean, we don't know--okay, just looking at the cohort
study, we can't tell whether these stool cultures represent transmission
between the patients or that health care workers were a vector between
patients. But the epidemiologic study,
I think, clinches it, because though, again, looking back at that, those that
were exposed to powdered formula were the only ones that had the stool cultures
or cultures at other sites. Of all the
patients who did not get powdered formula, none of those had E. sakazakii. And I think that sort of--although there
might have been something confounded exactly with powdered formula, I think
that really leans against nosocomial infection. So that's what this investigation--overall, I would say that it
isn't a common nosocomial pathogen. I
mean, I mentioned that NIS surveillance, and we just don't see it. And we've also done some case finding, which
I didn't have time to present, both clinical microbiology laboratories,
ClinMicroNet, and also infectious disease consultants, and over a three-year
period, there were very, very few isolates from NICUs. So, overall, it's probably pretty unusual,
and I think the epi really points to the powdered formula rather than
nosocomial transmission.
DR.
ACHOLONU: Thank you.
DR. LEE:
Hi. This is Ken Lee, from Ohio State.
There was
almost an offhanded reference to competing flora, and I'm just wondering, is
the competing flora a significant factor in E. sak infection, or is it even
present? What's your best guess about
other microorganisms that might be present?
DR.
KUEHNERT: Right. Well,
"guess" is a good word as far as what I'm going to say on that, as
far as what the significance of it is.
I mean, like the lab studies that I mentioned, I mean that was
inoculating E. sakazakii into sterilized formula. We have not done studies yet, but I think it's important that we
do, looking at sort of the in vivo environment of powdered formula, and how E.
sakazakii behaves in that environment.
We don't know if it's going to make it grow more slowly, grow
faster. We don't know if other
organisms are deleterious or actually whether it augments growth.
It
obviously is a complicated situation when you have, you know, multiple bacteria
in the formula. And this also gets back
to the difficulty in growing E. sakazakii out.
I mean, again to just emphasize the obvious here, I mean formula is not
sterile. When you try to grow--when you
try to isolate E. sakazakii you get all kinds of organisms growing from it--you
know, from bacillus, to other enterobacters, to other gram-negatives. And so the difficulty, actually, is in
picking the E. sakazakii out. And what
we don't know is whether the bacterial flora--what role the other bacterial
flora play in the growth of E. sakazakii.
DR. TARR:
Phil Tarr, Washington University.
Is there
any chance that these culture-positive cases are the tip of the iceberg? Was there any evidence of clustering of
sterile CFS with the presence of cliacytosis that might be represented in
partially treated cases of E. sakazakii?
DR.
KUEHNERT: Yes, that's a good question.
We actually went back in the hospital and looked over a multi-month
period at nosocomial infections of any kind; those due to enterobacter cloacae,
because we thought, well, the point you bring up or touch on is that if formula
has multiple organisms, could there be other organisms causing infection that
maybe we don't notice because they're not as striking as a sentinel organism as
E. sakazakii.
We didn't
see any association of nosocomial infections in general, or nosocomial
meningitis associated with E. cloacae or with--sorry, let me back up. We didn't see any association of powdered
formula with either nosocomial infections or specifically nosocomial meningitis
over the time period that we looked at at this hospital.
I think
it would be an interesting idea to look at that in a multi-center fashion. Again, it gets back to the difficulty and
challenge of how hospitals record powdered formula. But if there were a way to get over that hurdle, I think this
would be a very good idea to look at.
Because we just don't know.
DR. TARR:
What particularly--I'm thinking of missed E. sakazakii, if it was partially
treated prior to obtaining the CSF.
Have sera been obtained from the survivors?
DR.
KUEHNERT: Ahh--well, we have--when we confirm organisms, we get the CSF and I'm
not sure if we have sera. We have the
CSF stored, but I'm not sure if we have sera.
DR. TARR:
Thank you.
DR.
BLUMBERG: This is Hank Blumberg from Emory University.
There
have been previous reports of E. sakazakii causing necrotizing enterocolitis,
and I was wondering, in the outbreak that you investigated in Tennessee, were
there any cases of necrotizing enterocolitis in the patients--in the
neonates--who had positive stool cultures?
DR.
KUEHNERT: Yes. Thanks for that
question, because it reminded me that--no, the answer is no, we didn't see any
NEC. And also, when we looked at that
association between nosocomial infection and powdered formula, we also looked
at NEC over the previous few years, and did not find an association.
DR.
BLUMBERG: Thank you.
DR.
TOMPKIN: Good morning. This is Bruce
Tompkin, and I'm the industry representative t this meeting.
You
mentioned a statistic that I hadn't heard of, where you'd gone back to 300
hospitals over the past 10 years. Would
you please repeat that, because I'm not sure I heard it correctly.
DR.
KUEHNERT: Oh, yes. Sure.
Umm--this
was not published anywhere. We did this
case finding after this case, so this may be the first time you've heard
it. Let me just go through it again.
We have a
national nosocomial infection surveillance system, and this is a voluntary
participation of hospitals in the United States. We looked back at--and this is only nosocomial infections, you
know, acquired in the health care setting--and we looked back for E. sakazakii
infection. This was during a 12-year
period of surveillance. And we found
one case of E. sakazakii meningitis in that time period.
DR.
TOMPKIN: And did you say it was 300 hospitals--approximately?
DR.
KUEHNERT: Yes, it's about 300 hospitals.
DR.
TOMPKIN: Thanks.
DR.
KUEHNERT: Are there any other questions?
DR.
HEUBI: One comment. Jim Heubi.
We would
actually--Dr. Busta and I were discussing the fac that we would actually
appreciate it if you could provide us with the slide of the log growth for us
to look at, if you have that.
DR.
KUEHNERT: Oh, sure.
DR.
HEUBI: I think that would be very valuable for us. And just to clarify--this was E. sakazakii that was placed in
sterile formula, and then hung and then cultured repetitively thereafter--is
that correct?
DR.
KUEHNERT: That's right. I'm sorry--so
you were asking for me to e-mail it now, or--
DR.
HEUBI: Right--we'll actually--someone from the FDA will contact you and we'll
make arrangements to get this up here so we can have it for our books.
DR.
KUEHNERT: Okay.
DR.
HEUBI: Thank you for your presentation.
[Recess.]
DR.
HEUBI: I think we'll reconvene.
Dr. Karl
Klontz is going to present other relevant investigations. Where's Dr. Klontz.
Other Relevant Investigations
DR.
KLONTZ: Good morning. Karl Klontz,
here.
I want to
spend the next 20 minutes or so--oh, by the way, I'm with the Epidemiology Team
at the Center for Food Safety and Applied Nutrition.
The title
of my talk today is "Enterobacter Sakazakii Case Reports and Outbreaks
Involving Infants, As Reported in the Peer Reviewed Medical Literature."
Next
slide, please.
[Slide]
Now,
before I go any further, you have probably--you on the panel have a more
extensive slide set than the one I'm covering here. But I will, in the
interests of time, just cover the most salient ones. And if there are questions on the others you have, I'll be happy
to entertain those.
Okay. For the purposes of summarizing the
literature, we defined a case of Enterobacter sakazakii infection as an infant
meeting at least one of the following criteria. First, E. sakazakii was recovered from one or more of the
following normally sterile sites: blood, cerebrospinal fluid, brain tissue or
urine.
Second,
an infant could have been classified or defined as a case of E. sakazakii if
that infant was involved in an outbreak of necrotizing enterocolitis, and E.
sakazakii was recovered from blood, stool or stomach aspirate of more than one
infant in that outbreak.
A third
possible criterion was that the infant had bloody diarrhea and E. sakazakii was
recovered from a stool specimen in pure culture; that is to say, no other
pathogen was in the stool that could have accounted for the bloody diarrhea.
So any
infant that met one or more of these definitions, we called a "case"
of Enterobacter sakazakii.
Next
slide, please.
[Slide.]
This
slide summarizes the initial reports of Enterobacter sakazakii in the
literature in chronological fashion.
The first thing I'd like to point out is that the first published report
occurred in 1961, by Ermenye, et al.
This was a report that described two fatal cases of neonatal
meningitis. And, by the way, in some of
these early reports, the name Enterobacter sakazakii, of course, wasn't
used. That term was
introduced--proposed--in 1980 by Dr. Farmer, et al. Some of the terms used earlier in these reports were
"yellow-pigmented Enterobacter cloacae" and some other terms. But, nevertheless, for our purposes today,
some of these following publications that came after the Ermenye, et al., study, described also
infections in neonates or very young infants, and it really wasn't until 1982
that Jimenez, et al., reported the first case of Enterobacter sakazakii
infection in an adult--a 76 year old man with a history of rectal
adenocarcinoma who had urosepsis and recovered after antibiotic therapy.
Now the
first case series was by Muytjens--and, by the way, I'm pronouncing this
"Moyt-yens," with a little bit of counseling from Dr. Muytjens
himself. We were fortunate enough to
reach him by e-mail in the last couple of weeks, and one of our questions,
among other technical issues--
[Laughter.]
--was how
to pronounce his name. And he was very
kind to tell us "Moyt-yens," although he did say, curiously, that his
colleagues in the Netherlands, even in his area, have trouble pronouncing this
name. So I'm sure I'm not doing a very
good job here.
But
nevertheless, he published the first case series, and this was the first study
to propose a possible link to powdered formula. And we'll go over this study in more detail, but--let's go to the
next slide.
[Slide]
I would
like to stand back a bit now, and look at the literature as a whole, and point
out that there were 27 references in the literature that described Enterobacter
sakazakii infections, encompassing a total of 58 cases. As you can see, the majority of the cases
were in infants; 17 percent in children over the age of one, or in adults. And in the bottom half of the slide, we've
broken down these cases by age group; the number of cases, and then the
percent. And, as you can see, the majority,
again, were in the first year of life--83 percent. But even in that group, the majority were in the first month of
life, underscoring--at least from the literature standpoint--that this appears
to be largely a neonatal infection.
That's not to say there aren't older cases. For example, there were six cases in individuals over the age of
four. And if you look at their median
age, they were actually at the other end of the spectrum--median age 74 years
of age.
Okay. Let's go to the next slide, please.
[Slide]
Now this
slide summarizes the 48 cases of Enterobacter sakazakii infection in infants,
and selected clinical features of these cases.
About
half the cases were in males; half in females.
Meningitis, however, was the predominant syndrome, accounting for 58
percent of the cases. Sepsis and
bacteremia, 17 percent; and necrotizing enterocolitis, 29 percent.
Overall,
the case fatality rate was 33 percent.
And, again, in the bottom half of this slide we've broken down for you
the birth weight, where reported, for these infants, and the number that were
in each category, and then the case fatality weight. And, as you can see, there was really no increasing or decreasing
trend of case fatality by birth weight.
It's notable here that one of the stratum had a higher case fatality
rate, but I think that's largely because a number of the cases in this group
came from one paper--and that's the Muytjens article that we'll discuss in just
a minute--which was a retrospective survey in the Netherlands, and probably
more likely to have identified the most severe cases.
Let's go
to the next slide, please.
[Slide]
Now, this
is the Muytjens article of 1983, the case series I mentioned. And it was an analysis of eight cases of
neonatal meningitis and sepsis due to Enterobacter sakazakii. What they did here is they re-analyzed 20 Enterobacter
strains that had been isolated from cerebrospinal fluid, going back six years;
and 25 strains from blood, going back two years--all in the Netherlands.
Now,
their hypothesis was that Enterobacter sakazakii may not have been originally
in this group of organisms because this organism produces its characteristic
yellow pigment at temperatures generally lower than 36 degrees centigrade,
which was the temperature that these strains were identified. So what they did is they used various
biochemical and growth parameter systems, including a lower temperature, and
they were able to identify eight of these organisms as actually being
Enterobacter sakazakii. These were
confirmed as being E. sakazakii by the Centers for Disease Control
subsequently.
And so
the investigators then went back to medical records to obtain clinical data,
retrospectively.
Next
slide, please.
[Slide.]
This
slide now summarizes the clinical features of these eight cases of neonatal
meningitis and sepsis. The first thing
to point out is that three of the eight cases were delivered by Cesarean
section, and this is notable, because in the literature, until this point,
there had been some discussion about the possibility that Enterobacter
sakazakii was acquired from the vaginal canal during the normal birthing
process. But here we have three of the
eight cases that had been delivered by Cesarean section, and one of which was
delivered to a woman who gave birth less than 45 minutes after the membranes
were ruptured.
Now, as a
rule, these infants progressed well in the first few days of life. The first signs of illness occurred between
days four and eight. Two of the cases actually had necrotizing
enterocolitis along with meningitis.
The case fatality rate was 75 percent, and even the two survivors had
significant neurologic sequelae.
When
death occurred, it did so within hours to several days after onset of
illness. And the investigators actually
used the term "hemorrhagic encephalitis" to describe the illnesses,
because on autopsy, a number of these infants had large, swollen brains that
were soft and showed signs of intra cerebral hemorrhage.
Next
slide, please.
[Slide.]
Same
study--now let's look at some of the epidemiologic features of these eight
cases. Six out of eight had birth
weights of less than 2,500 grams, with the lowest being 850 grams. Five of the eight cases were premature; that
is, their gestations were less than or equal to 36 weeks.
There was
geographic clustering in this case series, in that five of eight cases occurred
at the same general hospital in the Netherlands, whereas the other three cases
were born at three other facilities.
But there was also temporal clustering in this series, in that three of
the five cases at the general hospital became ill within three months of each
other, and even at the other hospitals, two of the three cases became ill
within two months of each other.
Next
slide, please.
[Slide.]
Because
of this geographic clustering, Muytjens and his colleagues did environmental sampling
at the pediatric ward of the general hospital where five of the eight cases
occurred, and they were able to recover Enterobacter sakazakii from prepared
formula, from a dish brush, and from a stirring spoon--but not from powdered
formula itself, nor the water that was used to prepare that formula.
Now, let
me just say a word about the methods they used to test the powdered formula,
because one of the questions we asked Dr. Muytjens in the last couple
weeks--because it's not stated in the article--how much powdered formula did
you look at? And he responded by saying
it was somewhere between four and 10 grams of powdered formula--which, in terms
of today's methodologies is a relatively small amount.
They did
do plasmid profile analysis, and they concluded that "three or four of the
five isolates from the patients at the general hospital were probably the same
strain."
Now, it's
important to note, though, that these plasmid: profiles differed between the
isolates from the cases, versus the isolates from the prepared formula, but
that may not be surprising, because keep in mind that this was a retrospective
study. At the time of testing the
prepared formula, some of the cases had become ill months, if not years,
earlier
Now,
evidence that the formula, however, may have served as a vehicle for
transmitting the infection was found in a 1990 letter to the editor by
Muytjens, et al., to an infectious disease journal, in which he stated:
"No cases of Enterobacter sakazakii were observed at the general hospital
since the powdered formula was replaced by liquid formula eight years
ago." Again, liquid, of course,
being a sterile product.
Next
slide, please.
[Slide.]
All
right. Before I turn to three outbreak
investigations, I would like to summarize, in this slide, some of the key
developments that took place regarding Enterobacter sakazakii in powdered
infant formula for the period 1961--that is, the date of the first
publication--through the '80s.
The first
thing to note is in 1980 Farmer, et al., in studying 57 isolates of
Enterobacter sakazakii, note in one of their tables that one of these isolates
came from a "pan of dried milk."
This is a sample that came from a central public health laboratory in London,
but there's no other details about it.
We've seen from the 1983 Muytjens article that Enterobacter sakazakii
was recovered from prepared formula, and that prompted the investigators to
recommend, in fact, that sampling of "milk" take place as part of
future investigations of Enterobacter sakazakii infections.
And then
in 1988 Muytjens and colleagues obtained 141 powdered infant formula products
from 35 countries, tested them for the presence of Enterobacteriaceae and found
52 percent of the samples were positive; 14 percent of 20 powders were also positive
for Enterobacter sakazakii.
Important,
thought, is to underscore that the concentration of Enterobacteriaceae in these powdered formulas was uniformly less
than or equal to one colony-forming unit per gram.
Next
slide, please.
[Slide.]
Okay. What I'd like to do now is use the second
half of this talk to go over three particular outbreak articles that I think
are salient to today's discussion. The
first one is by Biering, et al., 1989.
It was entitled, "Three cases of Neonatal Meningitis Cause by E.
Sakazakii in Powdered Formula."
These
illnesses occurred in 1986 and 1987 in Iceland--two within a month of each
other. Two of the infants were normal
at birth. Their gestations are listed
there. One baby was a Downs syndrome
baby. All three did well until day
five, when they became ill. The Downs
baby died. The two other infants
recovered, but with several neurologic sequelae. The investigators found that all three had been fed powdered
infant formula, and this was the first study to show, at least in an illness
setting, that Enterobacter sakazakii could be recovered--albeit in low
numbers--from freshly prepared formula from a previously unopened can.
Now, the
investigators did not recover, or were not able to recover, the organism from
the environment; specifically not from the milk kitchen, nor the utensils, nor
the ward.
Next
slide, please.
[Slide.]
Staying
with this same article by Biering, et al., they note that in addition to three
ill cases, there was an infant with colonization, and that these four
Enterobacter sakazakii strains that were isolated from the neonates had the
same plasmid profile as 22 strains recovered from the formula. They also point out, however, that there was
anecdotal evidence that the formula bottles had occasionally been kept at
fairly warm temperatures--35 to 37 degrees centigrade--for extended periods in
bottle heaters. However, they also note
that in one instance they were able to recover the organism via direct plating
from a bottle that had been refrigerated for an unknown amount of time.
From this
outbreak investigation, the investigators concluded: "Milk powder can be
the mode of transmission for Enterobacter sakazakii meningitis or sepsis in
neonates."
Next
slide, please.
[Slide.]
The final
two studies I'd like to discuss with you I bring up because I think,
importantly, can be discussed in the context of what we call "historical
cohort studies" in epidemiology, which are very useful because they allow
one to calculate rates of infection by exposure status--whether infants ate the
formula or did not eat the formula.
And the
two in particular I want to discuss with you are the Simmons, et al., article
in '89, and the Van Acker in 2001. But
keep in mind--we heard this morning from Dr. Kuehnert, the Tennessee outbreak
of 2001 published in the MMWR, that too was handled as a historical cohort
study.
Okay,
Next slide, please.
[Slide.]
The first
study is the one by Simmons, et al., 1989, entitled, "Enterobacter
Sakazakii Infections in Neonates Associated with Intrinsic Contamination of
Powdered Infant Formula." These
illnesses occurred in February and March of 1988. There were two cases of bacteremia, one urinary tract infection,
and one case of blood diarrhea.
Enterobacter sakazakii was recovered from the stool of all four infants,
which prompted the investigators to go back and look at the feeding practices
in that neonatal intensive care unit.
And they found that all four had been fed the same powdered formula.
This
formula had been prepared in a blender which was then rinsed with tap water
between uses. On culturing, the blender
yielded a heavy growth of Enterobacter sakazakii, but once a policy of
sterilizing the blender was instituted, no further clinical isolates were
obtained.
They then
conducted a historical cohort study in the neonatal intensive care unit to
assess risk factors for infection and colonization. And, as you can see, this historical cohort study took place--or
covered a period of about five weeks in February and March of '88.
Next
slide, please.
[Slide.]
All
right. Here, then, are the results of
the Simmons, et al., historical cohort study.
And it's just the standard setup for a two-by-two table, where we've got
"infection and colonization" up on the top--"yes/no;" whether
the infants ate the implicated formula, "yes/no" on the left.
And, as
we can see from the slide, in this outbreak setting, four of the five infants
who consumed the implicated product developed infection or colonization, as
opposed to zero of 40 infants who did not eat the implicated product. That yielded a highly statistically
significant association between the implicated formula and
infection/colonization.
Next
slide, please.
[Slide.]
Staying
with the same outbreaks, they note that the implicated elemental formula had
been given primarily to the most premature infants, largely because it required
relatively little digestion. They
obtained samples from an opened yet un-mixed can of powdered formula, and they
cultured this powdered formula using the 1988 methods of Muytjens, et al.,
which called for at least 100 grams of powder to be sampled in each sampling
frame, and then repeated three times for that matter.
They were
able to recover Enterobacter sakazakii from the powdered formula at a concentration
of approximately eight colony-forming units per 100 grams. In addition, they cultured--they recovered
Enterobacter cloacae at a concentration of 48 colony-forming units per 100
grams. And, with respect to the
Enterobacter sakazakii, they had the same plasmid and multi-locus enzyme
profile--both the isolates from the formula as well as the isolates from the
patients.
Next
slide, please.
[Slide.]
All
right, the final outbreak I would like to discuss with you is the 2001 Van
Acker paper, entitled "Necrotizing Enterocolitis Associated with E.
Sakazakii in Powdered Formula." In
this study there were 12 cases of necrotizing enterocolitis that had been
diagnosed in June and July of 1998 in Belgian neonatal intensive care
unit. In this period--June and July--a
total of 50 neonates were admitted to that neonatal intensive care unit, but
with respect to the 12 cases of necrotizing enterocolitis, all 12 had birth
weights of less than 2,000 grams, and had been fed powdered infant formula. Six of the 12 neonates with necrotizing
enterocolitis had positive cultures for Enterobacter sakazakii--and the
specimens are listed up here--versus zero of the 38 without necrotizing
enterocolitis, and that was a statistically significant difference.
In
addition, 10 of the 12 neonates with necrotizing enterocolitis had been fed the
same powdered formula, and the article states that this was a product called
Alfare, a semi-elemental formula with a low osmolarity.
Next
slide, please.
[Slide.]
Here,
using the same setup that we used before, are the results of the historical
cohort study in this outbreak. And, as
we can see, six of 14 infants who were fed the implicated formula developed
Enterobacter sakazakii-positive necrotizing enterocolitis, versus zero of 36
infants who did not--were not fed that implicated formula. Again, a statistically significant
association between the implicated formula and the development of necrotizing
enterocolitis.
Next
slide, please.
[Slide.]
Van Acker
and his colleagues noted that Enterobacter sakazakii was recovered from the
prepared formula, as well as from unopened cans. They did molecular typing, using arbitrarily primed polymerase
chain reaction, and they confirmed a partial strain similarity between the
powdered formula and the patient isolates.
There
were no further cases of necrotizing enterocolitis observed after the
implicated powdered formula was stopped in that neonatal intensive care
unit. But it's interesting to note that
during the outbreak period there was an inadvertent challenge test that took
place as follows.
The
implicated formula was stopped on July 10th of 1998, as soon as the
investigators had a suspicion that there was an association between illness and
that formula. But then the formula was
released again 10 days later, when all cultures to that date had been
negative. However, three days after
releasing that formula, there was a new case of necrotizing enterocolitis
linked to the same formula, and it was on that same day that the cultures came
back showing intrinsic formula contamination.
Next
slide, please.
[Slide.]
In the
same article, they also provide information regarding the manufacturers quality
control data for the implicated formula, and that is as follows. Of five samples analyzed, one yielded 20
coliforms per gram; four yielded less than one coliform per gram. And these results fulfilled the requirements
of Codex Alimentarius, which call for a minimum of four of five control samples
to have less than three coliforms per gram, and a maximum of one in five
samples that may have more than three, but less than or equal to 20 coliforms
per gram.
However,
the implicated formula did not meet Belgian law, which called for less than one
coliform per gram in all samples, and therefore the product was recalled.
Next
slide, please.
[Slide.]
I think
this is my final slide, and I want to end it, again, on the Van Acker
publication, because they note in the article that the facility that produced
the implicated formula in the Netherlands was upgraded. More stringent standards were adopted for
products; specifically, calling for less than .3 coliforms per gram, and zero
Enterobacter sakazakii isolates per 10 grams.
After
these changes were instituted at the production facility, the implicated
powdered formula was reintroduced to the very neonatal intensive care unit
where the outbreak had taken place.
Reintroduction took place in 1999, and Van Acker, et al., note that as
of the publication date--2001--no further cases of necrotizing enterocolitis
associated with E. sakazakii had taken place--or had been diagnosed--in that
neonatal intensive care unit where the outbreak occurred.
All
right. That's my last slide, and I'll
stop at this point.
Do we
want to take questions now? I saw by
the schedule Dr. Alexander is coming up, but I defer to you as to what you'd
like to do.
DR.
HEUBI: I think we're going to take some questions now.
DR.
KUZMINSKI: Dr. Klontz--Kuzminski, here--just a question on your very last
slide, please, if you could replace that on the screen. Thank you.
Do you
have information on the nature of the upgrades at the production facility?
DR.
KLONTZ: No. The article doesn't get
into the specific upgrades that were made.
The reference to this is in the discussion section, and it's fairly limited. No, I don't know exactly what took place
there.
DR.
BEUCHAT: Larry Beuchat.
Out of
these cases that you've summarized here, or other information that you might
have, what is the minimum infectious dose of E. sakazakii, in terms of
eliciting illness?
DR.
KLONTZ: The literature--I don't think the literature answers that
question. Certainly, not in the
articles I read could one ascertain a definitive infectious dose. There are a lot of complicating factors
here, as I'm sure you're aware--how much is in the powdered formula that leaves
the plant, versus what sort of treatment that product received in the hospital
setting, in terms of hang time and so on.
So those are a lot of complicating factors. I don't think the literature tells us what the infectious dose is
here.
DR.
ACHOLONU: You said that--Alex Acholonu, from Alcorn State University in
Mississippi.
You said
that you recovered the bacterial organism in unopened cans. My question is: what is the longevity of the
bacterial organism; how long was it in the can when it was checked? And when we consider the fact that you're
dealing with a powder in an unfavorable environment for the bacteria, is growth
sustained while the organism is in the can?
DR.
KLONTZ: Again, the articles don't go into those details. That information is just not there. I saw no discussion whatsoever that would
answer any of those questions--how long the organization was in the can, or
whether it reproduced in the can, how the environment could have affected its
numbers and viability--none of that discussion is in these articles. So I just can't answer that question.
DR.
ACHOLONU: But you are aware of the fact that they don't grow very well in dry
conditions.
DR.
KLONTZ: That's what I hear. Right. That's what I hear.
DR.
ACHOLONU: So it is necessary to find out what is keeping them alive. They are not spore-forming bacterial
organisms, as far as I know. So how do
they stay in the can--indefinitely--until it is opened? Maybe this is food for thought; something we
may have to think about.
DR.
KLONTZ: Yes. And I think as the day
goes on we're going to have some more microbiologically-oriented discussions,
and this would be a very relevant question to arise then.
DR.
NEILL: Peggy Neill.
I was
just trying to quickly go back through this, but it's probably faster just to
ask you. In your literature review,
then, are all the cases--are any of the cases reported associated with
non-powdered formula preparations?
DR.
KLONTZ: Let me start to answer that by saying--no, I didn't see any cases that
were linked to non-powdered formula preparations. However, a number of these articles don't even comment on source
at all. There's just a question mark,
when one finishes reading the article, as to the source.
The ones
I've presented, I picked out largely because they focused, they commented on,
or at least took effort to check the source.
And, of those, they were all powdered produces that were implicated.
DR.
FISCHER: [off mike]
DR.
HEUBI: Please identify yourself.
DR. FISCHER:
Larry Fischer.
You
mentioned that several of the publications indicated that they fixed the
problem by either using a sterile liquid, or they upgraded production
facility. But did any of them say that
they altered the procedures used to prepare the formula in the hospital and fix
the problem?
DR.
HEUBI: No. From my reading of the
literature, I didn't see any comment about that specific aspect of changing the
way that they prepared it. There was
actually relatively little discussion in these articles regarding--after the
outbreak, so to speak. And this is one
area that I didn't see any comment, really, in terms of what they did in-house,
in terms of their mechanism of dealing with powdered formula.
DR. TARR:
Tarr--Seattle--ah, St. Louis.
[Laughter.]
DR. TARR:
Did the incidence of NEC total go down after these outbreaks, or was there
inadequate data in the paper? And,
also, you talked about powdered formula versus no powdered formula. Among the "no powdered formula,"
was that heterogeneous or homogeneous, with respect to breast milk, or not
eating anything at all?
DR.
KLONTZ: Regarding the first question,
did necrotizing enterocolitis decrease?
Do you mean in this particular outbreak setting, or just as a
literature--sort of an aggregate view?
DR. TARR:
Total cases of NEC in these facilities.
DR.
KLONTZ: Yes. The only article that commented, to my knowledge, on that was Van
Acker, who said following the changes made at the firm, product reintroduced in
1999, and then until 2001, no further cases of necrotizing enterocolitis
diagnosed in the facility.
There's
relatively--there's very little sort of
post-outbreak surveillance that's discussed in these articles.
DR. TARR:
[off mike] I'm wondering about NEC not associated with--
DR.
KLONTZ: They do not discuss that. I can't answer that.
DR. TARR:
And the second question is: the no powdered milk group, that was supposedly
protected from the disorder, do we know--is that NPO--children no eating
anything? Versus breast milk?
DR.
KLONTZ: They don't--again, unfortunately, there's a lack of details on
sort of other feeding practice attributes of the unexposed group. So I can't be too specific about that. Nevertheless, they shared the same
environment. They were in the same
place, over the same period of time that the exposed infants became
infected. That's about as far as one
can say.
DR.
KUZMINSKI: Thank you. Larry Kuzminski.
Dr.
Klontz, in one of your earlier slides of the Muytjens paper from 1983, there
was a bullet that says, "E. Sakazakii recovered from prepared formula but
not from powdered formula and water."
And you commented on that four to 10 grams of the powdered
formula--which you indicated was a small amount--relatively small amount--was
used for the sampling here.
And I
relate that observation to what you have in the last slide here, on the second
bullet: "More stringent standards adopted for products. Zero E. sakazakii per 10 grams of
product."--a relatively small amount when you relate it back to your
observation on the Muytjens sampling--environmental sampling paper published in
'83.
So I'm
just wondering: is, indeed, 10 grams, the more stringent sample--quantity for a
more stringent standard, when historically the literature would indicate it
wasn't found with that amount sampled in the past, and perhaps not being found
now? Just your comment.
DR.
KLONTZ: Yes, that's an interesting point.
That's an interesting point.
And, in fact, today, as some of the talks that we get into shortly on
the microbiological aspect will underscore is that generally 100 grams of
powder are taken, or 111 in three different sub-samples, and repeated three
times. So much larger amounts than 10
grams are used today , you know, in the process of looking for this organism.
So one
could argue that 10 grams is--you're right--a relatively small amount. And certainly today all I can say is larger
amounts are used, in part because this organism may be uniformly distributed in
dried powder. And if one is going to
detect it, if it's indeed present there, one needs to look at larger amounts.
It's a good point.
DR.
TOMPKIN: This is Bruce Tompkin.
During
the public open comments, there will be some information relative to the
specific plant that was involved in the outbreak as Muytjens has reported. Okay.
And could you clarify again, then, you're essentially summarizing all
the data of cases reported internally, from across the world--you mentioned
48--but within the United States, there's a little confusion as to the number
of cases, or clusters, that have occurred in the U.S. Could you--
DR.
KLONTZ: In the--I don't have the actual count.
I believe you have a line list in your--or, if you don't--in your
packet, that actually goes through each of the outbreaks and each of the single
cases, as well, as to what country they were from and so on. So, if that's not in there we can get it to
you.
