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Low Frequency Nonnucleoside Reverse-Transcriptase Inhibitor—Resistant Variants Contribute to Failure of Efavirenz-Containing Regimens in Treatment-Experienced Patients

  1. Elias K. Halvas1,
  2. Ann Wiegand2,
  3. Valerie F. Boltz2,
  4. Mary Kearney2,
  5. Dwight Nissley3,
  6. Michael Wantman4,
  7. Scott M. Hammer5,
  8. Sarah Palmer2,7,
  9. Florin Vaida6,
  10. John M. Coffin2 and
  11. John W. Mellors1
  1. 1Division of Infectious Diseases, University of Pittsburgh, Pittsburgh, Pennsylvania
  2. 2HIV Drug Resistance Program, Frederick, Maryland
  3. 3Basic Research Program, SAIC-Frederick, National Cancer Institute, National Institutes of Health, Frederick, Maryland
  4. 4Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, Massachusetts
  5. 5Division of Infectious Diseases, Columbia University College of Physicians and Surgeons, New York, New York
  6. 6Department of Family and Preventive Medicine, University of California at San Diego School of Medicine, La Jolla, California
  7. 7Department of Virology, Swedish Institute for Infectious Disease Control, Karolinksa Institute, Solna, Sweden
  1. Reprints or correspondence: John W. Mellors, M.D., University of Pittsburgh, Dept of Medicine, Div of Infectious Diseases, 818 Scaife Hall, 3550 Terrace St, Pittsburgh, Pennsylvania 15261 (jwm1{at}pitt.edu).

  1. Presented in part: 11th Conference on Retroviruses and Opportunistic Infections, San Francisco, California, February 2004 (abstract 39).

 
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Abstract

Background. The contribution of low frequency drug-resistant human immunodeficiency virus type 1 (HIV-1) variants to failure of antiretroviral therapy is not well defined in treatment-experienced patients. We sought to detect minor nonnucleoside reverse-transcriptase inhibitor (NNRTI)-resistant variants at the initiation of multidrug efavirenz-containing therapy in both NNRTI-naive and NNRTI-experienced patients and to determine their association with virologic response.

Methods. Plasma samples at entry and at time of virologic failure from patients enrolled in the AIDS Clinical Trials Group study 398 were analyzed by standard genotype, single-genome sequencing and allele-specific polymerase chain reaction (K103N and Y181C) to detect and quantify minor NNRTI-resistant variants.

Results. Minor populations of NNRTI-resistant variants that were missed by standard genotype were detected more often at study entry in NNRTI-experienced patients than NNRTI-naive patients by both single-genome sequencing (8 of 12 vs 3 of 15; P = .022) and allele-specific polymerase chain reaction (>1% Y181C, 5 of 22 vs 3 of 72, respectively; P = 0.16). K103N variants at frequencies >1% were associated with inferior HIV-1 RNA response to efavirenz-containing therapy between entry and week 24 (change in HIV-1 RNA level, +0.5 vs −1.1 log10 copies/mL; P < .001).

Conclusions. Minor NNRTI-resistant variants were more prevalent in NNRTI-experienced patients and were associated with reduced virologic response to efavirenz-containing multidrug regimens.

Human immunodeficiency virus type 1 (HIV-1) diversity within infected individuals arises from continuous, high-level virus turnover (∼ 1 × 1011 virions and 1 × 108 infected cells per day), from nucleotide mis-incorporations by error-prone reverse transcriptase during viral DNA synthesis, and possibly from misincorporations by host cell RNA polymerase II during viral RNA synthesis [1–3]. Many of these mutations do not have a large negative effect on viral fitness and, thus, accumulate during successive rounds of viral replication, generating a diverse population of variants termed “quasispecies.” This diversity supports the hypothesis that drug-resistant HIV-1 variants exist within an infected individual before antiretroviral drug therapy is started [3, 4]. The frequency of such pre-existing drug-resistant variants can increase rapidly with partially suppressive antiretroviral therapy and become dominant in the virus population. Following removal of drug selective pressure by cessation of therapy, levels of drug-resistant variants often decrease such that they become a minor fraction of the virus population but exist at higher frequencies than in drug-naive individuals [5]. Such variants can rapidly re-emerge after restarting antiretroviral therapy with drugs to which the variants are resistant or cross-resistant [5].

