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 
1
 
Guidance for Industry
Role of HIV Resistance Testing in  
  
Antiretroviral Drug Development 

 
 
 
 
This guidance represents the Food and Drug Administrations (FDAs) 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 Administrations (FDAs) 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.  
1 
 Contains Nonbinding Recommendations 
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.  
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe the Agencys 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 DAVPs 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 naive 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.  
2 
 Contains Nonbinding Recommendations 
   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 drugs 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. 
3 
 Contains Nonbinding Recommendations 
Optimally, a comprehensive evaluation of a new drugs 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 (EC  for cell-based assays; IC  for 
50 50
biochemical or subcellular assays) should be determined.  The use of the EC  value for 
50
determining shifts in susceptibility is preferred because it can be determined with greater 
precision than an EC  or EC  value.  A well-characterized wild-type (WT) HIV laboratory 
90 95
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 
4 
 Contains Nonbinding Recommendations 
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 
(CC  or CCIC ). The relative effectiveness of the investigational drug in inhibiting viral 
50 50
replication compared to inducing cell death is defined as the therapeutic or selectivity index (i.e., 
CC  value/EC  value). It is desirable to have a high therapeutic index giving maximum  
50 50
antiviral activity with minimal cell toxicity.  We recommend determining CC  values in both 
50
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 EC  value should be determined (see 
50
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 -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 
5 
 Contains Nonbinding Recommendations 
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 EC  value for the parental virus. 
50
 
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 EC  value) for recombinant virus relative to WT should be determined (see section 
50
IV.G., Characterization of Genotypic and Phenotypic Assays).   
 
2.  Phenotype 
 
Drug susceptibility (EC  values) for resistant variants and the fold change in EC  values 
50 50
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.  
6 
 Contains Nonbinding Recommendations 
 
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  
7 
 Contains Nonbinding Recommendations 
 
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 EC  values for the isolate and a WT standard virus done under the same  
50
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 drugs 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 
8 
 Contains Nonbinding Recommendations 
development of new antiretroviral drugs with unique resistance profiles in treatment-experienced 
patients, we also encourage concurrent clinical development in antiretroviral-naive patients as 
appropriate. 
 
Epidemiological data suggest that transmission of drug-resistant HIV is on the rise, meaning one 
cannot assume treatment-naive 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-naive 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-naive 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 
9 
 Contains Nonbinding Recommendations 
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 drugs resistance profile.  
Analyses for treatment-experienced patients are more complicated than for treatment-naive 
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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 Contains Nonbinding Recommendations 
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 log  decrease from baseline, and mean and/or median 
10
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-naive patients.  However, circumstances may arise in 
treatment-naive 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-naive 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 
11 
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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 
Virologic Response Rate 
Baseline RTI  MUTATION PRESENT  MUTATION ABSENT 
Mutations*  Drug X    Control  Drug X   Control 
(n =)  (n =)  (n =)  (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  Virologic Response Rate  
Mutations  Drug X   Control 
(n =)  (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 
12 
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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  Number of Mutations 
Primary  Drug X   Control 
PI  < 4 PI Mutations  > 4 PI Mutations  < 4 PI Mutations  > 4 PI Mutations 
Mutation  %  %  %  % 
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 
13 
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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 
  Drug X (n =) 
Baseline Drug X Susceptibility   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 drugs 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  N  Median-Fold Change in Susceptibility from 
New Entry Inhibitor  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  Drug X   Control  Baseline  Drug X  
Baseline  Phenotype  Susceptibility 
Mutations 
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, C ) are relevant to a given 
trough
virologic outcome.  In addition, sponsors are encouraged to evaluate the proportion of 
responders to a given drug regimen by inhibitory quotient (IQ), steady-state C  values, 
min
and other methods, as appropriate.  An IQ value is the ratio of C /EC  (protein-binding 
min 50
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 patients 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-naive 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-
naive 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 EC  value (see 
50
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 drugs 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 drugs 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 DAquila 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. 
20 
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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)4 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.5  In treatment-naive 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. 
 
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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 Contains Nonbinding Recommendations 
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-naive 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 of PI 
Isolate  V-82  N-83  I-84  I-85  G-86  R-87  N-88  L-89  L-90 
Number   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 EC  value for investigational product 
50
	   Baseline EC  value of reference strain for investigational product 
50
	   Fold resistant change of baseline EC  value compared to EC  value of reference strain 
50 50
of investigational product 
	   EC  value at time of endpoint assessment or failure for investigational product  
50
	   Fold change in EC  value at time of endpoint assessment or failure compared to 
50
reference strain for investigational product 
	   Fold change in EC  value at time of endpoint assessment or failure compared to baseline 
50
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 EC  value of baseline compared to reference strain for each of the 
50
approved and other investigational anti-HIV products (if available) 
	   Fold change in EC  value at time of endpoint assessment or failure compared to 
50
reference strain for each of the approved and other investigational anti-HIV products (if 
available) 
	   Fold change in EC  value at time of endpoint assessment or failure compared to baseline 
50
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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 Contains Nonbinding Recommendations 
3. 	  Information on approved and other investigational products (if available) outside the 
investigational products class with same target protein (e.g., NRTIs and NNRTIs)  
	   Fold change in EC  value of baseline compared to reference strain for approved and 
50
other investigational products (if available) outside the investigational products class  
	   Fold change in EC  value at time of endpoint assessment or failure compared to 
50
reference strain for each of the approved and other investigational products (if available) 
outside the investigational products class 
	   Fold change in EC  value at time of endpoint assessment or failure compared to baseline 
50
for each of the approved and other investigational products (if available) outside the 
investigational products class 
 
4.	   Information on other antiretroviral products in the regimen 
	   Fold change in EC  value of baseline compared to reference strain for other 

50
antiretroviral products in the regimen, one column per product 

	   Fold change in EC  value at time of endpoint assessment or failure compared to 
50
reference strain for other antiretroviral products in the regimen, one column per product 
	   Fold change in EC  value at time of endpoint assessment or failure compared to baseline 
50
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  
 
       
  Agent X  Other Agents in the  Other Agents Outside 
  Same Class*   Agent Class*  
  EC   Ref      resis   resis   resis   resis   resis 
50 
  value  strain  resis  from BL  from ref  from BL  from ref  from BL 
Sample  Agent  EC   from   Agent X  Agent Y  Agent Y  Agent A  Agent A 
50 
X  value  ref 
Agent  Agent 
X  X 
Baseline                 
Endpoint                 
Agent X = candidate agent 

 resis = fold resistance change,  e.g.:    EC  value of sample with Agent X 
  
50
EC  value of reference (or baseline) strain with Agent X 

50
Ref strain = reference strain (or  WT) 

Endpoint = predefined time point for endpoint assessment (e.g., week 24, week 48, failure or discontinuation) 

*Note: The  resis from ref and   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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 Contains Nonbinding Recommendations 
	   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): 
 
	   Patients C   
min 
	   Serum adjusted IQ (inhibitory quotient = C /serum adjusted EC  value) 
min 50
 
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