Role of HIV Drug Resistance Testing in Antiretroviral Drug Development

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Guidance for Industry
Role of HIV Resistance
Testing in Antiretroviral
Drug Development
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
October 2007
Clinical Antimicrobial

Guidance for Industry
Role of HIV Resistance
Testing in Antiretroviral
Drug Development
Additional copies are available from:
Office of Training and Communications
Division of Drug Information, HFD-240
Center for Drug Evaluation and Research
Food and Drug Administration
5600 Fishers Lane
Rockville, MD 20857
(Tel) 301-827-4573
http://www.fda.gov/cder/guidance/index.htm
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
October 2007
Clinical Antimicrobial

TABLE OF CONTENTS
I. 	INTRODUCTION............................................................................................................. 1
II. 	BACKGROUND ............................................................................................................... 2
III. 	HIV RESISTANCE TESTING — GENERAL.............................................................. 3
IV. 	NONCLINICAL STUDIES.............................................................................................. 4
A. 	Mechanism of Action ..................................................................................................................... 4
B. 	Antiviral Activity in Cell Culture ................................................................................................. 4
C. 	Cytotoxicity and Therapeutic Indexes ......................................................................................... 5
D. 	Protein Binding .............................................................................................................................. 5
E. 	Selection of Drug-Resistant HIV-1 Variants in Cell Culture..................................................... 5
1. Genotype .......................................................................................................................................... 6
2. Phenotype......................................................................................................................................... 6
F. 	Cross-Resistance ............................................................................................................................ 7
G. 	Characterization of Genotypic and Phenotypic Assays.............................................................. 7
1. Genotypic Assays ............................................................................................................................. 7
2. Phenotypic Assays............................................................................................................................ 8
V. 	CLINICAL STUDIES: USE OF RESISTANCE TESTING IN CLINICAL
PHASES OF DRUG DEVELOPMENT ......................................................................... 8
A. 	General Considerations ................................................................................................................. 9
B. 	Data Collection ............................................................................................................................... 9
C. 	Methods and Types of Analyses.................................................................................................. 10
1. Baseline Genotype and Virologic Response .................................................................................. 11
2. Development of HIV Mutations ..................................................................................................... 13
3. Baseline Phenotype and Virologic Response................................................................................. 13
4. Genotypic and Phenotypic Correlations: Changes in Susceptibility from Baseline...................... 14
5. Cross-Resistance............................................................................................................................ 15
6. Additional Analyses ....................................................................................................................... 15
D. 	Other Considerations................................................................................................................... 16
1. Role for Supporting Initial Activity and Dose-Finding Studies ..................................................... 16
2. Data Collection from Dose-Finding Trials.................................................................................... 16
3. Use of Resistance Data to Establish an Indication........................................................................ 17
4. Use of Resistance Data for Study Enrollment Criteria, Background Regimen Selection, and
Stratification Factors ..................................................................................................................... 17
5. Nonclade B Subtypes...................................................................................................................... 17
E. 	Monitoring during Phase 4 ......................................................................................................... 18
VI. 	SUMMARY ..................................................................................................................... 18
GLOSSARY................................................................................................................................. 19
REFERENCES............................................................................................................................ 20
APPENDIX A: GUIDANCE FOR SUBMITTING HIV RESISTANCE DATA .................. 22

Contains Nonbinding Recommendations
Guidance for Industry1
Role of HIV Resistance Testing in
Antiretroviral Drug Development
This guidance represents the Food and Drug Administration’s (FDA’s) current thinking on this topic. It
does not create or confer any rights for or on any person and does not operate to bind FDA or the public.
You can use an alternative approach if the approach satisfies the requirements of the applicable statutes
and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for
implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate
number listed on the title page of this guidance.
I. 	INTRODUCTION
This guidance is intended to assist sponsors in the clinical development of drugs for the
treatment of human immunodeficiency virus (HIV) infection. Specifically, this guidance
addresses the Food and Drug Administration’s (FDA’s) current thinking regarding the role of
HIV resistance testing during antiretroviral drug development and marketing and serves as a
focus for continued discussion among the Division of Antiviral Products (DAVP),
pharmaceutical sponsors, the academic community, and the public. The goal of this guidance is
to stimulate the generation of more complete resistance data and analyses for antiretroviral drug
products.
This guidance uses a broad definition of the term drugs including, but not limited to, small
chemical entities, biologics, monoclonal antibodies, synthetic oligonucleotides, and siRNA, and
focuses on resistance to antiretroviral agents as manifested by mutations in the HIV viral genome
that result in reduced phenotypic susceptibility to a given drug product. Although mechanisms
of cellular resistance to antiretrovirals exist, a discussion of these mechanisms is beyond the
scope of this guidance. In addition, loss of susceptibility to drugs is highlighted, rather than
hypersusceptibility. However, we acknowledge the potential for results to show increased
susceptibility of the virus to one or more antiretroviral drugs and we encourage sponsors to
report such observations to the FDA.
Although this guidance focuses on characterization of resistance and cross-resistance during drug
development, we recommend application of these principles to currently marketed antiretroviral
agents; therefore, we recommend ongoing resistance testing in the postmarketing setting.
1 This guidance has been prepared by the Division of Antiviral Products in the Center for Drug Evaluation and
Research (CDER) at the Food and Drug Administration.
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This guidance does not imply one type of resistance testing is more useful than another type of
resistance testing in the clinical management of HIV infection. This guidance addresses how
serial assessments of both genotype and phenotype can be useful in antiretroviral drug
development. For characterizing the utility of an antiretroviral drug, both phenotypic and
genotypic resistance testing have strengths and limitations as discussed in this guidance.
For information on trial design and endpoints in phase 3 antiretroviral drug development, see the
related guidance for industry Antiretroviral Drugs Using Plasma HIV RNA Measurements —
Clinical Considerations for Accelerated and Traditional Approval.2
Because the field of HIV resistance is evolving, we intend to revise this guidance as new
information accumulates.
FDA’s guidance documents, including this guidance, do not establish legally enforceable
responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should
be viewed only as recommendations, unless specific regulatory or statutory requirements are
cited. The use of the word should in Agency guidances means that something is suggested or
recommended, but not required.
II. 	BACKGROUND
The primary sources for the recommendations in this guidance are as follows:
• 	Data analyses and input from the HIV Resistance Collaborative Group (an international,
multidisciplinary group consisting of representatives from academic institutions, U.S. and
European health regulatory authorities, governmental clinical trial organizations, the
pharmaceutical and diagnostics industries, and HIV patient community groups). Data,
analyses, and opinions compiled by this group were presented at a 2-day session of the
Antiviral Drug Product Advisory Committee, convened November 2-3, 1999, to address
issues relating to HIV resistance testing.
• 	The DAVP’s experience with reviewing resistance data for antiretroviral drugs in new
drug applications from 1999 to the present, including subsequent analysis and
presentation of resistance data to the Antiviral Drug Product Advisory Committee.
• 	Additional input from pharmaceutical sponsors and the HIV community.
Presentations during the November 1999 advisory committee meeting included the following
topics:
• 	Performance characteristics of genotypic and phenotypic assays
• 	Prevalence of resistance in antiretroviral naïve patients
2 We update guidances periodically. To make sure you have the most recent version of a guidance, check the CDER
guidance Web page at http://www.fda.gov/cder/guidance/index.htm.
