Guidance for Industry
In the Manufacture and Clinical Evaluation of In Vitro Tests
to Detect Nucleic Acid Sequences of Human Immunodeficiency Viruses Types 1 and 2
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(Internet) http://www.fda.gov/cber/guidelines.htm
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Biologics Evaluation and Research (CBER)
December 1999
TABLE OF CONTENTS
- INTRODUCTION
- INTENDED USE
- MANUFACTURING
- Rationale and Design
- Assay Optimization
- Sample Preparation
- Primers and Probes
- Reaction Buffers
- Enzymes
- Controls and Calibrators
- Other Test Kit Components
- Detection and Quantitation of Amplicons
- Instrumentation and Software
- Sterility/Bioburden
- Kit and Component Stability
- CLINICAL VALIDATION OF ASSAY PERFORMANCE
- Preclinical Studies
- Clinical Trials: General Issues
- Specificity and Sensitivity Studies for Test Kits with
a Proposed Labeling for Screening of Blood and Plasma Donors
- Studies to Validate Intended Use as Additional, More
Specific Tests
- Clinical Prognosis and Management of Patients on Therapy
- Perinatal Diagnosis
- CONCLUSIONS
- REFERENCES
Guidance for Industry
In the Manufacture and Clinical Evaluation of In Vitro Tests
to Detect Nucleic Acid Sequences of Human Immunodeficiency Viruses Types 1 and 2
This guidance document represents the agency's current thinking on in vitro testing to detect specific nucleic acid sequences of HIV. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of the applicable statute, regulations, or both. |
- INTRODUCTION
In March 1985, the U.S. Food and Drug Administration (FDA)
licensed the first screening test for the detection of
antibodies to Human Immunodeficiency Virus (HIV) in serum and
plasma from infected individuals. As of October 1999, there
were 27 licensed kits and 5 premarket approvals (PMAs) for
detection of antibodies to HIV-1 or HIV-2 in blood, saliva, or
urine, which included: 19 Enzyme-Linked Immunosorbent Assays
(ELISAs), 3 Western Blots, 1 Particle Agglutination Assay, and
1 Indirect Immunofluorescence Assay (IFA) for detection of
antibodies to HIV-1, as well as 3 ELISAs for detection of HIV-1
p24 antigen, 2 of which are for use in donor screening. The
ELISAs include 4 combination tests for detection of antibodies
to HIV-1 and HIV-2 in blood specimens.
FDA recently licensed the first test for the detection of HIV-1
antibodies in urine specimens. HIV ELISAs have been approved to
screen blood and plasma donors. The IFA, the Rapid Latex
Agglutination Assay, and the colorimetric Single Use Diagnostic
System (SUDS), may be used to screen blood donors in urgent
situations. They are primarily used for urgent testing in
hospitals, laboratories, medical clinics, physician's offices,
emergency care situations, blood banks, or other health care
settings when a routine ELISA is unavailable or impractical.
Repeatedly reactive results from screening assays are further
evaluated by additional, more specific tests which include
Western Blot and IFA. In December 1994, FDA approved the first
oral fluid collection device for professional use with a licensed
HIV-1 antibody test kit and in June 1996, a supplemental Western
Blot test to further evaluate the presence of antibodies in oral
fluid was approved. Two PMAs for home blood sample collection
kits, labeled as part of a HIV testing system, were also approved
by FDA in 1996 (one has been voluntarily discontinued by the
manufacturer). The first PMA for a HIV-1 nucleic acid test,
a quantitative HIV-1 ribonucleic acid (RNA) test based on
amplification of deoxyribonucleic acid (DNA) sequences using
the polymerase chain reaction, to measure viral load in plasma
as an aid in determining patient prognosis, was approved by FDA
in June 1996.
In recent years, several technical advances have been made in
methodologies for direct detection of viral nucleic acid. This
document provides guidance on manufacturing and clinical trial
design issues pertaining to the validation of tests based on
nucleic acid detection either in the presence or absence of an
amplification step. Concerns regarding the procedures used for
detection of amplified products are also addressed. As may be
the case with new technologies, issues may be identified during
the review process, unique to the particular methodology under
review or the specific configuration of the assay that will need
to be addressed on a case-by-case basis. It is also recognized
that this area of science is in a state of rapid technological
development. As advances are made, this document will be
reevaluated and revisions or modifications made as necessary.
The criteria outlined below address both general and specific
concerns for nucleic acid based detection techniques for HIV.
This document is intended for products regulated by the Center
for Biologics Evaluation and Research (CBER). The FDA uses mandatory
language, such as shall, must, and require, when referring to
statutory or regulatory requirements. The FDA uses non-mandatory
language, such as should, may, can and recommend when referring to
guidance.
The reader is referred to the Points to Consider in the Manufacture
and Clinical Evaluation of In Vitro Tests to Detect Antibodies to
the Human Immunodeficiency Virus Type 1 (1989) (Ref. 1) for general
information on filing of the Investigational New Drug Applications
(INDs), Product License Applications (PLAs), Establishment License
Applications (ELAs), and content of applications for approval and
licensure of retroviral kits. General regulations related to these
products are located in 21 CFR parts 312, 600-680, and 800. Other
documents that may be pertinent to this topic include the "Review
Criteria" document (Ref. 2) issued by the Center for Devices and
Radiological Health (CDRH), and the Guidance for Industry: Content
and Format of Chemistry, Manufacturing and Controls Information
and Establishment Description Information for a Biological In Vitro
Diagnostic Product (Ref. 3) issued by CBER.