A quick
count? I know that in terms of
outbreaks, there was the Tennessee 2001 Dr. Kuehnert described. Simmons outbreak was four neonates. That was also the United States outbreak
there. And then there have been some
sporadic individual cases reported in the United States.
But I
think the bottom line is that this is truly an international phenomenon. And the Muytjens culture study of 1988
showed that, you know, formula from 35 different countries he tested and found
a 14 percent positivity for E. sakazakii.
So I think international is really sort of the bottom line here.
DR.
HEUBI: Any additional questions?
Thank
you.
Now, Dr.
John Alexander is going to discuss clinical consequences of E. sakazakii
infections.
Clinical Consequences of E. Sakazakii Infections.
DR.
ALEXANDER: : Good morning. My name is John Alexander. I'm an ID trained pediatrician who works in the Division of Anti-Infective Drug
Products, in the Center for Drugs at FDA.
And I was asked to give a presentation on what the clinical consequence
of Enterobacter sakazakii infection in infants is.
Next
slide.
[Slide.]
So what
I'm going to do briefly is give a presentation talking about the three major
manifestations of infection that have been identified in infants: neonatal
meningitis, necrotizing enterocolitis and bacteremia and sepsis. And I'm going to spend most of the time
talking about neonatal meningitis, because that's--we have the clearest
information about it, in terms of the consequences of the disease and the
infection.
Next.
[Slide]
Now,
overall, neonatal meningitis is a disease that occurs with an incidence of
about .25 to 1 per thousand live births.
But most of this is from the usual pathogens: Group B streptococci and
Escherichia coli, with a small proportion cause by listeria monocytogenes, as
the three major pathogens.
There are, of course, a lot of other
bacteria, other gram negative organisms, other types of bacteria that can cause
neonatal meningitis. In one survey that
was done in Dallas, Enterobacter species--and I use "species" because
of the fact that this was done before sakazakii was even separated from other
Enterobacters--accounted for less than 4 percent of the organisms. So this is talking about a rare organism in
what is a rare disease.
Next
slide.
[Slide]
Now, the
clinical manifestations of neonatal meningitis are often difficult, just as it
is often difficult in neonates, to separate any type of infectious disease one
from the other. There are a lot of
findings that are non-specific and may represent multiple different infections,
including fever or temperature instability, lethargy or just poor feeding, and
some infants, as they become more ill, develop respiratory distress.
The more
specific findings of neonatal meningitis are infrequent, including a bulging
fontanelle--so the soft spot on the top of the head starts to raise up. Stiff neck, or epistotonus--some type of
posturing, or convulsions that develop from the infection.
Next
slide.
[Slide]
Now, the
information that we have on E. sakazakii itself causing neonatal meningitis is
based on the collective literature. And
there is a predisposition for this occurring in neonates that are less than
2,500 grams, as a symbol of prematurity.
So that it occurs more often in premature infants. And about half of the pediatric cases that
have been reported, that were summarized by Lai, et al., in 2001, occurred in
children that were less than one week of age, and almost 75 percent of all
cases occurred in less than one month of age.
But I
would point out that this just represents what is collected in the literature
as case series, and this predisposition may be just the fact that we are identifying
through epidemiologic outbreaks in NICUs that might sort of push you towards
thinking that this is much more of a disease in neonates and isolated to the
neonatal intensive care unit. But there
were a couple of cases that were reported in otherwise health term newborns,
including a case that was reported of a five-week old who was healthy, left the
hospital on time, who at five weeks of age developed neonatal meningitis. So that is important to keep in mind, that
we aren't just talking about a NICU disease.
Next
slide.
[Slide]
One of
the important manifestations that needs to be kept in mind with Enterobacter
sakazakii infection is a predisposition towards developing these large cystic
lesions that are believed to be brain abscesses in the brain. And this is important because in--this is
much more likely to represent severe disease, as opposed to other causes of
meningitis. Other causes of neonatal
meningitis that were listed--the Group B, strep, E. coli and such--actually
cause brain abscess in a minority of cases, but from the cases that we have
identified so far in the literature, approximately 75 percent or so are cases
that involve brain abscess. And this is
something that's recognized. There are
other bacteria, including one called Citrobacter diversus, that's recognized to
cause--to have a predisposition to causing brain abscess, so that when it's
there in the brain you have to look at CT scans to see whether these brain
abscesses are present.
Next
slide.
[Slide]
Now, for
neonatal meningitis, the outcome that we're concerned most about is certainly
fatality. And for gram negative
meningitis--so, gram negative organisms of any variety that cause meningitis
there's approximately a 17 percent case fatality rate. For E. sakazakii meningitis, from the
reports, Lai, et al., had given about 45 percent, and the different reports
that looked at the collective literature ranged from 40 to 80 percent.
Now, it's
a little difficult to take this as an indicator of what the overall case
fatality rate is, because what you're doing is collecting the literature
reports. But I do believe that we are
talking about something that's at least 17 percent, and probably greater.
Therapy
for infants who have neonatal meningitis is usually at least three weeks of IV
antibiotics, and would be longer for persistent positive cultures, which would
be expected in those cases where you have brain abscesses. So you are talking about prolonged IV
antibiotic treatment.
Next
slide.
[Slide]
Now, this
study by Onhanand, et al.--and I didn't check beforehand about how to pronounce
that name--
[Laughter.]
--gives
the sequelae for gram-negative meningitis overall in a his institution. And they had sequelae in approximately 58
percent of those children who survived.
You have children with developmental delays, seizure disorders, cerebral
palsy, hydrocephalus, and hearing loss as the main sequelae that are recognized
from gram-negative meningitis. And,
again, these are from a variety of other organisms, and I think it is appropriate
to show these numbers because, if anything, they would probably be an
underestimate of what Enterobacter sakazakii that causes these brain abscesses
could do.
Next
slide.
[Slide]
So now we
move on to necrotizing enterocolitis.
And as an ID-trained pediatrician, I never thought I'd be speaking to a
bunch of gastroenterologists and nutritionists about this disease, but here
goes.
It's a
disease of the GI tract that is seen mostly in premature infants. Up to 10 percent of NICU admissions, and approximately
10 percent of affected infants, though, are term infants. So this is, again, mostly a disease that is
seen and recognized in premature infants.
It does, though, occur in some term infants, as well.
And this
is a multifactorial disease, so that there are other factors besides just the
presence of organisms that are believed to play a role in this disease. But the reason that infection in some cases
is considered important is because of the fact that there are the outbreaks
that occur in association with infections.
Next
slide.
[Slide]
Now, it's
important to remember that Enterobacter sakazakii is only one of a number of
organisms that have been associated with necrotizing enterocolitis. These are lists from a pediatric infectious
diseases textbook of the different bacteria and viruses that have been
associated with outbreaks of necrotizing enterocolitis. So, in response to some of the questions
that had been raised earlier about the effect on the incidence of NEC, I don't
think it's that surprising that there may not be a change in the incidence of
NEC overall from changing whether the exposure to E. sakazakii occurs.
Next
slide.
[Slide]
Now, the
clinical manifestations of this disease--again, there's a wide spectrum of
disease, ranging from a sudden or to an insidious onset. Again, there are nonspecific findings, as
you expect with neonates, of a bunch of different symptoms of infection or
disease: feeding intolerance, temperature instability, lethargy. Some develop apnea and respiratory
distress; evidence of metabolic acidosis, unable to maintain their blood
glucose, and then a bunch of more specific GI findings, where you have infants
who develop abdominal distension, tenderness and erythema, bilious emesis,
blood in stools that's either grossly visible or microscopically tested for;
and then a bunch of radiologic findings--pneumatosis intestinalis being sort of
a specific finding of air within the wall of the intestine that indicates that
necrotizing enterocolitis is occurring.
And then when you get to portal venal gas or pneumo turg, in the end
you're talking about a--usually a rupture of the intestinal wall and leakage of
the gas into the systemic circulation or into the peritoneum.
Next
slide.
[Slide]
Briefly,
therapy includes discontinuing feedings and nasogastric decompressions
following serial radiographic examinations; blood cultures and antibiotics are
usually given. IV fluid and supportive
care when there are systemic manifestations of disease, so when the infant
starts to appear shocky; and then for advanced disease, surgical intervention
to remove part of the bowel that's infected.
Next
slide.
[Slide]
Now, the
outcomes from both textbooks of neonatal medicine and a recent surgical article
give overall mortality rates to NEC of around 9 to 28 percent. The survival is around 98 percent for
medical management--and medical management, again, is the less severe
cases. It's the more severe cases that
usually end up going to surgery.
The
surgical mortality rate was approximately 45 percent in the article by
Grosfeld, et al., and ranged up to 60 percent from various sources. And, again, surgical mortality is inversely
related to gestational age and size, so it's the younger and smaller infants
that are going to have more problems overall and a higher risk of mortality.
These are
other complications of necrotizing enterocolitis: GI strictures occurring in 25
to 35 percent, whether after medical or surgical therapy; other
gastrointestinal dysfunction; neuro-developmental sequelae---whether that's
directly related to the NEC or related to prematurity and NEC is difficult to
tease out; and then short-gut syndrome, which is a syndrome where children have
problems and require other forms of alimentation because of the fact that they
don't absorb quite enough food from the feedings that they get.
Next
slide.
[Slide]
Now, for
bacteremia and sepsis, just to give you a separation here, bacteremia is
talking about bacteria in the blood, and it can occur with many different
infections. A lot of the bacteremia
that is reported with Enterobacter sakazakii is actually in association with
the meningitis, which isn't surprising because you have to get the organism
into the blood to get it to the brain; but also in cases of NEC.
Sepsis is
actually a clinical syndrome where you're talking about fever, systemic illness
progressing to shock, and it is associated with morbidity and mortality. And from bacteremia to sepsis, there's
actually a spectrum of disease, and different organisms can cause different
things. Some organisms--for instance
pseudomonas--are more likely to present with sepsis, even in normal hosts. Other organisms, like Enterobacter
sakazakii, are more likely to be organisms that would be identified as
bacteremia and in populations that are populations that are predisposed to
developing the infection.
Next
slide.
[Slide]
So, what
we're talking about here is basically an opportunistic pathogen; an organism
that isn't usually something that's identified in children who are otherwise
normal and healthy. The predisposing
factors that have been recognized include the neonate and premature infants,
where meningitis and NEC cases have been seen.
And one of the--what I'm going through here is basically the reports
that we have from the literature of infants who were septic, so there were the
many reported cases with meningitis.
There was also a separate report of a seven-day-old infant who developed
fever and sepsis, but not meningitis, that was hospitalized and, I believe, recovered
with antibiotic treatment.
There are
also other--two other reports of children with bacteremia. One was a three-year-old with a
rhabdomyosarcoma who recovered with antibiotics and removal of the lung from
which the organism was identified, and what his exposure is is unknown.
There's
also the issues of altered host defense.
The other case report of bacteremia is in a six-month-old who had had an
intestinal resection. So that's
probably a potential site of entry for the organism from the intestine. And this patient also recovered with
antibiotics.
So it's
overall, in terms of talking about bacteremia, we have to recognize that it's
difficult to just take the incidence of sepsis from other organisms and apply
it to this disease. We are talking about,
here, bacteremia with an opportunistic infection, so that you're talking about
infants that are sickened in some way, have altered host defenses, or altered
immunity that sort of predisposes them to developing infection with this
organism.
In conclusion,
Enterobacter sakazakii is a cause of meningitis, NEC and bacteremia--all
diseases that are associated with serious morbidity and mortality, and it's
found especially in neonatal disease but not exclusive to that population.
DR.
HEUBI: Questions?
Heubi. My first questions would be: the last
two--the last slide you showed, the patient with rhabdomyosarcoma, and one with
short-gut, were either of those patients on powdered formula during the course
of their therapy; specifically, the short-gut patient?
DR.
ALEXANDER: I don't know.
Actually,
for the--you're talking about the six-month-old who had the surgery?
DR.
HEUBI: Right. Right.
DR.
ALEXANDER: She had the surgery, for a jejunal atresia, actually. And I believe that she was receiving
powdered formula. I'd have to go back
and check that.
DR.
HEUBI: Thank you.
Virginia?
DR.
STALLINGS: Stallings.
We
haven't discussed antibiotic coverage.
Is this a particularly difficult infection to treat once you've
identified it? Because the mortality
rate seems to be high, in addition to the brain injury. What is it usually sensitive to, and why are
the kids dying so fast?
DR.
ALEXANDER: Well, part of it is that meningitis, and especially meningitis that
causes brain abscesses in premature neonates is more likely to be something
that's going to be associated with mortality.
Part of
it, again, is that collecting this information from the case reports, we're
more likely to be getting a snapshot of those patients with severe
disease. We don't necessarily have a
case series for the experience at one or a bunch of hospitals with this disease
where we could feel more comfortable that we are sort of collecting all of the
cases. So, there may be some of
the--there may be some bias towards seeing reports of higher mortality, and
that's why we're dealing with that.
In terms
of the susceptibility of the organisms, it is susceptible to most of the
immunoglycosides, which are typical treatment for neonates. So, I think that I saw one report for
Enterobacter where gentamicin susceptibility for the genus as a whole is
approximately 94 to 95 percent. And
from the case reports, I didn't see anything that really spoke to anybody
having a problem with treatment, other than a very recent report, about a month
or two ago, of an adult who had an Enterobacter sakazakii infection that was
resistant to immunoglycoside.
DR.
HEUBI: Margaret? Oh. I thought you were going to ask a question.
DR.
BAKER: Baker.
I wanted
to just think a little bit about the susceptible populations. The original--the FDA "Dear
Doctor" letter was specifically for NICUs, and we've seen that several of
these outbreaks have been in NICUs, so our sort of--the feeling was that this
is a neonatal and maybe specifically for premature kids. On the other hand, we had--there's been two
things that have sort of contradicted that, and that is--there was a slide
earlier this morning which didn't show any greater susceptibility with
decreasing birth weight. And then you talked
about a five-day-old who was supposedly full term and was infected with E.
sakazakii.
DR.
ALEXANDER: It was actually a five-week-old.
DR.
BAKER: Five-week-old--so--and then there have been some of these sporadic cases
of older children and adults with E. sakazakii. So can we identify a susceptible population, and are there other
susceptible populations outside the NICU?
That's my question.
DR.
ALEXANDER: Well, I guess that for neonatal meningitis, and for the meningitis
manifestations of Enterobacter sakazakii it really does seem to be something
that is isolated to those infants that are within the first months or two of
age, because for the reports that we've received--for the reports that I've
seen, at least, of Enterobacter sakazakii in anybody that's older than a month
or two of age--which is around the cutoff where you start transitioning from
what would be considered the normal neonatal meningitis pathogens to
community-acquired meningitis pathogens.
After
that transition period, you don't really see any reports of people who have
meningitis, other than one report where I think there was an infection of the
central nervous system in somebody who had some type of congenital malformation
of the central nervous system. So,
neonatal meningitis itself is something that is more likely to be seen early
on, and more likely to be seen in more premature infants, and that may be why
what we're seeing is mostly reports that are associated with the NICU.
For the
bacteremia, I think that we are talking about a population that has some
altered host defense, so that you're not really talking about children who are
otherwise normal and healthy, but it can be as simple as children who have had
previous surgery, as well as children who have immunosuppression, or some other
altered host defense system.
DR.
HEUBI: Phil?
DR. TARR:
Tarr.
I'd like
to probe again about the issue of potentially missed cases. Are there data in NICUs, on a population
basis, of culture-negative meningitis that could conceivably have been caused
by E. sakazakii, or another organization?
And are there cases of brain abscess caused by this organism in which
the CSF was sterile?
DR.
ALEXANDER: I think, actually--I
wouldn't necessarily know from the literature whether there are going to be
cases of culture-negative meningitis, or culture-negative brain abscess that
could be related, because you wouldn't figure that out unless you got a culture
that identified this organism. I do
think that in most cases where somebody has a culture-negative infection,
whether the spinal tap is negative or whether the source of an abscess doesn't
grow our any bacteria, you do start looking for other potential causes,
including tuberculosis, parasitic infections, different fungal infections that
can manifest in that manner. So it's
difficult to say how much of these other culture-negative things would be
related to that.
The other
difficulty that I have is, again, we're dealing with the case reports. And we don't necessarily know whether that
represents the tip of a large iceberg, or whether what we're seeing as a report
is a lot, or a majority, of the cases that have been identified. I would think that it's more likely to be
the tip of the iceberg, because I don't think that the literature reports represent
all of the sporadic cases, and we certainly don't get investigations of
individual sporadic cases to start looking at things like powdered formula, and
what the source of infection would be.
DR.
STALLINGS: Stallings.
Would you
concur with the previous speaker that if you have properly obtained biological
specimens--the blood, the CSF, pathological tissue--that we should be able to
grow this bug and identify it, you know, in a U.S./Canadian setting?
DR.
ALEXANDER: I mean, I think that the cases where you're talking about isolation
of the organism from CSF, or isolation of the organism from blood should be
possible. I don't think that this
Enterobacter sakazakii as a subspecies is going to be any more fastidious than
normal Enterobacter, and our hospitals have plenty of experience with isolating
those.
The only
caveat may be whether the testing is specific enough to separate Enterobacter
sakazakii from other Enterobacter cloacae.
So whether you're getting some cases that are reported out as
Enterobacter cloacae that are actually this organism.
DR.
STALLINGS: But the issue of how
useful--if we wanted to go back and look at cultured-negative CSF, I mean in a
neonatal setting, if you're working up a baby who's become ill, you're going to
get, in the U.S. anyway, you're going to get blood and urine and CSF, and those
will be managed in a way we ought to be able to identify that it was bacterial,
at least, and at least to the species.
DR.
ALEXANDER: And certainly the other things that you look at in the CSF--the
white count, and glucose and things like that--should be pointing you in the
direction as to whether this is a likely bacterial infection or not.
DR.
BEUCHAT: Beuchat.
The
information that you and other speakers have given this morning on surveys of
the dried powder for the presence of E. sakazakii indicate, at least in one
instance, up to 52 percent of the samples were positive.
Given
that number of servings per annum is probably a billion or more, the question
is why aren't we seeing more cases?
What do we know about the level of virulence of these strains? Are some avirulent under any conditions,
while others are highly virulent under all conditions? What do we know about the virulence and
pathogenicity of E. sakazakii?
DR.
ALEXANDER: Well, I'd say that overall what we can tell is that the virulence of
the organism is likely low, until you get to a particular patient that may be
predisposed. And I'm not sure that
there's any way, really, to identify all of those patients ahead of time. And then when you have an isolate that in
neonates and young infants, specifically--so children that we're probably
talking about less than four to six weeks of age--that they may be predisposed
to developing meningitis. And I think
that has something to do with the mechanism of neonatal meningitis overall,
which we still have a lot of--we still really aren't very clear about.
DR.
BEUCHAT: Are then all the strains equally virulent? Do we know that?
DR.
ALEXANDER: I don't know. I mean, so far all we've done is sort of
identify this organism as a particular species. If you're comparing the species of E. sakazakii to other species
of Enterobacter cloacae, there are certainly indications that Enterobacter
cloacae and other Enterobacter species may be more likely to cause bacteremia
and sepsis in an older age population because of the fact that it's just
isolated so infrequently. But, again,
it's a question--is it that it's isolated infrequently because of the fact that
you need a certain exposure and you're only getting it from powdered formula? Or is it because of the fact that the
organism is less pathogenic? Can't
tell.
DR.
HEUBI: Larry?
DR.
KUZMINSKI: Larry Kuzminski.
I think
you've answered partially, in your answer to the previous question, but I look
back to part of our charge here--based upon your survey--and I apologize if I'm
asking a question that's been at least partially ask before--but, in your
opinion, what do you think are the populations of infants at risk to this?
DR.
ALEXANDER: Well, again, I certainly think that the infants who are in neonatal
intensive care units, who are premature, are probably a population that's at
the greatest risk. But I do think that
there's also a somewhat lesser risk, but a risk of a fairly serious fatal disease--neonatal
meningitis--that goes to term infants and probably out to a month to six weeks
of age, which is the period that we see neonatal meningitis in. And then, again, for the older children and
te bacteremia, it's certain children with altered host defense,
immunosuppression, that are likely to be the ones who are at risk of developing
bacteremia from the organism.
So you
have a population of infants that are susceptible because of cancer, because
they have central lines for some other reason, that are fed powdered formula
and then could develop a bacteremia due to the organism.
DR.
HEUBI: I think we're going to bring the rest of the presenters up, and we could
actually pose these kind of questions to them collectively--although if you
have a question--so if the remainder of the presenters from the morning, except
for Dr. Kuehnert, could please come forward, we'll organize this, and have a
little question-and-answer period.
DR.
BLUMBERG: Henry Blumberg.
One
question I had for you: do you know if FDA or CDC, or other groups are trying
to do any surveillance to look at rates of necrotizing enterocolitis,
specifically, you know, since the CDC report came out in the MMWR, at least at
my institution, and I think probably others, there's been a shift away from
using--in the neonatal intensive care unit-from used powdered milk, and I think
there's a lot more use of the liquid, sterilized product.
And I was
just curious if anybody has any data, is that impacting rates of NEC?
DR.
ALEXANDER: I'm not aware of any.
DR.
HEUBI: So I think at this point--thank you.
I think at this point the committee members can pose questions to any of
the presenters from this morning. And I
think Larry's question is pretty relevant, because I think we're trying to
define what populations may be at risk, because ultimately we're going to be
asked to provide some counsel to FDA about which populations should be
protected.
DR.
STALLINGS: Stallings. So, actually,
thinking about that, and thinking about the clinical practice setting, I was
sitting here going--we've talked about the pre-term infant in the NICU. And just to be sure that all of the people
at the table may not be aware of how many term infants are NICUs, because of
the different kinds of presentation. So
not everybody there is a premature infant with all of the things that go with
that, including less well-developed immune systems.
So then I
was thinking, well, the other couple of groups--and I would just like for your
response--there's a whole subset of infants and lung children with poor gut
function. And some of these are the
ones that have got motility problems we don't understand. Some of them are post surgical. Some of them may have been the babies who
had NEC and now have short-bowel syndrome--and, again, a group that we've often
used special formulas in.
And then
the third group are children who we would recognize as immunosuppressed. So, discounting the premies, children who
might have HIV, or cancer, or even of the new biological agents that are coming
out to treat all sorts of diseases we're not sure. So there's a whole spectrum of children who might be at risk, as
we open the umbrella from the case presentation, which was--it was sort of a
classic premie presentation.
So, for
the committee, just as we start to think about that, what do you think about
those different scenarios, or other clinical ones you might think of from your
experience, as well?
DR.
ALEXANDER: Well, again, I think it's difficult to try and define well a
population that might be at risk. Now,
it's easier to define the populations that are at risk for neonatal meningitis
or for NEC, because what you are usually talking about are more often premature
infants, but can occur in term infants.
And then there's a defined window of time that we're usually looking at,
something along the line of four to six weeks of age, which is when the
neonatal meningitis would usually manifest--whether early-onset, which is
defined as just less than seven days, versus later-onset.
And so
for those two diseases--and certainly neonatal meningitis being the most
serious manifestation that we've seen of Enterobacter sakazakii--you have what
would be an easily defined group that's based on what their chronological age
is: about six weeks of age, or exposure within NICUs and prematurity. So it's potentially the case that you're
talking about an infant who is very premature, who might go beyond that four to
six week age period and then develop NEC at some point later on because of
exposure.
For
bacteremia, when you get beyond that four to six week age population, you are
really talking about populations that may have some form of immunosuppression,
whether it's due to cancer treatment, whether it's due to an unrecognized
immunologic deficiency--and there are several different forms that it could
take. And then altered host
defenses. So if what's happening is
that this organism is entering through the intestine, then potentially anybody
who'd had prior GI surgery or resection of either congenital anomalies or for treatment of NEC could be a population
that's potentially exposed. And then
also other populations that, for whatever reason, require a central line for
treatment.
DR.
KLONTZ: Karl Klontz here. I'd just like
to comment briefly on that issue.
In
looking at the literature again, one of the things I did was to go through the
line list of all the cases that were reported--in infants--n=48 cases.--just to
look at gestations. And of those 48,
there were five reports that don't comment on how old the baby was, in terms of
gestation. Then I think there were 13
cases that were described as either--quote-unquote--"term," or
gestations greater than 36 weeks, or--some of the older reports--"normal
baby" at birth. So there were 13 of
those, leaving 30 who fell into the category of prematurity.
But
beyond that, the articles really don't get into the sort of the molecular
biology, so to speak, of immune systems.
It's just insufficiently detailed to--in my opinion--to decipher
clearly, from an immune status, who exactly is at risk. I don't think the literature does that for
us.
DR.
ALEXANDER: Right. I think my comments,
in terms of the bacteremia and sepsis are meant to be just an extrapolation of
what you would think of as the populations at risk for an opportunistic
pathogen to cause infection.
DR.
BEUCHAT: Beuchat.
I tried
to find it in the handouts, but can't.
I'll ask--I think it was Dr. Klontz--you had indicated that four cases,
median age 74 years--what was the
DR.
KLONTZ: Six cases, I believe it was.
DR.
BEUCHAT: What was the vehicle--do we know what the food of the vehicle was,
that may have been associated with those cases?
DR.
KLONTZ: No, I don't think any of the articles described what the vehicle
was. So, no, I don't know the answer to
that.
DR.
BEUCHAT: Presumably it wasn't infant food, but--we don't know that.
DR.
KLONTZ: We don't know that. I mean, the
gentleman, 76-year-old, with a history of rectal adenocarcinoma, presumably he
was doing reasonably well, but then suddenly came down with, you know,
urosepsis. No comment on dietary
factors in that article nor, to my knowledge, the other five. I don't recall. I'll check during the break, but I don't recall any distinct
dietary information given in those cases.
DR.
BEUCHAT: I guess what I'm really asking: are there other foods that also may be
vehicles of E. sakazakii, other than the powdered infant formula.
DR.
KLONTZ: I don't have enough information to answer that question. I don't know. I don't know the answer.
DR.
HEUBI: Dr. Fischer?
DR.
FISCHER: Fischer.
I'm not a
physician, so I can ask this question.
Why would you fed the powdered formula in the hospital instead of using
the liquid formula? Under what circumstances would you want to take the risk of
feeding the
powdered formula?
DR.
ANDERSON: There are certain formulas that are only available in powdered
form. In particular, the formulas for
infants with metabolic disorders, the amino acid-based formulas, and some
formulas for infants with special medical conditions are only available in
powdered form.
In
addition, human milk fortifiers, which are added to human milk for premature
infants are available in powder form.
There is also one product on the market that's available in fluid form. The advantage to added the powder in this
situation is that there is a minimal increment in volume that the infant must
consume, and that's an important consideration for pre-term infants.
DR.
FISCHER: Well, let me follow up by asking what is the percentage of times that
you need to use these special formulas, as opposed to the non-special powdered
formula? You're saying the reason
you're doing it is because it's a special case and special diet. What's the percentage of time that has to be
used?
DR.
ANDERSON: I don't have specific information.
It would be small, but I can't say further than that.
DR.
THUREEN: I can tell you that having talked to many of my colleagues across the
country, that the use of powdered formulas has evolved over the last five to 10
years. Many infants can take the
regular prepared formulas for preterm infants, but there's a perceived notion
that better nutrition can be supplied if you supplement the existing liquid
formulas with powdered formulas. Many
times it's given to increase calories or proteins, calcium--whatever--but many
units, individual, without any protocols, concoct a variety of different
formulations using powdered formulas added to liquid formulas. And they don't just use more liquid formula,
because many of these infants have fluid volume restrictions. So it's a widespread practice. And I contacted a number of units around the
country when this first occurred, and about half of them stopped using powdered
formulas, or had never really used them much and always used ready-to-feed
formulas.
But many
other units have, and are continuing, to make their own concoctions, and it's
highly variable as to what exactly they're making up, but they individualize it
to different infants. So it's a very
common practice.
And why
would they do it? Well, it's just the
belief that if you strategically want a certain type of preparation for infant,
you can make it using what's available on the market, and just mixing up what
you think makes sense. It's a
widespread practice.
DR.
HEUBI: Margaret?
DR.
BRILEY: [Off mike.]
DR.
HEUBI: Please identify yourself.
DR.
BRILEY: [Off mike.] Margaret Briley.
DR.
HEUBI: Speak up, please.
DR.
BRILEY: Margaret Briley.
In regard
the population that might be involved with this organism, there's been some
part of our American adult group, as well as children, that are still trying to
find raw milk products to consume, even though we do not advocate that, and we
have rules against that. But there are
rules in Texas where people can produce raw goat milk, for example, and sell it
on their place. And we've had some real
serious situations about that.
Does
anybody here know if this organism is present in the raw state? Is that not where it's, maybe possibly,
coming from?
DR.
ALEXANDER: I'm not sure that we have clear idea about the source. I know that one of the reports--I think it
was Muytjens, et al., that tried to look at a bunch of environmental sources;
so, looking at soil, looking at a bunch of sources, didn't isolate it from
anywhere.
I think
there was something else that I had also seen about rice paddies as an
environmental source of where this organism is able to grow. But I'm not sure how that would be involved,
and whether that would be the source at all, of the infant formula exposures
that we're talking about.
I don't
know--I can't speak to whether it's something that's identified from cow milk
or not. But I don't think that that was
considered to be the source, ultimately, of the organism.
There was
another interesting report, I think, for one of the adults, where it was
actually found within the hospital environment, but it wasn't clear as to
exactly where. So that there's the
possibility that with the older adults, you may be talking about some sort of
exposure through environmental--through an organism that was present in the
environment, but it's not clear how that happened, and there are certainly
other reports with the different NICU outbreaks that didn't find the organism
in the environments.
DR.
HEUBI: Dr. Stallings?
DR.
STALLINGS: [Off mike.] Stallings.
I was
just going to add [inaudible] so that just so--there are some children with
some diagnoses that would have to be on these things almost for their whole
life. If you looked at something like
PKU, or MSUD. But there are many
children with GI diseases outside of infancy where we use all of these
products, as you were saying, in whatever creative way that the group
decides. So I think there is a lot of
use. There's a lot of use beyond sort
of the original intent. And, you know,
again, the case, if I recall correctly, was a product that wasn't available in
a liquid or ready-to-feed product.
The other
thing--it has been my impression, although I don't know the data and maybe the
manufacturing people will help us with this--is there was an element of the
dried products are cheaper and easier to store. It just takes up shelf space.
So if you have a big setting that you're willing to have a formula room
and that sort of thing to mix some of these things up.
So there
are a lot of different issues going on about who's exposed. But even if we trained all the
neonatologists to do this perfectly, there are a lot of other people in
pediatric practice who are using all these formulas to put together whatever
they think they need for the patient.
DR.
ACHOLONU: Alex Acholonu.
Has any
survey been done on neonates outside the hospital environment, who are breast
fed? And the reason why I ask this
question is that they may be getting colostrum from their mother, and that is
believe to have agglutinates that may be able to control the disease.
DR.
ALEXANDER: Again, I think that what we're dealing with in terms of what we can
report about Enterobacter sakazakii is only the information that we have on
these sporadic cases from the literature.