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• 	The ability of baseline resistance testing to predict subsequent virologic response
• 	Clinical factors that might influence the results of resistance testing
Summaries of presentations at this meeting have been published in Antiviral Therapy (Richman
2000; Hammer and Pedneault 2000; DeGruttola et al. 2000; Laessig et al. 2000), and the
transcripts of the FDA advisory committee can be found on the Internet.3
III. 	HIV RESISTANCE TESTING — GENERAL
Because of its high rate of replication (109 to 1010 virions per person per day) and error-prone
polymerase, HIV can easily develop mutations that alter susceptibility to antiretroviral drugs. As
a result, the emergence of resistance to one or more antiretroviral drugs is one of the more
common reasons for therapeutic failure in the treatment of HIV. In addition, the emergence of
resistance to one antiretroviral drug sometimes confers a reduction in or a loss of susceptibility to
other or all drugs of the same class.
The application of laboratory technologies, such as gene amplification, automated nucleic acid
sequencing, and nucleic acid hybridization, and the availability of recombinant viruses for testing
phenotypic susceptibility have permitted advances in HIV resistance testing. Many clinicians
and investigators are currently using these technologies in the clinical management of HIV.
However, performance characteristics (e.g., sensitivity, specificity, and reproducibility) for many
of the assays in investigational use have not been fully established. In addition, the clinical
significance of many mutations or mutational patterns has not been defined completely for many
antiretroviral drugs. Likewise, the quantitative relationship between reductions of cell culture
susceptibility and loss of clinical activity has not been established for most drugs. Consequently,
many of the current package inserts are deficient in the amount and type of resistance data
describing the utility of a drug in the setting of resistance or reduced susceptibility.
Despite limitations of resistance assays and their interpretation, several randomized controlled
studies have demonstrated that virologic outcome, at least over the short term, may be improved
when genotypic or phenotypic data are used to guide choice of drug regimens in patients with
loss of virologic response to prior regimens (Baxter et al. 2000; Cohen et al. 2002; Durant et al.
1999; Melnick et al. 2000; Meynard et al. 2000; Tural et al. 2002). The FDA recommends that
characterization of resistance and cross-resistance be a part of antiretroviral drug development so
that clinically relevant information is available at the time of approval. An efficient way to
accomplish the goal of having clinically relevant information available at the time of approval is
to include resistance testing in all phases of drug development. As discussed below, assessment
of resistance should not be delayed until phase 3 or post-approval. We recommend that, before
or during phase 1 and phase 2 studies, investigators begin assessing the potential of a drug to
select resistant viruses and the drug’s activity against HIV isolates resistant to other antiretroviral
agents. During early development, a wide range of doses should be evaluated and
pharmacokinetic data should be collected, providing information to investigate the relationship
between drug exposure and resistance.
3 See http://www.fda.gov/oashi/aids/advisorycom.html#geno.
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Optimally, a comprehensive evaluation of a new drug’s resistance and cross-resistance profile
will promote more rational use of antiretroviral drug combinations in the future.
IV. 	NONCLINICAL STUDIES
Cell culture studies can provide useful information for the design of in vivo studies and may be
predictive of the development of resistant viruses in vivo. This section identifies studies relevant
to resistance issues in the development of antiretroviral drugs for the treatment of HIV infection.
The FDA also issued a guidance that outlines general nonclinical studies for antiviral drugs (see
the guidance for industry Antiviral Product Development — Conducting and Submitting Virology
Studies to the Agency).
Sponsors should complete nonclinical studies (i.e., mechanism of action, antiviral activity in cell
culture, cytotoxicity and therapeutic index, and effects of serum protein binding on antiviral
activity) before the initiation of phase 1 clinical studies. Cell culture drug combination activity
studies for drugs that are used in clinical trials should be completed before initiation of those
trials. Selection of resistant HIV-1 variants in cell culture, the phenotypic and genotypic
characterization of resistance viruses, and cross-resistance analyses should be examined before
initiation of clinical studies in HIV-infected patients. We recommend sponsors be consistent in
the assay used for any particular analysis or measurement in phase 3 studies. Sponsors should
provide data supporting the implementation and use of the new or improved assays that become
available during drug development.
A. 	Mechanism of Action
A well-characterized mechanism of action for a new antiretroviral drug can provide insight into
the regions of the HIV genome where mutations that confer resistance may develop. These
regions are not limited to the site of action (viral-encoded target) of the investigational drug and
can include the enzyme substrate(s) (e.g., Gag and Gag-Pol cleavage sites for protease inhibitors)
or another viral-encoded protein(s) existing in a quaternary complex with the target protein (e.g.,
gp120 and gp41). Any metabolite that exerts inhibitory activity should be delineated and its
specificity for the target shown. For example, reverse transcriptase (RT) inhibitors should show
selectivity for RT over cellular DNA and RNA polymerases.
B. 	Antiviral Activity in Cell Culture
The antiviral activity in cell culture of a compound indicates that it effectively inhibits
replication and forms the basis for defining phenotypic resistance (detected by reductions in
susceptibility to the investigational drug (see below)). The concentration of an investigational
drug required to inhibit virus replication by 50 percent (EC50 for cell-based assays; IC50 for
biochemical or subcellular assays) should be determined. The use of the EC50 value for
determining shifts in susceptibility is preferred because it can be determined with greater
precision than an EC90 or EC95 value. A well-characterized wild-type (WT) HIV laboratory
strain should serve as a reference standard. Sponsors should obtain some susceptibility data with
a limited number of clinical isolates grown in peripheral blood mononuclear cells (PBMCs). The
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amount of susceptibility data in PBMCs needed to support an application should be discussed
with the division.
The antiviral activity of drugs can vary greatly because of factors such as genetic variation in
isolates, host cell type, and multiplicity of infection assay used for measurement of virus
replication. Because of genetic variation, determination of antiviral activity against a broad
spectrum of viruses of well-characterized HIV laboratory strains and clinical isolates sufficient to
assess the breadth of antiviral activity is recommended. The antiviral activity should be assessed
in multiple clade B and nonclade B isolates, T-cell tropic HIV-1 and monocyte/macrophage
tropic strains, HIV-2, and well-characterized drug-resistant laboratory strains and clinical
isolates.
C. 	Cytotoxicity and Therapeutic Indexes
It is important to establish that an investigational drug has antiviral activity at concentrations that
can be achieved in vivo without inducing toxic effects to cells. Furthermore, in a cell culture
model, apparent antiviral activity of an investigational drug can be the result of host cell death
after exposure to the drug. Cytotoxicity tests use a series of increasing concentrations of the
antiviral drug to determine what concentration results in the death of 50 percent of the host cells
(CC50 or CCIC50 ). The relative effectiveness of the investigational drug in inhibiting viral
replication compared to inducing cell death is defined as the therapeutic or selectivity index (i.e.,
CC50 value/EC50 value). It is desirable to have a high therapeutic index giving maximum
antiviral activity with minimal cell toxicity. We recommend determining CC50 values in both
stationary and dividing cells from multiple relevant human cell types and tissues to ascertain the
potential for cell-cycle, species, or tissue-specific toxicities. Studies determining cytotoxicity
and therapeutic indexes should be conducted before the initiation of phase 1 clinical studies.