As set forth in the Intercenter Agreement of 1991 between CBER and
CDRH, in vitro tests for HIV, that are recommended for blood donor
screening and related blood bank practices, are licensed under the
Public Health Service Act (PHS Act) through the IND/PLA/ELA/Biologics
License Application (BLA) mechanism. In vitro tests for HIV, that
are not performed in relation to blood bank practices (e.g.,
quantitative HIV assays and diagnostic tests that evaluate specimens
other than blood), will be regulated by CBER under the Medical
Device Authorities through the Investigational Device Exemption
(IDE)/Premarket Approval Application (PMA) mechanism. In vitro
diagnostics for pathogens other than HIV are regulated by CDRH
under the IDE/PMA mechanism.
The scientific and regulatory concerns pertaining to validation of
in vitro diagnostic/screening test kits can be broadly classified
into three main categories: 1) intended use; 2) manufacturing; and
3) clinical validation of assay performance.
Table of Contents
- INTENDED USE
The sponsor should state clearly in the application (IND, PLA/ELA,
BLA, or IDE/PMA) the intended use, the labeling claims, and the
clinical utility for the product. The proposed clinical trial
design should be capable of demonstrating assay performance at a
level that is sufficient to validate the intended use claim in the
target patient population and the specific test setting. It is
recommended that the sponsors/manufacturers meet with CBER to
obtain guidance early in the development process in order to resolve
any issues with regard to an approvable claim for the product or
special concerns related to the product, and to address any
questions the manufacturers may have. This should include a
discussion of the proposed claim for clinical utility and the
clinical studies that will be performed to validate the proposed
claim, including equivalence or superiority to existing methods or
licensed tests, if available, for detection and/or quantitation of
the same agent.
Table of Contents
- MANUFACTURING
The manufacturing issues that may have an impact on product design
and performance are: 1) rationale of assay design; 2) assay
optimization; 3) sample collection, extraction, storage and
stability; 4) manufacture of primers, probes, reagent buffers,
enzymes, calibrators, controls, and quantitation standards;
5) anchoring components, i.e., beads, plates, chips; 6) kit
stability; and 7) instrumentation and software. This document
addresses concerns pertaining to each of the points outlined above.
- Rationale and Design
The sponsor should provide in the application, the rationale
for the specific indication and for use of the specific test
methodology, and type of nucleic acid target (DNA or RNA)
for detection of the infectious agent and for the specific
indication. A detailed description of all aspects of the
technique including sample preparation, assay optimization,
amplification, and detection methods should be provided.
Validated quality control procedures that are state-of-the-art
should be used to assure manufacturing consistency. The
sponsor should provide details of assay optimization and
establish the format of the final product during the
preclinical stage of development. Changes in assay format
may lead to a recommendation for new studies. The rationale
for assay design should address the following aspects:
- Selection of target sequence(s) in the template including
the degree of nucleic acid sequence conservation,
guanidine:cytosine (GC) ratio, and length. For products
intended to detect more than one virus subtype or species
(e.g., a "multiplex" design) define the number of target
nucleic acid sequences and the rationale for their selection;
- Assay format (e.g., sample type, conjugate, detector);
- Selection of primer and probe sequences (e.g., degree of
nucleic acid sequence conservation); and
- Design and nature of the quantitation standards for a
quantitative assay.
- Assay Optimization
This phase is critical to product development and can have a
significant impact on product performance. The sponsor should
address the various aspects of optimization for nucleic acid
extraction, target sequence, amplification, detection,
quantitation, and instrumentation for these processes, and
set specifications for performance. The application should
contain information on the details of:
- The length, region, specificity, and efficiency of primer
and/or capture sequence;
- Methods of extraction, amplification, hybridization,
detection, and quantitation;
- Percent recovery of nucleic acid for the total assay and
for each significant step in the process of sample
preparation;
- Optimization of reaction conditions and kinetics of
amplification with multiple primers or hybridization with
multiple probes or both (e.g., for a multiplex format);
- Internal and external assay calibrators/controls; and
- Procedures to prevent cross-contamination.
During this phase of assay optimization, the sponsor should
determine and define the optimal assay conditions, reaction
kinetics and the lower bounds of reliable assay performance.
For qualitative assays the assay cutoff, or reporting
threshold, is the lowest concentration of HIV RNA copies per
ml that the assay can reliably distinguish from HIV negative
samples (> or = 95% detection rate). For quantitative assays,
the lower limit of reliable assay performance may be defined
by two potentially distinct values; the lower limit of
detection (LOD) and the lower limit of quantitation (LOQ).
For the purpose of this document the LOD is defined as the
lowest concentration of analyte that can be distinguished
from a negative specimen with a predefined level of assay
sensitivity. The LOQ is the lowest concentration of analyte
that is distinguishable from a negative specimen with the
same degree of sensitivity as the LOD that is also
quantifiable with an acceptable degree of precision and
accuracy (e.g., CV of < or = 35%).