I think it would be very difficult to try and identify and tease out
specific factors as to whether children who are breast fed are less likely to
have this organism, or more likely to have this organism--and how do you
necessarily separate that out from the fact that those children who are breast fed
are less likely to be receiving powder or any type of formula at all.
So, I
don't think that there's anything out there that's going to be able to help use
in terms of a survey, because I think we are still talking about a fairly rare
organism.
DR. BUSTA:
Frank Busta.
When I
look at the Tennessee cohort study, which was given to us indirectly, seven of
the nine were on continuous feeding, which I assume is tube feeding. Is there ever any discontinuous tube
feeding?
VOICE: Uh-huh.
DR.
BUSTA: So that the other two could have been a discontinuous tube feeding. Also, that same--seven of the nine never
received any breast milk.
Is there
a possibility that the infants that are infected are not exposed to the normal
inoculum flora, like the bifidabacter, or lactobacilli, that normally-fed
infants are exposed to, and colonize their digestive tract? Or is there any similar type of data in the
other studies that you did internationally that would show that it's tube
feeding, and maybe a lack of good competitive flora?
DR.
KLONTZ: No. Karl Klontz here.
From the
published literature there is really not that depth of discussion--even in
terms of how the formula was fed. I'm
thinking back to the Van Acker study, some of the major--large outbreak
publications, and I don't recall details on mechanism, or mode of delivering
the prepared formula, nor were was there--certainly, there were no features on
sort of the microbiology of the intestines, to my knowledge, in those
outbreaks.
DR.
ALEXANDER: I think part of the problem
that you're going to get in trying to look at the idea of whether it might be
associated with something like the tube feedings, or the plastic tubing, or
things like that is when it comes to the disease of NEC, the infants that
you're talking about that are more likely to develop that disease are also the
infants who are going to be receiving this almost exclusively through some type
of gastric tube feeding, and they're not the infants who are going to be
sucking on a bottle.
So it's
not going to be possible for the NEC cases that were identified to see whether
it's--to try and tease out the differences between whether it may be related to
the plastic tubing, versus the powdered formula itself.
With the
meningitis cases, I'm certain that, for instance, that the five-week-old infant
that I was talking about was most likely a child who was normal and healthy and
wasn't receiving any kind of gavage feedings, so that some of those cases of
meningitis, if we could find more information from--you know, from the cases
themselves, would probably indicate that those were children who were on
otherwise normal feedings, and not related to gavage.
But it's
not something that's clear, that we clearly know at this point.
DR.
HEUBI: Dr. Fuller.
DR. FULLER:
Changing topics a little bit, could you refresh my memory or give us a
little--go back over--what information do we have in E. sak in the ingredients
used to formulate the powdered formula?
Or do we have that information?
DR.
ANDERSON: I believe information on that topic may be part of the talks this
afternoon.
DR.
HEUBI: Rob?
DR.
BAKER: Baker.
I just
had two comments. One was about breast
feeding. And just sort of a priori, you
would sort of expect breast feeding or breast milk to be protective, in that
the information did sort of suggest. So
I would think that would be something to look at.
The other
thing I wanted to mention is that there is another population that we haven't
really talked about that may be at risk, and that's the graduate of the
NICU. And babies are graduating from
the NICU at earlier and earlier stages, so there are some quite premature
infants that are being cared for in other parts of the hospital or even at
home, and they may be at risk for this.
DR.
HEUBI: Last question
DR.
LEE: Yes, going back to the earlier
question--
DR.
HEUBI: Please identify yourself..
DR. LEE:
This is Ken Lee. And going back to Dr.
Busta's question about feeding by gavage and establishment of favorable flora,
is there any attempts in a neonate to try to establish a helpful flora. Obviously the line of questioning is that if
friendly flora is established, then there's less opportunity for an
opportunistic pathogen like E. sak to establish itself. And that kind of thinking leads you to the
idea that well, maybe, that ought to be put in the powder itself to help the
patient.
DR.
THUREEN: I'd like to just make a comment on that. This is Thureen.
I would
have to say that most of these infants that are preterm have very altered
flora. They've gotten antibiotics
staring at birth--often many courses.
They've gotten medications that have altered the pH of their GI
systems. They've undergone many
insults. So their GI flora is anything
but normal.
I think
there are a lot of studies that are now underway, looking at probiotics as a
means of establishing a more normal flora.
But I think this population is so vulnerable, just because they're set
up, for many, many reasons, for infection from this organism.
DR. LEE:
We had a little conversation over here--the idea, of course, is--that's a very
good point, in that perhaps there are things that--any growth condition that
would lead to an outbreak of the pathogen E. sak could also lead to the growth
of something that's non-pathogenic. And
if one could find a non-pathogen that would inhibit E. sak, then that would
also be a helpful thing to be able to do.
DR.
THUREEN: Thureen--one more comment.
[Laughter.]
DR.
THUREEN: You know, I wonder if the E. sakazakii's not just sort of a marker
that's given us an interesting epidemiologic chance to study these infants,
because also my understanding is in any infant formula there's very low levels
of contamination with other coliforms, staph aureus, and other organisms, and
those are very common pathogenic organism for NEC, sepsis, etcetera--and that
we are just focusing on E. sak because it's such an unusual organism. But, in fact, this could be a contributor to
a lot of the other infections that we see going on.
DR.
ALEXANDER: Certainly, it seems like there is--it's easier to focus on E.
sakazakii because of the fact that there is what appears to be a more clearly
epidemiologic association between its presence in the infant formula and lack
of identification in other settings. I
mean, for these other organisms that you're talking about--the coliforms, E.
coli, klebsiella, the other enterobacter species--the problems that you run
into are those are part of normal human GI flora, and so how do you tease out
the fact that formula would have been what introduced the organism that caused
the disease, as opposed to just passing through the birth canal, or some other
source, whether related to what's on the health care worker's hands, or what
would be normally the process of sort of GI colonization that, for whatever reason
in this infant, leads to a more serious infection?
DR.
HEUBI: I'd like to thank the presenters and the committee members for the
lively discussion. The committee
members and the speakers are invited to lunch, behind us. The guests are on their own.
[Laughter.]
DR.
HEUBI: And, I forgot--if you haven't found this out already, the restrooms are
out here. If you didn't know already, I
think you're probably in trouble.
[Laughter.]
[Luncheon
recess.]
A F T E
R N O O N P R O C E E D I N G S
DR. BUSTA:
I f you will take a seat we will get started with this afternoon's
presentation.
The
committee has received the information requested from Atlanta. You see the two graphs that we were
sent. This is just for the committee,
not for public distribution. It's
unpublished data, not for further dissemination . I don't know how much more
that can be emphasized. It's right
across the top of the slide, but it's to help us understand the discussion this
morning. So, please bear that in
mind--not for public distribution.
We also
have a table to supplement the presentation this morning, that you received.
Thank you
for the prompt return to the committee.
Brief luncheon period, but we will get on our way. We'll do our best to stay on time this
afternoon. It's a little tighter. And we had the good fortune of having a
little extra time this morning. We'll
have to be tighter this afternoon.
The first
presentation is on the general microbiology of Enterobacter sakazakii. This is from Dr. Maria Nazarowec-White.
DR.
HEUBI: As your co-chair, I'd like to remind everyone to
speak--into---the--microphone.
[Laughter.]
General Microbiology--Ecology, Pathogenicity,
Subtyping, Etc
DR.
NAZAROWEC-WHITE: How to speak into the microphone. Can everybody hear me?
First of
all, I'd like to thank you for inviting me to speak about Enterobacter
sakazakii. This is an organism that I
spent a number of years studying during my doctoral research. And at that time--and I'm talking about the
early '90s, because I went back to school quite late in life--there was very
little information available. And it
would have been wonderful to have had a meeting like this where different
people were presenting different bits of information that they have.
I do
notice, though, that in the last couple of years, you do hear people talking
about Enterobacter sakazakii. I work
for the Canadian Food Inspection Agency in Canada, and we are already in work
planning meetings talking about maybe putting in a monitoring program. Health Canada is planning to include a
method for isolation of Enterobacter sakazakii in its official analytical
methods. CODEX is also talking about
this organism, through its committees on food hygiene, as well as the Committee
on Nutrition and Foods for Special Dietary Uses.
Next
slide, please.
[Slide.]
Now, I'm
just going to briefly touch on a number of things, give you a little bit about
the history of this organism, and what we actually don't know bout the ecology
or the environment that this organism can be found in. I did a bit of a study on the incidence on
the Canadian retail market. I want to
describe some growth studies, looking at generation time and lag time of this
organism in reconstituted infant formula.
I also
did a little bit of work on the phenotypic and genotypic characterization of
the strains that I worked with. And,
finally, a number of people have asked the question--a number of people have
asked a lot of questions this morning--a little bit about the initial work on
pathogenicity and virulence factors. The paper is actually just out in the Journal
of Food Protection, on the pathogenicity.
Next
slide.
[Slide]
Okay. As we know, until 1980, Enterobacter
sakazakii was called "yellow-pigmented cloacae." However, there was a proposed name change in
1980 based on differences between Enterobacter sakazakii and Enterobacter
cloacae, using DNA hybridization studies, biochemical reactions, and pigment
production.
Enterobacter
sakazakii has a biochemical profile very similar to Enterobacter cloacae, but
unlike Enterobacter cloacae, it's always sorbitol negative, and positive for
deoxyribonuclease.
Next.
[Slide]
It think
it's time we saw a picture of this organism that we've been talking about all
morning. And someone this morning has
said that the yellow-pigmented colonies were difficult to identify. Well, the yellow pigment has a stronger hue
when the organism is grown at 15 degrees Celsius, rather than at 36, and often
growth or isolation is done at 36.
The other
thing that we found--and other researchers, as well--that there are two
morphologically different colony types.
This slide illustrates a colony that is mucoid, or dried, and it has a
slightly--well, it doesn't really show the scalloped edges, but it's a
three-dimensional thing. And when you
are trying to touch it with the loop on the actual plate, it's very rubbery,
and it's sort of almost difficult to get off the plate.
It looks
sort of like a raspberry upside down, sitting on the plate.
Next
slide, please.
[Slide.]
Now, the
second colony type is a typical smooth and soft colony. It is easily removed with a wire loop. And what also we found is the colonies--the
rubbery, scalloped kind--revert sometimes to this type of colony on
sub-culturing. At one point in time,
when we first saw this, we were wondering whether differences in virulence or
any other phenotypic characteristics would be different between these two
morphology types.
Next
slide, please.
[Slide.]
Here's a
picture--an electron micrograph--of an individual bacterium. The blue color, of course, has been
added. And, as you know, it is a
motile, gram-negative rod. It grows
very easily on laboratory medium. And
another interesting observation was that after 24 hours of growth, all the
strains that we had produced a large amount of sediment. It appeared that--it was really quite
interesting--and it contained sort of clumped cells and masses that were sort
of stuck together. I have no idea why
that was. But that's something that
could be explored a little further.
Next
slide, please.
[Slide.]
Ahh--the
environment. Little is known about the
environment. There was this one
study--or it was mentioned in a letter to one of the journals--and it was--now
I call him "Mutagens," but I guess it's "Moyt-yens?" Am I pronouncing it correctly? He was the one who showed that it could not
be isolated from surface water, mud rotting wood, bird dung, rodents, domestic
animals--I'm not quite sure which ones they mean--and cows milk.
Now,
aside from the infant formula that we have seen the association there, there is
a paper where sakazakii was isolated from a UHT tetrapack box of milk. I have heard that bottled water--this is
certainly not a scientific study--someone mentioned, "Oh, yes, by the way,
we did find it there"--and raw ground beef. Then there were the studies where they were looking at the actual
illnesses and meningitis, I guess, cases, where they talked about the dish
brush and the blender that the infant formula was reconstituted in.
So this
is certainly an area where research is needed.
When we're looking at the infant formula, I certainly did not look at
any of the specific ingredients that go into the formula. We know that the formula is not sterile, but
is the milk? Does it go
through--sometimes the milk will go through a pasteurization process prior to
being put into the formula, and sometimes not.
And they are--because it's going for further processing--meaning that it
has to go through the powder--to be made into a powder, through the drying, so
we don't know whether it's coming from the milk or other ingredients. Are they sterile?
Next
slide, please.
[Slide.]
At the
beginning of my research, I contacted children's hospitals and general
hospitals across the country to see if their microbiology departments
maintained Enterobacter sakazakii strains in their culture collections. I managed to obtain nine clinical
strains. There was a St. Joseph's
Health Center, affiliated with the University of Western Ontario, they sent me
two isolates. These isolates are from a
one-month-old child that had had meningitis with cerebral abscess
formation. I received another strain
from the Montreal Children's Hospital, and then the Toronto Hospital for Sick
Children sent three specimens. Two
isolates were isolated from cerebrospinal fluid, and one strain from a blood
culture.
Each of
these strains are from different patients, isolated in different years. The Laboratory Center for Disease Control in
Ottawa kindly provided three clinical Enterobacter sakazakii isolates, but they
did not provide any history as to where these strains came from. So these were my clinical strains that I
worked with.
There are
a number of dried infant formula available on the Canadian retail market. We don't have very many actual manufacturers
in Canada. And although there were no
incidents reported in the literature of E. sakazakii meningitis related to
infant formula in Canada, in 1990, two incidents of infection were reported to
Health Canada. In one incident,
analysis of two cans of formula obtained from the home of the one-month-old
infant showed no microbiological contamination. However, the original can was discarded, and therefore could not
be evaluated.
The
second incident involved a neonate in a hospital from a different city. It appears that in this case, there was a
misconception that all powdered formula are sterile. And, again, we don't know whether the less developed intestinal
flora or, as someone this morning described, the totally different flora in
these children that are ill. Even--it
sort of emphasizes that we should be very careful in all the procedures we use
to ensure as low a microbial load as possible.
Samples
of dried infant formula were obtained from the manufacturers or from
retail. And strains of E. sakazakii
were isolated. Five cans, a retail
unit, from five different lots manufactured on five different days were used in
my study.
Next
slide, please.
[Slide.]
This
slide shows the prevalence of E. sakazakii in the formula we evaluated. The incidence varied from zero percent to 12
percent for Company A. And we already
know that there was the study that was in the literature talking about the 141
cans from 35 countries, and they found sakazakii in 14 percent of them.
The
levels of Enterobacter sakazakii were low, at 0.36 coliform-forming units per
can. However, there was no information
on the pathogenicity of this organism, and we wanted to see--learn more about
the growth of sakazakii.
Next
slide, please.
[Slide.]
Farmer et
al., in his initial paper, tested 57 strains for growth. He found that all 57 grew at 25, 36 and 45
degrees Celsius; 50 strains grew at 47, but none grew at 4 or 50. Ten Canadian strains--five clinical and five
food isolates, were selected, and growth was observed over a temperature range
of 4 to 50 degrees.
Temperature
gradient incubator was used. Laboratory
media was spike with a thousand cells, and growth was observed over a 15 to
20-day period.
Next.
[Slide]
This
slide shows the minimum growth temperature for both the clinical and food
isolates. None of the tested strains
grew below 5.5 degrees Celsius. Now this would indicate that Enterobacter
sakazakii would not grow at refrigerator temperatures, which we consider 4
degrees Celsius. However, in looking at
studies of in-home refrigerators, they are well above that 4 degrees
Celsius. They have shown that the
temperature range between 7 to 10 is probably what you're going to find in most
in-home refrigerators. And this, in
turn, could help Enterobacter sakazakii to grow.
And this
morning we were hearing how there was a study that was done which made up the
formula, spiked it, refrigerated overnight, and then put it to hang for four
hours. Well, depending on what
temperature that refrigerator was at, if it's in there for 24 hours, would
allow the sakazakii to grow.
In order
to evaluate growth in reconstituted dried infant formula, three formulae were
selected. These three had the highest
market share on the retail market in Canada at the time. A cocktail of five clinical strains and five
food isolates were evaluated in three different formulae. The formulae were spiked with 103
CFUs, and incubated at three different temperatures: 4 degrees, 10 degrees and
23 degrees. Four was considered the
refrigeration temperature; 10 was considered a slightly abusive temperature;
and 23, room temperature.
Sampling
was done at timed intervals, using direct plating, which provided colony counts
for determination of generation time and lag time. After calculating generation time and lag time, results were
subjected to analysis of variants to calculate and determine if there were any
significant differences.
[Slide]
This
graph shows the generation time. The
dark blue is 10 degrees, and the light blue is 23 degrees. And there was no statistically significant
differences found, either among the tested formulae--in other words, formula 1,
formula 2--or between the clinical isolates and the food isolates.
At 10
degrees, the generation time varied from 4.18 hours to 5.52 hours. Food isolates have a slightly lower
generation time--although, again, it's not statistically significant.
At 23
degrees, the generation time was around 0.67 hours, which translates into 40
minutes.
The data
for 4 degrees Celsius is not shown here.
It was found that it remained at the initial levels that were--that the
formula was spiked with, or declined over time. And, again, this certainly confirms the importance of storing
reconstituted infant formula at the correct temperature.
When
prepared bottles are stored overnight at room temperatures, we looked at the
data and you could get at least 105 CFU per mil, and this is easily
obtained.
Next
slide, please.
[Slide.]
And this
is the lag time. Again, there was no
statistically significant differences among the three formula for both clinical
and food strains. The lag time varied
for from 19 hours--is it backwards?--19 hours for formula in the food, to 47
hours for formula in the clinical.
Again, no significant difference.
And,
again, you can see the generally shorter lag times for the food isolates when
compared to the clinical strains. And this is really just sort of initial work,
and so only using a very few strains.
Next
slide, please.
[Slide.]
Okay--typing
systems, of course, are based on the premise that clonally related isolates
share characteristics by which they can be differentiated from unrelated
isolates. Methods for identification
and discrimination of bacterial isolates have always been divided into two
broad categories: phenotype and genotype.
The traditional micro-techniques, phenotypic typing based on the
secondary characteristics of bacteria, including biochemical reactions,
antibiograms, serotyping, bacteriophage typing.
We used
the API 20 miniaturized biochemical test strip to identify Enterobacter
sakazakii. as we isolated it from the infant formula. And the 18 strains in our studies were grouped into three
biotypes.
Biotype
I, included nine strains, and that includes the type strain. Six strains out of the 18 were inositol-negative,
and three strains were VP-positive.
Farmer, et al., also found that 98 percent of his 57 strains tested to
be VP-positive, and 25 percent were inositol-negative.
Next one.
[Slide]
Somebody
this morning was talking about antibiotics and antibiotic resistence. Again, this is very preliminary work. And, as you know, all clinical laboratories
do antibiotic susceptibility testing routinely. |We used just the disk diffusion
method, using commercial antibiotic disks to determine antibiotic resistence in
the 18 strains. Now, these 11
antibiotics are commonly administered in gram-negative infections, and were
evaluated using the Enterobacter sakazakii isolates that we had.
Included
in this list are the gentamicin and ampicillin which, if you look through those
literature reports that many people were describing this morning, if you look
at the actual antibiotics that were given to some of these infants, the
ampicillin/gentamicin combination is often considered the gold standard. I think it was Robinson that said that.
Next
slide, please.
[Slide.]
Now, what
I'm showing here is the resistence to the certain antibiotics. We found that there was one--it was a food
strain--that was resistant to ampicillin.
The other interesting thing is that there were 17 strains that were
resistant to cephalothin, and there was only one clinical strain that was
susceptible to it. The
sulfisoxazole--all 18 strains showed resistence to this antibiotic.
So when
we looked at the antibiotics, 14 strains fell into Biotype I, and this was
including the type strain. One strain
in Biotype II--antibiogram II--and two strains that fell into the third
category, and one strain was antibiogram IV.
Now,
antibiograms are not a very discriminatory test, and there have been reports in
the literature, by Clark, et al., in 1990, that found this same thing; that the
strains that she tested were categorized into--there were many strains in
certain categories.
The other
interesting thing was if you took colonies that were supposedly clonal on one
plate, if you picked five colonies, sometimes you would get two or three
different antibiotic patterns.
So--don't know what's going on with that, where we're assuming they're
the same organism, where the same antibiogram should come up, it doesn't.
Next
slide, please.
[Slide.]
I'm not
going to talk about the actual methodology here, but I do want to mention that
we did use ribotyping, pulse field gel electrophoresis, and random
amplification of polymorphic DNA. It's
come a long way since the early '90s as to some of the work that I did and what
is being done now.
And the
next slide shows the diversity among those 18 strains. It you look at the ribotyping, it was
grouped into 10 different ribotypes--those 18 strains. In both pulse field electrophoresis, and the
RAPD, it depending on the restriction enzyme that was used in the pulse field,
because you can see with XB-A1, we had 18 different pulsovars, and with SP-E1,
we had 17. And the same thing happened
with the random application for polymorphic DNA. It depending on which primer was used.
Now, the
interesting thing--if you look at the ribotypes--see these three here? All Number III--these are the three isolates
from the one hospital--Toronto sick kids hospital--but they were isolated in three
different years. So, with ribotype,
they fell into the same profile.
And here
we have MNW III, IV and V--these are food isolates. These are all the ones from one company. Interestingly, they also fell into one
category. However, if you go to where
we have only 17 different profiles, again, these two strains--MNW III and MNW
IV, both in the pulse field as well as the RAPD, fell into the same
category. So, it was interesting. And whereas the third isolate from that
particular company did not fall into the same category as it did in the
ribotyping. So, really, what I'm
showing here is the heterogeneity among the strains we looked at. There were only 18 strains, but they all
appear to be genetically heterogeneous.
Okay--the
next slide, please.
[Slide.]
Virulence
factors. Now, I could find nothing that
reported anything with respect to Enterobacter sakazakii. There was one study, where it was a
retrospective in a Danish hospital, that looked at enterobacters--not
particularly Enterobacter sakazakii.
And it stated in that paper that a portal for entry for enterobacter
species, infection can be the gastrointestinal portal.
So we
took that statement and thought, well, we can also find Enterobacter sakazakii
in infant formula and, of course, it goes through the GI tract, and in that
paper, again, it said that they did find that Enterobacter cloacae produced
exotoxins, and E. sakazakii and Enterobacter cloacae are kind of related, so it
was decided to explore a little bit on the pathogenesis or virulence factors of
this organism.
We used a
suckling-mice assay to test for enterotoxin production, and then three cell
culture lines to look and see what the impact was on the morphology of those
cells.
Pathogenicity
was assessed with the use of suckling mice challenged intraperitoneally and
orally, with both the clinical and the food isolates.
Next
slide, please.
[Slide.]
For the
enterotoxin production, we found of the 18 strains, four produced and
enterotoxin. Three were clinical, and
they were all three from three different hospitals, and in three different
geographic regions in Canada. One food
isolate also produced an enterotoxin.
And the type strain was negative.
Next
slide, please.
[Slide.]
As far as
the tissue culture, we only looked at three isolates. There was the type strain, and two clinical strains were
used. We used three cell lines: Y-1,
CHO, and vero cells. Once clinical
strain was toxic to all three cell lines.
On the Y-1 and vero cells, approximately 75 percent of the cells died. On the CHO cells, there was cell shrinking
and vacuation.
We also
boiled the cells to see if there were any differences there, and the boiling
produced the same results on the Y-1 and the Chinese--the CHO cells, however
the boiling decreased the impact on vero cells.
The other
clinical strains--the type strain and one from one of the hospitals in
Montreal--had no effect on all three cell lines. Again, these are very preliminary and initial work. But we wanted to see whether we could, you
know, sort of at least see what was going on.
Next
slide, please.
[Slide.]
I'm going
to talk just a little bit about the infectivity. IP injections were lethal at 108 CFU per mouse for all
strains tested. Death usually occurred
within three days of dosing, and the lowest level causing death by IP injection
was two strains: one food and one clinical.
The clinical one was at 105--both of them were actually
at 105. And the clinical strain also produced an
enterotoxin.
By the
oral route, only two strains caused death: one clinical and one food. These results are the first to be reported
with respect to any virulence factors in Enterobacter sakazakii. As you can see, much work remains to be
done.
The paper
on the infectivity has just been published in this month's issue of Journal of
Food Protection. So details and tables
are in that particular paper.
Next
slide, please.
[Slide.]
Just in
summary, incidence in dried infant formula in the Canadian market was found to
be about 6.7 percent. We've seen values
of 14 percent in the one Dutch study.
Minimum growth temperature is between 5.5 and 8 degrees, and emphasizes
the importance of refrigeration.
Generation
time at room at that temperature of 40 minutes. If we thinking of putting that bottle at the bedside table so
you don't have to get up in the middle of the night, or taking bottles with you
when you're going shopping and keeping it in the stroller--looking at that
time/temperature relationship.
Four of
18 strains produced enterotoxin. The
enterotoxin was not characterized.
Infectious dose--we have some initial results that maybe we could use in
further work--certainly needs to be done in this area.
And when
we're looking at the molecular level, we see that very heterogeneous strains.
Next
slide, please.
[Slide.]
So it was
very interesting working on this organism, because anything I did was new at
the time. I'm glad to see that there is
more interesting in it on the one hand; on the other hand much remains to be
learned about this organism.
And I look
forward to the discussion over the next couple of days. Thank you.
DR.
BUSTA: Thank you very much
This
afternoon, being that we have a fairly tight schedule, I would like to restrict
this to questions of clarification, and then we'll have all the speakers up at
3:50 for questions in general.
Are there
questions for clarification.
VOICE:
[Off mike.]
DR.
BUSTA: The question is: a definition of lag time?
DR.
NAZAROWEC-WHITE: Oh, lag time is the time once we spiked the formula--and, as
you know, bacteria grows sort of in that S-curve way, well, it's that beginning
of that S, before it begins to double, and the doubling is the generation time.
DR.
BUSTA: For my clarification--this is Frank Busta--for my clarification, what
was the shortest lag time? I couldn't
tell that from the--
DR.
NAZAROWEC-WHITE: Oh, I know, because it was very--there was no significant
difference.
DR.
BUSTA: It looked like one of the food ones was very small column, and I was
wondering what the shortest lag time is--for an idea of--now, you were using
cocktails in each case, so that you in fact, were measuring both generation
time and lag time, you're measuring the fastest strain of the five strains.
DR.
NAZAROWEC-WHITE: That's right.
DR.
BUSTA: While you're looking for that, can you handle another question and look
at the same time?
DR.
NAZAROWEC-WHITE: No--lag time--let's see.
I don't have the numbers, but I will be able to supply them for
you. They're in the paper--the actual
numbers. I don't have them.
DR.
BUSTA: All right. Dr. Fischer?
DR.
FISCHER: What was the dose used to kill animals when given orally? The oral dose?
DR.
NAZAROWEC-WHITE: Oh, the oral dose? We did--se started off with, you know, 103,
104, 107.
DR.
FISCHER: What dose killed the animals.
You said it killed two strains--I mean--killed the animals
DR.
NAZAROWEC-WHITE: Oh, I'm sorry--107. Yes. Orally. And it was the two strains.
DR.
BUSTA: Any other questions for clarification?
[No
response.]
DR.
BUSTA: All right. Thank you very much.
The next
presentation will be by Dr. Burr; Microbial Detection--Clinical and Food--from
the FDA.
Microbial
Detection--Clinical and Food
DR. BURR:
I want to thank you for inviting me here today. One of the nice things about going at this time of the afternoon
is that essentially everyone's covered just about everything I'm about to
say. So it makes it kind of nice. But you're not going to learn very many new
things; hopefully a few pictures will come in.
But most of the stuff that I'll talk about has been briefly touched upon
already.
I've been
given the assignment of discussing the microbial detection of Enterobacter
sakazakii, both in food and clinical.
And--
Can I
have the next slide?
[Slide]
--what
I'd like to do is provide a summary of the methods that are available for
isolating and quantifying levels of E. sakazakii in food and clinical
samples. But I am not planning on
covering everything that is in the White Papers. I'll try to go over the high points of the methods. And as you'll see in your papers, there's a
bit more information that's provided.
Next
slide.
[Slide]
In terms
of presentation, I'd like to divide it into two aspects: one, talk a little bit
about the initial isolation reports of E. sakazakii, and then turn our
attention more to the development of the quantitative methods for E. sak.
Initially,
the first isolation was in 1980. And,
again, you've heard about all this from previous speakers. 1983--I think we'll go now to just Dr. M--at
this point--
[Laughter.]
--and in
1984, we had Dr. Postupa and Aldova.
Next
slide.
[Slide]
The
initial isolation, or the initial description of E. sakazakii being associated
with a can of dried milk came in Farmer's initial description of E. sak as a
new species, and that was in 1980. But
like the other papers that will talk about the initial ones, there's no real
isolation detailed. Okay? They talk about it's just being an isolate,
but they don't describe how they got it, where it really came from, or what it
took to get it.
The next
slide, again, is Dr. Muytjens in 1983, and this is the study that Karl and
Maria talked about, where eight cases of neonatal meningitis associated with E.
sakazakii. They isolated the organism
several times, as has been pointed out, from the prepared formula, but never
from either the powdered formula itself, or the water used in preparing the
formula. Again, no information was
reported on the quantity of powdered formula analyzed. However, as Karl has pointed out this
morning, we have had several conversations or e-mails, and we have been
informed that the quantity analyzed in this 1983 paper was the 10 gram sample.
Next
slide.
[Slide]
In 1984,
Postupa and Aldova--again, I apologize if I've done something to the names
here--described four strains of E. sakazakii from powdered milk, two strains
from powdered milk infant formula. The
only details provided was that it was isolated on deoxycholate-citrate agar,
incubated at 37 degrees for 48 hours, but no details on anything in terms of
the quantity analyzed, again.
Okay, and
now I'd like to turn attention to the development of the quantitative methods,
and essentially we'll deal with three methods, bringing us up to the current
one that the FDA is using. And sort of
for ease of discussion, we'll call the first one the "European," then
we'll move to the "Canadian" and then we'll move to the FDA method.
Each one
of these--the '97 and the FDA method--is essentially a minor modification of
the 1988 paper. Sample size,
sensitivity remains the same and, for the most part, the change has been in the
ease of being able to do the method.
As we
spoke about, this method was first described in 1988. And in referring back to their 1983 paper, they commented that
although it was not cultured from the formula powder itself, this might have
been due to an unequal distribution in the powder, or it was present in such a
low concentration that it escaped detection by conventional methods. And for a long time we were sort of hindered
by, you know, what exactly does "conventional methods" mean? And that sort of put us on trying to get in
touch with Dr. Muytjens as quickly as we could, and that's when we got the
information about the 10 gram sample.
So, based
on their early results, in the 1988 work they decided to culture a large
quantity of breast milk substitutes for the presence of all Enterobacteriaceae,
including E. sakazakii.
Next
slide.
[Slide]
So
essentially what their method is--and a method paper is probably not the best
thing after lunch--but hopefully we'll go through it. In triplicate, they mixed 100, 10 and 1 gram samples with 900, 90
and 9 mils, respectively, of buffered peptone water, 45 degrees, until
completely dissolved, and this gets incubated overnight at 36 degrees. From these overnight cultures, you
innoculate 10 mils from each of the flasks into 90 mils of Enterobacteriaceae
enrichment broth--EE broth--and, again, this is incubated overnight at 36
degrees.