D. 	Protein Binding
Protein binding of an antiviral drug to human serum proteins may result in reduced antiviral
activity. The effects of 45 percent to 50 percent human serum on the cell culture antiviral
activity of the investigational drug should be evaluated for at least one well-characterized
laboratory or clinical isolate, and the serum-adjusted EC50 value should be determined (see
section V.D.2, Data Collection from Dose-Finding Trials). A series of human serum dilutions
(e.g., 5 percent, 10 percent, 20 percent, 40 percent) can be used to extrapolate the effect of 100
percent human serum. In addition, an examination of the protein binding effects of α-acid
glycoprotein and human serum albumin at physiological concentrations is recommended.
E. 	Selection of Drug-Resistant HIV-1 Variants in Cell Culture
Selection of resistant viruses in cell culture may indicate whether development of resistance to a
drug is likely to involve a few (1 to 2) or several (more than 2) mutations. The ability of an
investigational drug to select HIV-1 variants with reduced drug susceptibility (phenotypic
resistance) should be determined in cell culture systems. Selection of variants resistant to the
investigational drug should be repeated multiple times (e.g., with different strains of WT, with
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resistant strains, under high and low selective pressure) to determine if the same or different
patterns of resistance mutations develop.
Two basic methods have been developed to identify mutations conferring a reduction in
susceptibility to a drug.
1. 	In the first method, a high initial virus inoculum is propagated for several passages at a
fixed drug concentration, using multiple cultures to test different concentrations.
2. 	In the second method, a low initial inoculum of virus is passaged in the presence of
increasing drug concentrations starting near the EC50 value for the parental virus.
Virus production is monitored to detect the outgrowth of resistant virus by characterizing
intermediate and ending isolates with respect to genotype and phenotype.
1. 	Genotype
Mutations responsible for reductions in susceptibility to a drug can be identified by DNA
sequence analysis of the relevant portions of the virus genome. The complete coding sequence
of the gene for the target protein should be determined in the early stages of characterization of
mutations associated with reduced drug susceptibility. Once mutations are identified, their
ability to confer phenotypic resistance should be evaluated in a recombinant virus system (e.g.,
by using site-directed mutagenesis or polymerase chain reaction (PCR) amplification of relevant
portions of the virus genome to introduce these mutations into a standard laboratory HIV genetic
background) or other suitable system, such that the mutations necessary to reproduce the
resistant phenotype are identified. If site-directed mutagenesis experiments within the target
gene fail to recapitulate the resistance phenotype, then the potential effects of mutations
elsewhere in the viral genome should be examined. In the case of studying mutations in the
envelope gene, which is highly variable, a possible option is to introduce mutations into the
parental envelope to assess their contribution to the resistance phenotype. Recombinant virus
should then be tested for drug susceptibility in cell culture. Shifts in drug susceptibility (fold
increases in EC50 value) for recombinant virus relative to WT should be determined (see section
IV.G., Characterization of Genotypic and Phenotypic Assays).
2. 	Phenotype
Drug susceptibility (EC50 values) for resistant variants and the fold change in EC 50 values
relative to the parent virus should be determined (see section IV.G., Characterization of
Phenotypic and Genotypic Assays).
A number of drugs targeting gp120, gp41, and the CCR5 and CXCR4 chemokine co-receptors
are in development. A potential concern with CCR5 inhibitors is that loss of effectiveness may
develop by the virus switching to the CXCR4 co-receptor. The evolution of HIV to a CXCR4
utilizing virus has been proposed to result in a more virulent virus. Therefore, sponsors should
monitor co-receptor usage in cell culture drug selection experiments and in clinical trials
evaluating drugs targeting the co-receptors gp120 and gp41.
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F. 	Cross-Resistance
HIV variants resistant to one drug in a class of antiretroviral drugs may be resistant to another
drug in the same class. Recombinant viruses containing resistance-associated mutations to an
investigational drug should be tested for susceptibility to all approved and any investigational
drugs (where possible) of the same class and other classes with the same target protein or protein
complex. Conversely, laboratory strains and well-characterized clinical isolates containing
resistance-associated mutations for each of the approved and investigational members (where
possible) of the same class should be tested for susceptibility to the investigational drug.
Clinical isolates should be representative of the breadth of diverse mutations and combinations
of mutations known to confer reduced susceptibility. A standardized panel of virus strains and
isolates with diverse resistance-associated mutations and combinations of resistance-associated
mutations for the different drug classes can be helpful for profiling investigational drugs and
allowing for comparison with approved and other investigational drugs. The panel should be up
to date and include mutational changes in current circulating viral populations caused by the
introduction of newly approved drugs.
G. 	Characterization of Genotypic and Phenotypic Assays
Well-characterized genotypic and phenotypic assays can provide the basis for the analysis of the
emergence of resistant virus during the development of investigational drugs. Phenotypic and
genotypic assays used in clinical practice need more extensive validation than exploratory assays
used for the characterization of antiviral activity and/or the resistance profile of the
investigational drug. Commercially available assays that are routinely used should be identified,
but it may not be necessary to provide the performance characteristics. The amount and nature
of validation necessary for an assay and mechanisms of submitting assay performance
characteristics should be discussed with the division (also see the guidance for industry
Antiretroviral Drugs Using Plasma HIV RNA Measurements — Clinical Considerations for
Accelerated and Traditional Approval).
1. 	Genotypic Assays
The performance characteristics of genotypic assays should be described, including elaboration
of the following characteristics:
• 	Minimum plasma viral RNA level with a standard isolate to assess PCR sensitivity
• 	Purification methodology for viral nucleic acids
• 	Amplification methodology and primers
• 	PCR controls
• 	Clade differences
• 	Nucleic acid sequencing methodology
• 	Description of sequencing primers
• 	Range of mutant and WT ratios detectable
• 	Interpretation criteria for mutant scoring
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The entire coding sequence of the gene for the target protein should be determined in the early
stages of resistant variants analysis. Once the mutations leading to resistance are identified, only
the relevant portions of the genome need to be sequenced. The pattern of mutations leading to
resistance of an investigational drug should be documented and compared with the pattern of
mutations of other drugs in the same class.
Reporting the details of methodologies is important. Sponsors should identify sequencing
primers and state how many bases from them can be read accurately. Sponsors also should
define the sensitivity of the genotypic assay used for detecting minority viral subpopulations.
2. 	Phenotypic Assays
The performance characteristics (accuracy, precision, limits of detection and quantification,
specificity, linearity, range, robustness, stability) of an investigational phenotypic assay should
be well documented. The sources of viruses (e.g., blood, plasma), their storage and stability, and
cell culture procedures should be described. For definitions on assay validation, refer to the
guidance for industry Bioanalytical Method Validation. An additional reference is the ICH
guideline for industry Q2A Text on Validation of Analytical Procedures. Sponsors are
encouraged to use a previously characterized and validated assay.
The utility of a phenotypic assay will depend upon its sensitivity (i.e., its ability to measure shifts
in susceptibility (fold-changes) in comparison to baseline clinical isolates) and on the drug
concentrations achieved. Shifts in susceptibility for a clinical isolate are measured by
determining the EC50 values for the isolate and a WT standard virus done under the same
conditions and at the same time. Simultaneous testing provides for absolute comparisons
between assays. Readout of phenotypic assays can be detected with any standard virus assay,
such as p24, viral RNA, RT assay, MTT cytotoxic assay, and reporter gene expression.