The assay cutoff/reporting threshold or LOD should be well
defined in terms of copy numbers and the unit of sampling.
This limit should be validated by an established form of
independent characterization (e.g., an (approved)
amplification technique or a combination of more direct
measurements such as particle counts, EM scanning and
quantitation of RNA by optical density). The assay cut-off
or LOD can be established by in-house testing then further
defined or verified based on the data from clinical trials.
If the assay is quantitative, additional studies should be
conducted to examine:
- Linearity in the readable range in order to ensure
accurate interpolation of unknown specimens. This
range should also be clinically meaningful to
demonstrate the clinical utility of quantitation; and
- Accuracy and reproducibility based on quantitation of
analytical specimens on a standard curve (analytical
sensitivity). These studies should also provide a
preliminary estimate of the LOQ.
- Sample Preparation
The sponsor should specify the type of specimen (e.g., cells,
plasma, whole blood, dried blood spots) and the template for
amplification (DNA and RNA) and hybridization, as appropriate.
The composition of the buffers, reagents, and detergent or
chaotropic agents used for nucleic acid extraction should be
clearly specified. The effect of anticoagulants and any
potential inhibitors present in the sample or extraction
buffers on assay performance should be evaluated.
Controls that monitor the efficiency of the extraction and
reverse transcription (when the template is RNA) procedures
should be included and whenever possible, these controls
should simulate the actual sample type. Spiked controls
are also acceptable.
The reproducibility of the sample preparation method should
be determined under the specimen processing conditions
including sample handling, storage, and shipping conditions.
The sponsor should also verify possible interference of
specimen processing reagents with reverse transcription,
amplification, hybridization, detection, and quantitation.
For pool testing, sample stability during pooling and the
subsequent processing steps should be determined.
- Primers and Probes
The primers and probes are the main components of a nucleic
acid based detection system and the performance of the assay
is highly dependent on the quality of these reagents. The
sponsor should provide the:
- Rationale for selection of primers and probes including
specific sequences used;
- Justifications for alignments made to generate consensus
sequences or best-fit modifications made to existent
sequences, e.g., to permit maximum homology to several
strains; and
- Information on size, GC content, melting temperatures,
hairpin or other secondary structures if any, and the
nucleotide position on the genome map of the primers
and probes.
For assays designed to detect or quantitate multiple HIV
subtypes or variants, data should be provided to demonstrate
that the primers and/or probes chosen are effective for all
of the subtypes or variants identified in the label.
If synthetic oligonucleotides are used as primers and probes,
details of the manufacture and purification should be provided.
In addition, the following information should also be included:
- The yield and composition for the first 3 lots (at a
minimum) by absorbance and DNA fingerprinting, restriction
endonuclease mapping or nucleotide sequence analysis;
- A description of the chemical nature of the modification,
for modified oligonucleotides and procedure(s) to insure
lot to lot consistency of ligand content;
- Nucleotide sequence analysis to establish the fidelity of
the procedure for oligonucleotide synthesis;
- The purity of the final product should be analyzed by an
appropriate state-of-the art analytical technique (e.g.,
reverse phase high performance liquid chromatography,
electrophoresis or ion exchange HPLC), that has been
validated according to ICH guidelines;
- Potency of primers and probes. This may be addressed by
dilutional analysis comparing lot-to-lot consistency in
functional efficiency or other methodology appropriate to
the technology under development.
The analyses listed in 6-8 should be conducted on each lot of
oligonucleotide manufactured as a routine part of new product
development and characterization. If a high degree of
consistency is demonstrated over time the sponsor may request
a reduction in the frequency of required monitoring.
- Reaction Buffers
The sponsor should demonstrate the identity and purity of reagents
used in the preparation of reaction buffers that are employed in
amplification, hybridization, and detection reactions. The
potency and stability of the reagents on storage and under
cycling conditions should be verified. If reagents are obtained
from vendors, the quality system regulations (21 CFR 820.50 and
820.80) require the manufacturer to establish and maintain
procedures to ensure that all received reagents conform to
specified requirements. The extent of control necessary will
be related to the nature of the reagent, taking into account
the effect of the reagent on the finished product. A
certificate of analysis should be provided for purposes of
verification and the criteria used for acceptance/rejection of
specific reagents defined. If deviations from component
specifications could result in the product being unsuitable
for use, a sponsor may be expected to sample and test
components.
- Enzymes
The source and function of all enzymes used in the assay should
be identified and clearly defined. The identity, purity,
potency, and specific activity should be demonstrated and
criteria for acceptance established.
For rDNA-derived enzymes manufactured by the sponsor, the master
and working cell banks should be characterized for cell and
genetic stability, and freedom from adventitious agents.
Plasmid stability should be monitored by assays that include
restriction mapping or DNA sequencing. If restriction mapping
is used for plasmid monitoring confirmation of enzyme amino
acid composition and sequence by peptide mapping and amino acid
sequencing should also be considered.
Enzyme preparations should be tested for other enzymatic
activities, e.g., exonucleases and DNA and RNA dependent
polymerase activities and specifications should be established.
For enzymes obtained from vendors, the certificate of analysis
should be provided. In addition, functional testing designed
to assure that the component is suitable for its intended use,
should be performed as part of establishing the acceptance
criteria.