This
first incubation is non-selective, and this is the first area where you get an
enrichment broth which has some selectivity to it. In duplicate, innoculate from these cultures in duplicate;
innoculate 1 mil from each of the enrichment broths into 20 mils of fluid,
violet-red bioglucose agar. And again
incubate this overnight at 36 degrees C.
Suspect
colonies sub-cultured to sheep blood and eosin-methylene blue agar, and the
strains were identified via the API 20-E system. And, as I said, this initial paper was for all of the
Enterobacteriaceae, so they did additional testing for E. sakazakii by looking
at the production of yellow colonies on nutrient agar after 48 hours at 25
degrees, production of extracellular DN-ase. And a positive alkaglucocytase
reaction.
Next
slide.
[Slide]
For those
of you that are not familiar with what an API system is--and I know Maria
talked a little bit about it--API system is a rapid identification process,
where essentially you have plastic strips that have 20 small wells consisting
of dehydrated media. You then add your
bacteria suspended in saline to each of the wells, and then incubate for 16 to
24 hours, and then look at the color reaction.
So, essentially, you have each one of these wells is an individual
biochemical reaction. And so after
incubation, you then end up with--and this is a series of four different
bacteria--four different examples--of what you see following 24, or 16 to 24
hour incubation.
Next
slide.
[Slide]
What you
then do is, based on the color of each of the reactions, it will be scored as
either a plus or a minus and so on. So
you very easily just score.
Next
slide.
[Slide]
This is
just a typical reaction of what E. sakazakii would look like in an API strip.
Next
slide.
[Slide]
So then
what happens is that you take your score here, and you transform the
biochemical reaction; so you had a plus here, a plus reaction, a negative
reaction. You then transform that into
a numerical profile. And that numerical
profile you then just take to either a computer program that they provide, or a
book that they provide, and that then comes up with an identification of E.
sakazakii. And you have different
levels, based on the variation; you have good identification, you have
excellent identification; good identification, acceptable.
Now,
sometimes this numerical profile is not considered sufficient enough to be able
to identify the organism, so API then recommends additional testing. And for E. sakazakii, generally the
additional test would be testing for the yellow pigmentation, which API has,
and I think the Journal of Clinical Micro has listed as 98 percent of the
isolates are yellow--produce yellow pigmentation.
So this
is basically what you end up with after doing an API.
Next
slide.
[Slide]
Now, in
terms of the levels of E. sakazakii in the sample, in this method and, again,
in all the later ones that we're going to talk about, the actual level is
determined by the most-probable-number procedure.
Next
slide.
[Slide]
And I
don't want to go into a lot of detail, because I'll confuse myself quicker than
I'll confuse you, but it's a statistical method, and it assumes that the
bacteria are separate, and the conditions of incubation such that every
inoculum that contains even one viable organism will produce detectible
growth. And it's based on the number of
positive samples from each of the series of triplicate cultures of the three
inoculation levels. So essentially what
you're doing is you're scoring the number of positives in the three 100 gram
samples, in the three 10 gram, and in the three 1 gram. And the tables that you go--the standardized
table--and I'll show that in a minute--provides you an MPN number--most
probable number--plus a 95 percent confidence interval.
Next
slide.
[Slide]
This is a
typical MPN table. And I think this is
actually taken out of the BAM manual, and this is for a three-tube at an
inoculation of .1, .01 and .001 gram.
And what it give you--as I said, it gives you an MPN per gram in the 95
confidence level. So, essentially, you
look here and you're saying how may tubes of the three were inoculated with the
.1 gram are positive? So here you have
one positive, zero positive, and this gives you an MPN number of 3.6 MPNs per
gram, and then here is your 95 confidence level.
Now, down
here it describes how we will go from this standardized table to go into an E.
sak of an MPN per hundred gram. And
essentially what you do is you take the MPN per gram from this table, you
divide by 1,000 to adjust for the larger sample size. So that gets you down to these sort of values here. And that would be giving you an MPN per
gram. And then you simply multiply 100
to obtain per 100 gram.
So, in
this case, if you had 1.00, you would have .36 MPNs per hundred grams.
Next
slide.
[Slide]
The
results of the European survey--and these, I think, have been talked about I
think twice already, maybe three times.
But essentially, from the 35 countries, 141 powdered formula samples
were analyzed. E. sak was isolated from 20 samples, from 13 of the countries. And the levels recovered using the MPN was
.36 to 66 colony forming units--or CFUs--per 100 gram.
And the
lowest level of detection reported in this method was 0.36 CFU per 100 grams.
Now, on
the next slide I want to show a little bit about where that comes from here,
and I want to distinguish sort of a 0.36 per 100 gram detection limit, versus a
0.31 per 100 gram--actually, the lowest possible limit.
[Slide]
If you
take this chart, in order to get this .36 per 100, that's essentially you have
one tube out of the--of the largest sample.
So one of the 100-gram sample is positive, and then the 10 gram and the
1 gram are both negative.
It's
theoretically possible that you could have zero out of three in the 100 gram,
zero out of three in the 10 gram, and one out of three in the 1 gram, giving
you the lowest possible level of a .3 per 100 grams. But most of the papers that reported, this is the number--where
they're getting that one particular number that it's coming from. Okay?
Next
slide.
[Slide]
In 1997,
Maria--as she discussed--and this we'll call the Canadian method--made
modifications of this initial method.
And, essentially, they just--the dried infant formula was suspended in
sterile water. Suspect colonies from
the VRBG plate sub-cultured to TSBYE agar.
And, again, they're just very minor modifications. And as in the previous study, the API 20 was
used for confirmation, and no additional biochemicals were used. The levels were again determined by MPN, and
again you're left with the same sensitivity: .36 per 100 grams.
The
results of this study: E. sak was isolated from eight of 120 cans; this
representing five different manufacturers.
And, again, the level reported is the .36
Next
slide.
[Slide]
Now, in
2002, the FDA was charged with developing its method. And rather than reinventing anything took from the Canadian
method and, again, just made some minor modifications. And what we did was went to direct spreading
or streaking over the overnight EE broth, rather than the pour plates. The pour plates are a bit laborious in order
to set up, so this was a much easier method of getting the isolated colonies.
Five
presumptive colonies are sub-cultured
to tryptikase soy agar, and incubated at 25 degrees C for 48 and 72 hours. And here, again, at the lower temperature
where we're looking for only yellow-pigmented colonies from the TSA plates are
then further worked up in the API 20 system.
Like the Canadian method, we do not do any additional biochemical tests,
and we just rely on the results from the API 20.
The level
of detection by MPN, again we report as the most likely one as the .36 per 100
gram. And we can detect levels of E.
sak much lower than recommended--by the recommended FAO level of 3 CFU per gram
of powdered infant formula.
These are
just some pictures that have been provided from Sharon Mallow who was c--author
in producing this method. This just
shows the mixture of the powdered milk in the flask.
Next
slide.
[Slide]
These are
an example of the typical colonies of E. sak on the VBGP. And, again, you're looking for the purest
purple colony, surrounded by a purple halo of precipitated bio-acids.
Next
slide.
[Slide]
And this
is just the description of the yellow pigmentation that you get at room
temperature.
Next
slide.
[Slide]
Just
briefly want to touch on clinical isolation, and this was one of the questions
was brought up earlier this morning. E.
sakazakii is isolated from clinical samples using standard methods for the
isolation of Enterobacteriaceae.
There's no special media has been developed for E. sak, and it grows well
on all the standard media that is used in clinical laboratories. And for the most part, when you read the
papers, they don't even describe the method.
They just say "cultured from blood," "cultured from
spinal fluid." And in talking to
several friends who do clinical microbiology, again their comment is you just
follow the normal routine for blood cultures, and that there's nothing
different, nothing unique.
You can
confirm with either API 20, as we've done, or there's an enterotube 2
system. But, again, it just follows the
standard methods for identification.
And essentially because you're dealing for the most part with supposedly
sterile sites, again much easier identification process.
So then,
finally, and just in conclusion then, the procedures E. sakazakii from powdered
formula and clinical samples: follow the standard microbiological methods for
the isolation of other members of the family Enterobacteriaceae. Normally, sterile clinical samples pose no major
problems for isolating E. sak.
Because
of the very low levels of E. sakazakii in powdered formula samples and its
non-random distribution in the powder, larger quantities and sub-samples should
be cultured for isolation. And in both
clinical and food microbiology laboratories, appropriate incubation times and
temperatures should be applied if the diagnostic tests were precise. And, again, this appropriate temperature
would be going down to the lower temperature for pigment production.
I think
that's the last slide.
So,
hopefully that--as I said, a lot of the information was covered in some of the
earlier talks, but hopefully this gives it a bit more of a clear picture of
what exactly is done to get it out of these food samples.
Thank
you.
DR.
BUSTA: Questions--preferably for clarification.
Dr.
Blumberg.
DR.
BLUMBERG: Yes--Henry Blumberg.
Do you
know--generally in the clinical micro lab they're not using an API strip to
identify gram-negative bacteria, and most of the identification of
susceptibility is done by automated methods like Vitec or Microscan--things
like that.
And do
you know how well those identify Enterobacter sakazakii, versus, you know,
other enterobacter species, like cloacae or things like that?
DR. BURR:
I don't, actually, because the only thing that I got from the two friends that
I had was just that, "We just do the standard way." So I'm assuming that it's easy, and that the
automated ones do pick it up, but I don't have the experience with those.
DR.
BUSTA: Other questions?
Dr.
Baker.
DR.
BAKER: Baker. This morning people
talked about difficulty picking out these organisms among a whole lot of other
organisms. Your slide certainly didn't
show that. Is that a problem or not?
DR. BURR:
Well, in this only, hopefully it's the combination of the EE broth plus the
selective plate that actually is cutting down on whatever else is in
there. So we're really trying to do
this--the selectivity of the method removes a lot of the other bacteria.
DR.
BUSTA: Other questions?
Dr.
Fuller.
DR.
FULLER: I apologize for what may be a very stupid question. When you started with the European method, I
think it said triplicate runs using 100, 10 and 1 gram. Again--pardon what may be a pretty stupid question.
Are we
talking about--
DR. BURR:
If I can't answer it, you're really setting me up here, now.
[Laughter.]
DR.
FULLER: Well, I'm just asking--is this from a single can, or are these nine
different--you know, is this for a lot?
What are we sampling?
DR. BURR:
It's for--you take--out of one can you would take--
DR. FULLER:
Each of those.
DR.
BURR: --for each of those.
DR.
FULLER: Okay.
DR. BURR:
So you would take a total of 333 grams out of one can--
DR.
FULLER: One can. Okay.
DR.
BURR: --and do it in the separate
things. Oh, that was a good one.
[Laughter.]
DR.
BUSTA: Dr. Acholonu.
DR.
ACHOLONU: You said that there is no special medium or media for isolating E.
sak. Is there any special need for
developing one, since you have indicated that it grows in different kinds of
media?
DR. BURR:
It doesn't appear that there is a need for one. As I said, for the clinical, it will grow on any of the media
that's used for enterics. And so
there's been no need to actually get one that's just specific for E. sakazakii.
DR.
BUSTA: Dr. Tompkin.
DR.
TOMPKIN: Yes--Tompkin.
Due
to the low prevalence of this organism
in the product--you make a statement--or a statement is made that it is
"non-randomly distributed."
How can you differentiate low prevalence and just failure to detect it,
versus whether, in fact, it is non-randomly distributed. Does this have to do--did you actually go
into a large quantity of a given lot and try to determine random distribution,
or is this just an assumption.
DR. BURR:
It's just a generic way of--
DR.
TOMPKIN: It's an assumption.
DR. BURR:
Right.
DR.
TOMPKIN: Okay.
DR.
BUSTA: I have a question--this is Busta.
When
you're doing a normal Enterobacteriaceae enrichment, and then you go on to an
Enterobacteriaceae agar, what portion of the colonies that you're selecting
would be sakazakii? What portion of the
colonies? Do you run all colonies from
that enrichment onto the API?
DR. BURR:
No, as you--you're just taking--when you go to that second plate--okay--when
you're going from the broth to the VRBG plate, then you're taking--you're looking
for the purple colonies with the haze around them. So, again, you're picking what you think are going to be those
colonies. Okay? So you're looking for typical colonies of E.
sak on the selective plate.
DR.
BUSTA: Dr. Tarr.
DR. TARR:
I have two questions that straddle the question and clarification border.
First,
when you state your sensitivity is 3 or 3.6 colony-forming units per gram--
DR. BURR:
100 grams.
DR.
TARR: --per 100 gram--do you ever
normalize that to the ambient flora? I
can imagine one E. sakazakii in a kilogram being detected if there's nothing
else there when you amplify it as if it were coming our of a blood
culture. And I can imagine many E. saks
being swamped by ambient flora in the grow-up stage.
That's my
first question.
DR. BURR:
I'm not sure if I--again, I'm not sure if I get that question.
DR. TARR:
Because, again, you're growing it up--
DR. BURR:
Remember, you're looking for just growth in that one particular tube. So you're not looking for any particular
level within that tube when you're doing the MPN procedure.
DR. TARR:
But you have a subsequent step when you have a sample--the sampling comes out
on your plates, looking for the yellow colonies. If you have many additional organisms there, you might miss the low
frequency E. saks. If you have few
other organisms there, you might get them.
DR. BURR:
Right. But again--
DR. TARR:
It's a variable that's not--is that taken into account?
DR. BURR:
Right. Remember you're doing that--the
hundred grams is first going in to a non-selective, and then you've got
selective. So you've got two, really,
grow-outs--
DR. TARR:
Right.
DR.
BURR: --that are occurring before
you're going to any plates. So that's
supposedly going to bring up the levels of any low levels of bacteria, to bring
it up to where you've got lots in those cultures. So, essentially, you've got 48 hours' growth is what you're
actually putting on plates.
DR. TARR:
But that will be limited by the other organisms in there, won't it?
DR. BURR:
And everything should grow. In at least
the first one, you're essentially allowing everything to grow. That initial one is non-selective. Everything is going to grow out there. And then when you take the smaller portion
of that to the enrichment, then you're more selective for the E. sak colony.
DR. TARR:
Okay. And that laterals into my second
question.
Have you
or anyone else explored the thermal tolerance of this organism, as an initial
selective step, where the first incubation is perhaps done in 45 degrees--
DR. BURR:
We have not net--
DR.
TARR: --to give competing flora a
penalty/
DR. BURR:
Yes--we have not yet.
DR.
BUSTA: Thank you very much.
To
continue on, with Dr. Buchanan, talking on resistence--thermal and otherwise.
Resistence--Thermal
and Other
DR.
BUCHANAN: Thank you. Okay. I've got a microphone and I'm armed.
[Laughter.]
What I'd
like to do today is talk mostly about thermal resistence of Enterobacter
sakazakii. I have a few additional
items that I want to mention in passing; one that may help address one of the
questions that came up earlier today.
And in talking about resistence, I'm talking now about resistence to the
type of things that this organism might encounter during its manufacture or
distribution. I'm not going to be talking
about antibiotic resistence, which was covered earlier.
Next
slide.
[Slide.]
And I'm
going to break this up into the--a quick look at a number of studies that have
been done, first at a laboratory level, and then at a pilot plant level. I am going to be focusing a great deal on
the latter study, that has been completed.
It has been submitted for publication, but will be relatively new data
to the members of the committee.
We have
had two studies done--laboratory trials.
They were done by Maria, who talked to you earlier. And then Sharon and I did one using a
somewhat different technique. And just
so you have an idea of what are covered in these, Maria's used a stainless
steel tube and a constant water bath as the heating system. It used five pooled food strains and five
pooled clinical strains, in separate trials.
And in that kind of a study, the most resistant organism is what comes
out at the end.
We used a
submerged coil apparatus .and 12 individual strains. A submerged coil apparatus is a fairly sophisticated instrument
for determining thermal resistence, allowing holding times in factions of a
second.
Next
slide.
[Slide.]
Before
going on, I'm going to be mentioning a couple of terms. And, knowing that this is a mixed group, for
the food microbiologists, I'm sorry to bore you--the others, two terms that I
will be using: "d-value," is the time at any given temperature that
would be needed to reduce a microbial population by 90 percent. And it's a common term that's used for
measuring the relative thermal resistance of an organism.
A second
term that's used commonly is referred to as the "z-value." This is a value that--what change in
temperature, what increase in temperature, you would have to have in the
processing in order the decrease the d-value by 90 percent.
So there
are two ways of describing the time-temperature relationship of the organism's
resistence.
Next
slide.
[Slide.]
And,
typically, when we do a thermal resistance curve what we do is we do a survival
curve. These are two examples. They cover the extremes that we saw with 12
individual strains. The organism on
that has a d of 591 is the most resistant strain that we ran into. And the one on the left, with a d of 30.5,
is the least resistant strain. And the
d-value is simply the slope of those inactivation curves. That's what we're really measuring in this
case.
So those
are what I'll be referring to as I go along.
Next
slide.
[Slide.]
We looked
at the 12 individual strains and they fell into two distinct groups as we
examined them. They fell--half of them
fell in the group there on the far left.
Those six strains had d-values actually less than 35 seconds. So they were very sensitive. The group on the right all had d-values
greater than 300. They range from 320
up to about 590. And so we found two
distinct populations--in itself is very interesting.
Next
slide.
[Slide.]
Trying to
put this in perspective so that you can sort of judge this against other
pathogens of a similar nature, we did have some exact data that was generated
under the same exact conditions, and so I plotted these so you can get a
relative idea. The Enterobacter
sakazakii 607 was the most resistant strain we ran into. And, like I said, it had a d-value of around
590. So it bracketed at one end. And the Enterobacter sakazakii 51329 was
very heat sensitive and it bracketed the others. The other strains that are of note here: this is E. coli 0157,
Klebsiella pneumoniae, Salmonella Hartford, which is a relatively heat
resistant salmonella; a biotype-1 E. coli, and Enterobacter aerogenes. And then this, here, is the pooled strains
from Maria's study. So, again, it
covers a wide range and sort of behaves like other Enterobacteriaceae do. They, themselves, often have a distribution of
thermal resistences.
Next
slide.
[Slide.]
Now, this
is the--when you do a thermal resistence determination, and you do it at
different temperatures, and then you plot the d-values you calculate on a log
scale. Typically, if it follows the
rules that everyone says it's supposed to follow, it winds up producing a
straight line.
Well,
Enterobacter sakazakii--this is strain 607--did a very nice job of following
the rules. It's just--I've never--
[Laughter.]
--I
couldn't draw a better line if I did it on purpose. Each of these points represent at least three independent
trials. I mean, it just fell out
beautifully. So this is--its d-value's
over a--almost a 15-degree range.
If you
then--well, you're not supposed to--
Can I get
the next one? Next slide? Oh, that's this one. Okay.
Just to
show you that the gods do shine down on you sometimes when you're doing
experimental work, when Maria did her pooled food isolates, and pooled clinical
isolates, She found that there were no significant differences between the two
groups, and that they had an overall d-value of 5.8 degrees Celsius. And when we did it by an entirely different
method four years later we came out with 5.6.
I mean, you can't get much closer than that.
Next
slide.
[Slide.]
Going back
to the z-value--and while you're not supposed to extrapolate z-values past your
experimental ends, we did anyway as a way of predicting how the organism should
behave when you go up to even higher temperatures. And I might note going above higher temperatures in the apparatus
we used is virtually impossible, because you're down at fractions of a
second. So we did just simply
extrapolate this starting at the 70, which was our upper end, and it basically
suggested that when you get above the temperature of 70, inactivation becomes
almost instantaneous.
But
following the linear relationship, for example, to get a 5-d
inactivation--which is something we typically would shoot for with these kinds
of treatments--a pasteurization-type treatment--you can see that by the time
you're up to 80 degrees, to get a 5-d kill you should be, at this point, at
about .7 seconds. And if you go up even
higher, you see it becomes literally a small fraction of a second to get that
kind of inactivation.
So one of
the things we wanted to check out was whether or not this actually occurs in
reality, when you're dealing with an infant formula, and so we designed a
simple experiment where what we did is we took a commercial dried infant
formula. We inoculated it with a
concentrated culture of Enterobacter sakazakii 607, which was, again, our most
thermally resistant isolate. We did it
under a condition where we maintained the dry nature of the product. We pelleted a colony--basically, we pelleted
about 1015 cells and then distributed it throughout a large volume
of formula, so we had a dry formula with approximately a million cells per
gram--or would produce a million cells per gram when it was rehydrated.
We then
distributed that inoculated formula into standard plastic baby bottles,
following the manufacturer's instruction on how to weigh it out, with the
appropriate volume. We then added
preheated water to those bottles; capped it, agitated the bottles--the
formula--as one would normally do, shaking it periodically; and we allowed it
to stand for 10 minutes, and then we sampled the bottle and determined the
remaining cells to see how much inactivation we got.
Next
slide.
[Slide.]
Just to
show you what the heating looks like, we did a series of temperatures in
approximately 10 degree Celsius ranges, starting at boiling water and then
going down to 50 degrees Celsius. And
then our control was just simply room temperature water that was added to it.
Next
slide.
[Slide.]
So,
basically, we have the control is on the far left. This is room temperature water added to the formula. You can see that when we added them back we
had approximately a million cells per gram.
And at 50 we got a very small degree of inactivation. At 60 we got approximately a little over one
log cycle inactivation. And then, as
predicted by our original studies in model systems, at temperatures at 70
above, we got greater than a four log kill.
We basically got below the limit of detection that we could use. We didn't try to go into an NPN at this point,
but certainly we got a very substantial inactivation.
Next
slide.
[Slide.]
We also
wanted to have some information on what would be the impact of this type of
heating on nutrient content. And so we
did have the same procedure done by our nutrition analysis center in
Atlanta. They basically did the same
procedure s we did, except they only used boiling water as the single
temperature they examined. They did
that because that would have been the worst case in the scenario we set
up. We did the analysis in triplicate,
using four different commercial infant formulas. And I might note that--I'm going to have to explain how the
results are expressed, but they were all normalized against units per hundred
calories, depending on what nutrient we're analyzing.
Next
slide.
[Slide.]
Now, this
is a very busy series of slides, but I do want to point out what you should be
looking for. This is the nutrient being
analyzed. This was the four commercial formulas being analyzed. Within each of the boxes there are four
values. This is the value on the
label. This is the value that was found
when the product that was rehydrated with room temperature water was analyzed. This is the value that was found when it was
rehydrated with boiling water. And this
is the change between this and this.
So you're
basically looking for two things when you're looking; you're looking for the
differential between these two and then you're looking for whether or not what
was remaining, versus what was on the label.
So, in this case, the addition of boiling water produced a 3.7 percent
decline in the nutrient, but it's still well in excess of the reported value on
the label.
We've
done this now for a series of them, and basically here very little change in
vitamin A, very little change in vitamin D, E, K, thiamin.
Next
slide.
[Slide.]
Riboflavin--again,
you know, we see some fluctuation plus or minus between the two groups, but
basically little change; little change would be 12. Niacin--again, little change; folic acid, little change.
Next
slide.
[Slide.]
Pantothenic
acid--actually, a small increase pretty much across the board. Biotin--little change. The only nutrient we found that was a
significant decrease, in terms of the concentration across the board was with
vitamin C. This would be expected. This is the most thermally sensitive of the
nutrients. In some cases it did fall
below the label value. In other cases,
even though there was a decrease, it was still above what was indicated on the
label.
So,
overall, with the exception of vitamin C, the addition of boiling water had
really not much impact on nutrient content.
Next
slide.
[Slide.]
There
have been only, as far as we can tell, only one study that was done at a pilot
plant level on Enterobacter sakazakii.
This was done by Maria also. It
used pooled strains, we think--and, Maria, if this is a question you can help
answer that. It was unclear. And it was done with an HTST pasteurizer.
Next
slide.
[Slide.]
It was
done in a manner that bracketed the different times and temperatures associated
with pasteurization. And I've tried to
take what was a great deal of information and reduce it down to a single
table. But basically, this value you
see here is the percent surviving population of the initial inoculum. So you can see here with a three-second
heating at 63 degrees C you got about a 60 percent reduction, down to, you
know, several log cycles when you get down here--and that it follows pretty
closely what we would expect in normal time-temperature relationships. That is, it's not a very heat sensitive
organism, and normal pasteurization would certainly knock it out.
Next
slide.
[Slide.]
Okay. Let's switch to other treatments or
resistences. And when we're talking
about Enterobacteriaceae as general--and it's important to keep in the back of
your mind that Enterobacter sakazakii is a normal Enterobacteriaceae. It is one of the fecal coliforms that we
have a great deal of experience with in general terms.
Next
slide.
[Slide.]
and we
know, in general Enterobacteriaceae are not heat resistant. They can be moderately acid resistant,
particularly if they have been pre-adapted.
Conversely, they can become moderately alkaline resistant if
pre-adapted. They have low to moderate
chlorine resistence; low to moderate irradiation resistence. They will remain viable in refrigerated and
frozen products for extended periods, particularly if the pH is neutral. And then, finally, they have moderate to
good resistence to drying.
Next
slide.
[Slide.]
Now, when
you get specifically to Enterobacter sakazakii there is virtually no
information available in the literature specifically related, and there's very
little when you start looking at the distributions of resistences the way we
did with the 12 individual strains.
Just
summarizing very quickly what we found, the isolation of it from a variety of
dried foods indicates that it's probably resistant to drying. And I'll come back to that. We did find one paper where it tends to be
found after treating seeds that are going to be used for sprouting with
chlorine. This is one of the organisms
that remained on the dried seeds, and it would be indicative of at least having
moderate resistence to chlorine, though this would have to be confirmed. And, just an odd thing, it seems to be
pretty sensitive to kitazans, which is used as an inhibitor.
Next
slide.
[Slide.]
The one
additional piece of experimental information that we do have and can share with
you is this slide--when we prepped up the samples--the
dried infant formula samples that we used for the
baby bottle experiment I showed you--we did keep the leftover formula, and we
have been periodically sampling it over the course of almost a year now. And this is the survival in that infant
formula.
Again, we
started with about a million per gram upon rehydration, and then we've been
following it over the course of nine months.
There was an initial die-off in the first four months of about two log
cycles--two-and-a-half log cycles.
Since that time, it has basically maintained itself as a steady-state
level. We anticipate if its behavior is
like most other Enterobacteriaceae that this value will change little over the
next year or two. And it's worth noting
for any of you that follow archeology, they have opened up tombs in Egypt that
have been basically sealed for 10,000 years, and they have found fecal
coliforms. So, in a very arid and dry
environment, don't expect that it's going to disappear. It will probably hang around for at least
the shelf life of an infant formula.
And I
think that's the last slide.
Summary--this
is not a particularly heat resistant organism.
There seems to be a substantial diversity in the thermal resistence
among the strains, and this seems to be in keeping with the substantial genetic
diversity that you've heard discussed in other attributes today.
There's a
good agreement among the studies that have been done. There's been a limited number of studies but, I mean, they've
matched up, despite the fact that they were done in different ways, the
agreement is great.
Inactivation
at temperatures above 70 degrees occurs almost instantaneously, and certainly
within a few seconds. And then specific
information on the organism's resistence to other factors or stresses it might
see in a food-processing type environment, or food preparation environment are
generally lacking, though I would say that every indication is that it will
survive dehydration for extended periods.
Thank
you.
DR.
BUSTA: Thank you, Dr. Buchanan.
Are there
questions for clarification?
DR.
FULLER: We asked it here, and I just
want to make sure. On your resistence to dehydration slide, that was stored at
room temperature?
DR.
BUCHANAN: Yes.
DR.
FULLER: Is that a correct assumption?
DR.
BUCHANAN: Yes, that's sitting on a lab bench in the lab. It's sitting at room temperature.
DR.
FULLER: Right.
DR.
BUCHANAN: It would probably show it down if we refrigerated it, but this is--we
tried to mimic conditions that would occur in the home.
DR.
BUSTA: Dr. Moyer-Mileur.
DR.
MOYER-MILEUR: Moyer-Mileur.
I have a
question concerning your results and how they relate to the USDA letter that
went out to health professionals where it advised not using boiling water to
reconstitute powdered formula, one, because of loss of heat sensitive
nutrients, which your results would say is not the case, except for vitamin C,
as well as the inability to assure adequate destruction of E. sakazakii.
From your
findings, would you say that those are no longer concerns?
DR.
BUCHANAN: What I would suggest is when that original was put out, there was a
lack of data upon which we could evaluate the efficacy of that treatment. Based on that, these experiments were
done. I would say that the results of
these suggests that one, nutrient loss does not appear to be an issue--or not a
substantial issue, and that at temperatures above 70, it is a viable means of
reducing the level that may occur within a dried infant formula.
DR.
MOYER-MILEUR: Okay. Thank you.
DR.
BUCHANAN: The only issue that was not there is that we still have not completed
studies on the extent of clumping that might occur. Those are being evaluated.
Our problem right now is we don't have a standard for what
"clumping" means for actually measuring clumping. We have empirical observations at this point
that it does not appear to be a major factor, at least in most formulations,
but we don't have an objective measure of the extent of clumping.
DR.
BUSTA: Dr. Acholonu.
DR.
ACHOLONU: Just for curiosity--Alex Acholonu--I saw a slide--I think it was an
electromicroscope slide by one of the previous presenters. And then I've seen a slide that you showed
of E. sak. Some look like they have--
DR.
BUCHANAN: By the way, that wasn't E sak.
Actually, that was E. coli.
DR.
ACHOLONU: Oh, I see. Okay.
DR.
BUCHANAN: Okay? That was just a general
Enterobacteriaceae.
DR.
ACHOLONU: Okay. Well, anyway, my
question still is: do the E. sak organisms have flagella? Are they flagellated?
DR.
BUCHANAN: Mm-hmm. Yes.
DR.
ACHOLONU: Okay. That's what I wanted to
find out.
Also, are
they supposed to be facultative anaerobes, or just anaerobes?
DR.
BUCHANAN: They're facultative anaerobes.
They grow best in the presence of oxygen; can grow quite nicely in the
absence of it, though.
DR. ACHOLONU:
Thank you.
DR.
BUCHANAN: They have all the normal characteristics of an enterobacter.
DR.
BUSTA: I have a question, Bob.
How did
you inoculate the infant formula with that 106?
DR.
BUCHANAN: How did we--okay. What we did
is we grew up a very large culture of Enterobacter sakazakii. We spun it down into the centrifuge. We took it up into a small volume of
water. We then added it, dropwise, to
the infant formula that was in a--I think it was a four-liter flask, as I
remember. After adding a drop, we would
then shake the material to get it transferred as beset we could. We then continued that process until we
added the entire inoculum to make sure that we had a reasonably distributed
material. We had added a small amount
of a blue dye to the inoculum so that we could see how the color was
distributed throughout. And while I
can't say that it was homogeneously distributed, it had to be pretty close,
because we got a nice flat color across the entire lot.
DR.
BUSTA: Still could have been clumps, though.
DR.
BUCHANAN: There could have been very small clumps, but we got it distributed as
beset we could. And it's interesting to
note that as we pulled samples out over time, the amount of fluctuation or
differences between the levels we saw was very, very small.
DR.
NEILL: Dr. Fischer?
DR.