V. 	CLINICAL STUDIES: USE OF RESISTANCE TESTING IN CLINICAL
PHASES OF DRUG DEVELOPMENT
Before advances in resistance testing technologies, resistance and cross-resistance data were
often obtained late in drug development or during postmarketing. However, given the current
availability of resistance testing in clinical practice and the need to give health care providers
information about an antiretroviral drug’s resistance profile, comprehensive resistance testing
should be undertaken in all phases of drug development. Crucial decisions in protocol design
and drug development hinge on resistance and cross-resistance data.
Cell culture resistance and cross-resistance studies of an investigational drug can focus the scope
of drug development. For example, drugs that exhibit extensive cross-resistance with approved
drugs of the same class are unlikely to be suitable for studies in treatment-experienced patients
harboring resistant isolates to that class. Conversely, for drugs that demonstrate a
nonoverlapping or unique resistance profile, the division strongly encourages sponsors to
develop clinical protocols studying treatment-experienced individuals. Although we encourage
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development of new antiretroviral drugs with unique resistance profiles in treatment-experienced
patients, we also encourage concurrent clinical development in antiretroviral-naïve patients as
appropriate.
Epidemiological data suggest that transmission of drug-resistant HIV is on the rise, meaning one
cannot assume treatment-naïve patients harbor WT virus (Little et al. 2002). In this regard, the
division strongly recommends the collection and storage of samples for baseline resistance
testing (preferably for both genotype and phenotype) from all HIV-infected, multiple-dose study
patients, regardless of treatment history. Refer to section V.C., Methods and Types of Analyses,
for further information regarding settings where analyses on baseline and follow-up samples are
recommended. Knowledge of the genotype and phenotype at baseline can aid in the
interpretation of unexpected antiviral responses, particularly in smaller dose-ranging studies.
A. 	General Considerations
The goals of resistance testing are to:
• 	Determine the effect of an antiviral drug on the evolution of the virus
• 	Identify the baseline genotypic and phenotypic determinants of virologic success or
failure (or clinical success or failure) in the study
We strongly recommend resistance testing in all phases of development and, in most cases, as
soon as the drug is introduced into HIV-infected patients. Data from nonclinical studies and
phase 1 and phase 2 clinical trials should provide a preliminary idea of the genotypic mutations
that confer reduced drug susceptibility and a lack or loss of virologic response. Phase 3 trial
designs should incorporate this information and expand on it, thereby aiming to further
characterize drug resistance.
In general, the type of information collected and the types of analyses conducted should be the
same for all phases of development. However, the amount of data collected and the types of
analyses performed may differ for treatment-naïve versus treatment-experienced patients as
described below. Whenever possible, resistance analyses should be prospectively defined.
However, since it is not possible to define a priori key mutations or susceptibility breakpoints,
retrospective analyses can provide important information in characterizing resistance and cross-
resistance. The following sections provide recommendations on the type of resistance data that
should be collected during drug development and the types of analyses that should be conducted.
Appendix A provides guidance for submitting HIV resistance data. Information about the
specific assays and mutational algorithms used in protocols also should be provided to the
division.
B. 	Data Collection
To characterize drug resistance during development, sponsors are strongly encouraged to collect
samples from both treatment-naïve and treatment-experienced patients. Overall, the extent and
type of resistance testing should be discussed and agreed upon with the division throughout drug
development. Discussions with the division are particularly important for new classes of
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antiretroviral agents as the data and technologies are evolving. The following data should be
collected and analyzed:
• 	Baseline phenotype and genotype samples on all study patients. Samples can be analyzed
at a later date as appropriate.
The reasons for obtaining baseline samples for phenotype and genotype on all clinical
trial patients are twofold. First, the prevalence and rate of transmission of drug-resistant
virus is increasing (Little et al. 2002), and may continue to increase, as the HIV
population becomes more treatment experienced. Second, collection of baseline data
provides an opportunity to examine the relationship between genotype and phenotype and
virologic response to drug, particularly in treatment-experienced patient populations. Use
of resistance testing in study protocols may help in choosing appropriate combination
regimens for treatment-experienced patients (see section V.C.4, Genotypic and
Phenotypic Correlations: Changes in Susceptibility from Baseline, for further details).
• 	Post-baseline phenotype and genotype samples on all study patients, regardless of
treatment history who demonstrate a lack or loss of virologic response during the trial to
determine mutations that may contribute to reduced drug susceptibility.
Collecting samples for resistance testing when patients are still on study drug, or as soon
as possible if study drugs are discontinued, is important. Studies have shown WT virus
may outgrow resistant HIV viral strains in the absence of selective drug pressure
(Devereux et al. 1999; Halfon et al. 2003). In addition, continuation of resistance
monitoring on subsequent regimens can provide useful information regarding cross-
resistance and sequencing of therapy. We recognize collection of resistance data on
subsequent regimens is not feasible for all clinical trials or patients; however, sponsors
are encouraged to consider such studies in their development plans as appropriate (see
section V.C.5, Cross-Resistance, for details) and are encouraged to discuss continuation
studies with the division.
C. 	Methods and Types of Analyses
Several types of resistance analyses can be used to characterize a drug’s resistance profile.
Analyses for treatment-experienced patients are more complicated than for treatment-naïve
patients. Some analyses are possible only when larger datasets are available. In phase 3, clinical
trial datasets can be sufficiently large to study the effect that mutations confer upon drug
susceptibility and outcome. Pooling data from several trials can be appropriate (provided the
study populations, endpoints, and assays are similar), but should be discussed in advance with
the division. To facilitate pooling data, sponsors should attempt to use similar, if not identical,
assays during phase 3. Whenever possible, resistance analyses should be prospectively defined,
with the caveat that prospectively defining key mutations or susceptibility breakpoints is not
always possible. In some cases, retrospective analyses can provide important information in
characterizing resistance and cross-resistance.
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Because of a large number of potential comparisons, statistical testing can be problematic for
analyses of resistance testing and outcome. Sponsors are encouraged to provide resistance
analysis plans to the division in advance.
Analyses of virologic outcome by baseline genotype or phenotype should be based on a censored
population to assess the effect of baseline resistance on outcome without confounding factors
such as early discontinuation because of adverse events. Therefore, patients who discontinue
study treatment while suppressed or who discontinue study treatment before confirmed
suppression for adverse event, noncompliance, protocol violation, pregnancy, or withdrawal of
consent should be censored. Rules for censoring patients who appear to have a virologic
response before discontinuation should be discussed with the division. We encourage sponsors
to analyze the baseline resistance data by the primary and secondary endpoints used in the trial.
Virologic response parameters recommended for analyses include the following, but are not
limited to: proportion of HIV RNA below the limit of quantification (e.g., less than 400 or less
than 50 copies/mL), proportion less than 1 log10 decrease from baseline, and mean and/or median
change from baseline or time average change from baseline at the protocol-specified time points.
Sponsors are encouraged to discuss endpoints with the division in advance. All patients should
be included in the dataset until the time of censoring. The datasets should include variables for
reasons for censoring patients. Refer to Appendix A for further details.