- Controls and Calibrators
Controls are important tools that allow the operator to verify
that the assay has performed within accepted specifications and
are, therefore, a vital component of any test kit. Controls
should be separate from, and in addition to, reagents used to
estimate the concentration of an unknown sample (i.e., standards
or calibration reagents).
In nucleic acid analysis, there are several steps in the testing
process, as outlined above, that should be monitored and verified.
It is therefore advisable to include multiple controls or
controls that serve multiple purposes in the final kit. The
controls should reflect the specific technology under development
but will typically allow for monitoring of ultracentrifugation,
extraction, amplification, hybridization, quantitation,
contamination, etc. These controls should be similar to the
specimen type whenever feasible although spiked controls may be
acceptable, particularly for labile analytes.
Sponsors are strongly encouraged to include a minimum of two
positive controls to monitor assay performance. A control at or
near the LOD or assay cutoff/reporting threshold should be
incorporated into any assay that will be read in a qualitative
fashion. For the validation of individual assay runs with a
diagnostic assay, it is recommended that this control be within
three standard deviations of the assay cut-off/reporting threshold.
For quantitative assays the low concentration positive HIV RNA
control for validation of individual runs should be within 3
standard deviations of the LOQ. The second positive RNA control
may fall anywhere within the linear range of the assay. In the
event that the assay LOD is different from the lower limit of
quantitation sponsors are strongly encouraged to include an
additional control at the LOD to allow laboratories to monitor,
on a routine basis, their ability to detect RNA at that level.
Assays that have or are seeking a label claim for quantitation
of multiple viral subtypes should make a subtype specific
positive RNA control available for each subtype.
Multiple negative controls should be included such as non-target
sequences and nucleic acid free controls to monitor for false
positives resulting from contamination. Due to the high
sensitivity of amplification assays, it is highly recommended
that sponsors include control measures for prevention of
contamination events.
Specifications for both positive and negative controls should be
provided, as well as validation data supporting the proposed
assay cut-off/reporting threshold value or LOD of the assay.
The sponsor should define the source of the controls and
calibrators, and have a plan for their continued renewal.
Controls should be non-infectious, and validation of viral
inactivation should be provided.
For quantitative assays, validation data should be provided for
all quantitation standards and calibrators. Specifications and
acceptance criteria should be established for each
control/calibrator and for the collective set of controls.
Quantitation should be based on co-amplification of a
heterologous internal control and/or a competitive RNA
template or co-hybridization, as indicated by the technology
under development. For RNA assays, the efficiency of reverse
transcription should be determined for the specific assay format.
- Other Test Kit Components
The sponsor should provide a description of the anchoring
solid phase component (e.g., plates, beads, filters),
concentration of antigen or oligonucleotide on the component
method of conjugation, or binding to the component, and a
demonstration of lot-to-lot consistency of manufacture of bound
component.
If more than one component is used for coating (e.g., two
oligonucleotides) a description of the validation of coating
methods including ratios used and acceptance criteria for the
coating process should be provided. If the sponsor purchases a
solid phase component (e.g., beads, plates, chips) a
description of the source, quality assurance methods, and
acceptance criteria should be included.
- Detection and Quantitation of Amplicons
A detailed description of the chemical/biochemical nature of
capture probes, conjugates, detectors, quantitation standards,
etc., which are part of the assay system should be provided.
This should include:
- The chemistry and limits of detection of system of choice
(e.g., chemiluminescence, fluorescence);
- Chemical and biochemical characterization of the ligand,
chromophore, fluorochrome, including stability under
reaction conditions;
- Quality control and assurance of conjugation to detect or
capture oligo- or polynucleotide sequence, including
functional testing; and
- Nature and copy numbers of the quantitation controls and
standards.
Validation data should be provided for controls and quantitation
standards. Specifications should be established for the
individual and/or collective set of controls/quantitation
standards used to detect/quantitate nucleic acid.
- Instrumentation and Software
Any dedicated equipment used in the amplification, detection,
and quantitation of the amplified product should be validated
for its use. These may include devices such as thermal cyclers,
waterbaths, luminometers and cycling ovens.
Validation of thermal cyclers should include demonstration of the
accuracy of temperatures of individual wells during the cycling
process, specify limits for well-to-well variation, if any, as
well as any impact there may be on test results. If software
is utilized for amplification, detection, and calculation of
quantitative or qualitative results, validation of such software
for the intended function should be provided.
For non-dedicated instruments, the premarket notification (510k)
submission number should be cited for review. If previously
approved under a PMA, a supplement for use with the product
under review should be submitted.
If special specimen collection, storage and/or transport devices
are used, specifications should be provided for conditions of
collection, storage, and transport. Criteria should be
established for suitability and adequacy of the specimen for
the test.
- Sterility/Bioburden
Refer to the "Points to Consider in the Manufacture and Clinical
Evaluation of In Vitro Tests to Detect Antibodies to Human
Immunodeficiency Virus Type 1" (1989) for guidance in this area.
- Kit and Component Stability
The stability of the final kit and individual components should
be tested using a panel of specimens, including weak reactives
(e.g., near cut-off, middle, and upper end of the readable
range). A reference panel consisting of plasma spiked with
known amounts of virus is acceptable for this study. The real
time stability at storage and shipping temperatures should be
evaluated using specimens with varied reactivities in the
readable range.