FISCHER: You did this experiment where you inoculated infant formula and then
looked at the rate of killing?
DR.
BUCHANAN: Mm-hmm.
DR.
FISCHER: The strain that you used, was that the most resistant--thermally resistant
strain--or-
DR.
BUCHANAN: Yes.
DR.
FISCHER: It was.
DR.
BUCHANAN: Yes--607 has been consistently the most resistant strain we have in
our collection now. And that was the
strain we used for all subsequent experimentation.
DR.
BUSTA: Dr. Tompkin.
DR.
TOMPKIN: Yes. Tompkin.
Do you
have any idea as to the code date and the freshness of the four formulas that
were used for the vitamin studies?
DR.
BUCHANAN: No, but I can get that information, if it would be helpful to the
committee. We have the details of all
of that. I just didn't try to put it
all on one slide.
DR.
BUSTA: Dr. Kuzminski?
DR.
KUZMINSKI: Kuzminski.
If you'd
had clumps, though, wouldn't your death-time curves have reflected the presence
of clumps by the changing slopes?
DR.
BUCHANAN: The question was posed to me.
We tried to get as homogeneous of a distribution as possible in adding
it to it. All indications, by the
methods that we used, both using the dye to see that it was evenly distributed,
by the linear nature of the death-time curve, and the fact that we didn't get
substantial shoulders, and by the fact that we were able to take samples
throughout this large lot and they all pretty much came out within .2 log
cycles, are indicative of the fact that we did a pretty good job.
DR.
KUZMINSKI: I agree.
DR.
BUSTA: Other questions?
We have a
point in the agenda, now, where I'd like your feeling. I arbitrarily postponed the preliminary
subcommittee discussion on clinical presentations because I felt these last two
had some implications on some of the clinical questions this morning. We can have that discussion now and then go
on to the item on the agenda for the production situation, by Dr. Zink, or we
can have Dr. Zink's presentation, take a break and come back for an overall
preliminary discussion, and then have the public comment, and then have another
discussion.
What is
your preference? The other alternative
is you really don't care.
[Laughter.]
Next
speaker. As close a consensus as we
need.
Is Dr.
Zink ready? He's ready. Here he comes. And Dr. Zink will give us the FDA field survey of powdered
formula manufacturing.
FDA Field Survey of Powdered Formula Manufacturing
DR. ZINK:
Can you hear me? I figure, if nothing
else, I'll try to speak up.
What I
want to do is accomplish two things with this presentation. I want to take what you've learned this
morning and put it into an industrial scale, using the information we gained
from our field survey to talk to you about manufacturing practices; give you a
real good idea of how formula is made; start you thinking about potential
opportunities of how the organism could get into formula; and then talk to you
in some detail about our findings.
Could I
have the next slide, please?
[Slide]
The
objective of our field survey was to describe manufacturing processes that were
used--excuse me. This is presentation
objections--talk about the manufacturing practices used, summary micro-criteria
used in various jurisdictions and by various governing bodies, and to review the
results of our survey.
Next
slide.
[Slide.]
Powdered
infant formula manufacturing , we can roughly talk about two kinds of
processes. One is dry blending, and the
other is where all the ingredients are mixed together into a homogeneous
liquid, and then spray dried to produce a dry powder. Each of these processes has different challenges and
benefits. I wouldn't characterize one
as being preferable or inferior to the other.
Among dry
blending, certainly it minimizes the use of water in the processing area. Bacteria need water to grow. Anything that minimizes the use of water
aids your ability to control the growth of bacteria in the environment of a
manufacturing plant. However you're
vulnerable to the contamination of ingredients. If your buying dry ingredients from various suppliers, and you're
not, you know, hearing those ingredients in any way, pretty much what you get
in the finished product is whatever you start with in these dry
ingredients. Also, if you're going to
use microbiological testing of the incoming raw materials to qualify them,
that's a difficult thing to do because of the limitations of lot testing, the
potential for non-random distribution of organisms, and the difficulty of
getting a microbiological sampling plan that truly detects a positive organism.
Wet
mixing, on the other hand, you have the advantage that you're hydrating all
your ingredients and you can pasteurize them to destroy all harmful bacteria,
including organisms like sakazakii, but
this requires frequent wet cleaning of very large equipment items. And also this use of water in the plant
promotes the growth of bacteria in the plant and makes it a little harder to
control. So you see some pros and cons
with these.
Next
slide.
[Slide.]
This is a
typical wet mixing, spray drying type process.
This does not show any one manufacturer's process. It's sort of a stylized, simplified summary
of the types of processes used by manufacturers in the survey.
In
general, you begin with the protein, mineral oil and carbohydrate--often corn
syrup--components. These all go into a
large tank. It can be three to five
thousand gallons in this tank. They're mixed
to homogeneity, and then they go through the pasteurizer. I should add that in some cases,
manufacturers homogenize before they pasteurize, and some pasteurize before
they homogenize. But, at any rate,
knowing from Bob's presentation, you're dealing with pasteurization
temperatures that could be anywhere from 170 to 260, this is a process that
effectively destroys, completely, Enterobacter sakazakii. The holding time on this can range from, oh,
15 seconds to more than a minute.
Then the
product is usually cooled down to something like 170 degrees Fahrenheit--maybe
a little bit less. Some heat-sensitive
ingredients, such as vitamins and perhaps some mineral component, fatty acid
component, could be added here. Then
it's homogenized. This is generally
done at about 170. Again, remember
Bob's presentation; 170 is pretty close to 70 degrees centigrade, so you're still
doing some killing here, even though you're not documenting it.
Then it
goes to a holding tank--if it goes to a holding tank. This is optional. If it
goes to a holding tank, it's usually cooled down to about 40 degrees
centigrade, and may be held for some period of hours at that temperature. And then it's preheated before it goes into
a spray dryer.
This
spray dryer--and I'll show you some pictures of them later--can be a very large
structure; seven, eight, nine stories tall.
And it's sprayed into that structure at the top as a fine mist, very
small droplets. It can have a
temperature anywhere between 160, 200 degrees Fahrenheit. When the liquid goes in here the air temperature
of the spray dryer can run anywhere from maybe 260 Fahrenheit to over 400
degrees Fahrenheit.
So, up to
this point the chances that Enterobacter sakazakii would have been in this
material, survived this process and be alive and be contaminating product at
this point--not very great. The
lethality steps are considerable up to this point.
However,
after this point, that is where, should the organism be present in the
manufacturing environment it could certainly contaminate the powder. As this powder falls through the spray
dryer, the swirling hot air, it very rapidly dries the droplets into particles
of dry powder. By the time the powder
falls to the bottom of this funnel-shaped spray dryer, you have a temperature
of maybe 170, 180 degrees Fahrenheit; maybe as high as 200 degrees Fahrenheit
in the powder. And it goes into what's
called a "fluidized bed." And
you can think of a fluidized bed as a kind of a churning conveyer of powder, if
you will, which has air flowing through it.
And usually these fluidized beds are two stages: one that will pump hot
air in there to do some additional drying, and then maybe several stages of
cold air to cool it down.
When the
powder comes out of the fluidized bed it goes through a sifter. And at this point the powder is about room
temperature. The sifter is to remove
large clumps. And from the sifter it
can go directly to a can-filling line, where it's filled, purged with nitrogen
and hermetically sealed; or, it can be stored temporarily in totes or large
bags for, say, quality assurance analysis, and then scheduled for filling.
This is
fairly typical.
There's
more than one kind of spray dryer. I've
diagramed a kind of a vertical funnel-shaped dryer here. You can also have box type dryers which are
more room-shaped. But the principle of
operation is the same.
Next
slide.
[Slide.]
In a dry
blending type of process, actually most--the formula manufacturers that use dry
blending more heavily, it really should be thought of as a combination of wet
mixing-dry blending. They'll take some
dry materials, particularly the protein fraction, and the oil components, and
they will wet mix those, again in a perhaps several thousand gallon tank,
pasteurize them, cool them, homogenize them as before. Homogenization, if you're not familiar with
it, is simply a step where the product is raised to several thousand pounds of
pressure and forced through a small orifice to make a uniform distribution of
the components, particularly the oils.
The oils are reduced to very small micro-droplets so that they stay uniformly
in suspension and don't settle out at the top of the product.
Then,
again, the holding tank, the pre-heater, and this protein and fat fraction is
then spray dried. Now, this
is--percentage-wise, this is a smaller amount of the formula, and this process
is advantageous to manufacturers, because they're not having to put water in
and take water out by expenditure of energy into such a large quantity of
powder.
They then
take this dry protein and oil fraction.
It goes through the same sort of fluidized bed and sifter process, and
into drums or totes or bags for storage.
And then what happens is additional raw materials, particularly the
carbohydrate fraction, but also potentially other kinds of fractions, are added
to this pre-mix, if you will, in a ribbon blender. Again, it gets sifted and it goes to the can-filling line. The purpose of the ribbon blender here is to
blend all of the materials to homogeneity.
These are
basically the two kinds of processes that are used.
Next
slide, please.
[Slide.]
This is a
diagram, a little bit more detail about spray dryers and fluidized beds. First, I want to point out to you: this is
the bottom of a funnel-type vertical spray dryer. That little figure right
there is a man. Okay? It's a large piece of equipment. And this piece of equipment has
doors--access doors into it. They have
gaskets. It's--we're not talking about
a smooth tube here. So periodically
these things have to be cleaned. And
they have to be wet cleaned. And
sometimes you have to open an access door, and people have to go into them, or
people dangle in a harness in them, trying to clean it. This is not what we would call a
clean-in-place type operations, where you just flush some water through there
and away you go. It's a challenging
thing to clean, with lots of little nooks and crannies, when you think about
it.
So what
you see here is this portion of a diagram.
So here you have a large spray dryer.
Hot air is generated here. It
passes through high efficiency filters that filter out any microorganisms, and
it goes in the top of the spray dryer, along with the heated liquid
formula. The whole mixture swirls
around and dries and falls to the bottom, goes into the fluidized bed. This is a two-stage fluidized bed shown
here, with a hot air stage and cold air state.
Again, this air is filtered when it goes in there to prevent microbial
contamination--and comes out of the fluidized bed through the sifter, as I
showed you in the diagram.
Things
are always more complicated than you typically can draw them and show
them. And, in fact, you get some very
fine particles of powder which don't readily settle out to the bottom. These can be taken out of the spray dryer,
and they go through what are called "cyclones," which is, in essence,
a centrifugal type of centrifuge. And these
very fine particles can be separately collected. And those may be used for infant formula, or they may be--excuse
me. Those may go to waste as animal
feed, or they may be recycled back into formula through a wet-mixing
process. It varies.
Next slide.
[Slide.]
This is
the inside of a box dryer. And a box
dryer is just a square spray dryer.
It's hard to see here--it's dimly lit--but these are--there's nozzles
and hot air inlet up here. Imagine this
room was a box dryer. You have hot air
and sprayed liquid coming in at this end, swirling around and falling to the
floor, where it's collected. This
picture showed liquid being sprayed in a very fine mist, and it dries. This detail here shows a chain drive. And how do you get the powder off the floor? Well, you just have a bar that's pulled by a
chain that scrapes it over to one side, and then you have an auger--if you go
to the--yes, you can see the auger here, I think, in this slide--and then you
have an auger in the corner there against the wall that carries the powder
away. So that's the other type of spray
dryer used.
Next
slide, please.
[Slide.]
This is a
diagram of a fluidized bed--not a small piece of equipment, either. If a person were standing here, they would
be about so tall--okay? So if you're
standing on the floor, certainly the apparatus is taller than you are. It gives you some idea of the scale. If you look inside of it--and, of course,
this is clean and not operating right now--you see this kind of a porous bed
here that vibrates and that air can come up through, and the powder just moves
along like it's flowing in a turbulent stream over the bottom of the fluidized
bed.
And you
can see, there's some nooks and crannies in here, too, that pose cleaning
challenges. And it is--it's a large
piece of equipment. So there's
opportunity for contamination to occur here if the equipment is not perfectly
clean.
Next
slide.
[Slide.]
On to
microbiological standards. Before
talking about our findings in formula, we thought it would be good if we could
review with you what we knew about the various microbiological standards
currently being applied to powdered infant formula.
This is
the CODEX standards. You heard it referred to earlier in one of the
presentations, in relation to an outbreak that occurred in Belgium. It's permissible--they take five samples of
a lot. It's permissible to have one of
those samples with as many as 20 coliforms in it, but you should have fewer
than 3--and "fewer than 3" in this case means no positive tubes in a
three-tube MPN--but you should have fewer than 3 in the remaining four samples;
n, number of samples, c maximum number of samples that can have a number in
excess of m, and M is the "no single sample shall exceed" limit. Okay?
Next
slide.
[Slide.]
This is
the Canadian standards, and I'm going to focus in here. They don't specifically have one for
coliforms or Enterobacteriaceae, but Escherichia coli comes close. Here they're taking 10 samples. One of those would be permitted to have as
many 10 organisms in it, but the remaining nine would have to be less than
1.8--essentially, probably a negative results.
Next
slide.
[Slide.]
This is a
standard currently being used in China: aerobic plate count less than 30,000
per gram. They have a coliform standard
of less than 40 per 100 grams.
Next
slide.
[Slide.]
This is a
standards that was proposed--or, guidelines, really, that were proposed I guess
in our--I don't know the exact term we would use for it--but it was proposed as
criteria that we would use in judging the acceptability of a lot of infant
formula. And it was less than 3.05 MPN
per gram coliforms. There were similar
separate standards for fecal coliforms.
Next
slide.
[Slide.]
In our
survey of infant formula plants we surveyed all of the plants that produce
powdered formula in the United States.
There were 15 such plants. Some
of these plants produced concentrates and ready-to-feed only. Some produced--I think seven of the plans
produced powdered only. Some plants
produce ready-to-feed and powder, and I think a total of 10 plants each
produced powdered formula and all were included in this assignment. So this is a complete survey, if you will,
of the U.S. powdered formula market.
Next
slide.
[Slide.]
What we
wanted to do with this study was to get an official sample of powdered infant
formula and raw materials to assess the prevalence and the number of E.
sakazakii in the samples. And the
sampling protocol that we devised was based on prior literature reporting
numbers as few as .36 per 100 grams, and we wanted to be certain to have a
sampling protocol that could quantitate down to that limit; also to conduct an
inspection at each major domestic infant formula producer in order to
understand any possible relationship between manufacturing practices and the
prevalence of E. sakazakii.
Next
slide.
[Slide.]
Here you
can see the 10 plants that were studied. The total number of samples collected
was 92. Of this 92, you'll probably be
most interested in the finished product samples, of which there were 22. We also collected separately samples of
carbohydrate fraction, fat, if it was present as a dry powder, and protein
samples.
These
samples were collected regardless of the process used. In other words, in some cases this carbohydrate
was directly dry blended into the finished product without further heat
treating. And, in some cases, this
carbohydrate was part of a wet mixing-spray drying operation. It went in into a wet mix and then it got
pasteurized before it was spray dried.
But we wanted to look at prevalence in raw materials as well.
Next
slide.
[Slide.]
Looking
at finished products by the types of products--again, there were 22 finished
product samples. Fourteen of these were
formulas for full-term infants; four were formulas intended for pre-term
infants; and two were metabolic formulas, and two were hydrolysates.
Next.
[Slide.]
I want to
talk to you a little bit about our procedure.
If you've gotten the impression everybody does their own thing, sort of,
when they develop these that's kind of the nature of microbiology. We have essentially, as we said, used the
procedure of Nazarowec-White, with very
little modification. Okay? However, it's important to note that we
started out with 20 cans here. In the
study Don talked to you about earlier, they were actually looking at 330 grams
in a given lot or can of sample. We're
looking at 1,332 grams, and here's how we come to it.
Twenty
cans were collected, and each of these cans had at least eight ounces of powder
in it, and a composite was constructed by sorting these cans into four groups
of five; 100 grams came out of each can in the group, so we now had four groups
of 500 grams. And those four groups of
500 grams were each treated as four separate three-tube MPNs. So you had 4 times 3 times 100, times 10,
times 1, for a total--out of any given lot sampled--of 1,332 grams. That's important to note when you consider
the prevalence of the organism reported by other investigators looking at
smaller quantities of powder versus our results.
The
theoretical limit of detection here would be one in 1,332 grams. That could occur, provided you had optimal
conditions; you didn't have competing microorganisms, etcetera. We don't know what the actual limit of
detection is, of course, because we don't know what the true contamination
was. The minimum limit of
quantitation--as Don Burr explained to you--is 0.31 most probable number in 100
grams.
I want
tot diverge just a minute so you don't get to where you misunderstand what's
meant by "most probable number method." It has limitations, and you need to think of those limitations,
and you need to think about the statistics associated with it. First thing, the most probable number, to be
a valid method of quantitating something, the main assumption you have to meet
is that whatever it is you're measuring has to be uniformly distributed in what
you're measuring. And that is almost
certainly not the case here.
If you
remember the paper reporting the outbreak in the Belgian hospitals, they
referred to the fact that they analyzed five samples; four of them were
negative and one of them had 20 per gram.
Well, that shows you what I think a lot of microbiologists--food
microbiologists--know, is that this organism is not uniformly distributed in
the powder. Contamination theoretically
could occur from sloughing off of a equipment.
That could happen sporadically.
So, in our MPN analysis, we're already violating one of the assumptions
of the MPN, simply because we had no other alternative way to do it that was
do-able. And you should keep that in
mind. The confidence interval for this
result--say you get a result of 0.31 MPN in 100 grams, the confidence interval
for that--95 percent confidence interval--is anywhere from 0.15 per 100 grams,
all the way up to 1 per 100 grams.
Now, the
only way to really understand--I think it's important to think about what might
actually go into a bottle of formula.
On average, I guess an eight ounce bottle of formula has something
like--what?--34 grams of powder, something like that, in it. And the only way we could really find out
what might actually be going into a serving would be to do an experiment like
this where we culture everything in little 28 or 34 gram aliquots. But when we report this result of 0.31, or
0.36 per 100 grams, you could very well have a gram of that powder with 8, 10
or a dozen organisms in it, and then many aliquots of that powder with nothing
in it. Okay? So this is a statistical
result, if you will.
Next
slide.
[Slide.]
What we
found, overall, in finished product, we found five positive samples, for a
total of 22.7 percent of the sample--finished product sampled--were
positive. In every case, when you
analyze this by the three-tube MPN tables, we found 0.36 per 100 grams--at or
very close to the limit of detection--22 samples tested.
In the
carbohydrate fraction, we found only one positive result. Now, I should say we only did quantitative
MPN test on finished product, not the components of it. And we found one protein fraction that was
positive.
Next
slide.
[Slide.]
If you
break this down by type of formula, four of 14 were full-term formulas; one of
four was a pre-term formula. The
metabolic and hydrolysates weren't positive.
There's no significant difference in that result.
Next
slide.
[Slide.]
All
positive finished product samples were at the lowest limit of
quantitation. So we're talking about
low numbers here. There was no
correlation between the test results and manufacturing practices. In other words, you know, we got positives
from people that wet-mixed, and we got positives from operations that
incorporated dry blending, without apparent difference.
And there
was no relationship between the results and product type. We had soy products that were positive; we
had milk-based products that were positive .
So there's no reason to believe that this has got any particular type of
association.
I believe
that's it. Thank you.
DR.
BUSTA: Are there questions for clarification for Don?
Dr. Fuller. Oh, I'm sorry. Dr. Moyer-Mileur.
DR.
FULLER:
. DR. MOYER-MILEUR: Moyer-Mileur.
Could you
just clarify--for the pre-term formulas, did that include your transitional
feeding formulas for pre-term babies, or just the in-hospital 24
calorie-per-ounce products.
DR. ZINK:
I believe that the pre-term did include what you would call transitional.
DR.
BUSTA: Dr. Briley.
DR.
BRILEY: Margaret Briley.
Could you
clarify for me the differences--you said there were no differences in cleaning
the vats when they were dry or wet milled.
Would you say that possibly it would be more difficult in one, or that
one would be a slight bit more toward the possibility of contamination, or--
DR. ZINK:
Okay--talk about equipment cleanability?
DR.
BRILEY: Yes.
DR. ZINK:
Dry versus wet blending? All right.
In the
wet blending and spray drying, since everything has to be spray dried, you're
generally dealing with some very large capacity spray drying systems. And those spray dryers, on average, probably
have to be cleaned once a week by wet cleaning. And that can take a day or two, you know. It's a difficult process. The fluidized bed has to be cleaned, and all
of that associated equipment with it has to be thoroughly dried out. That's a challenge as well.
In a dry
blending operation--like I said, most people still have some wet blending and
spray drying going on. But they're
generally not having to clean as large a piece of equipment. And on the dry side of it, you can go quite
a long time between cleaning, or you can use strictly dry cleaning methods for
your brushing, vacuuming--that sort of thing.
So, on a purely dry side of the process, oh you can go weeks, perhaps
months without using much water. And
keeping it dry is a huge advantage in controlling these organisms. You can't deny them food, but you can
certainly deny them water, and they need that water to grow and without it they
don't grow.
DR.
BUSTA: Dr. Fuller?
DR.
FULLER: Given the--you mentioned the potential for clumping in equipment to
release chunks of bacteria, if you will.
Did you all do any looking at--do any environmental sampling? And, if not, was there any reason for not
doing that at the same time?
DR. ZINK:
We've had limited ability to go back and do those kinds of investigative samples. I believe it was done in one case. I believe that organisms were recovered from
the environment. Perhaps some of these
investigations will still be ongoing. I
don't know what the status of that is.
But by
the time we get a sample, analyze the sample, and interpret the results--this
wasn't done to be in real time. So a
sample simply went in and they were enqueued with the labs other work. Oftentimes, by the time we got that result,
it might be a month or more from the time the sample was collected, and by that
time the equipment has gone through many cleaning cycles, and the manufacturers
themselves were monitoring equipment and environment. So I think going back in and doing a detailed equipment survey in
the timeframe we were working in here, that was clearly a secondary or tertiary
objective.
So I
don't think we sought anything meaningful out of that or got anything
meaningful out of it.
DR.
MOYER-MILEUR: So let me just--let me ask my question a little bit differently.
The
assumption, then, is that the primary source of the E. sak is coming from the
ingredients, not from equipment contamination.
You feel that there's enough cleaning going on to prevent that? Or--I mean, I was just trying to get a
better understanding.
DR. ZINK:
I think if I had to offer you my opinion, I would say the organism is probably
coming from the equipment, post spray drying.
I think that the organism, by whatever means unknown, gets into the
plant and can contaminate that equipment.
And the difficulties of cleaning this equipment, you can never make that
equipment completely sterile. And so I
think that what low levels of contamination do occur are probably coming from
that handling equipment.
DR.
MOYER-MILEUR: Okay. So we've got that
assumption, but no data to back that up yet.
DR. ZINK:
Nope.
DR.
MOYER-MILEUR: Okay.
DR. ZINK:
I'm--that's going to have to be the subject for further study.
DR.
BUSTA: Dr. Blumberg.
DR.
BLUMBERG: Henry Blumberg.
Did you
look at other organisms besides Enterobacter sakazakii? Or did these studies that you were doing
just focus on that?
DR. ZINK:
We were specifically looking for Enterobacter sakazakii. They were specifically picking yellow
colonies.
When they
first started this study--and going over the work sheets of the labs, I think
they were more liberal in picking colonies as they were trying to become
familiar with this organism. So I think
they cast a wider net at first. And,
certainly, we did see, in some of those early samples, some things like
Serratia, Panatea--other organisms cropped up.
But, for the most part, after the first few positive samples, they were
zeroing right in and just picking sakazakii.
DR.
BUSTA: Dr. Tompkin.
DR.
TOMPKIN: This is Tompkin.
You
didn't do MPNs on the carbohydrate and protein fractions, the ones that tested
positive. What was the amount of
product, or ingredient?
DR. ZINK:
We were looking--I'm trying to remember.
You would ask me that. I believe
we looked at 500 grams as one sample.
Is that right? Don, do you
remember? Is Don Burr back there?
DR. BURR:
No, I don't remember.
DR. ZINK:
I'm pretty close to that. I'll
check. But I believe it was 500 grams.
DR.
BUSTA: Dr. Neill?
DR.
NEILL: Peggy Neill.
Don, do
you recall what month or season of the year the sampling was done?
DR. ZINK:
I think we began collecting in August and continued through January, or maybe
even February.
DR.
BUSTA: Dr. Tarr.
DR. TARR:
Tarr.
What
governs the interval between cleanings?
Is it tradition, or is it driven by colony counts or other data?
DR. ZINK:
It's driven by need. You begin to see
accumulation of material on the walls of the spray dryer. Or else they need to switch from product
type to some other product type. That's
another thing that would drive it--say, going from milk to soy, or soy to milk.
DR.
BUSTA: Dr. Briley?
DR.
BRILEY: Margaret Briley.
Did you,
in your survey, by any chance make record of any of these plants that had HACCP
procedures in place?
DR. ZINK:
The question was did we, in our, surveys make a record of any of the plants
that had HACCP procedures in place. No,
I don't recall that we were specifically looking "Did you or did you not
have a HACCP plan?"
The
nature of how these plants operate is it's HACCP by any other name. In other words, all of these manufacturers
had detailed procedures. Every one of
them had SOPs for cleaning the equipment.
They all identified critical control points in their process. It was--you know, there was a remarkable
sameness as you read through all the reports about the methodology of
controlling the process.
DR.
BUSTA: Dr. Stallings.
DR.
STALLINGS: Stallings.
We've
really been waiting to see some of these data.
So, to clarify the one the FDA field survey results--you did have
starting product--carbohydrate and protein--that had positive cultures.
DR. ZINK:
Yes.
DR.
STALLINGS: And then the finished product.
And so you were saying, though, that you really felt like the source of
the organisms was during the manufacture.
So the protein and carbohydrate--I mean, just explain that.
DR. ZINK:
I can talk about those in a little bit more detail.
DR.
STALLINGS: Okay.
DR. ZINK:
In the case of the protein fraction, that was used in a product which went
through a traditional wet mixing-spray drying process. So all of the protein was hydrated into
water with the other ingredients, and then it went through pasteurization. I remember doing a calculation on the
lethality, taking that processor's exact time and temperature conditions, and
then only at the pasteurizer--okay?--not considering other heating
contributions. And I think I came up
with a theoretical lethality of around 200 log cycle reduction. Very substantial. No way that organism could possibly have survived that
process. So, I do not believe that that
protein contributed--you know, although it was contaminated, it would not have
been a factor in contaminating the other product.
You
always have to consider, when raw materials come into a plant and they're
contaminated, even though you're going to mix them wet and pasteurize it, if
they get opened and tracked around the plant, or--you know, that could be a
problem. But in the case of the
contaminated carbohydrate, it did go into a product through a dry blending
process. So, most certainly, what was
in it would have been expected to also show up in the formula.
DR.
BUSTA: Dr. Kuzminski.
DR.
KUZMINSKI: Thank you. Larry Kuzminski.
A number
of questions. In your survey, did you
get a chance to review--and it sounds like you did--the QA practices---the
standard quality assurance practices--during the manufacturing process?
DR. ZINK:
Yes. All of those procedures were
documented and recorded, and there was nothing remarkable or deficient showing
in any of that documentation.
Bear in
mind, you know, when all of that field survey material comes back to us, they
were following specific instructions to collect certain kinds of information,
and collect certain kinds of documentation.
I think one of the things you have to ask is: do these traditional kinds
of critical control point documentation systems and SOPs--do they adequately
address risk of contamination with an organism like this? I mean, certainly, in looking at the data as
it came in, you know, it seemed to meet, you know, all of the requirements one
would expect of a manufacturer of that product--as documented on those reports.
If you
were standing there in those plants, I think you have the ability to assess
things, and consider things, you know, that might not be part of formal control
programs that might be contributing factors.
DR.
KUZMINSKI: One of the points that you made was the potential for contamination
post--in the process flow, past the spray dryer, once heat's stopped being
applied to components of other process, back to ribbon blending and to addition
of the minor ingredients; the vitamin and mineral pre-mixes.
I noted
that--you perhaps omitted this, but did you get a chance to take a look at the
vitamin components in the pre-mix for microbiological quality?
DR. ZINK:
They did not sample any of the vitamin components in this survey.
DR.
KUZMINSKI: There must--also from the data on the distribution of the nutrients
that, I think, Dr. Buchanan showed, it looks like the efficiency of the ribbon
blender is a pretty efficient blender, in terms of getting distribution pretty
homogeneously throughout a batch, even up into a 5,000 pound batch--quite a
size batch.
Would you
think, though, that if something if something is sloughed off from the
equipment--a chunk of dried material, caked on material, from something that's
been running for awhile, a number of shifts, a number of days, especially in
the dry process, that the efficiency of the ribbon blender would be there to
get that distributed through the batch also?
DR. ZINK:
Well, to the extent that--I mean, not all formulas go through that kind of
ribbon blender dry blending process.
Some are strictly the wet mix-spray dry, more of a continuous stream
type of thing.
Yes, I
would think the ribbon blender would tend to take non-random contamination and
randomize it to a degree. I'm not
familiar with any kinds of studies--and I don't have any experience to say how
effective in doing that it would be.
But I would expect that the ribbon blender, on those dry blended
products, would tend to randomize the contamination a bit--yes.
But in
the other process, you really don't have that at work. So you would maybe expect more
non-randomness in the wet mix-spray dry type of process.
DR.
KUZMINSKI: And, lastly, there have been a couple of recalls--voluntary recalls--in
the industry due to E. sak presence.
Very responsible companies are involved. And in the follow-up investigation to these recalls, in terms of
process analysis, the efficacy presence perhaps and the line flow of
ingredients, right back to the raw materials--individual raw material
supplier--to the discharge end of the packing line--can you share with us any
information that has been gained from those recall incidents, as to the
occurrence?
DR. ZINK:
I can say--you know, did someone--did an investigator or a company come forward
with the smoking gun in each of those cases?
No. Okay. I mean, I am familiar--some manufacturers
shared the fact that, indeed, they were finding the organism in the food
processing environment, and that controlling the organism in the food
processing environment is a real challenge.
And perhaps we'll hear more about that later.
DR.
BUSTA: Dr. Neill
DR.
NEILL: Don, can you tell me--I have two very different question, one of which
is how analogous is this process for the production of the powdered formula to
the production of powdered milk? And
then, kind of a--
DR. ZINK:
Virtually .identical.
DR.
NEILL: All right. And then perhaps
somewhere in the later batch of questions, one of the individuals who spoke can
tell us whether any similar work has been done trying to analyze, microbiology
speaking, any powdered milk for this.
DR. ZINK:
Her question was how similar is the infant formula manufacturing process to,
say, what would be used to produce spray-dried milk. And my answer is it's virtually identical; same types of
equipment, same types of processes, probably even similar temperatures
involved.
We
certainly haven't done a survey of spray-dried milk plants for this organism.
DR.
NEILL: And then the second question has to do with the derivation of the
sampling plan. Since, in looking at
that I notice that there's quite a difference in the distribution of the sample
types--finished product versus component ingredients, etcetera--across the 10
plans, and was this derived on the basis of volume of product, or what?