In general, we recommend analyses of baseline genotype and phenotype for all treatment-
experienced patients to evaluate baseline predictors of virologic response. In addition, genotypic
and phenotypic analyses of samples obtained at baseline and at the time of virologic failure are
recommended for all treatment-experienced patients to characterize resistance and cross-
resistance.
The following examples reflect suggested analyses for studies in treatment-experienced patients.
Sponsors are strongly encouraged to conduct additional analyses and should discuss these
analyses with the division before submission of a new drug application (NDA). In most cases,
we do not anticipate baseline genotype and phenotype and virologic outcome analyses, as
described below, for all treatment-naïve patients. However, circumstances may arise in
treatment-naïve patients where analyses of baseline resistance and virologic outcome data are
warranted, particularly in the event of unexpected efficacy results. In addition, in select cases,
sponsors can propose to collect samples at baseline and at the time of failure on a subset of
treatment-naïve patients, for example, when cell culture data indicate that acquisition of a
specific single mutation results in a high degree of phenotypic resistance.
We also acknowledge for various analyses the number of patients in certain subgroups may be
limited and as a result definitive conclusions regarding specific baseline factors will not be
possible.
1. 	Baseline Genotype and Virologic Response
Analyses should be conducted to evaluate HIV RNA response according to the presence and
absence of baseline mutations. These analyses can help assess the association between a specific
mutation or mutational pattern and virologic response rates. For example, for a new nucleoside
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analogue, virologic response rates would be determined for patients with and without clinically
relevant mutations associated with resistance to other nucleoside analogs. Table 1 shows one
example of virologic response rates for a hypothetical nucleoside analog by the presence or
absence of zidovudine-associated mutations at baseline.
Table 1. HIV RNA Response by Baseline RTI Mutations at Endpoint
Baseline RTI
Mutations*
Virologic Response Rate
MUTATION PRESENT 	MUTATION ABSENT
Drug X
(n =)
Control
(n =)
Drug X
(n =)
Control
(n =)
M41L 	27% 	6% 	79% 	7%
D67N 	65% 	3% 	63% 	2%
K70R 	72% 	3% 	54% 	2%
L210W 	17% 	6% 	72% 	5%
T215Y/F 	38% 	3% 	80% 	7%
K219Q/E/N 	60% 	9% 	58% 	7%
* Patients included in these subgroups may have other RTI mutations or mutations in addition to the baseline mutations listed.
Table 1 shows response rates according to the presence or absence of specific mutations. In
clinical isolates, however, mutations often occur in patterns, some of which are considered
primary and others compensatory or accessory. Exploratory analyses should be conducted to
define sets of mutational patterns with the largest effect on subsequent response rates.
For some drugs, defining specific mutational patterns that best correlate with a reduction in
treatment response can be difficult. In these cases, another approach can be to investigate the
number of baseline mutations that affects overall response. We encourage sponsors to explore
how the number of baseline mutations correlate with maximal, reduced, or minimal virologic
responses. For example, the response rate (less than 400 copies/mL) in patients with 1 to 2
protease inhibitor- (PI) associated mutations at baseline may be 80 percent, 45 percent if 3 to 4
PI-associated baseline mutations are present, or 10 percent if 5 or more PI-associated baseline
mutations are present. For some drugs, the number and types of mutations may be important for
overall clinical response. Therefore, we recommend sponsors conduct analyses as suggested in
Tables 2A and 2B. Sponsors should discuss in advance with the division the specific mutations
to be included in the overall mutation score. Additional exploratory analyses may be
recommended to further investigate the effect of certain mutations on virologic response.
Table 2A. HIV RNA Response by Number of Baseline Mutations at Endpoint
Number of Baseline
Mutations
Virologic Response Rate
Drug X
(n =)
Control
(n =)
No mutations 	78% 	80%
1-2 mutations 	74% 	76%
3-4 mutations 	45% 	41%
5 or more mutations 	10% 	3%
Note the number of PI mutations (4) used in Table 2B is for illustrative purposes. The number of
mutations that affect virologic response rates vary for each drug; therefore, the number and type
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of mutations used for analyses of HIV RNA response should be discussed with the division. We
recognize the limitations of this type of analysis in that an interaction between two or more
mutations may not be detected. However, this analysis may provide insight into baseline
predicators of response and may provide useful information regarding relationships between
number and type of mutations for further exploratory analyses.
Table 2B. HIV RNA Response by Number and Type of Mutations at Endpoint
Baseline
Primary
PI
Mutation
Number of Mutations
Drug X 	Control
< 4 PI Mutations
%
> 4 PI Mutations
%
< 4 PI Mutations
%
> 4 PI Mutations
%
46 	77% 	32% 	38% 	26%
54 	75% 	24% 	67% 	31%
73 	67% 	34% 	75% 	50%
82 	53% 	31% 	50% 	31%
88 	75% 	44% 	58% 	25%
90 	70% 	40% 	56% 	9%
2. 	Development of HIV Mutations
We strongly recommend genotypic testing on all patients who meet the protocol-defined
definition of a lack or loss of virologic response, preferably while on study drug or as soon as
possible after discontinuation of study drug. Studies have shown WT virus may outgrow
resistant HIV strains in the absence of selective drug pressure. For this reason, it can be useful to
collect and store samples for resistance testing at the same time points HIV RNA testing are
done. These samples can provide important information on the development of resistance,
especially for drugs that may have more than one possible resistance pathway.
The proportion of patients who develop any nucleoside analogue reverse transcriptase inhibitor-
(NRTI), nonnucleoside reverse transcriptase inhibitor- (NNRTI), or PI-associated mutation and
the time to development of these mutations (measured as time to virologic failure) should be
presented. Both primary and secondary mutations should be evaluated. For example, for
patients receiving a new PI, it is important to evaluate the development of primary and secondary
PI mutations along with any other changes in the PR (protease) and RT gene, when applicable.
It is also important to assess the genotypic basis of drug susceptibility changes attributable to
extragenic sites, such as the protease cleavage sites.
3. 	Baseline Phenotype and Virologic Response
Analyses also should be conducted to define the decrease in phenotypic susceptibility that
adversely affects virologic response (i.e., susceptibility breakpoints). Rather than a single
breakpoint, we encourage sponsors to explore incremental subgroups associated with maximal,
reduced, or minimal response rates. However, we recognize a single breakpoint may be more
appropriate for some drugs. Importantly, the breakpoints determined for a given study are not
meant to represent definitive clinical susceptibility breakpoints for all patient populations. Often
the data in an initial NDA submission are based on a select patient population. Data displayed in
package inserts are provided to give clinicians information on the likelihood of virologic success
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based on pretreatment susceptibility to a given agent. Additional data are needed to determine
definitive susceptibility breakpoints for a given drug. An example of this analysis is presented in
Table 3.
Table 3. HIV RNA Response to Drug X by Baseline Susceptibility
Baseline Drug X Susceptibility
Drug X (n =)
HIV RNA < 400 copies/mL
0-5 	56%
>5-10 	33%
>10 	10%
4. 	Genotypic and Phenotypic Correlations: Changes in Susceptibility from Baseline
Assessing changes in susceptibility over time on treatment is an important factor in the
characterization of a drug’s resistance profile. For patients who meet the protocol-specified
definition of a lack or loss of virologic response, evaluation of mean and median-fold changes in
susceptibility from baseline for the investigational drug and other approved drugs from both
inside and outside the same class is important. In addition, analyses should be conducted on
patients who develop a particular new mutation during treatment, and the median-fold change in
susceptibility from baseline should be presented. Table 4 shows an example of how data from
this analysis can be displayed. Efforts also should be made to define relationships between
genotype and phenotype as shown in Table 5.