Table of Contents
- CLINICAL VALIDATION OF ASSAY PERFORMANCE
- Preclinical Studies
Preclinical studies, performed either in-house or at field sites,
provide preliminary information on assay performance. These
studies should be designed to assess the sensitivity, specificity,
and reproducibility of the test kit, as well as to identify the
lower bounds of reliable assay performance (i.e., the assay
cut-off/reporting threshold, LOD and LOQ, as appropriate for
the assay under development). In general, preclinical testing
should be performed prior to initiation of clinical validation
studies, particularly if prospective, large scale clinical
trials are planned. Clinical validation of these preliminary
assessments is then accomplished through field testing of
appropriate clinical specimens to provide final specifications
for test kit performance characteristics.
- Specificity and Sensitivity Studies for Preclinical
Testing (statistical determination of false positive and
false negative rates)
- For donor screening assays or diagnostics, specificity
should be established by testing samples from a minimum
of 500 random blood or plasma donors. For quantitative
assays, a minimum of 100 samples should be tested in
the preclinical development phase; and
- For both assay types, sensitivity should be established
by testing at least 300 seropositive repository specimens.
Testing should be performed in parallel with appropriate
licensed comparator assays (i.e., antigen or antibody assays
for blood screening or diagnosis and a nucleic acid based
test for quantitation).
- Analytical Sensitivity
Analytical sensitivity should be evaluated by testing a
dilution series of at least 10 distinct HIV positive samples
obtained from different individuals (clinical specimens).
Prior to initiation of these studies, the starting
concentration of viral RNA in each sample should be
determined by an appropriate independent technology (see
section III.B, above). The evaluation of patient isolates
should be run in parallel with samples from a recognized
reference panel (e.g., reference HIV panels produced by the
WHO, CBER or the ACTG VQA).
For quantitative assays the dilution series should cover the
full range of accurate quantitation for the assay under
development. For pool testing, the lowest concentration in
the dilution series must be at or below the minimum
sensitivity level required for these assays (i.e., 100
copies/ml in a plasma pool or 5,000 copies/ml in an
individual sample, based on a 95% detection rate). The
highest dilution reproducibly and consistently detected
and/or quantitated by the investigational assay should be
defined in copies per unit sampling until recognized
international standards for viral quantitation are available
for the target virus, at which time, analytical sensitivity
can be expressed in international units/ml. Comparator
assays should include an antibody, antigen, or other
state-of-the-art amplification/probe technology. The
selection of an appropriate comparator assay will be based
on the technology under development and the proposed
indication.
For products seeking a labeling claim for quantitation of
HIV that includes non-clade B viruses, analytical
sensitivity should be demonstrated for each subtype or
variant proposed for inclusion in the label. A minimum of
10 distinct HIV positive specimens obtained from different
individuals would be advisable for each subtype/variant.
In the event that 10 distinct clinical specimens of a
specific viral subtype are not available, culture derived
specimens may be used to supplement clinical samples.
- Clinical Trials: General Issues
Clinical trials designed to assess clinical sensitivity,
specificity, and reproducibility should be performed at clinical
trial sites by qualified independent investigators. Refer to
section VI. in the "Points to Consider in the Manufacture and
Clinical Evaluation of In Vitro Tests to Detect Antibodies to
the Human Immunodeficiency Virus Type 1" (1989) for additional
general guidance on clinical trial design issues.
Common components of a clinical development program for any
specific intended use include studies designed to assess the
precision, reproducibility, and non-specificity of the
investigational assay.
- These studies are designed to assess the coefficient of
variation for the test results for each sample and for the
various lots tested.
- Proficiency Testing
A panel of samples similar to that described in section
IV.B.1.b (below) should be tested by a number of
operators on multiple days, at all clinical testing
sites. This study is designed to assess operator
proficiency.
- Reproducibility and Precision
A panel of 10 or more samples including low reactives
should be tested at all clinical trial sites. This
could be a series of samples spiked with the analyte or
human specimens with known reactivity. Samples should
be tested in duplicate or triplicate. Testing should be
performed on a minimum of three different lots, on
multiple days and by at least two operators. The
operators chosen to conduct these studies should have
demonstrated a high degree of proficiency with the
assay. Reproducibility studies should be designed to
assess variability intra- and inter-site, intra- and
inter-assay and intra- and inter-lot, as well as total
variability for both qualitative and quantitative
assays. Assay precision may be established by
performing multiple tests using multiple operators and
multiple kit lots on a panel of specimens. Testing may
be performed in-house and at least one clinical site.
- Instrumentation
Instrumentation effects on product performance should be
evaluated using the sample panel employed for
reproducibility testing and a minimum of three different
machines.
- Non-specificity studies
Most assays are subject to some inherent non-specific
reactivity resulting in false positive reactions or
interference resulting in false negative reactions or reduced
accuracy of quantitation. This effect may be due to specific
assay components or the nature of the sample being tested.