DR. ZINK:
I think probably others could talk about that sampling plan more, but--correct
me if I'm wrong--we wanted to get a good sampling of the various different
kinds of products that were out there.
I don't believe there was aan attempt to bias it in any way. It was really what we could get at within
the timeframe allocated for the study, really.
And we wanted to look at some of each thing, but--you know, some of
these formulas are produced infrequently--once or twice a year. And that had a lot to do with it,
particularly some of the speciality formulas and stuff--our ability to get
those samples.
DR.
BUSTA: I'll take three more questions.
Dr.
Acholonu.
DR.
ACHOLONU: Doctor, you discussed the microbiological standards in Canada, in
China and as used by FDA. And each of
them has aerobic plate count. Since we
have been told that E.sak is a facultative anaerobe, can that method be used?
DR. ZINK:
It will certainly grow up as a colony on the aerobic plate count. A lot of other things will too, of
course. You would have to, at that
point, pick the colonies and identify them to differentiate it and detect it.
To a
microbiologist--the ideal method to a microbiologist is that you take a whole
lot of the sample, throw it in some magic broth and only what you are
interested in grows, and it's not the least bit inhibited by anything in there,
but everything else is inhibited--and out comes the bug. We don't have that method for this organism,
unfortunately.
Infant
formula is very clean, microbiologically.
Although you saw aerobic plate count limits up there of 10,000,
30,000--things like that, most manufacturers are producing more in a norm of
about 500 per gram.
DR.
BUSTA: Dr. Beuchat.
DR.
BEUCHAT: Beuchat.
My
question concerns the physical structure, or the layout, of a typical wet
mixing-spray drying process. Is the
area in which the raw ingredients handled separated physically and/or by air
flow, ventilation and so on, from the post-spray dry area?
DR. ZINK:
In the plants I have been in--I'll answer that question by saying yes. In some plants, it's outstandingly well
separated. And in somse plants, there's
room to improve--okay? But I think all
manufacturers strive to achieve segregation, and all of the manufacturers I
know of have very strict physical environmental isolation once they're dealing
with a finished powder. You just can't
stroll from one area of the plan to the other.
DR.
BUSTA: Thank you, Don.
We will
now take a 20 minute break. Is that
long enough for you? A 20 minute break,
so we'll aim at 3:25 reconvening, and we'll ask any questions of the three
speakers so far this afternoon first.
[Break.]
DR.
BUSTA: Will all of you that are asking questions--now that's how to make a
point--right? You're asked to talk
loudly, as well as into the microphone.
But even with the microphone, talk loudly, because all of this is being
recorded and someone down the line has to try and transcribe it all. And that's--we're trying to make their job
not as terribly difficult as it might be.
At this
point we have about, maybe, 20 minutes if we need that to ask questions for any
of the four speakers from this afternoon.
And we're open to questions.
Dr.
Tompkin.
DR.
TOMPKIN: Bruce Tompkin. This is for
Maria.
I don't
recall that you mentioned it in your presentation, but in the manuscript that
you had published, at 4 degrees centigrade, which is almost 40 Fahrenheit, you
found that E. sakazakii actually died?
But I don't recall that it stated how rapidly. Could you comment on the rate of death at refrigerated
temperature?
DR.
NAZAROWEC-WHITE: At refrigerated temperatures we took samples, I think it was
every two days. And there was
nothing--in some of them it would be 103 and 103, and
then after about--I don't know--two weeks, it was down.
DR.
BUSTA: Dr. Thureen.
DR.
THUREEN: Thureen.
If this
committee--this is a hypothetical question--if this committee can agree that
there should be a standard above which there should be no higher level of E. sakazakii
contamination, and we decide that this is an index marker, because it has such
virulence, and because it's easy to detect, and we establish that standard as
being the limit above--or at which we feel comfortable of having to be a slight
contamination rate, do you think then that we will also eliminate risk for
serious infection from other organisms that are found within the powdered
infant formulas? Would that be a good
benchmark to set to provide overall safety, not just against his particular organism. And this is for any of you.
DR.
BUCHANAN: You're asking a really big question.
You've posed a hypothetical question that--the best that I could give
you is a hypothetical answer--with all the disclaimers, caveats, etcetera.
The
suggestion is that if you could have a standard that controlled this organism,
would you control others? And the real
question here is: is their mode of action the same? Is their mechanism of pathogenicity the same? Is the impact of the interventions or
standards that you would use the same?
If you
could fulfill all those criteria--probably.
And I hesitate to say anything more than probability. If their method of control was different,
then I would say that you'd have to actually go through and make the case. And, gee, if you'd ask me, you know, are
there infections due to Enterobacter cloacae, and would controlling
Enterobacter sakazakii control Enterobacter cloacae? Yes, I think you'd probably have a good chance. Salmonella?
I'd be less sure of.
DR.
THUREEN: And so, along that line, do you think it should be E. sakazakii, or do
you think it should be enterobacter in general that should be sort of the
target organism for eradication? Or do
you think it makes a difference.
DR. ZINK:
I think I'd be more comfortable answering if I knew a little more than I know
right now. I agree with Bob. If you choose to pick an organism as the
focus of your control, and it happens to be part of a family of organisms that
have a similar mode of contaminating the product, and similar resistance or
susceptibility to control measures, then I think picking the most onerous of
the family probably would be a good way to go.
Talking
about Enterobacteriaceae, as you've seen, they're all pretty similar in their
susceptibilities and resistences to control measures. So that may be an approach you could use. The trouble is that I'm not--I don't think
any of us have a lot of data on the diversity of organisms in formula and how
they get there, you know.
DR.
NAZAROWEC-WHITE: That's what--I
agree. I think that we don't know
what's in there yet. We do know that
there are some salmonella, maybe, in the dried formula. So you have one option of sort of setting
that standard is a possibility. On the
other hand, are there any intervention strategies--can we do something with
that final product to inactivate organisms?
DR.
BUSTA: Dr. Tompkin.
DR.
TOMPKIN: I didn't look it up, Don, but maybe you might remember. In the '96 ANPR for FDA for listeria
monocytogenes, do you recall what the amount of product it's--your slide says
it's negative, but is it negative in a 25 gram, or 50 grams? Do you remember?
DR. ZINK:
I don't remember.
DR.
TOMPKIN: Or was it specified?
DR. ZINK:
It would be specified, if by reference to a method. Okay? I don't remember
whether it was actually spelled out in the ANPR. If it wasn't, it would have been referred back to the method we
used. I don't know--Ada, do you know? 220?
250? 250 grams?
You know,
you hardly ever see listeria in these dry powder plants. It just doesn't like dry powder plants. I don't think I've ever recalled it being an
issue.
DR.
BUSTA: Dr. Fuller.
DR.
FULLER: This is Fuller.
Not being
particularly knowledgeable of manufacturing practices, etcetera, is it
possible--or maybe it's cost prohibitive--but to apply a heat step at the--you
know, just prior to filling the cans?
Is there--I mean, does it make any sense? Can you use dry heat? Do
you have to--you know, is there anything that would--could be done at that
step?
DR.
BUCHANAN: I'll jump in here for a
minute, because I looked at dry heating for a number of issues.
The big
thing about dry heating is whether or not your product is a mixture of protein
and carbohydrate, which infant formula are.
In some instances where you have a single entity, and you don't have a
browning reaction taking place, you can actually get away with dry
heating--long enough and hot enough that you would inactivate
microorganisms. A classic example is
salmonella control in egg white. But
egg white is interesting because they have to de-sugar the egg white before
they can dry it because if not, it turns brown.
The same
thing would be expected in infant formula.
You'd get a lot of browning reaction products.
DR. ZINK:
One of the problems is these bacteria get a lot more heat resistant when they
dry. I guess this is best studied with
salmonella. And there are garden
variety salmonella strains that can resist 200 degrees Fahrenheit for
hours. And that's a real problem--when
they're dry, when you dehydrate them.
So, I
think that applying heat to the dry powder would be--it would be challenging to
get significant log reductions.
DR.
BUSTA: Dr. Tarr.
DR. TARR:
Since looking for low counts of specific pathogens can be frustrating and quite
variable, let's go to the more robust total colony counts, or
Enterobacteriaceae counts. Are there
trends over time that you are observing?
Are they generally going down year by year in industry, and are they
associated with any new practices that could be either accelerated or amplified
in your production line?
You're
clearly exceeding the CODEX and the FDA guidelines, but you're still getting
some counts coming through. Are there
trends in those that would be informative for making a safer product?
DR. ZINK:
That might be a better question for industry.
I don't know that we have--at least not in a readily analyzable
form--data that would say, "In 2003 we're better than we were in
1985"--in terms of counts.
I know
there are specific instances within industry where--going back to paper on the
Belgian outbreak, citing the plant that was the source of that powder. I do know there are specific instances where
improvements in manufacturing process were introduced that had dramatic
improvements in the microbiological quality of the product.
DR.
BUCHANAN: Phil, I'd also caution you to be a little careful about the use of
indicator and index organisms. Because
there's no guarantee that the reduction that you see in total aerobic plate
counts are equivalent reductions taking place in the pathogens of concern in
the product. In fact, you could, for
example, eliminate large numbers of vegetative cells from a product, and if the
organism of concern was a spore former, it would have no meaning in terms of
reducing the level of risk.
DR. TARR:
I agree with that, but at least with Enterobacteriaceae, if we got to zero
Enterobacteriaceae we would get to zero E. sakazakii. And that should be a target.
Maybe not this year.
DR.
BUSTA: Dr. Stallings.
DR.
STALLINGS: Stallings.
One of
the overriding principles we're still dealing with is that this is a rare
disease. You know, that if--that it's a
rare bacteria in the product, and that it's rarely pathogenic, even in the
setting we're using.
And I go
back to the concern that someone brought up earlier about the changes in
clinical microbiological methods, in that many things have become more
automated. And I don't know if any of
you know, or if it's something that we can just get for the committee--just
some reassurances that the systems that are now being used, where basically
they're put in banks, and heated, and no human eyes see them until, you know,
there's something that says something's growing--that for this fairly rare bug,
that we would still be picking it up.
Because if--we know the neonates are getting smaller and smaller, and
sicker and sicker, and there would be some reassurance if we really knew that
we were doing very good clinical microbiological surveillance, and not picking
this up, and that it was being identified properly when it happened, and that
sort of thing.
Do any of
you have any information in really what's going on in clinical hospital
settings?
DR.
BUCHANAN: I think we can try to find that out.
But the only comment I would offer is that the most important thing for
getting a clinical sample done is having the physician ask for it.
DR.
STALLINGS: Well, to address that, I think any baby--if we're talking about the
high risk nursery setting, the signs and symptoms you're going to see were
described. It's temperature
instability, it's food intolerance if they've been eating, and--it's very soft
signs. But in a level 3 neonatal
nursery, at the drop of a hat you do blood, urine and spinal fluid. That's just the way we manage the highest
risk babies. So, in this one rare
setting, I think we probably do--we don't do stools, but stools would not be
the specimen of choice for this.
So--I'll
defer to my neonatologist, but it's usually a pretty--if you are at all worried about these babies, you do
the drill and you do it comprehensively.
DR.
THUREEN: One of the problems is that, in theory, you'd like to do lumbar
punctures on all these infants, but they're usually too unstable to have that
done. Anda I think in many nurseries,
just signs of NEC would not be enough to trigger spinal sampling being
done. And it varies from nursery to
nursery.
While I
have a microphone, I have a question.
DR.
BUSTA: Dr. Thureen.
DR.
THUREEN: Thureen--for the record. And
I'm sure there's a good reason, but why isn't irradiation a solution to this
problem?
DR.
BUCHANAN: I'll take that one on. And,
again, it's the characteristics of these organisms in a dry environment.
Just as
the thermal resistence of a normally sensitive organism goes up drastically
when you put it in a dry state, so does the irradiation resistence. So you would probably exceed the level of
irradiation that's allowed at this point.
Now, I'd
have to say that I don't know of any specific information on the irradiation
tolerance of this organism in an infant formula and, for that matter, in just
about anything. It certainly is an area
that could be looked at. But,
traditionally, as you decrease the water activity of a product its resistence
to radiation goes up dramatically.
Because the way that you kill organisms by irradiation--there's two;
there's direct and indirect effects.
The indirect effects are due to the formation of free radicals because
of, actually, the irradiation of the water.
And if you eliminate the water, you eliminate all of the secondary
effects, which is the greatest one for these organisms, and you go back to
single hit targets on DNA. And it gets
to be much more difficult.
And I
think if I remember--your Chair knows a lot about that.
[Laughter.]
DR.
BUSTA: Don, are you just holding the
phone?
[Laughter.]
Dr.
Kuzminski.
DR.
KUZMINSKI: Thank you. Larry
Kuzminski. Just a brief question to Dr.
Nazarowec-White.
You
mentioned in your introductory comments to your presentation that Health Canada
was considering a monitoring program.
Can you share any more details about that, please?
DR.
NAZAROWEC-WHITE: I guess we've talked about it for the past couple of years,
and it's the Canadian Food Inspection Agency.
At this point in time, we are set up very similar to what you have here
in the U.S. And we do not have any
infant formula companies producing powdered formula at this point in time. But monitoring the, you know, prevalence of
E. sakazakii on the formula that we do import, for instance, is something that
they are considering doing, but looking at other programs that right now have a
higher priority.
So I
think that depending on how the meetings go, and what we learn, as far as that
importance, we'll see what happens with next year's programming.
DR.
BUSTA: Dr. Beuchat.
DR.
BEUCHAT: Beuchat. This question is
directed to Maria.
You made
a comment, almost in passing, about the observation of some of your strains
clumping or sedimenting. There's some
evidence to make us think that some enterics that are capable of producing capsules--exopolysaccharide-are
more heat resistant, are protected from the heat assault, are perhaps more
resistant to harsh environments--as simple as acid conditions that would be
occurring if the cells were taken into a body.
And perhaps also virulence.
Do you
have any indication of those strains that you did observe that did have this
trait of sedimenting, of being in the category--or among those strains that
you've reported on in your paper on infectivity and pathogenicity?
DR.
NAZAROWEC-WHITE: We found that all of the strains actually produced this
sediment. And there were only the
infectivity--you know was not that--you know, the strains that cause
infectivity also produce sediment. But
strains that did not cause infectivity also produced sediment. So we don't know at this point in time. Further research, I guess, to look at how
many other strains that are in various culture collections--what's happening
with them, what they're doing.
DR.
BEUCHAT: And, along that line, do we know anything about biofilm formation by
E. sakazakii? And also, these strains,
if you transfer ad infinitum, do they lose this capability? This is something we need to be aware of if research is conducted in this area.
We've
observed some things along that line with listeria.
DR.
NAZAROWEC-WHITE: We haven't done anything.
And, at this point, I'm not sure what the plans are for the work with
this organism.
DR.
BUSTA: Dr. Heubi:
DR.
HEUBI: Heubi.
I had a
side conversation with Dr. Buchanan during the break, and I actually wanted to
raise this for the group as a whole, because I was a little confused about this
myself.
According
to the proposed standards of the FDA for microbiological standards, it doesn't
include any comment about E. sakazakii.
And are there new standards that are not printed here that specifically
relate to E. sakazakii? And can you
specifically comment on that for the group?
DR.
BUCHANAN: Yes, there seems to be a little confusion about microbiological
standards, or the way we work.
For the
standards that you've been talking about--the CACFH, etcetera, those are
largely good manufacturing, or good hygienic standards; this is what we would
expect of an industry that's producing a product under, you know, what would be
good conditions.
Our
standard for a ready-to-eat food for a pathogen is the absence, usually
specified by some sampling protocol, some standardized method. But here, sakazakii was considered--this was
considered a ready-to-eat food.
Sakazakii was considered a pathogen, and the level of sensitivity of the
test determined whether or not we were finding it or not.
This is
basically the same standard that we have for, essentially, all ready-to-eat
food: absence based on the detection using a certain method.
Does that
help clarify?
DR.
BUSTA: Okay. Chris.
DR.
TAYLOR: Thank you.
Just to
add a little bit to what Dr. Buchanan has said, in the 1996 proposal, there was
an effort to update some of the microbial standards and reopening the comment
period on this rule in order to make it a final rule, there will be the
opportunity again to talk about specific microbial standards for infant formula
specifically.
I just
wanted to add that.
DR.
BUSTA: Thank you.
Dr.
Briley.
DR.
BRILEY: Margaret Briley. I wanted to
clear up something.
It seems
that this pathogen is suspected--
DR.
BUSTA: Dr. Briley, could you speak louder?
It's on, it's on, but you just have to speak a little louder.
DR.
BRILEY: This pathogen, from what I understand--maybe you need to correct me--is
suspected to be in the equipment, possibly, in the manufacturing stage. Did I understand that to be a possibility?
Can you
make some comment about any improvement in the cleaning of the equipment, or in
the process of moving it through the equipment that industry might take that
would allow this to be corrected?
I don't
know enough about what's inside the big funnel. I can see a little man with a bonnet and a robe and covered shoes
climbing up and scrubbing the walls.
But I just would like to know if there's some comment that you could
give, based on your observations?
DR. ZINK:
I think maybe some from industry could comment on it better than I could. In my capacity at FDA, you know--that was
another life, a prior life for me. But,
you know, you can always design something a little better, or clean something a
little better than you have in the past.
Things come along, and we're required to raise the bar. I mean, were it not for the Titanic, would
we have life preservers on cruise ships.
I'm sure
that the discovery of this organism and its significance has caused a
reexamination of cleaning methods and procedures. I think the question becomes to what extent, by procedures and
equipment-tweaking alone can you get to some other level, and what is that
other level. And that I don't know.
DR.
BRILEY: Hopefully, zero tolerance maybe.
DR.
BUSTA: Dr. Thureen? Okay, Dr. Tompkin.
DR.
TOMPKIN: I sensed a perception that the frequency of cleaning could be an issue
with these facilities. And I'd like to
have some comment, probably from Don, on this.
But, in
operating a dry operation it's better to run it, I think, as long as you
can. And actually, from my own
experience, I've seen situations where your problems occur as a result of
cleaning, and that this non-random distribution of certain bacteria can occur
in the first product through the system.
So sampling the first product through the system may, in fact, pick up a
contamination that may have occurred in the process of cleaning, or while the
equipment was sitting and still wet, allowing for some growth.
So, in
terms of recommendations or strategies, one that the group may come up with is
clean more frequently, but I don't feel comfortable with that, and I would like
to know what your view is.
DR. ZINK:
I think most manufacturers would agree with you, that they'd rather not break
and clean very often; that if they could run longer and drier, they would. The fact is that if you've got one spray
dryer, and you make four different kinds of formula, you know, and you have to
do these changeovers, that will dictate some need to clean.
Also,
these formulas have a fair bit of carbohydrate or sugar in them, and that can
tend to make them form a cake or adhere to the walls of the spray dryer. There's other operational things that you
have to go in there, such as, maybe, the accumulation of some small number--you
know, burnt particles and things like that might dictate a need to get in and
clean.
But for
the spray dryers, I seem to remember that somewhere between once every five to
12 days seems to be the usual frequency at which they're cleaned. And I think cleaning more often--I agree
with you, it might cause more trouble, you know, by the additional water it
puts in the system.
DR.
BUCHANAN: Also, I'd like to also respond or expand your question out a little
bit, because I have some concern about misinterpreting what is meant by
"low levels of contamination," versus an nonhomogeneous distribution
of a sample. And while I have not seen
a rigorous evaluation--I haven't seen a chi-square test yet--on looking across
a large number of cans out of a single lot that's known to be contaminated, I
do have to reflect that we have examined numerous cans from several of the
recalled lots. And I think we're more
in an issue of the level of contamination, at least with those lots, than we
are with "It's in the first three cans and it's not in the next 400 cans,
and then it's in the next three."
Because we have found it.
You also
have to keep in perspective, when we're talking about a classic one-pound can,
and you're down in an MPN of .3 per 100 grams, that basically means that you're
dealing with one-and-a-half in every can.
No, it's not even that. It's one
per every three cans. So you're already
in the type of distribution that you're--it's a rare event. You're not going to find it in every
can. But that doesn't mean that it's
not homogeneously distributed within the lot.
DR.
BUSTA: Dr. Lee.
DR. LEE:
Perhaps a generic question--as I understand it, E. sak wouldn't be a problem if
we are rehydrating with boiling water.
And certainly, one can formulate, in anticipation of that, by just
putting in more vitamin C to offset any potential losses.
So I'd
like to know what the contraindications are to formulating this product with
boiling water?
Well, as
far as I can tell, there are two contraindications--or two factors that haven't
been taken into account. And first I
want to emphasize--you don't have to use boiling water. That's 30 degrees too high. It really only has to be at 70. It needs to be hot water.
Second,
is we still don't have sufficient data on clumping. And while we don't think it is a large problem, it's material
that has to get through enteral feeding tubes.
And so clumping is something that certainly needs to be examined.
And
then--and probably the most significant thing--when you do heat, the formula
retains its temperature for quite a long time.
I showed you a cooling curve of the first ten minutes following the
heat. And there is--realistically, you
have to be careful. You can't feed 100
degree formula to an infant. They'll
get burned.
So it has
to be balanced by the fact that if you use hot water to rehydrate the formula,
there does have to be adequate cooling.
DR. LEE:
Just to follow up on clumping--if clumping does occur, is this formula normally
turbid? Is it clear if it's fully
dissolved? No?
DR. ZINK:
No. I mean, it looks kind of like, you
know, milk. And you can get coagulation
of protein that's fairly significant there without it's being obvious. You know, I can see it going into an enteral
feeding tube and clogging that tube up, and outwardly not looking like it had a
precipitate or clumps in it.
DR. LEE:
But it will pass through cheesecloth or some kind of filter, if that's needed?
DR. ZINK:
Well, I think it would--you know, when it clumps it clumps. You would get some of it retained on
cheesecloth, probably.
DR.
BUSTA: Dr. Fischer.
DR.
FISCHER: Fischer.
I was
surprised to hear that--no, I'm not surprised to hear that
powdered--formulation of powdered milk, or the production of powdered milk was
very similar to the production of infant formula.
If that's
the case, have you looked in powdered milk for the organism? And if it is there--and if it isn't
there--mightn't this not be clue as to where it might be coming from, in terms
of the raw ingredients involved?
First of
al, have you looked in DR. TARR: ?
DR. ZINK:
We haven't specifically run a survey of powdered milk. And having just recently joined the agency
from industry, let me say that in this job your capacity to cause mayhem by
your actions far exceeds that of what it was when I was in industry.
[Laughter.]
You
know. No, we haven't gone out and
looked at, you know, producers of dry milk for this organism. And while that might seem like a good idea
at the moment, I think I would want to think about that long and hard. You know, what am I going to do when I find
it, and what does that mean?
I really
would expect, if I did do that, to find it.
DR. BURR:
Actually, from the 1984, the Postupa and Aldova paper, they have four strains
coming from powdered milk.
DR.
BUCHANAN: Most normal analysis of powdered milk for gram-negative
Enterobacteriaceae is to determine whether or not there's E. coli in it. And so you have E. coli, and then you have
everything else, and you forget about everything else. No one tries to differentiate all
the--enterobacter serratia, klebsiella that may show up as fecal
coliforms. They're worried primarily
about E. coli, as an indicator organism.
So I'm
sure that it's been isolated from powdered milk. I just don't know of anyone that takes the time to try and
differentiate all those species.
DR.
FISCHER: Yes, it seems to me that's helpful--that information is helpful,
because then it would tell you it's not some substance you've added,
particularly after the drying process, like the vitamins, that is the source of
the organism.
I'm just
surprised that the source hasn't been stumbled on as yet. And it seems to me that is a key issue, that
would help everybody out, if the source could be found.
DR.
BUCHANAN: Can I answer this? And I
hesitate to do it because this is really a question that needs to be addressed
to the industry. But I know from past
experience of looking for the sources of listeria--and there's been a lot of
work on that, and people have literally torn their plants apart to find out
where a small micro-colony of listeria is harbored and causing problems.
These
plants are really complex places, and all you need is one little small niche
somewhere that's feeding into it. So,
it's not as simple a job as one might think to track down where in a plant
environment you actually have that organism residing.
DR.
BUSTA: Could I take the chair's prerogative and expand on that just a second?
Did I hear
correctly, in two presentations, that it's not in the environment?
DR.
ZINK: Oh no, it's in the environment.
DR.
BUSTA: Marie?
DR.
NAZAROWEC-WHITE: [Off mike.].
DR.
BUSTA: Marie,.you said in that list-
DR.
NAZAROWEC-WHITE: Right.
DR.
BUSTA: --that this was not the source.
DR.
NAZAROWEC-WHITE: Correct. That's right.
DR.
BUSTA: And that as quite an extensive list of all kinds of environment.
DR.
NAZAROWEC-WHITE: That's correct. And it
was not found in that particular--in those particular areas. But we have no idea whether they took five
samples, or whether they actually--you know, there has been no systematic
scientific approach to look at that environment.
DR.
BUSTA: So chances are where Enterobacteriaceae are, it is also.
DR.
NAZAROWEC-WHITE: Well, I would think so.
DR. ZINK:
I think this is a question for industry.
I believe, as Bob alluded to, is what went on with the meats industry
and listeria, I think this industry has disassembled their plants and done
extensive testing and has, you know, probably isolated the organism from many
places in their plant. And I suspect
that, you know, while as an agency we're not in possession of this kind of data
and information, I suspect that it's known, the types of plant environment
where the organism can be found.
DR.
BUSTA: Two more questions, then.
Dr.
Acholonu.
DR.
ACHOLONU: Alex Acholonu.
In one of
the background materials given to us, the title is "Powdered Infant
Formula: An Overview of Manufacturing Processes." It has a statement which I'm going to
read--this is for Dr. Zink.
"Typically,
finished product is held until it undergoes a final check for conformance to
specifications--"--and the next statement is "--including testing for microbiological contaminants."
My
question is what method would you use to check for microbiological contaminants
in a finished product?
DR. ZINK:
Well, what manufacturers do right now--what you want to do is you don't want to
produce value-added garbage. Okay. So if you can run your test for quality
conformance before you add value by putting it in a can and labeling it and
casing it, only to find out you have to throw it out, if they can they'll get
these tests done before they put it in the can. And they're typically testing to two kinds of standards. Each manufacturers has its own internal
standards; typically much more stringent than what a regulator would
require. So they're going to test for
conformance to their own internal quality assurance standards. And that could include things like aerobic plate
count, salmonella, listeria, coliforms, E. coli. They take the lead, it's been my experience, from what regulators
require. And they'll be also testing
for, you know, vitamin content, homogeneity--all those sorts of things.
Right
now, I think most manufacturers test for the kinds of things that a regulator
would, and they apply the same or more stringent criteria.
DR.
ACHOLONU: My understanding of a finished product is one that has been prepared
and may be canned and ready to go. Do
you understand--
DR. ZINK:
Well, you get into semantics.
Right. From a standpoint of
microorganisms, if you have that powder sitting in a tote or a big bag, it's
true that there is some potential for further contamination by the time it goes
through a can-filling line and into the can.
I'm
familiar with several manufacturers that will test product while it's in
bulk. Then they'll go ahead and put it
in the cans, and then they'll test it again.
DR.
ACHOLONU: They open the can and test it again?
DR. ZINK:
Yes.
DR. ACHOLONU:
Doesn't that give--make it get contaminated--
DR. ZINK:
Well, they open the can--usually, they'll have a biological cabinet to open the
can in. It's very common in these
plants to see laminar flow hoods, and--you know, they're able to open those
cans in a manner that precludes recontamination.
DR.
BUSTA: Thank you all very much for an extended question and answer
session. I appreciate this.
And now
we will go on to the public comment. We
have three presenters from the International Formula Council.
Public Comment
DR.
BUSTA: The first one that I have on the list is Dr. John Vanderhoff. He's vice president, Global Medical Affairs,
Mead Johnson Nutritionals, a Bristol-Meyers Squibb Company, speaking on
Overview and Risk Characterization.
[Pause.]
Will
there be a copy of the slides available, that you are using?
DR.
VANDERHOFF: If you'd like. I don't know
what we've decided to do about that.
I'm speaking for the entire group, so I'll have to defer that to you,
whether we want to leave these slides here or not. But they're--
VOICE:
[Off mike.]
DR.
BUSTA: All right. Thank you.
Proceed.
DR.
VANDERHOFF: Okay. I think we're waiting
on a little warmth from the projector here.
I very
much appreciate the opportunity to kick this off and try to give you what I
would like to call an industry overview of Enterobacter sakazakii, and how we
see it. And I think it's probably going
to be a rehash of all the different things that we've hear today. Hopefully we'll be up here in a jiffy.
I think,
as I recall, the first thing that we wanted to do was just to cover what is
Enterobacter sakazakii. And we know
that it's a gram-negative rod. We know
that it is--whoops. Now do we have to
wait for it to warm up again?
Okay. Tom, do you have the--that way, if it does
come up we won't be too far off track here.
But this
organism we've all said is an opportunist, which it's been probably in the
environment for some time, and has a predilection to cause central nervous
system infections. I think that's important. Because of that, these are not infections
that we usually miss when we treat children with infectious diseases. And so I guess the question is: are we
dealing with the tip of an iceberg? And
I think probably not. These things are
the sort of things that one usually sees and diagnoses.
The
epidemiology of this organism: we know that it's found in very low
concentrations in powdered infant formulas.
This seems to be a worldwide phenomenon. It's also been shown to be found in hospital environments. I know I reviewed one paper where numerous
swabs had been taken around hospital rooms, and several of the swabs
demonstrated the presence of this organism.
It's been found in numerous food products. It's even been found in the gastrointestinal tract of a number of
fruit flies.
So it's
probably--we don't know how common this thing is in the environment, but we
know that it's there. And we also know
that it's been--that it can occasionally be found in the stools of infants.
Yeah--go
ahead and bring us up to--next one.
[Slide]
And we
know that it's been cultured out of the stools of some infants who apparently
had no symptoms at all, as part of the epidemiological evaluation of outbreaks.
Slide,
please.
[Slide.]
Who's at
risk for the infection? Well, the
primary risk appears to be in premature infants, especially small premies that
are being treated in neonatal intensive care units. And these children are, of course, with some degree of impairment
of immunological function because of their developmental age. We also know that occasional term infants
have been reported to have become infected with this agent, but if you look at
the case reports, a fairly high percentage of these--and there were very few of
these--have some underlying condition, either immune or barrier function, or
some congenital anomalies or chromosomal anomalies and so forth.
And we
also know that immunocompromised children and immunocompromised adults appear
to be able to be infected with this organism.
So it preys on, basically, immunocompromised people, like most
opportunistic organisms.
Slide,
please.
[Slide.]
What does
it do? Primarily it causes meningitis,
ventriculitis, and maybe sepsis. We
know it has a predilection for the central nervous system. And we know that it can be present in the
gastrointestinal tract in asymptomatic children.
And as a
pediatric gastroenterologist, I would discourage you from drawing too close of
a connection between the reports of necrotizing enterocolitis and this
organism. Necrotizing enterocolitis
seems to be a condition that results when you aggressively feed a premature
baby, causing some degree of ischemia, and the bowel breaks down, and you get
an invasion with whatever organism happens to be there. And there's a long list of these. And everyone's thought they found the cause
of NEC, and that's never panned out before, and it probably won't with this
organism either. It can certainly cause
a problem if it's there and you have an ischemic bowel, but considering it an
etiologic agent for necrotizing enterocolitis is probably not correct.