HIV-1 drug resistance testing using genotype analysis of polymerase chain reaction (PCR)-amplified sequences from the virus population in plasma is recommended for management of antiretroviral therapy [6]. However, this standard method identifies only the dominant viruses present and fails to detect variants comprising less than 10%–20% of the virus population [7]. More sensitive techniques have been developed that can detect minor populations of drug-resistant variants that are missed by standard genotype, but their clinical utility has yet to be established [5, 7, 8, 9, 10]. Minor drug-resistant variants have been detected in treatment-naive individuals and are associated with higher risk of failure of initial antiretroviral therapy [9, 10]. In treatment-experienced patients, minor drugresistant variants may persist for months and even years during and after treatment [5, 11, 12]. It is not known, however, whether such drug-resistant variants influence response to subsequent treatment regimens. We therefore assessed the influence of minor nonnucleoside reverse-transcriptase inhibitor (NNRTI)-resistant variants on response to efavirenz-containing multidrug regimens.

AIDS Clinical Trials Group (ACTG) study 398 was a randomized, multicenter trial that tested the efficacy of 1 or 2 protease inhibitors in combination with abacavir, adefovir, and efavirenz, in patients receiving a failing regimen containing a protease inhibitor. All patients had prior exposure to a nucleoside reverse-transcriptase inhibitors and 44% had prior exposure to the NNRTIs delavirdine or nevirapine [13]. Treatment response was strongly associated with prior NNRTI exposure; NNRTI-experienced patients were more than twice as likely to experience a protocol-defined virologic failure end point, compared with NNRTI-naive patients. NNRTI experience predicted poor virologic outcome independent of detecting NNRTI resistance at entry by standard population genotype [14]. This finding led to the hypothesis that minor NNRTI-resistant variants, selected by prior NNRTI exposure but below the limit of detection by population genotype, were contributing to failure of the efavirenz-containing regimens. To test this, we compared the frequency of minor NNRTI-resistant variants between NNRTI-naive and NNRTI-experienced patients, determined if their detection was predictive of virologic response, and analyzed the genetic relatedness of NNRTI-resistant variants detected at study entry and at the time of virologic failure.

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Methods

Subject selection and samples. Plasma samples were obtained at study entry and at the time of protocol-defined virologic failure from subjects in ACTG study 398 [13]. For single-genome sequencing analyses, a total of 27 subjects (15 NNRTI-naive and 12 NNRTI-experienced) were randomly selected from enrollees meeting the following criteria: (1) entry sample negative for NNRTI resistance mutations by standard genotype analysis (ViroSeq platform; Celera); (2) reached a protocoldefined virologic failure end point by study week 24 [13], and (3) the virologic failure sample had ⩾1 major NNRTI resistance mutations (ie, L100I; K101E; K103N; V106A or M; V108I; Y181C or I; Y188C, H, or L; G190A or S; P225H; or P236L [15]) detected by standard genotype analysis.

NNRTI resistance at study entry by allele-specific real-time PCR was determined for 103 participants, consisting of 27 NNRTI-experienced and 76 randomly selected NNRTI-naive patients. Both groups had available entry plasma samples and a negative standard genotype for NNRTI resistance. ACTG 398 was approved by the institutional review boards of the participating centers, and all patients provided written consent for participation in the study and testing of samples.

Single-genome sequencing. Single-genome sequencing was performed as reported [8] with the following modifications: 500 µL of plasma (diluted to contain 5000 copies of HIV-1 RNA) was extracted, complementary DNA (cDNA) was prepared using the Superscript 1st Strand Synthesis System (In-Vitrogen), and cDNA was serially diluted 1:2 in 5 mmol/L Tris-HCl (pH, 8.0) to a maximum dilution of 1:16. Ten independent PCR reactions were setup in a screening plate for each cDNA dilution (1:2, 1:4, 1:8, and 1:16). Amplification products from cDNA dilutions yielding ∼30% positive PCR reactions were sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). All sequences were assembled using Sequencher software (Gene Codes) or by the automated Phrap program in conjunction with Phred and CLUSTAL W [16–19]. The Polyphred program was used to identify potential mixtures, which were excluded unless they were at a single nucleotide position [20]. Drug-resistance mutations in single sequences were identified using the Stanford database HIValg web-based program [21].

Phylogenetic analyses. Phylogenetic analyses were performed as follows: sequences were trimmed using Sequencher, aligned by CLUSTAL W (DAMBE), and phenograms were generated with HIV-1LAI as the outgroup using the Neighbor-Joining method (MEGA v2.1) with the p-distance model analysis preference [22, 23]. Distance was determined as the proportion (p) of nucleotide sites at which 2 sequences being compared differ and calculated by dividing the number of differences by total number of nucleotides compared. Bootstrap values were calculated using 1000 replicates and values ⩾60 are shown in the phenograms.