Table 4. Development of Mutations and Median Change in Susceptibility
from Baseline
Patients Developing
New Entry Inhibitor
N 	Median-Fold Change in Susceptibility from
Baseline
Mutations 	Drug X 	Control
Mutation A
Absent through week 24 	2.1 	3.2
Present by week 24 	10.5 	5.5
Mutation B
Absent through week 24 	1.6 	2.6
Present by week 24 	5.2 	2.2
All patients analyzed 	1.9 	3.2
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Table 5: Relationship between Genotype and Phenotype and
Response
Number of
Baseline
Mutations
Drug X 	Control 	Baseline
Phenotype
Drug X
Susceptibility
1-2 	78% 	75% 	0-5 	Susceptible
3-4 	43% 	45% 	>5-10 	Decreased
susceptibility
5 or more 	10% 	3% 	>10 	Resistant
5. 	Cross-Resistance
The evaluation of cross-resistance with other drugs in the same class is important.
Characterization of cross-resistance of a drug can provide health care providers and patients with
information on how to choose and sequence antiretroviral drugs. Evaluation of the effect of the
investigational drug on subsequent use of other drugs and how previous treatment with other
drugs may affect the response to the investigational drug is essential in drug development. The
former can be accomplished by designing rollover studies evaluating virologic response rates in
patients discontinuing study drug in clinical trials.
We encourage sponsors to incorporate prospective rollover designs to provide for assessment of
virologic responses in study patients administered subsequent antiretroviral regimens. When
possible, the design of a rollover study should include a randomized control. Every effort should
be made to capture as much information as possible from the original studies. Resistance testing
can be used to assess the genotype and phenotype of antiretroviral-experienced patients that
predict success or failure after exposure to previous therapies. This testing can involve longer
follow-up of study patients, perhaps continuing into the postmarketing period.
6. 	Additional Analyses
In addition to the analyses previously suggested, sponsors should consider conducting the
following investigations:
• 	Genotypic sensitivity scores (GSS) and phenotypic sensitivity scores (PSS) and virologic
outcome analyses can be investigated in all phases of development. Sponsors should
discuss with the division in advance methods to calculate GSS and PSS for the optimized
background regimens.
Sponsors are encouraged to conduct exposure-response analyses throughout the drug
development process, beginning with phase 2a studies in HIV-infected patients. One
goal of exposure-response evaluations is to aid dose selection for phase 2b and phase 3
studies. Results from exposure-response evaluations also can help determine whether
dose adjustments are warranted for special populations and whether therapeutic drug
monitoring may be helpful in some patients.
Exposure-response evaluations require the collection of exposure data (drug
concentrations) and response data (efficacy and safety). The sponsor should discuss
•
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exposure-response evaluation plans with the division and the Office of Clinical
Pharmacology before study initiation. Exposure-response evaluations should indicate
which drug exposure measures (e.g., area under the curve, Ctrough) are relevant to a given
virologic outcome. In addition, sponsors are encouraged to evaluate the proportion of
responders to a given drug regimen by inhibitory quotient (IQ), steady-state Cmin values,
and other methods, as appropriate. An IQ value is the ratio of Cmin /EC50 (protein-binding
corrected).
• 	Pharmacogenetic analyses can be conducted to determine genetic factors that may be
involved in virologic response (e.g., for co-receptor inhibitors targeting a host receptor,
genetic differences in the receptor may affect response).
D. 	Other Considerations
1. 	Role for Supporting Initial Activity and Dose-Finding Studies
Primary objectives of initial studies in HIV-infected patients should be to establish that the new
investigational agent has anti-HIV activity, to determine the magnitude of that effect, and to
determine the most active dose that can be taken forward in larger studies. Often, studies that
incorporate short periods of monotherapy (e.g., less than or equal to 2 weeks) or functional
monotherapy (when a drug is added to a failing but stable regimen) have been helpful in
accomplishing these objectives. Compared to combination studies, such protocols can more
clearly delineate the effect of the drug of interest. However, resistant viruses sometimes emerge
rapidly for certain drugs, such that periods of monotherapy can jeopardize a patient’s future
therapeutic options. Thus, some drugs are not candidates for use in monotherapy trials,
including trials of short duration. Drugs for which a single mutation is easily selected and able to
confer large reductions in susceptibility to the new drug and other drugs of the same class should
not be studied as monotherapy, particularly in treatment-naïve individuals. Data from cell
culture resistance studies, along with pharmacokinetic and safety data, should be used to
determine whether a new drug could be safely administered as a single agent for limited periods
of time.
2. 	Data Collection from Dose-Finding Trials
Sponsors should collect baseline genotype and phenotype information in HIV-infected patients
who participate in pharmacokinetic and dose-finding studies. As stated previously, we
encourage sponsors to analyze baseline resistance information and outcome in treatment-
experienced patients. Analysis of baseline resistance and virologic outcome data in treatment-
naïve patients may not be routinely needed; however, analysis of this information may be helpful
to interpret unexpected virologic results. Current evidence indicates virologic response is better
when drug levels can be maintained some increment above the serum-adjusted EC50 value (see
section III, HIV Resistance Testing — General). Study patients with baseline resistance
mutations may require higher drug concentrations of the antiretroviral drug to achieve an
antiviral response that is comparable to that in patients with WT virus. Patients with particular
genotypes and/or phenotypes of interest should be prospectively identified for inclusion in dose-
ranging studies.
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3. 	Use of Resistance Data to Establish an Indication
The amount of evidence sufficient to characterize a drug’s general resistance profile and the
amount of data to support a specific efficacy claim against a particular resistant strain of HIV are
not always the same. Well-controlled, randomized, prospective trials are preferred when
attempting to develop a drug product to obtain a specific indication for use in a select group of
patients with a particular resistance profile at baseline or antiretroviral treatment history. The
amount of evidence sufficient to establish an efficacy claim in a specific patient population will
be substantial, and studies of small numbers of patients are unlikely to accomplish this goal.
Sponsors are encouraged to discuss their development plans with the division in advance. In
addition, resistance data also can be considered for usage statements in a package insert.
4. 	Use of Resistance Data for Study Enrollment Criteria, Background Regimen
Selection, and Stratification Factors
As mentioned earlier, resistance testing at baseline can be helpful in selecting antiretroviral
experienced study patients with particular resistance profiles. For example, to evaluate the
efficacy of a new NNRTI in patients who have failed previous NNRTI regimens, sponsors can
elect to enroll patients with confirmed genotypic and/or phenotypic NNRTI resistance. We
strongly encourage sponsors to use all available phase 1 and phase 2 and nonclinical data to
define which mutation or mutations adversely affect response rate. Early determination of the
effect of baseline genotype and phenotype for new investigational agents can be important for
patient selection into phase 3 clinical trials, thereby restricting specific mutational patterns or
limiting the number of mutations to ensure all patients, regardless of randomized treatment, have
a reasonable chance of virologic response.