Slide,
please.
[Slide.]
What's
the risk involved in this particular infection? There's 50 r 60 cases reported worldwide. And this is what we're basing our
information on. Since we're dealing
with case reports, there are probably more cases out there than we know about,
but it still suggests that it's a relatively rare condition. Most of these occur in premies. It appears to be an extremely rare condition
in health term infants, despite the fact that there are billions of feedings of
term infants with powdered infant formula, we seem to have only a handful of
cases occurring in this age group.
Next
slide.
[Slide.]
Well,
then why is it so uncommon? The
organism itself appears to be a minimally pathogenic opportunist. The infective dose is probably high. It's probably much higher than we normally
see in fresh reconstituted infant formulas.
In fact, if you look at the various case reports, almost all of them
suggest that there has been some mishandling--I would call it mishandling--of
the infant formula from the standpoint of hang times, or preparation or use,
that has let the formula sit around and proliferate, so that there's an
increase over the background number that's quite significant in a number of
these cases in which infection has occurred.
And most
of these have also included something like environmental contamination or
breakdowns, or contamination of blenders and so forth.
Slide,
please.
[Slide.]
What,
then, could the infant formula industry do to deal with this condition? Well, I think one of the things we can
certainly do is work with the FDA, the CDC and other organizations to do
everything we can to help educate users and health care providers. And this would be to do things like minimize
hang time with enteral feedings, to minimize the use of refrigeration to 24
hours, to use liquid formulas in hospital setting whenever possible--especially
in neonatal intensive care units.
We've
hear a little bit about boiling, and the risks associated with that. And we an industry have sort of discouraged
that because we're very concerned about mishaps occurring when people might
give a formula that's too hot to a baby and cause burning in the esophagus or
even an injury to the preparer of the formula.
And there's also now been case reports--at least one--of vitamin C
deficiency from a mishandled infant formula.
So these
things can happen and I think probably the major efforts we can utilize are on
this slide. And then I think we have to
do everything we possibly can to maximize the capabilities to reduce the levels
to as low as possible--which our next speaker will discuss.
Slide,
please.
[Slide.]
So, in
summary, I think Enterobacter sakazakii poses a limited threat--again, because
it's a rare condition--to preterm infants and other immunocompromised
individuals in a hospital setting. And
because it's occasionally spread through mishandled reconstituted powdered
infant formula, the infant formula industry wants to take an active role in
supporting the FDA and the CDC efforts to educate consumers and health care
professionals, and to work on new policies and procedures to minimize the
exposure of these high risk patients to this organism.
So, now I
think Les Smoot is going to tell us a little bit more about the manufacturing
side.
DR.
BUSTA: Dr. Les Smoot is Director of Food Safety Quality Management at Nestle
USA, Nutrition Division.
DR.
SMOOT: Good afternoon. Is this on? Can you hear me all right?
Okay. I'm going to spend just a few moments to,
hopefully, follow on and answer a fair amount of the questions--or at least
provide some further insight on some of the questions that have been asked most
of the afternoon.
Next
slide, please.
[Slide.]
First of
all, some comments on what is not known about this organism. We know genetic diversity is still something
we're debating and learning as we go along.
It would be important for us to understand the incidence and behavior of
the organism throughout the entire food chain.
Still we're gripping, trying to get a hold of where this particular
organism--the origin of this; how does it get into the food chain? So the specific environmental niches is
something that we don't have a real clear handle on at this point. And then, finally, optimum methods for
control, detection and recovery. I
think we're still in the very early stages of the science involved with,
particularly, the detection and recovery phase. And, as an industry, we're also continuous ly improving the
methods for control.
Next
slide.
[Slide.]
What is
known about the organism--and this is very similar to what you've seen for most
of the day--just recently recognized in 1980 as Enterobacter sakazakii. As we saw this afternoon, it's not unusually
heat resistant. I think it's very fair
to say it survives well in warm, dry environments. We'll talk a little bit more about that in a minute.
We also
know, in industry, based on our internal studies, that it is not overly
resistant to typical food sanitizers; and also knowledge that it does appear to
have a narrower host range of salmonella, one of the common food pathogens that
we worked against in this particular food product category.
What is
known about ES ecology? This is getting
to some of the questions that have bene asked around the table, relative to
where we find this in our food supply.
A recent report by Cordier, et al., has shown we've found it in skim
milk powder, a lactose, starch, powdered banana flakes and powdered orange
flakes--all of these being typical ingredients for infant cereals and infant
formulas.
Now,
Castano, et al., found it in raw pork for a dry-cure bacon. This was a Spanish study. Soriano, et al.--I think this was a Spanish
study, as well--found it in raw lettuce.
From and
environmental standpoint, Kandhai, et al., in 2003--this is a paper that's
being submitted to Lancet and should be published this year--out of the
University of Wagenen in the Netherlands, they found this organism isolated
from the environment--well, there were actually five milk powder factories, one
chocolate factory, one infant cereal factory, a potato flour factory, pasta
factory and numerous households. So
we're starting to see that this is not necessarily an unusual organism to be
found in the environment where food preparation or food ingredients might be
prepared.
As
mentioned earlier, there have been reports of its isolation from insects--this
one on a Japanese dairy farm, another one from the gut of the Mexican fruit
fly, by Kuzina, et al., in 2001. Two
isolations have been reported in hospital settings; one by Masaki, et al., in
2001 in Japanese geriatric hospital environment, and then most recently the
Block, et a., a pediatric hospital kitchen in Israel. So this gives us a little
bit of the flavor of the type of environmental ecology, as well as food
ingredient and food material ecology, hopefully answering some of the questions
that have been raised this afternoon relative to the industrial process.
What I,
hopefully, have here for you is a representation of the partial steps within a
food supply chain for the preparation of infant formula. This is kind of a general step-wise
progression. What we're looking at here
is potential sources for E. sakazakii, and also roots of recontamination of the
product. And, as mentioned earlier, raw
materials--there needs to be a better understanding of the raw that raw
materials play as a potential vector, bringing the organism into the
environment of the factory. I believe
Don Zink had mentioned this earlier this afternoon. Also, we have been just
discussing, and throughout the day, the concept of handling practices during
reconstitution and consumption.
Ut we
want to spend a little bit of item, obviously, where there's some interest is,
what type of intervention strategies are applied in the manufacturing process,
when we look at these various steps throughout the process as were detailed by
Don Zink in the wet and dry blending operations. What types of activities is industry currently applying? What kind of good hygienic practices are we
currently using to reduce the incidence of this particular organism from the
factory environment.
Next
slide.
[Slide.]
Well,
it's not rocket science, really, but it is something that we have to do and
continue to do, and we are currently doing.
Key to this is the raw material supply.
And, as mentioned earlier, we adhere very much to the good manufacturing
practices and the HACCP principles. You
see here the term "prerequisite programs." These are programs that are conducted and embraced within the
manufacturing arena that are prerequisite for a successful hazard analysis,
critical control point system to be in place, assuring the quality and, most
importantly, the safety of the food.
So, as a
prerequisite program relative to HACCP on raw materials, it's very important
that you have a very rigorous supplier quality assurance program; auditing,
working with the supplier, improving their industrial process to ensure a very
clean supply of raw materials.
Secondly, though you want to verify how effective they are in their
industrial process. So it's very
typical a very rigorous raw material monitoring program.
All of
this supports the use of the HACCP--the hazard analysis critical control
point--principles, particularly for raw materials, hazard analysis on the raw
material. Is E. sakazakii, a
significant hazard, likely to occur in the various raw materials that we would
use within our manufacturing base? And
I believe Don Zink had answered earlier that from the perspective of the FDA,
they have evidence that all manufacturers are adhering to these principles in
detail.
Secondly,
the processing environment. This seems
to be a little bit of--there's kind of an open question, in terms of
understanding our ability to control recontamination of the product
stream. Prerequisite programs here that
are very important; obviously they're good manufacturing practices; the
standard sanitation operating practices--this is your cleaning practices that
were questioned earlier. The microbial
surveillance programs. These are the
programs that are in place to verify the efficacy of those cleaning programs;
in other words, are we using hygienic indicators, whether it be
Enterobacteriaceae, whether it be coliforms, so on and so forth.
And very
critical to the processing environment, and I think this goes a ways towards
answering some of the questions--this "hygienic zoning" concept. This is something that's embraced in great
detail in many, if not all of the infant formula manufacturers, where we have
very strict separation of incoming raw material to finished product handling
and filling; also very strict separation between the dry aspects of the factory
operations as well as the wet processing areas, because we know the
Enterobacteriaceae, as well as the salmonella and listeria all need water to
grow. So, as mentioned earlier, the
drier we can keep our environments, in and where we have finished product
handling, it's most important to minimize potential regrowth and then
recontamination of the product stream.
So these
are the cornerstones that we are continuously using to combat the potential for
recontamination. In the process--and
additionally a prerequisite program very critical to the ability to exclude
organisms like E. sakazakii out of the process--is the hygienic design of
equipment. And I believe this is where,
over the recent years, there has been significant improvement. And this is one of the areas that the
manufacturers themselves, in terms of the design--as Don was showing you the
various diagrams, the various pictures of the spray dryers, the drying tunnels
and so on and so forth. This is where
technology has started to make a difference in terms of the hygienic design.
In terms
of the HACCP aspect on the process, as mentioned very clearly, the application
of thermal related critical control points.
And, finally, how effective are these intervention strategies? Finished product testing is not an
intervention strategy. It's primarily a
HACCP verification program, to assure that these particular activities and raw
materials, process environment control and process control, have been effective
in assuring the highest quality and safety in the finished product. So the finished product monitoring that
we've been discussing about, in terms of the surveillance data, is really there
to verify the effectiveness of the HACCP program and these prerequisite
programs. And as mentioned in the White
paper on manufacturing, finished product monitoring is used before release of
products.
Everyone
has been talking quite at length today about the Van Acker 2001 report. This is a set of unpublished data. This is data from that particular factory in
the Netherlands. Shortly after the even
occurred in the summer of 1998, there's mentioned in the CODEX risk profile and
numerous speakers about the enhanced manufacturing practices were put in place
at this particular facility to assure improved safety and quality of the
product.
Here we
have four consecutive years of monitoring the finished product. These were 100 gram samples coming from that
particular facility. This was a
targeted study, above and beyond the ongoing monitoring criteria that were
utilized--cited in the publications.
But we have here the percent of these samples that were positive for EB,
and then the percent of those samples that were positive for E. sakazakii. And through the years, through continuous
improvement in areas, particularly in hygienic design of the process, as well as
environmental control, really learning how to manage the use of water in these
dry processing environments, through a relatively level number of samples over
those four years we can see a dramatic decrease in the percentage of
EB-positive finished product and, correspondingly, see a dramatic--or a
reasonably significant decrease in the presence of E. sakazakii in those
finished products.
So this
coming from a well-celebrated problematic factory can show how enhanced
environmental and process controls can make a difference, can drive this number
very low, but not necessarily to the point of eradication.
Next
slide.
[Slide.]
So, the
frequency in finished products has been reduced through environmental and
process control. And historically we
know that CODEX standards have been used to assure the safety of infant formula
for the intended population. And the
industry is proposing, at this point, a more stringent standard than the CODEX
standard that's currently based on coliforms.
Now we've seen those standards cited earlier. I think they were shown in Don Zink's presentation.
So this
next slide is the industry proposed sampling and testing program for powdered
infant formula. You'll not that we have
expanded--as has been mentioned in some discussion--this particular standard to
the entire Enterobacteriaceae family.
It's a sampling program on per lot basis, where the sample size would be
5 grams--the analytical unit would be 5 grams.
The number of units tested would be five, and an acceptable marginal
quality would equal 1, where if there were no samples--basically, what we have
here is a qualitative test, where you have five 5-gram samples; a total of 25
grams being tested. If all samples are
negative, then you would accept the lot unconditionally. If there were one sample that were to show
positive, then you would only accept the lot if that particular positive sample
was confirmed to be negative for fecal coliform. If it was found to be positive for fecal coliform presence, the
lot would be rejected. And then if
there was a chance where there were two of the lots found positive for
Enterobacteriaceae, then there would be a conduct of an intensive investigation
looking for root cause, as well as confirmation of presence or absence of fecal
coliforms, and this could lead to either acceptance or rejection of the
finished product lot.
Next
slide.
[Slide.]
So, in
summary, really, the industry's ability to control this organism in finished
product really comes to the implementation of a phased effort through raw
material management, and working with the suppliers, continually improving our
ability to control the organism in the environment; going after, looking at
niches in the environment. We do know
that this organism has the ability to survive in dry, warm environments, and in
that particular study, performed on the Netherlands factory, we did see a
higher prevalence, typically at the bottom of the spray dryer, in the
warmest--one of the warmer parts of the building.
Process
control--really, the key there is the hygienic design for us, in terms of
improving how the equipment is designed to minimize the possibility for
micro-niches to be development; and then, ultimately, then finished product
verification testing.
Thank
you.
DR.
BUSTA: Thank you, Les.
And the
third presenter is Dr. Russell Merritt, Director Medical Affairs, Nutritionals,
Ross Products Division of Abbot Laboratories, who will be speaking on
industry's response to FDA charge.
DR.
MERRITT: Thank you, Dr. Busta, Dr. Heubi.
And Thank you for the time to present hereto the committee today.
I'm
speaking to you from the perspective of a clinician who worked in nutritional
support, including neonatal support, for many years, and more recently have
been very involved in industry and our concerns with safety.
You have
heard this afternoon a clinical summary of the issues from the industry
perspective, and also a summary of the manufacturing issues and some of the
steps that we are proposing to move forward with. It's my goal to bring this industry perspective into the context
of the specific charges that FDA gave to this committee. So I will comment specifically on the FDA
questions, and this commentary is available to you as well in written form.
Next
slide, please.
[Slide.]
The first
charge was: given available information on E. sak and powdered infant formula,
is there a risk? Are there
sub-populations at risk? And what about
immune status, general health, etcetera, in regards to the susceptibility to
this infection.
Next
slide, please.
[Slide.]
For
preterm and immunocompromised infants, we've heard considerable material today,
and it indicates that there is a rare risk to such infant. Somewhere in the neighborhood of 75 percent
of the identified cases have a birth weight less than 2,500 grams, or the
infants were premature. There's some
suggestion--particularly in those cases where there were issues with formula
preparation, storage and delivery, that there may be a dose effect. It appears that it requires more than simply
the presence of an organism--one organism in two or three cans of formula--but
rather, the presence, a susceptible host, and then presumably some defect in
the environment, by which the formula gets from the can to the baby.
The
situation appears to be quite different in regards to healthy, term infants. The literature does not--particularly in
North America--support the evidence of a clear association between consumption
of a formula known to possess this organism, and the development of serious
clinical infection. If any risk exists,
it needs to be put in the context of the number of feedings of infant formula
that are used per year, and the extraordinary rarity of this infection. If you think about it, a baby probably gets
about 2,000 feedings over the course of a year, and maybe close to half of
those in the U.S. come from infant formula.
Then crudely, that gives you something on the order of four billion
feedings--or two billion feedings a year of the four million babies who are
born. And yet one rarely sees this
organism. We heard earlier about the
CDC screening program in 300 hospitals, where a case was identified, to my
knowledge, not associated with infant formula.
So part
of the critical task of the committee is to differentiate the possible presence
of E. sakazakii on occasion in infant formula from the risk of infection in
infants.
Next
slide.
[Slide.]
If there
is a risk, what intervention strategies can be used in infant formula
manufacturing processes and plants?
Well, you
just heard from Dr. Smoot that there is an opportunity in almost any situation
to enhance environmental monitoring and controls, using the HACCP
principles. And you have just seen an
excellent example of where not only E. sakazakii, but also enterobacter,
presence in infant formula was substantially reduced by adherence to these
principles.
Industry
is also proposing confirmatory final product testing for enterobacter. Again, this is a confirmation of the
effectiveness of the environmental and the incoming ingredient controls that
are placed on the manufacturing process, more than the method of controlling
the product. The testing is not the
method, it's the indication that those methods are working.
Next
slide.
[Slide.]
Are there
other intervention strategies? And I
think, clearly, we've heard that there are.
We can minimize the use of powder in at-risk infant populations to the
extent possible. We've heard from Dr.
Thureen's experience a little bit about how difficult this would be to
eliminate completely. And I believe Dr.
Bhatia may address this issue as well in his comments.
We can
promote the published guidelines from the CDC, from the American Dietetic
Association that emphasize appropriate hygienic practices within the
hospital. And, again, picking up on Dr.
Thureen's comment, I was impressed that perhaps if neonatologists understood
more readily that powdered formula is, in fact, not sterile, then the
motivation no their part to control the use of these products, both in terms of
quantity and in terms of practice, might in fact represent an opportunity for
us.
The hot
water reconstitution process has been described--also some of its limitations,
including possible incomplete bacteriocidal kill, vitamin C loss, protein
denaturation and resultant clumping, and injury that may result to personnel preparing
the formula, if we have boiling or hot water in the nursery environment.
In
addition, there is opportunity, I think, to limit hang time. And based on what we've heard today, the
four hours seems to make some sense, and more consistent labeling and
directions for preparation and use is another opportunity.
Next
slide.
[Slide.]
Continuing
the second charge: is it possible to identify lower levels of E. sakazakii in
powdered formula and, in effect, assign allowable levels?
Next
slide.
[Slide.]
For
premature or immunocompromised infants, the data are insufficient to really
specify an allowable level, given that there's at least some degree of risk
there. For healthy term infants a low,
non-zero level can be established, based on the historical exposure data--for
example, 5 to 15 percent of infant formula, based on a number of surveys. The extraordinary rarity of this infection
in term infants, and the data we have heard today about the numbers of organisms
that appear to be required in neonatal animal inoculation studies.
Next
slide.
[Slide.]
What are
the critical knowledge gaps and research opportunities?
Next
slide.
[Slide.]
Well,
certainly we need to understand more about the circumstances that need to
opportunistic pathogenicity; whether it's susceptibility factors in the host,
or the virulence factors in the organism itself. We need to understand of the incidence and behavior of the
organism in the food chain. I think Dr.
Smoot's comments were helpful in giving us some direction in that regard. The probability and circumstances under
which pathogenic levels--that is, levels capable of causing infection in
susceptible populations--occur. And
then, lastly, we need to focus attention to the methods that may potentially
give us environmental control, whether that's in ingredients a the level of
plant manufacture, or at the site of use.
So,
again, I thank you for the opportunity to provide these industry provisional
answers to the questions at hand.
DR.
BUSTA: Thank you, Dr. Merritt.
The
fourth presentation this afternoon is by Dr. Jatinder Bhatia, Medical College
of Georgia. The title is, "Use of
Powdered Additives in Neonatal NICUs."
DR.
BHATIA: Dr. Busta, Dr. Heubi, Ms. Latham, distinguished members and
guests. Thank you for the opportunity
to share my thoughts with you.
I come
from the perspective of being trained as a neonatologist and a nutritionist
who's been practicing his trade for a while.
Next
slide, please.
[Slide.]
The needs
have been highlighted because we truly don't have a gold standard for pre-term
babies. And the gold standard the AP
has put forth is that post-natal growth that approximates the in utero growth
of a normal fetus. Well, the question
that can be raised is: should you achieve maximal achievable gain without
adverse metabolic and other costs?
We know
from a whole body of literature that growth of most of these infants remains
lower that in utero, and that the nutrient intakes remain lower than the fetal
counterpart during much of the in-hospital stay, and possibly much beyond.
Now, what
are the consequences? And this is has
been known in the animal literature and the human literature for a long
time. Under-nutrition, especially
during these vulnerable periods of brain growth, can have permanent CNS
development, cognition, behavior and somatic growth effects. And these effects may be present, even if
there's sub-optimal growth, as shown in animal studies. For example, an animal doesn't grow, he may
have these side effects, but if the animal grows suboptimally, he may still
have the same effects. Therefore
premature infants are at high risk.
And I
don't need to remind this body that the extremely low birth weight infant has
additional needs for protein, calcium, sodium that far exceed those for the
term infant. And these, perhaps, may
not be completely met with the armamentarium that we have at present.
All this
information has ben reiterated by the NICHD-sponsored trials published
recently, which has heightened the awareness of practitioners of nutrient
intakes and growth curves. These were
something that we knew, but we did not have.
And I want to share with you the data on these three cohorts of very
small premature babies--24, 25, 26, 27 and 28, 29 weeks. In general, birth weight was regained by
about two-and-a-half weeks, with the larger babies gaining them a little
sooner. But the more important point is
the next one. By 32 weeks
post-conceptional age, growth curves of all cohorts fell below the 10th
percentile--and I'll show that to you in the next slide graphically.
The three
cohorts that you have here--the first one being the 24, 25--the largest
one--all three of them, even if you look at 36 weeks post conceptional age, all
fall below the 10th percentile, and the smaller babies may not catch
until almost the first year of life.
This is despite the maximal feeding that we can do currently. So we are taking appropriate-for--age
babies, and now making them small for gestational babies by the time they leave
the hospital.
So, at 32
weeks the average weight of a 24, 25 infant was 50 grams below the 50th
percentile; 26 to 27, by 780, and the larger cohort by 715 grams. And the postulated reasons, at least to
practicing neonatologists, is the expected post natal weight loss, and our
inability to feed babies to the maximum potential, and therefore under
nutrition.
So what
are the strategies that we use? Early
initiation of parenteral nutrition, starting on day one or day two; early
initiation of enteral feedings, also limited by fear of necrotizing
enterocolitis--although, as Dr. Vanderhoff has pointed out, and other people
have pointed out, that the starting or not staring of enteral nutrition early
has no association with it. There are
other associations, which I'll come back to later; increasing nutrient density
while maintaining relative fluid restriction, because we can only go so far as
far as fluid volume goes, and therefore the need for supplements.
What do
we have available? Liquid concentrate,
powdered formula--either the premature or transitional formulas--or single
nutrients. Or we have MCT, vegetable or
lipid emulsions.
Here's an
example of what you would do if you want to increase caloric density by either
using a single constituent or a balanced powdered formula. For example, on the left hand slide, a 24
cal premie has 81 calories, and draw your attention to the completely far right
hand corner, that if you add a proportionate amount of powder to the premature
infant formula, you can achieve 98 calories per 100 ml; you can increase the
protein intake and, more importantly, you can also increase calcium intake,
which is shown on the bottom right-hand corner; whereas, if you use corn oil,
as an example of vegetable oil, or polycose, all you do is increase calories,
you do not increase protein, and you don't affect calcium. So a balanced addition of nutrient is far
better than using single product.
Now, I'm
going to share briefly some disease-specific concerns, and why these are
important. For example,
bronchopulmonary dysplasia, a chronic lung disease, a fairly common entity
known to neonatologists. Anywhere
between 30 to 80 percent, depending on the cohort you study, will have
continuing needs. And there is need for
additional nutrients to compensate for the increased work of breathing, and
side effects of common drugs. And, more
importantly, these babies also need the same nutritional intervention after
they go home, because--several studies have been done. For example, one study has clearly
demonstrated that afterwards 73 percent of babies, after they leave the
hospital, now deviate in their growth.
And this can be obviated, to a certain extent, by using nutrient-rich
formulas, which then come from supplementation. We also have an issue of fluid restriction. And since they are also born prematurely,
they also have high needs of calcium, phosphorous and electrolytes.
Necrotizing
enterocolitis, which we've been hearing all day--the only two factors that I
know that have held the test of time have been a baby has to be premature, and
maybe the feedings have been advanced very rapidly, and this has been
reevaluated again as of this month.
More importantly, in the clinical arena, timing of NEC does not
always--or usually does not have--a temporal relation to the addition of
powdered nutrients. And there's a wide
range of onset of NEC, with less than 40 percent of them having concomitant
positive blood or other cultures. In
other words, you heard this morning as well, that there are whole host of
organisms that are associated with NEC, but there is also a high incidence of
NEC without any organism identified at all.
So what
are the long term consequences? We know
that very little catch up growth occurs.
I've already shown you the growth curves up to post-conception age of 36
weeks. We have been creating, and
continue to create, shorter, lighter infants, with a smaller head circumference
compared to the fetal counterpart, and now data as long as eight years of age,
because of the early growth restriction that occurs in the NICU.
And the
neurological outcome of these babies is not always explained by disease process
alone. So I believe that nutrition, or
under nutrition, has to have played a role as well.
And
here's a good example of that. These
data were published in 2001, and what they're showing, the left hand, at birth,
that babies who were above the 10th percentile, and by the time
they're discharged, the number of babies who are above the 10th
percentile. And you can see, between 70
and 85 percent were appropriate or above 10th percentile at birth,
and barely above zero--way above 10th percentile, by the time they
reached 36 weeks post-conceptual age.
And this has been shown, not only in England, it's been shown in other
places in the United States as well.
So,
post-discharge, which is now becoming a more and more important thing--evidence
demonstrates better growth with enriched formulas, i.e., the transitional
formulas, or using premature formulas at home.
These infants are predominantly fed powdered infant formulas due to
cost. And, with earlier discharge from
the hospital, calcium and phosphorous, and other nutrient needs cannot or may
not be met by term-infant formulas, and this statement is supported by the
American Academy of Pediatrics as well.
So in-hospital
practices--given the variety of scenarios in the NICU--and this is my own
anecdotal data, and I believe, if you surveyed, you'll find similar data. And Dr. Thureen can confirm that for me--up
to 50 percent of infants in a large NICU will have some form of feeding
modification--either addition, thickening due to feeding intolerance or
concentrating. However concentrating
inherently has a problem with changing osmolarity, which then limits the
feeding problems as well.
And
rather than asks the question of how have the in-hospital guidelines suggested
by CDC affect the current practices, what we're now seeing is while trying to
adhere to the CDC, ADA and FDA guidelines, that neonatologists and
nutritionists are reluctant to use appropriate measures to try and achieve the
maximal achievable gain that I said before.
So we've gone backwards, at least in ability to provide the nutrition we
believe we need to.
So how do
neonatologists weigh the risks? We need
to establish enteral feedings. We all
agree with that. We need to establish
adequate weight gain, because mortality, morbidity and long-term outcomes are
clearly related to weight and gestational age.
And the risk of E. sak--20 cases per 10 years, or however you want to
count it--should be put in a perspective that during the same period of time,
there were more than four million premature infants who were fed and delivered
during that period of time.
Thank
you.
DR.
BUSTA: Thank you. We have about 10
minutes to ask questions of these last four presentations, if there are any
questions.
Dr.
Stallings.
If the
four of you would come up, we'd appreciate it.
[Pause.]
You just
have a comment, Dr. Tompkin? All
right. Go ahead.
DR.
TOMPKIN: Yes. This is Tompkin.
The white
paper that was on the table in front of each of us is the white paper that the
industry has prepared and shared with us.
And, likewise, the information that Dr. Merritt presented, in terms of
the industry's perspective on the answers that may be the outcome--that also
was prepared by industry. So each of
you should have those documents.
DR.
BUSTA: Dr. Stallings.
DR.
STALLINGS: Stallings.
I have
almost a question--short question--for each of us. So, starting with John--if we were starting at the beginning of
this question--and in your slide you summarized that you felt that the
pathogen, it was a minimally pathogenic organism, and that the infective dose
was high.
And I
guess I'm not convinced of that. And
what kind of animal models, neonatal animal models--I mean, there should be
some pretty straightforward ways of testing that. Do you, or you guys, have any ideas? Because I would feel a lot more comfortable if I knew that from
something other than we've given, you know, four billion feedings and 10 babies
have gotten sick.
DR.
VANDERHOFF: I think at this point in time that is an assumption that we're
making. And it would be nice if we
could verify it with some animal models.
And I'm probably not the ideal person to tell you which ones would be
most valid, but I know there are some.
But this, I think comes from the epidemiological knowledge that there's
a huge amount of feedings--Jatinder just told us--of powdered infant formula to
pre-term babies; half the premies get added powdered. And, of course, all the term infants being exposed to low levels
of this stuff, 14 percent of the formulas appear to have low-level
contamination, according to the data yet, you know, I've never seen one of
these. I asked Jatinder if he'd ever
seen one? I don't know if you've seen
one.
But this
is a very uncommon infection. And it
would certainly suggest to us that there must be some other factors, such as
mishandling or host defense problems or so forth, involved in doing this. I agree, I think investigating it with an
animal model would be ideal. I'm not
sure we can a hundred percent draw the connection between the two, but it will
certainly give us an idea.
DR.
MERRITT: If you think about it, you're never going to have the dose response in
the human, because the event is going to occur sufficiently long after the
event, that the actual concentration in that formula will never be done. So that forces you to a dependence,
presumably, on an animal model to try and get that answer. And Dr. Nazarowec-White--I mean, we were
desperately searching for some of that kind of information. And the paper that came out this month I
think is the first clue in that regard.
And, at least, I believe, suckling animal model, it indicates that the
number of organisms is quite high, and if the practices we are suggesting that
we all adhere to in conjunction with the levels of detection that were
described were used, we shouldn't see those levels.
Now, I
don't think we have any way of knowing that the mouse is the same as the 800
gram baby, but that's the best information we have, I believe.
DR.
STALLINGS: But, really, it's not the 800 gram baby that's getting the
formulas. It's usually a little bit
later. But--okay.
So, the
next question--for Les. How big is a
lot? You know, when you were describing
what's been proposed, it was, I think, 5-gram samples times five samples. And I'm just not familiar enough to know
what is a lot, and, you know, really what kind of surveillance might that
represent?
DR.
SMOOT: Right. I can only speak from our
experience with Nestle. Typically, a
lot for us might range in about a 20 ton--
DR.
STALLINGS: How many?
DR.
SMOOT: 20 tons.
DR.
STALLINGS: Okay. That's a lot of cans
of baby food.
DR.
SMOOT: True. Yes.
DR.
STALLINGS: 20 tons, and we would be sampling about 25 grams--5-grams times
five.
DR.
SMOOT: For the family Enterobacteriaceae--correct.
DR.
STALLINGS: Okay.
And then
my last question is, are the HACCP regulations--are they regulations? Are they required, or are they voluntary at
this point? Because there was a lot of
reference to using that kind of evaluation of manufacturing and risk
analysis. And I'm--
DR.
SMOOT: I don't believe it's mandatory on FDA.
It might be infant formula regulations, the GMPs--we see this more of a
mandated effort. But in our
manufacturing base, typically HACCP right now, the use of these principles I
would say to be voluntary in these types of processes.
DR.
STALLINGS: So I just wanted to be clear that that might be required in the
future, but right now it remains voluntary.
Thank you.
DR.
BUSTA: Dr. Moyer?
DR.
MOYER-MILEUR: Moyer Mileur.
I think I
want to direct my comment to Dr. Bhatia, but then get response from all of you.
I think
your comments--I appreciate your comments, and I think you're preaching to the choir
when it comes to Dr. Thureen and myself, as far as the extremely small or
extremely premature infants not being served well by--in that growth is
minimized during their hospitalization, and continues to be minimized.