The presence of non-specific reactivity and the impact of
potential interfering factors on assay performance should be
assessed. Appropriate samples for these studies can be
obtained by spiking the agent or factor into known HIV
positive and/or negative samples, in addition to testing
original specimens or reference panels. Examples of
conditions, factors or sample characteristics that should be
considered in the evaluation of cross-reactivity or
interference include the following:
- Other infections including Human T-cell Lymphotrophic
Virus Type I/II (HTLV-I/II), Cytomegalovirus (CMV),
Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV),
Hepatitis C Virus (HCV), yeast infections, and
pneumocystis;
- The impact of different anticoagulants (i.e., heparin,
ACD, or EDTA) or other suitable collection tube/media
(e.g., plasma preparation tube);
- Hemolyzed, icteric, lipemic, and bacterially contaminated
samples;
- Samples treated with chemicals, drugs, heat or
detergents;
- Samples subjected to multiple freeze thaw cycles;
- Fresh vs. frozen samples, serum vs. plasma, and single
specimen vs. plasma pool;
- Samples from patients with autoimmune diseases including
Systemic Lupus Erythematosus (SLE), Anti-Nuclear
Antibodies (ANA), and Rheumatoid Arthritis;
- The impact of subject age, gender, race, or ethnic group;
- The impact of drug resistance mutations. This evaluation
should include a cross section of viruses that
demonstrate phenotypic and genotypic resistance to each
of the currently approved drug classes. Multiple drug
resistant viruses should also be evaluated;
- The presence of nucleic acid based drugs and metabolites
and binding substances; and
- The presence of drugs or biologicals that increase
circulating nucleic acid.
- Specificity and Sensitivity Studies for Test Kits with
a Proposed Labeling for Screening of Blood and Plasma Donors
FDA does not currently envision discontinuation of antibody
testing for donor screening. However, nucleic acid testing (NAT)
may be more sensitive than other methods currently available for
early detection of a virus during the pre-seroconversion phase
of infection and may, therefore, have added value in blood
safety. For NAT, clinical specificity should be established by
testing a large number of specimens from random U.S. blood and
plasma donors. The evaluation of clinical specificity should
include samples from at least 10,000 individuals or plasma pools,
based on the intended use of the product. Testing should be
performed at a minimum of 3 distinct clinical sites, including
areas of both high and low prevalence. Studies should be
performed in a manner that allows for donor identification
(i.e., "linked") to permit clinical follow-up. If positive
results are encountered, the donor should be deferred
temporarily and units held in quarantine until test results are
confirmed by repeat NAT testing and/or by clinical follow-up and
antigen or antibody-based testing over the subsequent 3-6 month
period. The basis for reinstatement of donors with
false-positive investigational test results should be defined in
the study protocol.
- Additional Studies to Establish the Clinical Sensitivity of
Tests Intended for Screening of Individual Blood Donations
A minimum of 1,000 specimens from seropositive individuals
including samples from various risk groups and different
stages of HIV-1 disease should be tested at 3 distinct
sites. At least 200 of these seropositive specimens should
be derived from persons with a clinical diagnosis of AIDS.
It is recommended that the study also include a gender-based
analysis with at least 20-30% of the samples derived from
females. Geographically diverse specimens representing all
known viral subtypes (a minimum of 20 of each) should be
included in this data set to evaluate the performance and
establish the sensitivity of the kit for detection of
variant HIV-1 strains. All of these samples should be actual
clinical specimens as opposed to culture derived virus
stocks or cloned species. However, if 20 distinct clinical
specimens of a specific viral subtype are not available,
culture derived specimens may be used to supplement clinical
samples.
A minimum of 200 samples known to be positive for HIV-2
should also be tested in the evaluation of clinical
sensitivity for single donation screening tests, including
a subset that contain both HIV-1 and -2. These samples may
be obtained from a repository.
Prospectively collected, freshly drawn specimens from
individuals at high risk for HIV-1 should be tested in
a linked study so that a minimum of 50 positive cases
are identified (by serology, p24 antigen or NAT) to
estimate sensitivity. A similar prospective trial in
a population at high risk for HIV-2 should also be
conducted, with sufficient enrollment to identify a
minimum of 30 seropositive cases. These studies should
include methods to objectively resolve discordances
between investigational and comparator assays that
include both antibody and antigen-based tests. This
may require follow-up testing of the study subjects.
A subset of these samples (100) should be tested by
another state-of-the-art test (amplification/probe test).
A comparison should be made between freshly drawn and frozen
specimens, as well as paired serum and plasma or other
specimens (e.g., Whole Blood). The purpose of this study
is to establish the comparability of the two storage
conditions and the two specimen types, respectively.
- Additional Studies to Establish the Clinical Sensitivity
of Assays Intended for Pool Testing
Clinical sensitivity testing for pool tests should include
the evaluation of 1,000 known seropositive specimens in a
minimum of 100 separate pools. Of these, at least 25 pools
should contain weakly reactive seropositive specimens
introduced singly into the pool. All pools should contain
at least several known negative specimens. The positions
of the seropositive specimens should vary from one pool to
another, in a random fashion. These studies should be
performed at 3 sites, one of which could be in-house. In
addition, the ability to detect known HIV subtypes/variants
and HIV-2 should be determined using a minimum of 20 samples
for each variant. These samples may be plasma pools spiked
with the appropriate subtype/variant or a virus preparation
diluted in seronegative plasma. Additional HIV-2 samples
may be required to support a label indication for detection
of HIV-2. These studies should demonstrate that the NAT is
capable of detecting a minimum of 100 RNA copies/ml in the
pooled sample or 5,000 copies/ml of virus in the original
donation, with a > or = 95% detection rate.