Some studies evaluate a new drug combined with an optimized background, meaning that the
concomitantly administered antiretrovirals were chosen based on data from resistance testing.
For trials that include an optimized background regimen, genotypic and/or phenotypic resistance
testing is used to guide the selection of the background antiretroviral regimen. In addition, some
studies have used genotypic and/or phenotypic susceptibility scores to quantify the number of
drugs to which a patient may still be susceptible. Sponsors also can consider using an external
expert committee to aid in the selection of background regimen, especially for patients with
limited therapeutic options.
Baseline resistance testing also can be used to stratify patients. Rationale for specific
stratification factors should be discussed with the division in advance.
5. 	Nonclade B Subtypes
Sponsors are also encouraged to evaluate baseline resistance data and response and the
development of resistance in patients with nonclade B viruses versus clade B viruses. Since
many sponsors are conducting global development plans and it is unknown if different HIV
subtypes develop resistance via different pathways, these data will add to the overall
characterization of an investigational drug’s resistance profile (Hirsch et al. 2003).
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E. 	Monitoring during Phase 4
Resistance should continue to be monitored and further described during postmarketing of an
antiretroviral drug (phase 4). As more drugs enter the market, additional cross-resistance studies
can help to further characterize the cross-resistance profile between new and existing
antiretroviral agents.
VI. 	SUMMARY
Resistance analyses provide information regarding which mutations may have an effect on the
therapeutic success of a given antiretroviral drug product. Such information potentially can be
included in drug labeling to facilitate appropriate prescribing of products and to maximize the
chance for therapeutic success.
The preceding sections of this guidance include recommendations for how and when to obtain
HIV drug resistance information. Appendix A provides guidance for submitting HIV resistance
data. The decision that any given mutation is clinically relevant and deserves inclusion in
product labeling will be determined during the course of an NDA review.
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GLOSSARY
Accessory or compensatory mutation: A mutation that by itself does not confer a decrease in
susceptibility to antiretroviral agents. Accessory or compensatory mutations can augment key
mutations and, perhaps, fitness mutations.
Fitness mutation: An amino acid change that compensates for the reduced virus growth
resulting from a drug resistance-conferring mutation.
Key mutation: A treatment-selected amino acid change that can cause a decrease in
susceptibility to one or more antiretroviral agents of the same class.
Polymorphism: Natural variation in the HIV-1 genome.
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REFERENCES
Baxter, JD, DL Mayers, DN Wentworth et al., 2000, A Randomized Study of Antiretroviral
Management Based on Plasma Genotypic Antiretroviral Resistance Testing in Patients
Failing Therapy; CPCRA 046 Study Team for the Terry Beirn Community Programs for
Clinical Research on AIDS, AIDS, 14(9):F83-93.
Cohen, CJ, S Hunt, M Sension et al., 2002, A Randomized Trial Assessing the Impact of
Phenotypic Resistance Testing on Antiretroviral Therapy, AIDS, 16(4):579-588.
DeGruttola, V, L Dix, R D’Aquila et al., 2000, The Relation Between HIV Drug Resistance and
Response to Antiretroviral Therapy: Re-Analysis of Retrospective and Prospective
Studies Using a Standardized Data Analysis Plan, Antiviral Therapy, 5(1):41-48.
Devereux, HL, M Youle, MA Johnson et al., 1999, Rapid Decline in Detectability of HIV-1
Drug Resistance Mutations After Stopping Therapy, AIDS, 13(8):F123-7.
Durant, J, P Clevenbergh, P Halfon et al., 1999, Drug-Resistance Genotyping in HIV-1 Therapy:
The VIRADAPT Randomised Controlled Trial, Lancet, 353:2195-2199.
Halfon, P, J Durant, and P Clevenbergh, 2003, Kinetics of Disappearance of Resistance
Mutations and Reappearance of Wild-Type During Structured Treatment Interruptions,
AIDS, 17:1351-1361.
Hammer, SM and L Pedneault, 2000, Antiretroviral Resistance Testing Comes of Age, Antiviral
Therapy, 5(1):23-26.
Hirsch, M, F Brun-Vezinet, B Clotet et al., 2003, Antiretroviral Drug Resistance Testing in
Adults Infected with Human Immunodeficiency Virus Type 1: 2003 Recommendations
of an International AIDS Society — USA Panel, CID 2003 July 1, 37:113-128.
Laessig, KA, JS Murray, and G Chikami, 2000, The Role of Resistance Testing in Clinical Trial
Design and Product Labelling: A Regulatory Perspective, Antiviral Therapy, 5(1):77-83.
Little, SH, S Holte, JP Routy et al., 2002, Antiretroviral-Drug Resistance Among Patients
Recently Infected with HIV, New England Journal of Medicine, 347(6):385-394.
Melnick, D, J Rosenthal, M Cameron et al., 2002, Impact of Phenotypic Antiretroviral Drug
Resistance Testing on the Response to Salvage Antiretroviral Therapy (ART) in Heavily
Experienced Patients, 7th Conference on Retroviruses and Opportunistic Infections, San
Francisco, CA, January 30 – February 2, 2000.
Meynard, JL, M Vray, L Morand-Joubert et al., 2000, Impact of Treatment Guided by
Phenotypic or Genotypic Resistance Tests on the Response to Antiviral Therapy (ART):
A Randomised Trial (NARVAL; ANRS 088), 40th ICAAC Abstracts, Toronto, Ontario,
Canada, September 17 – 20, 2000.
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Richman, DD, 2000, Principles of HIV Resistance Testing and Overview of Assay Performance
Characteristics, Antiviral Therapy, (5):27-31.
Tural, C, L Ruiz, C Holtzer et al., 2002, Clinical Utility of HIV-1 Genotyping and Expert
Advice: The Havana Trial, AIDS, 16(2): 209-218.
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APPENDIX A:
GUIDANCE FOR SUBMITTING HIV RESISTANCE DATA
Sponsors are encouraged to use the following sample format for submitting HIV resistance data.
One dataset combines patient data, endpoint data, genotypic data, and phenotypic data. There
are a number of ways datasets can be subdivided (i.e., by clinical study, baseline isolates, or
virologic failure isolates) and this should be discussed with the division before submission.
For each study, we recommend constructing datasets as SAS transport files containing the
following information:
• 	One record (row) per patient per isolate (e.g., baseline, failure, and other time points).
• 	Data in columns (with suggested column headings shown below) 	on all isolates.
Genotypic data should be provided on the corresponding record for each patient isolate for
baseline isolates of all patients in treatment-experienced studies and the endpoint isolates of
virologic failures and discontinuations in all studies.
4
•
In treatment-naïve studies, a baseline
sample should be collected and stored from all patients for future phenotypic and genotypic
analysis of virologic failures.
Phenotypic data should be provided on the corresponding record for each patient isolate for
baseline isolates and the endpoint isolates of virologic failures and discontinuations.5 In
treatment-experienced studies, it is recommended that baseline phenotypic data be obtained
for all patients.
5
•
The specific criteria for defining virologic failures should be discussed with the division and
may include multiple primary and secondary protocol endpoints. The endpoints for
clinical virologic and resistance outcome analyses should be consistent.