The
concern I have is that I don't see this as an--the issue of powdered formula in
these babies as an issue until later on in their neonatal period. And my personal experience with these babies
is that we really can't even get up to a full volume of enteral feeding until
at least 21 days of age, at which time we would either be using mother's milk
or a liquid premature infant formula and then go to 24-calorie, and then if not
able to get them to grow, then start to consider adding additives to those
products.
So my
bigger concern is that I think the message you had is that you're worried that
neonatologists will stop--will not provide sufficient nutrition because of the
worry about contamination from E. sakazakii.
And I don't know--and so I think maybe directing this critical need for education
to neonatologists, but not only neonatologists, but other care providers who do
see preterm infants or other infants who are immunocompromised, and that
includes pediatricians, that includes nurse practitioners, both neonatal and
pediatric nurse practitioners, as well as registered nurses.
I don't
know if you--
DR.
BHATIA: Well, I would have changed my slides if I'd known you two were going to
be here. Anyway--the point's well
taken. The approach we take is exactly
the same. But the point I think I was
trying to get across is that without these additives available, we will not be
able to even feed a human-milk fed baby or give the additional needs of
specific concerns. I can go on and on.
But these
are not additives you add in the first two weeks of life. These are much later. This is not to the period of time when you
see this organism affecting babies, so the temporal relationships seem
different. My issue was: these are the
only things we have left now to try and do better with what we have. The current formulas for premature babies
were made when survivors were above 750 to 800 grams. They were not designed for 500 to 800 gram babies. So the best we can do is use a balanced
additive, and that's the point I was trying to make--including the human-milk
fed preterm baby.
DR.
BUSTA: Dr. Heubi.
DR.
HEUBI: Heubi.
My
question is one that I think both John and Russ commented
about--immunocompromised and pre-term infants being potentially at risk.
What are
your thoughts about, number one, the issue of kids that have motility
disturbances, short gut, other kinds of problems, who are exposed to potential
"specialty formulas" that tend to be non-liquid in composition and be
powder instead, and whether you would consider them to be a potential risk from
exposure because of their underlying condition? And then I have another question after you answer that for me.
DR.
VANDERHOFF: I think I've seen one case report of a short-gut getting infected
with E. sakazakii, despite the fact that there are a lot of these, and they've
been fed with this stuff for a long period of time. And I've seen them get infected with almost every other enteric
organism over the years. So I think
it's relatively low risk.
The
motility disturbance kids--probably same argument. Anything you feed to these kids, because they have mucosal
breakdown, anything that grows in their gut can get into their blood
stream. But I don't think it's a big risk
for kids with GI tract disease.
Russ?
DR.
MERRITT: I think, clearly, there's a literature that indicates that short-gut
is a special situation immunologically, and these children are often not well
nourished as well. So it's hard to deny
some potential there. They become
somewhat more like the premature baby, in terms of having impaired
gastrointestinal function and barrier.
DR.
HEUBI: So, my follow-up question is this: what position will industry take in
terms of trying to educate physicians and health care providers--just like what
Laurie was pointing out--about the fact that these formulae that are powders,
are not absolutely sterile. Because I
think, in general, most clinicians don't understand this. Now, I don't know that we should scare people. But I think, on the other hand, it should be
clear that these preparations are not sterile, and that there are some
potential minimal risks, or risks associated with their use.
Can you
comment on that?
DR.
MERRITT: I think the point is well taken, and I think what the recent
experience with this organism has pointed out to all of us is that there are
differences in products, according to form.
And I think there probably has been a lack of education in clinicians,
even those most involved in the care of these kinds of infants, in this regard.
And so I
think that does create an opportunity for us, because I believe if clinicians
understand the risk they'll take all steps they can to avoid it. And if they really have not fully understood
the potential risk--even if small--up until this point, then I think that gives
us some cause for optimism.
DR.
VANDERHOFF: Yes. One other comment that
concerns me a bit is that--I think there's an awful lot that you can do to use
these formulas in a way in which the risk is very low. There's also a risk of getting people so
concerned about it that they utilize products that may not be as useful, simply
because they're in liquid form. And I
think this was a concern you brought out, Dr. Bhatia.
And so I
think the educational thrust needs to be toward how to use the products rather
than "don't use them" if they are, in fact, the most appropriate
product to use.
DR.
MERRITT: Clearly, many of the infants we're describing are within what appears
to be the critical period for developmental programming. And I think the neonatologist is in a
tremendous bind here, because he's, one, worried about the acute risks that
nutrition poses to the immune system; secondly, he is trying his best to
prevent the long-term sequelae, neurologically, of being malnourished for an
extended period of time. And now there's
concern that one of the tools that may be most effective in helping him deal
with these two issues has another side to it.
And that's why I think--I think there's such a need to put this risk in
perspective.
DR.
BUSTA: Dr. Thureen.
DR.
THUREEN: Thank you. Thureen.
I have a
two-part question about human-mil fortifiers.
Are they processed in the same way as powdered infant
formulas--breast-milk fortifiers?
DR.
MERRITT: In general, these are powdered products, and so the basic principles
would be the same. Their composition is
different, and that requires some modification of processing.
DR.
THUREEN: If you do do an educational process to inform clinicians about the
downsides of using powdered formulas, and they should be in a different way,
would that extend to human-milk fortifiers, too, in that that may be the most
valuable for the infant powdered product that you make.
DR.
MERRITT: I think that's another educational task, because I can tell you, in my
capacity, since these recommendations came out of CDC--and for a while there
was a recommendation to use hot water for reconstitution and the like--we saw
some hospitals turning to strange practices to achieve the desired result--in
an attempt, you know, to be compliant with infection policies within the
institution, or to develop new ones.
And so I
think, again, there is an educational need.
That's another product where there isn't a particularly good substitute
for the powder, because the concept is that the mother's milk is available,
with all of its potential benefits, including immunologic. One likes to dilute it as little as
possible, and get the nutrient content up.
And that becomes, then, the advantage of using a powdered as opposed to
a liquid isocaloric fortifier, which can bring the volume up but does not do
anything to increase nutrient value--or less to increase nutrient value.
DR.
THUREEN: So do you think that human-milk fortifiers should really be excluded
from this consideration because it has such an exceptional role and the volume
that's used is actually quite small?
DR.
MERRITT: [Nods affirmative.]
DR.
BUSTA: Dr. Fischer.
DR.
FISCHER: I'd like to ask about your recommendation on what to do according
to--when you monitor the finished product, what you're going to do. This has to do with the sample size equals
5, n equals 5, c equals 1. And we
have--when c equals 0, then we'll accept the lot unconditionally. When c equals 1, then accept the lot if
identification of fecal coliforms is negative; reject lot if identification for
fecal coliforms is positive.
And then
when c equals 2, then conduct--it says, "--then conduct additional
investigation, up to and including rejection of the lot."
I'd like
to know what the criteria would be, after the investigation, for rejection of
the lot in that case.
DR.
SMOOT: For that particular scenario, when we say "investigation"--and
it would also occur, I would assure and assume, even with the c equal 1. Typically, in manufacturing, when you have a
product that's going to be determined marginal, in terms of its release
criteria, you will typically commence an investigation to look at criteria that
have been developed relative to the monitoring of the environmental hygienic
condition. You're going to look at
process controls, in terms of process indicators in the actual line of the
process. You will look for evidence of
manufacturing practices where there might have been interventions on the line;
product stops, starts--things of this nature.
You're
looking for a root cause: why would you find something that's out of
compliance? If you find evidence that
you can--as mentioned earlier this morning--a smoking gun where you find that
there is clear indication that there was an event that occurred that would
allow the product to go out of compliance on that, you would typically do a
safety assessment at that point to determine with the level of contamination
that you have, indicated by this qualitative test--which now you would have two
out of five samples positive for an Enterobacteriaceae--you would look at the
confirmatory tests that are conducted on that, like the fecal coliform, very
possibly further to E. sakazakii, and from that you would have enough
information to determine is there a food safety risk associated with that
particular lot with the c equal 2. And,
from that point--you said up to and equal to the rejection of the lot.
DR.
FISCHER: Let me see if I have this straight.
I assume that you would do the analysis again, wouldn't you? Wouldn't you start out by making sure that
the c equals 2 is really correct? Or
not. You're saying--
DR.
SMOOT: Well, not provided in this particular monitoring specification. I think it might be very safe to say
individual manufacturers might have a secondary, more detailed examination that
you would go and subject the lot to.
So, you could very well go into a secondary examination, where there
might be a more extensive test, and that might be a part of the investigation
that's referred to--yes.
DR.
FISCHER: So, you--
DR.
SMOOT: But we don't try to test the product into compliance. I mean, that's not the intent.
DR.
FISCHER: I assume you would write down, for the FDA I suppose, the criteria you
would use to ferret out--
DR.
SMOOT: That would definitely be spelled out.
Correct. Yes. Just in an abbreviated form, in terms of
sample size, number of units, "this is what we're proposing at this
point." But, yes, there would have
to be more detail relative to the types of decision criteria on that c equals
2.
DR.
BUSTA: I'm going to limit this to two more: Dr. Lee, and then Dr. Tarr. Dr. Lee first.
DR. LEE:
Yes, this is Ken Lee.
I think
it was Dr. Merritt, but I'm not quite so sure--somebody made a casual mention
of the protein denaturation. If you're
using 70-degree or even 100-degree reconstitution, are you really changing the
quality of the proteins? Is there any
change in digestibility?
DR.
MERRITT: Well, certainly with some of these formulas, one gets substantial
clumping in the context of what we're describing. And therefore I think you introduce some uncertainty regarding
nutrient delivery. You may also have
the issue, as described, relative to passing a given feeding through a
nasogastric tube, or a nasojejunal tube, which is something one likes to change
as infrequently as possible.
DR.
BUSTA: Dr. Tarr.
DR. LEE:
Just to continue that thread, though--those proteins, if they are thermally
professed by a wet preparation process, they would have been denatured, then,
at the factory, would they not?
DR.
MERRITT: Well, I can tell you that our empiric data of utilizing the suggested
procedure results in some clumping.
DR.
BUSTA: Dr. Tarr.
DR. TARR:
Tarr.
I've
heard three reasons today to continue to have powder available for, presumably,
the high-risk premature infant: first, expense; second, bulk and storage; and,
now, most recently, the probable or possible nutrient value of being able to
deliver more assets to the child per unit volume.
I'm
concerned that that third approach will run the risk of administering
inappropriate amounts of solute--solute load to premature infants, in the goal
of attaining growth. And powdered
formula may not be medically the most appropriate way to address this--the
growth issue.
I do not
mean to minimize the growth issue, but I am worried about adding another
problem to that without definite benefit, and also retaining the small risk of
an infection.
DR.
BHATIA: Maybe I should restate that given the constraints of time, I did not go
into renal solute load, and all those calculations have been done.
If you
take the example Dr. Thureen about a purely human-milk fed baby, that
human-milk fed baby, under current guidelines, or any guidelines, will have to
have an additive added. The type of
additives I'm talking about is on the same order of magnitude. We're not talking about substituting an
entire proportion of formula, we're talking maybe to fine-tune the formula,
take it from 24 calories to 27 calories.
So you're talking about one-eight of a teaspoon. And we've actually done osmolarity and renal
solute load calculations. They are well
done by other places as well.
It just
is heightening the awareness--we need one step higher; just like heightening
the need for premature babies. We know
we cannot feed premature babies plain human milk at all.
So, the
nutrition risks, if you will, are far less than any other risks at this point
in time, infection notwithstanding.
DR.
MERRITT: If you take the example of the post-discharge formulas, which have
only a 10 percent difference in caloric concentration at recommended dilution,
and maybe a 20, 25 percent difference in protein content, there are at least
three studies now which show better growth in these infants when given such
formulas.
I think
what you'll find in many nurseries is a common practice of taking the feeding
up to 27 calories, more rarely 30. And
I think most clinicians are hesitant to go beyond that level, both because of
free water concerns, and also because of the osmotic concerns. So the usual modification is modest. But in light of the data that Dr. Bhatia
showed on the growth history of infants below 1,500 grams. In light of the data, for example, that Alan
Lucas has presented, that as little as three weeks of feeding
intervention--that is, the difference between a term and a pre-term formula--will
make a substantial IQ points different--or IQ substantially different, perhaps
as late as eight years, or now longer.
I think
there is a great deal of pressure on the health care provider--and probably
appropriately so--to try to assure better growth of these infants than we are
currently seeing.
DR. TARR:
As a follow on--can we not come up with a panel of liquid sterilized formulas
to meet those needs in the majority of children?
DR.
VANDERHOFF: Russ, maybe you can say more about this than I can, but there are
technological considerations--I hope I'm saying this correctly--that exist in
making a liquid infant formula that don't exist in making a powdered infant
formula. And when you push the nutrient
concentrations up to a certain level with a liquid formula it just doesn't
work. I mean, you can't make it.
So,
that's almost kind of a limiting step there.
And I think this is probably why this hasn't been done. I mean, I'm relatively new to this industry,
but I think that's--
DR.
MERRITT: You know, it's clearly an interesting challenge, from our perspective,
in this issue. To date, the technology
hasn't been there to do that, and if it were I think one would have sen such
products. But continuing to push that
is certainly a challenge on the industry side.
DR.
BUSTA: We need to start our preliminary discussion, but I'm getting all kinds
of pressure from this side of the table with some urgent questions.
[Laughter.]
So
if--there are two of you that have been pushing me, and if you do them real
fast, we will take three more minutes.
And Dr.
Blumberg was first on waving me down.
And then Dr. Kuzminski will have the last word--but it's real quick,
please.
DR.
BLUMBERG: I had a quick question on the per-lot testing. Why is the sample size five grams? What we heard earlier today was much larger
sample size, 100 grams in the CDC investigation of the Tennessee outbreak. They used 100 grams, and in some of the other
things. So why are you proposing five
grams for the per-lot testing?
DR. SMOOT:
Well, currently, as you've seen, the 1986 A&PR criteria for infant formula
production with coliforms less than 3 per gram as manufacturing criteria that
they're looking to regulate the industry at, as well as the CODEX coliform
criteria of 3 per gram.
We are
looking to increase that 3 from 3 to 25 grams, in terms of monitoring the
production verification of our HACCP principles, as an indicator that the
allowable--not the allowable levels, but the levels of this particular
bacterium would be at a very low level in the infant formula.
When you
look at and reason--in comparison, looking at other bacteria--Dr. Buchanan
indicated as contaminants or pathogens and zero tolerance--and relevance,
looking at listeria monocytogenes, even though mentioned, it's not found
frequently in manufactured infant formula.
The requirement there is on the order of only 250 gram-negative in 250
grams. And in light of the unknown
opportunistic pathogenicity of this particular organism, the fact that we have
epidemiological data that there are billions of feedings over the years, we
felt as a process-control verification, that a 25 gram sample--absence in 25
grams would be adequate to indicate the safety and quality--
DR.
BLUMBERG: Do you have data looking at whether, you know, you can pick it up
with 25 versus 100 grams?
DR.
SMOOT: There is industry data that has not been shared with you today that
would indicate--the data set that I provided on that one particular Dutch
factory, where there was absence in 100 grams, that was the data there.
There is
a data set that that particular factory's been operating since that time frame
under the monitoring criteria of less than .3 coliforms per gram, as indicated
in the risk profile for CODEX. And so
there shows--there's good correlation, that whether it's less--operating at
less than .3 coliforms per gram, or operating at negative in 100 grams, per
gram, for Enterobacteriaceae, there has not been evidence that that particular
manufactured powder has been implicated or presents a risk for infant
nutrition.
I think
we're just trying to resist the issue of going--keeping the risk of the
organism in relation to the sample size that's being monitored on the
production-lot basis.
DR.
BUSTA: Dr. Kuzminski.
DR.
KUZMINSKI: Kuzminski. Thank you. A quick question.
Did I
hear you correctly when you mentioned that the lot size was 20 tons?
DR.
SMOOT: Typically, an average, around 20,000 pounds--correct.
DR.
KUZMINSKI: And that--I guess--you must have a high degree of
confidence--statistical confidence, that the 25 gram sample is truly
representative of 20 tons.
DR.
SMOOT: Well, at this point what we're doing is verifying the HACCP principles,
the HACCP activities and the prerequisite programs relative to the family of
Enterobacteriaceae, and that representing the E. sakazakii. We feel that that gives us a very good
representation of the microbiological quality, relative to that group of
organisms.
DR.
BUSTA: Thank you very much.
We have
20 minutes to have our preliminary discussion.
Time goes fast when you're having fun.
[Laughter.]
First--well,
thank you all from the industry group.
I must
say, the bus will leave at 6:00 p.m.
Abe's shuttle leaves at 6:00 p.m.
So we've got to keep that in mind.
We won't want to dawdle when we conclude here.
Tomorrow
morning, the bus will arrive at 7:15, in case you really don't know what else
to do. But it will leave no later than
7:45. So, at 7:45, the bus pulls out,
and most of you, I would guess, will want to be checked out and have your bag
with you. I know I intend to do
so. Because when we conclude here,
there will be opportunities to go directly to the various locations--airports,
etcetera.
There
will be, at eight o'clock there will be a continental breakfast when we get
here in the morning. So much for the
mechanics there.
Preliminary Subcommittee Discussion on Clinical
Presentations
The
reason I want to start the preliminary discussion now is to let you start
thinking about tomorrow. Because
tomorrow we will have extensive subcommittee discussion in the morning, and
then in the afternoon we will attempt to have concluding deliberations and
recommendations from the subcommittee.
One of
the approaches that we have used in the past, on other committees, is to go and
ask each one of the representatives, the voting members on the committee, to
make concluding comments on what they see are the conclusions and responses to
the charges. We probably will round--do
Charge 1, and then we will do charge two.
So you can think about this evening, being that the President is not
speaking tonight.
VOICE:
[Off mike.]--starting right away in the morning?
DR.
BUSTA: Well, first we will have discussions.
So we will start discussing. And
those concluding remarks will presumably be in the afternoon, after lunch.
DR.
KUZMINSKI: [Off mike.]--addressing each of the charged questions, or--
DR.
BUSTA: The question from Dr. Kuzminski
was: will the concluding comments discuss each of the charges? And I would say we would go Charge 1, and
everybody make a comment. And then we
would go Charge 2. And I haven't
thought whether we do Charge 2(1), Charge 2(2), or look at them as a unit. But, in part, I think our discussion in the
morning, as we give our opinions and attitudes, will give us an idea of how we
will give the concluding remarks in the afternoon.
Would
someone like to start some comments on their thoughts on Charge 1: Characterize
the infants at risk?
Please
note that you had a new charge today, different from the one in your
book--slightly different. Charge 1 is
slightly different. So if you just look
at the one that was handed out this morning in your blue packet, that is the
current charge.
DR.
THUREEN: The distinguishing features?
DR.
BUSTA: The distinguishing features in number 1 are that--in the new one, it now
says "consideration of factors" in the middle line. The old one said "facts."
After
"status," age has been added.
So it's "immune status, age, and general health status, etc."
has been added. And then it "may
impact," rather than "impact."
So there are a couple of modifications--expansions and modifications on
the intensity of the recommendation.
So would
someone with a strong opinion like to say, given the available information on
E. sak and powdered infant formula, is there a risk?
Dr.
Thureen.
DR.
THUREEN: Thureen. I'll start.
Is there
a risk? Yes, I think we've identified
today that there is a risk. But is it
an acceptable risk? It seems like that
the number of infections with E. sak are very low, considered to other neonatal
infections. And we haven't put it in
the context of other infections. There
was some context with the number of doses of infant formula given that makes
the risk seem quite low. But just as a clinician, without having any data here
in front of me, I think, that as an infection source and risk, it's very low,
considering the actually worldwide number of infants that we have that are in
the pre-term infant group.
I think
the highest risk group--oh, do you have a question after that?
DR. BUSTA:
Well, the comment right after that, which you are probably going into
immediately, is identifying the populations of infants at risk. Maybe that's a second comment, but--
DR.
THUREEN: Right. And that second comment
is: I think the highest risk group, by far, are pre-term infants. And, as a group, they're at risk for so many
things, this one is really a drop in the bucket, compared to everything else
that they're exposed to. But it is a
risk to them.
I'm not
convinced by the data presented that term infants are at significant risk, when
you look at the risk and the association with infant formulas, and the data of
all term infants--not pre-term, but term infants--those cases that have been
identified, I think that that risk is exceedingly low, and that any limitation
of powdered formula should probably not extend to term infants, based on the
data that's there, unless more data becomes available. And I think it has to be an ongoing data
collection, with better surveillance.
But I
think the pre-term infant group is at risk, but is it an acceptable risk, or an
understandable risk, or a risk that can't be eliminated, looking at the
downsides of the alternatives of not using powdered infant formula
DR.
BUSTA: Is there--I'm trying to keep this balanced. Is there someone who has a counter comment to that?
Dr.
Stallings?
DR.
STALLINGS: The only concern that I have to that is I guess I'm still not
convinced that term infants that are truly sick enough to be in a level 3
nursery, may not still be at some risk.
Because if--I mean, we don't understand the pathogenesis, so we don't
know if it's really a dose effect yet, because we don't know if this is--we've
never had the opportunity to measure the dose in the product that actually got
the children sick. We've done
retrospective analyses.
So, I
still am concerned about term infants with various surgical and stressed--
DR.
THUREEN: Right. I agree with you
completely, Virginia.
DR.
STALLINGS: Okay.
DR.
THUREEN: I think this should be to the non-NICU population.
DR.
STALLINGS: Okay. Good. So that was my only--I don't think we know
enough the other stuff, and that environment refers--
DR.
THUREEN: Right. And I agree with
you. If they're sick enough there, they
should be included, because they would be immunocompromised--presumable, based
on some underlying illness.
DR.
BUSTA: Dr. Heubi.
DR.
HEUBI: I have a question, I think that I still need to have clarified from
Patti, and maybe from Laurie. And that
is, if we simply look at the risk-benefit, in terms of growth and neurological
development for the pre-term infant, is it appropriate to state that using
powdered fortifiers fall into the category of the risk of E. sakazakii is so
low that the benefit is so much greater, that this is not an issue. And then I think if you follow that, is
it--are the other formulas that we're talking about, in terms of constituted
formulas, such as any of the names that you want to name--are they a different
category?
DR.
THUREEN: Thureen. Do you mean the
constituted, ready-to-feed formulas?
DR.
HEUBI: No. I mean powdered formulas.
DR.
THUREEN: Powdered formulas.
DR.
HEUBI: E.g., the amino acid or protein hydrolysate formulas, etc.
DR.
MOYER-MILEUR: For the pre-term infant, I think that--
DR.
BUSTA: Laurie, would you identify yourself?
DR.
MOYER-MILEUR: Moyer-Mileur--sorry.
I think
that when you have a baby who is receiving mother's milk and that requires
fortification, that the fortifier itself is given in very small amounts, and
that the benefit from having mother's milk far outweighs the risk from the
powdered supplement.
The
concern I have in this high risk population is the use of other powdered
products in these infants. And I
would--you know, there are ready-to-feed liquid products available that should
be, for a hospital, readily available, that that could be substituted. Unless they have a metabolic--inborn error
of metabolism.
DR.
STALLINGS: Stallings.
Do we
have any data on culturing the breast-milk fortifier? I haven't heard anything that's been presented. Because you're hearing from all of us that
that's a very unique and important product to the care of these kids. So are
there any data from the powdered products, versus the liquid, sterile products?
DR.
BUSTA: I see a negative from the FDA presenter. Negative from Canada. Any
positives in the--
DR.
STALLINGS: Well, those would be interesting data for us to learn about, because
that is something that almost didn't exist in the '80s when, you know, all of
this was first starting.
The other
thing, for clarification--on this list that we got at the table today, of
"powdered form only," it said "amino acid based
formulas." And I just--those
really are the metabolic disease ones--I'm assuming. Because the protein hydrolysates are under the powdered and
liquid form. And, again, it would be
very difficult to do the work without the protein hydrolysates, but they are
available in a sterile liquid form.
So I
think if we're starting to focus on the highest risk is the premies, there
really are only a couple of places--and we eliminate the metabolic diseases,
because that's a different setting, it seems to be headed toward the
breast-milk fortifiers.
DR.
BUSTA: All right.
DR.
STALLINGS: Other comments?
DR.
BUSTA: Other discussions. Someone else
would like to--Dr. Neill.
DR.
NEILL: Neill.
Maybe
before we leave this particular aspect that we're talking about, I think one of
the things that has made me a little uneasy today si the issue of risk and
trying to categorize it along a spectrum of quantitation, when you're calling
it "low," and "very low," and words like
"negligible."
I think
we know that it's somewhere in the vicinity of low, but I am personally very
unsettled that we really have a good box that we can draw around this. I guess, maybe, kind of two points that I
would make to that issue would be that the NNIS hospitals--which have come up a
couple of times--and, Henry, help me out here if you know the answer. That acronym is NNIS, for the National Nosocomial
Infections Surveillance System. And it
is, as you know, the 300 hospitals. The
extent to which those participating hospitals have obstetrics, NICU or
pediatric services is unknown to me. I
think it's a natural, in terms of a question to ask: whether the NNIS system
has any data on E. sak, but it may not at all be the mechanism that could give
you the answer.
DR.
BLUMBERG: I think that's a good point, because--Henry Blumberg--not all the
NNIS hospitals may have a neonatal ICU.
And if they do, the neonatal ICU may not be contributing data to
NNIS. It's like a menu, and there's a
lot of different surveillance projects.
For
instance, we're in this hospital, but our NICU, we're not reporting NICU data,
we're reporting surgical site infections or other things. So you could be in this hospital and not
contributing anything from, say, the neonatal ICU.
DR.
NEILL: The second half of the point that I was going to make on that--because,
again, it gets to this idea of trying to quantitate "low," because of
where I think we're going to end up going, where we have this terrible clinical
conundrum of trying to weigh the benefit against the risk--and that has to do
with the fact that the data we've talked about today are published cases of
diagnosed infections, which everyone in this room recognizes is inherently
biased. And at this point I would not
know how many are diagnosed but have eluded formal surveillance mechanism, or
how many--to Dr. Tarr's point from this morning--of sepsis of unknown etiology,
or the patients got the antibiotics on board beforehand, so the CSF cultures
are negative. Then how many of all this
other category--a clearly very ill but un-microbiologically diagnosable illness
in fact could be due to E. sak? And I
don't know.
But I
thought we probably ought to at least get that one out on the table.
DR.
BUSTA: Dr. Blumberg?
DR.
BLUMBERG: Three's two things I wanted to mention.
One is, I
think it would be useful to query directors of clinical microbiology labs to
find out do the automated systems that they use pick up Enterobacter
sakazakii. Because I think that's
one--I just don't know the answer.
Because most of the work's been done with API strips, and what they're
using in the clinical micro lab is a different system for, you know, most of
these things.
The other
issue is whether there's other enterobacter species in the formula that could
be contributing to illness in the neonatal ICU setting. Because from Muytjens' study that's been
mentioned, 14 percent of the samples cultured positive for E. sakazakii, but in
that same study, 25 percent cultured positive for Enterobacter glomerans, and
21 percent for Enterobacter cloacae.
And those are very common, hospital acquired infections. And they don't jump out. Like E. sakazakii is unusual, it jumps out
at people. But every day hospitals are
having Enterobacter cloacae or glomerans infections, and those things would not
jump out. And I'm just wondering if, in
the NICU setting, the infant formula could contribute to those infections.
Maybe
that's beyond our charge.
DR.
BUSTA: Are we--should I look around and see if we've got an answer? Does anybody in FDA have any data on
those? On the other enterobacters that
might be coming in in the infant formula--other enterobacters--glomerans,
cloacae--that may be coming in in infant formula that possible could result in
a hospital infection?
DR.
ALEXANDER: Dr. Alexander.
As I
noted during my presentation on neonatal meningitis, I mean, there's certainly
data that speaks to the fact that other enterobacter species also cause neonatal
meningitis. They don't necessarily have the same predisposition to developing
brain abscesses that Enterobacter sakazakii does, but it is a cause of many
different types of neonatal infection.
And the
problem that you get into when you try and figure out is it coming from the
formula, or is it coming from someplace else, is that those organisms--the
glomerans, the Enterobacter cloacae--are organisms that have been identified as
part of normal enteric flora for adult humans that are interacting with these
infants. And also it's been identified
as part of the hospital environment, so it's difficult to tease out whether the
organism in formula would be the source of infection.
With E.
sakazakii, since the only place that we really seem to be finding it
consistently seems to be from the powdered formula, it seems to more of a
clearer direct association. Although,
even that, has some difficulties; whether there's the possibility that one
child gets contaminated and then hospital practices, or, you know, health care
workers carrying the organism to multiple people on their hands would be
another source that we just haven't been able to sort out.
DR.
BUSTA: Thank you. Dr. Baker? Did I see you--you have the microphone in
your hand, and I thought you--
DR.
BAKER: Baker. I had a couple of
comments.
I think
one is that I think in this list of formulas that they're talking about--the
amino acid formula--they're really referring to NeoKate as available as a
powder only.
The other
comment I had was about your comments about risk and unease about risk. Just to sort of think about that from a
philosophical point of view, risk is a measurable thing. That is something that you can put a number
on in science. Meaningful risk, or
safety, you can't put a number on it.
That's a societal decision. And
that's what we're being asked actually to do, is to tell FDA how meaningful
this risk that has been identified is, and it's not easy. Because there's no scientific way of doing
it. You just have to come up with it.
But
anyway, talking about the first charge, I felt that there definitely is a risk,
and the risk to the normal neonate is probably negligible, and not a meaningful
risk. But the risk to the premature or
the NICU baby is probably a significant risk that we need to at least
address. And I wonder about other
populations, like the immunocompromised normal baby, or the graduate of the
NICU--should be worrying about them as well?
I don't
think I have an answer for what we should say about that yet, though.
DR.
BUSTA: Dr. Thureen.
DR.
THUREEN: I suspect that any recommendations we come up with are going to have a
lot of provisions based on getting further data, and that we might not have
final conclusions and recommendations by the time we leave this meeting because
there are so many questions; and that perhaps part of what we should be
thinking about tonight is not only what recommendations we have based on the
information we received today, but what kind of further information we might
like to collect before final rule--we could at least set up provisional
guidelines, but that it may be changed based on important information that we
feel needs to be collected before we can come to definite conclusions.
And your
insights into how data is collected, and what some of the presentations are may
not reflect reality. That's important
information, and there are ways we could suggest gathering that kind of
information to at least ultimately come up with a good final solution, and we
may have to settle for provisional recommendations in the meantime.
DR.
BUSTA: Would someone like to have the last word of today?
[No
response.]
It's
me? My last word of the day is we have
10 minutes, according to my watch, to meet Abe's shuttle in front of the
building, which leaves at six o'clock.
Let me
thank each and every one of you, all the presenters, all the support staff, and
everyone else for an outstanding, intense, long and good day. Tremendous involvement by everyone on the
committee, and we appreciate all your efforts.
Think about this tonight. Come
back vigorous and ready to address the questions tomorrow morning.
Thank you
all. We'll see you on the bus.
[Whereupon,
at 5:50 p.m. the meeting was adjourned, to reconvene at 8:00 a.m. on March 19,
2003.]
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