- Additional Issues for NAT Intended for Screening of Plasma
Pools
If testing will be done on pooled specimens, additional
issues to be addressed include:
- Demonstration of equivalent or superior sensitivity of
the assay for testing donor pools compared to currently
licensed methods for donor screening by laboratory and
field testing;
- Rationale for the proposed pool size;
- The impact, if any, of possible interference or matrix
effects generated during pooling, on test performance;
- Sample stability during collection, storage, pooling and
transport , including a comparison of assay performance
and sample stability with freshly drawn versus frozen
specimens;
- Procedures for logging and tracking of specimens in a
given pool, including validation of the pooling process
(i.e., procedures included to verify that appropriate
test samples are obtained for all donations included in
a pool and that all donations intended for a specific
pool are actually included;
- Specimen retrieval procedures to identify a positive
specimen in a positive pool;
- Quality assurance in computing and reporting test results;
- Validation of instrumentation and automation; and
- Validation of software that may be used in conjunction
with any of the procedures listed above.
Testing of specimens from appropriate primate models may
also be useful towards establishing utility for detection
of infection in the pre-seroconversion phase.
- Clinical Validation of Assay Performance for Blood Screening
Tests
Because of the limited availability of specimens in the
antibody negative, pre-seroconversion phase, the agency urges
the use of specimens from the following categories for
clinical validation of assay performance for all blood
screening tests: ongoing cohort studies and retrospective
investigations; as well as a large number of seroconversion
panels and specimens from high risk individuals enrolled
in prospective studies being conducted in areas of high
prevalence. These data should be analyzed on the basis of
mathematical models that estimate the timing and duration
of the window period. Testing should include a head-to-head
comparison with a licensed assay for HIV p24 antigen. These
studies are particularly important for NAT, where the value
added may be in the ability to reliably detect early infections.
- Studies to Validate Intended Use as Additional, More
Specific Tests
These tests are used to further evaluate the accuracy of the
positive test results of a screening assay. Gene based tests
may be developed as an alternative to Western Blot (WB), Strip
Immunoblot Assay (SIA), or Immunofluorescence Assays (IFA)
currently in use for this purpose. In some instances, the test
may be used to resolve the indeterminate patterns seen on
additional, more specific tests including those referred to above.
In specificity and sensitivity testing for confirmatory tests,
random donors should be tested at two or more sites. At least
500 samples should be tested to assess specificity.
Approximately 300 specimens from random donors that are
repeatedly reactive (RR) by licensed screening assays should be
evaluated along with other additional, more specific tests to
establish clinical sensitivity. In addition, a minimum of 300
known positive specimens should be tested to determine clinical
sensitivity.
For tests that are used to resolve indeterminate results of other
confirmatory assays, a minimum of 300 samples from persons with
such indeterminate test results using licensed tests should be
tested. Testing should be performed on a combination of WB,
SIA, and IFA indeterminate specimens from random donors.
Sponsors should demonstrate that the sensitivity of the
second, more specific test is equivalent to or better than
the screening assay.
In all cases, a plan for resolving discordant/discrepant
results should be included. In addition, a minimum of 500
random donor specimens and 300 known positive specimens should
be tested to establish clinical specificity and sensitivity.
- Clinical Prognosis and Management of Patients on Therapy
- Clinical Specificity
Clinical specificity should be established by testing at
least 500 specimens from healthy, random donors. This
test series is distinct from the preclinical evaluation of
specificity and should be conducted at an appropriate
clinical trial site.
- Clinical Sensitivity
Clinical sensitivity should be established by testing samples
from seropositive individuals and from high risk groups in a
head-to-head comparison with a licensed or approved nucleic
acid detection test. Performance should be evaluated in
cross-sectional studies involving HIV positive individuals
stratified by CD4 counts and clinical history.
Category |
No. of subjects |
|
CD4 < 200 |
200 |
CD4 200-500 |
300 |
CD4 > 500 |
300 |
Studies designed to assess variability in consecutive viral
nucleic acid measurements in an individual over time should
also be performed. A minimum of 40 adult and 20 pediatric
(age < or = 12 years) patients with stable viral loads in
the ranges listed above should be followed on a bi-weekly
basis for a period of 16 weeks or weekly for a minimum of
8 weeks. The data should be evaluated with respect to
short-term variability (1-2 weeks) and at 8 week intervals
(typical monitoring frequency for routine patient care).
The distribution of male and female participants in each
adult study cohort should be sufficient to allow for the
evaluation of gender based differences in assay variability.
The information obtained from the studies listed above will
be important in establishing the significance and validity
of changes observed after initiation of a specific therapy
and the utility of these tests in patient management.
- Performance in Patients Undergoing Therapy
The clinical utility of an assay in treated individuals
should be demonstrated in prospectively conducted drug
efficacy trials by direct comparison to an approved nucleic
acid quantitation test. Randomized, concurrently controlled
clinical trials that compare different drug combinations or
investigate the utility of new drugs as adjuncts to existing
therapies based on their effects on plasma virus RNA are
acceptable for assay comparator studies.