Information to Include with Suggested Column Headings4
I. Patient Data:
• 	Patient identification number (ID number should be unique for all studies)
• 	Isolate (e.g., baseline, week 24, week 48, discontinuation. Multiple isolates should be
numbered.)
• 	Date of isolate
• 	Study day (number of days since the patient started the study product)
• 	Previous therapeutic products where available
• 	Treatment group
• 	Censored for analysis (yes or no)
4 In the SAS transport files, column headings can be given abbreviated column names to fit the SAS format;
however, it is suggested that a description of column names be provided to the reviewer in the submission.
5 Treatment and endpoint samples should be collected when the patient is still on the study product.
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II. Endpoint Data:
• 	HIV RNA (copies/mL) at baseline (viral loads less than LLOQ should be listed as “<LLOQ
value” and values greater than ULOQ should be listed as “<LLOQ value” and not left blank)
• 	HIV RNA (copies/mL) at predefined time points (e.g., week 24 and week 48), one column
for each time point including baseline (i.e., the viral load throughout the course of the study
is provided for each sample)
• 	HIV RNA (copies/mL) at other times (e.g., loss of virologic response or discontinuation
because of adverse event)
• 	Endpoint assessment (e.g., mean log change in viral load from baseline)
• 	Other endpoint assessments (e.g., DAVG)
• 	Indication of data were censored for reasons other than virologic failure (e.g., discontinuation
because of adverse event)
• 	Outcome (i.e., responder, virologic failure, discontinuation while suppressed, discontinuation
before achieving viral suppression)
• 	Reason for discontinuation (i.e., adverse event, pregnancy) or failure (i.e., never suppressed,
rebound)
• 	HIV RNA (copies/ml) from additional time points can be included
III. Genotypic Data:6
• 	Clade
• 	Genotype for the RT, protease, and gp160 (for products targeting entry only), one amino acid
per column with the WT amino acid as column heading. Changes from WT standard
sequence indicated (i.e., blanks indicate no change).
• 	Column with total number of PI mutations in patient isolate (for baseline and endpoint
isolates). The specific mutations to include should be discussed with the division in advance.
• 	Column with total number of NRTI mutations in patient isolate (for baseline and endpoint
isolates). The specific mutations to include should be discussed with the division in advance.
• 	Column with total number of NNRTI mutations in patient isolate (for baseline and endpoint
isolates). The specific mutations to include should be discussed with the division in advance.
Example (Table A highlights how genotype information can be displayed, but does not include
all column headings previously suggested.)
6 Genotypic data should be provided for baseline isolates of all patients in treatment-experienced studies and the
endpoint isolates of virologic failures and discontinuations in all studies. In treatment-naïve studies, a baseline
sample should be collected and stored from all patients for future phenotypic and genotypic analysis of virologic
failures.
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Table A. Example of Genotype Information Display
Patient
Number Isolate 	V-82 	N-83 	I-84 	I-85 	G-86 	R-87 	N-88 	L-89 	L-90 Number of PI
Mutations
001 	BL 	S 	M/L 	2
001 	WK48 	V 	S 	M 	3
002 	BL 	A/T 	V 	D 	M 	4
002 	WK48 	T 	V 	M 	3
003 	BL 	T 	V 	2
004 	BL 	V 	M 	2
BL = baseline
WK48 = week 48 for investigational product
IV. Protease Cleavage Sites (for protease inhibitors only):
• 	NC/p1 Gag cleavage sites: show amino acid and position of cleavage site of WT in column
headings (as above for genotype) and indicate amino acid change if mutant
• 	p1/p6 Gag cleavage sites: show amino acid and position of cleavage site of WT in column
headings (as above for genotype) and indicate amino acid change if mutant
V. Phenotypic Data:7
1. 	Information on the investigational product
• 	Baseline EC50 value for investigational product
• 	Baseline EC50 value of reference strain for investigational product
• 	Fold resistant change of baseline EC50 value compared to EC50 value of reference strain
of investigational product
• 	EC50 value at time of endpoint assessment or failure for investigational product
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to
reference strain for investigational product
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to baseline
for investigational product
• 	Replication capacity (if available)
2. 	Information on approved and other investigational anti-HIV products (if available) in the
same class
• 	Fold change in EC50 value of baseline compared to reference strain for each of the
approved and other investigational anti-HIV products (if available)
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to
reference strain for each of the approved and other investigational anti-HIV products (if
available)
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to baseline
for each of the approved and other investigational anti-HIV products (if available)
7 Phenotypic data should be provided for baseline isolates and the endpoint isolates of virologic failures and
discontinuations. In treatment-experienced studies, it is recommended that baseline phenotypic data be obtained for
all patients.
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3. 	Information on approved and other investigational products (if available) outside the
investigational product’s class with same target protein (e.g., NRTIs and NNRTIs)
• 	Fold change in EC50 value of baseline compared to reference strain for approved and
other investigational products (if available) outside the investigational product’s class
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to
reference strain for each of the approved and other investigational products (if available)
outside the investigational product’s class
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to baseline
for each of the approved and other investigational products (if available) outside the
investigational product’s class
4. 	Information on other antiretroviral products in the regimen
• 	Fold change in EC50 value of baseline compared to reference strain for other
antiretroviral products in the regimen, one column per product
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to
reference strain for other antiretroviral products in the regimen, one column per product
• 	Fold change in EC50 value at time of endpoint assessment or failure compared to baseline
for other antiretroviral products in the regimen, one column per product
Example (Table B highlights how phenotype information can be displayed, but does not include
all column headings previously suggested.)
Table B. Example of Phenotype Information Display
Sample
EC50
value
Agent
X
Agent X
Ref
strain
EC50
value
Agent
X
Δ
resis
from
ref
Agent
X
Δ resis
from BL
Agent X
Other Agents in the
Same Class*
Δ resis
from ref
Agent Y
Δ resis
from BL
Agent Y
Other Agents Outside
Agent Class*
Δ resis
from ref
Agent A
Δ resis
from BL
Agent A
Baseline
Endpoint
Agent X = candidate agent
Δ resis = fold resistance change, e.g.: 	EC 50 value of sample with Agent X
EC50 value of reference (or baseline) strain with Agent X
Ref strain = reference strain (or WT)
Endpoint = predefined time point for endpoint assessment (e.g., week 24, week 48, failure or discontinuation)
*Note: The Δ resis from ref and Δ resis from BL should be included for all approved anti-HIV products
VI. Co-Receptor Usage (for all agents targeting co-receptors):
• 	Co-receptor usage of baseline isolates. Indicate R5, X4, D for dual-tropic, M for mixed-
tropic, or D/M if the assay cannot distinguish between dual or mixed, in a column.
• 	Baseline R5 tropism assay value (e.g., RLUs).
• 	Baseline X4 tropism assay value (e.g., RLUs).
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• 	Co-receptor usage of virologic failures and end-of-study isolates (on therapy). Indicate R5,
X4, D for dual-tropic, M for mixed-tropic, or D/M if the assay cannot distinguish between
dual or mixed, in a column.
• 	R5 tropism assay value at failure or end of study (e.g., RLUs).
• 	X4 tropism assay value at failure or end of study (e.g., RLUs).
VII. Therapeutic Drug Monitoring Data (when available):
• 	Patient’s C min
• 	Serum adjusted IQ (inhibitory quotient = C min /serum adjusted EC 50 value)
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