A possible alternative approach to testing in the context
of a prospectively conducted clinical study is the use of
well-characterized, repository specimens from cohorts for
which clinical outcome is known. However, if a retrospective
analysis is planned, the study employed should be consistent
with current treatment regimens and practices. In addition,
it is essential that specimen adequacy requirements be
confirmed prior to initiation of the study so that meaningful
results may be evaluated and discordant results resolved.
Prior to the conduct of a retrospective analysis of clinical
utility, sponsors are strongly encouraged to consult with the
division about the suitability of the study(s) selected.
Regardless of the approach taken to demonstrate assay utility
in patient monitoring a minimum of 300 patients who underwent
therapy with regular monitoring of virus load should be
evaluated. This study population should include a subset
of patients whose therapy was modified as a result of
clinically defined disease progression, changes in virus
load and/or the development of drug resistance. In the
context of these studies, an effort should be made to
address gender and age-related (i.e., pediatric versus
adult) differences in kit performance.
Sponsors interested in pursuing a claim for patient monitoring
are encouraged to consult the FDA Draft Guidance for Industry:
Clinical Considerations for Accelerated and Traditional
Approval of Antiretroviral Drugs Using Plasma HIV RNA
Measurements (Ref. 4) for additional information on clinical
testing strategies and accepted use of plasma RNA measurements
in clinical therapeutic trials.
- Clinical Prognosis
A number of different approaches may be taken to establish
the prognostic value of a nucleic acid quantitation assay. A
prospective study may be conducted involving patients at
different stages of disease with monitoring of their disease
free time interval as the primary endpoint. Data may also be
derived from well-characterized, retrospectively collected
samples obtained from appropriate clinical endpoint trials.
In some instances the prognostic utility of an assay may be
demonstrated by direct comparison to another nucleic acid
technology that is currently approved for prognosis.
When prognostic utility is defined by evaluation of clinical
trial samples with clinical outcome data, whether
prospectively or retrospectively identified, a statistical
analysis should be performed to determine the relative
predictive value of nucleic acid levels as it relates to
disease progression. This analysis should be based on a
combination of longitudinal and cross-sectional studies that
includes information on clinical outcome. The cross-sectional
study should employ a design similar to that described above
for clinical sensitivity. For the longitudinal study, a total
of approximately 500 patients at different disease stages
based on the Centers for Disease Control and Prevention
classification scheme should be followed. Clinical outcome
may be defined as the time to first AIDS defining event or
duration of disease free survival. The studies outlined above
should include a sufficient number of female participants to
allow for the evaluation of gender based differences in
prognostic utility. An independent evaluation of pediatric
patients is also strongly encouraged.
A comparative approach to demonstrate equivalent or superior
performance may be acceptable in combination with supporting
preclinical and clinical data for modified versions of
currently approved assays, or for new tests that approximate,
in a clearly definable manner, the RNA values generated with a
nucleic acid technology approved for prognosis. A minimum of
150 clinical samples spanning the full linear range of both
the experimental and reference assays should be evaluated.
In addition, a set of sequential clinical specimens from 30
individuals with demonstrable changes in viral load (a minimum
1 log10 change), over time should be evaluated to permit a
comparison of the assays based on individual readings and on
sequential changes in those readings. A comparative approach
is not sufficient for assays that do not demonstrate an
acceptable degree of correlation with an approved assay based
on both the individual readings and on sequential changes in
those readings.
- Perinatal Diagnosis
Nucleic acid tests may be useful for early diagnosis of
infection in infants born to seropositive mothers. The
specificity of tests intended for this purpose should be
established by testing at least 300 healthy (adult) donors
and approximately 100-200 samples from infants born to healthy,
seronegative mothers. Clinical sensitivity for early diagnosis
should be evaluated by testing at least 200 infants born to
seropositive mothers. Testing should include the first 0-6
weeks after birth and the results should be compared with
licensed antibody and antigen tests. Long term follow-up may
be needed to resolve test results in some cases.
Table of Contents
- CONCLUSIONS
Gene based tests for viral agents are regulated by FDA either as
biologics or devices. Although this document outlines some of the
major regulatory and scientific issues concerning gene based tests
for HIV-1 and HIV-2, these considerations may also be applicable
to tests for other transfusion transmitted viruses including HCV,
HBV, and HTLV-I and II. Sponsors are advised to consult with the
agency (CBER or CDRH, based on the assay and indication for use)
during the development phase so that product specific issues in
manufacturing and clinical trial design may be addressed early in
the validation phase.
Table of Contents
- REFERENCES
- "Points to Consider in the Manufacture and Clinical Evaluation
of In Vitro Tests to Detect Antibodies to the Human
Immunodeficiency Virus Type 1" (1989).
- "Review Criteria for Nucleic Acid Amplification-Based In Vitro
Diagnostic Devices for Direct Detection of Infectious
Microorganisms" (1993).
- "Guidance for Industry: Content and Format of Chemistry,
Manufacturing and Controls Information and Establishment
Description Information for a Biological In Vitro Diagnostic
Product" (1999).
- Draft "Guidance for Industry: Clinical Considerations for
Accelerated and Traditional Approval of Antiretroviral Drugs
Using Plasma HIV RNA Measurements" (1999).
Table of Contents
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