Allele-specific PCR for NNRTI-resistant variants. Allele-specific PCR was performed as reported elsewhere [5, 24] with the following modifications: 250–1800 µL of plasma containing ⩾10,000 HIV-1 RNA copies were analyzed at codons 103 and 181 with use of 3 discriminatory primers for 3 different mutant alleles (AAC and AAT for K103N and TGT for Y181C). A first round of real-time reverse transcriptase PCR generated a 637-bp pol amplification product, which was diluted to 1 × 107 copies and reamplified by a second round of real-time PCR utilizing both nondiscriminatory and discriminatory primers specific for codons 103 and 181 in reverse transcriptase. Amplicon generation was monitored by Sybr Green fluorescence and specificity determined by thermal denaturation analysis. Results were reported as percentage mutant or wild-type.

Statistical analysis. All analyses were performed using the R statistical software package [25]. The association between presence of NNRTI resistance and virologic failure by week 24 was assessed using Fisher's exact test. The association between a history of NNRTI experience and virologic failure by week 24, controlling for baseline NNRTI resistance mutations, was determined using a logistic regression model (Wald test, odds ratios, and confidence intervals). The association between NNRTI experience and the detection of NNRTI-resistant variants at study entry by single-genome sequencing was determined using a Fisher's exact test. The association between NNRTI experience and the proportion of sequences containing NNRTI resistance mutations at entry or virologic failure were determined by a logistic regression. A Gehan-Wilcoxon rank test was used to compare changes in HIV-1 RNA between subgroups with and without NNRTI-resistant variants detected by single-genome sequencing, stratified by baseline NNRTI experience and accounting for HIV-1 RNA censored below the limit of detection. Seven of 27 subjects experienced virologic failure before week 24; thus, their available HIV-1 RNA data were imputed (week 16 for 5 cases and week 8 for 2 cases). HIV-1 RNA values below the limit of detection (200 copies/mL) were replaced by 200 copies/mL. Statistical analyses of data from allele-specific PCRs were performed using mutant frequency cutoffs of >0.5% and >1.0%. The association between NNRTI experience and the detection of K103N or Y181C was determined using a Fisher's exact test. The association between mutant frequency and change in HIV-1 RNA level from entry to week 24 was determined using a nonparametric log-rank test. Fifteen of 103 subjects experienced virologic failure prior to week 24; therefore, their available data were imputed (week 16 for 7 cases, week 8 for 4 cases, week 4 for 3 cases, and week 2 for 1 case). The association between mutant frequency and virologic failure (HIV-1 RNA level >200 copies/ml at week 24) was determined by a logistic regression.

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Results

Influence of prior NNRTI experience on virologic response to therapy. As reported elsewhere [13], virologic failure in ACTG study 398 was strongly associated with prior NNRTI exposure to either nevirapine or delavirdine (efavirenz exposure was not permitted). By study week 24, protocol-defined virologic failure had occurred in 83% (176 of 212) of NNRTI-experienced patients versus 58% (156 of 269) of NNRTI-naive participants (intent-to-treat analysis, P < .001) [13]. Standard genotype analysis was performed on baseline samples from 452 of the 481 study participants. NNRTI resistance mutations were detected in 165 participants (9 of 246 NNRTI-naive and 156 of 206 NNRTI-experienced patients), and the presence of resistance was associated with virologic failure by week 24 (P < .001). In the current study, we found that a history of NNRTI experience was significantly associated with virologic failure at week 24 after controlling for baseline NNRTI resistance (odds ratio, 2.07 [95% confidence interval, 1.1–3.88]; P = .024, by Wald test of logistic regression). To explore this observation further, we examined the change in plasma HIV-1 RNA level from baseline to week 24 among the following 3 subgroups: (1) NNRTI-naive with baseline standard genotype negative for NNRTI resistance (N = 235), (2) NNRTI-experienced with NNRTI resistance detected at baseline by standard genotype (N 156), and (3) NNRTI-experienced with baseline standard genotype negative for NNRTI resistance (N = 48). The fourth group of NNRTI-naive subjects with NNRTI resistance at baseline (N = 9) was too small to include in this analysis.

Figure 1 shows that the change in HIV-1 RNA level from entry to week 24 in the NNRTI-experienced subgroup without baseline NNRTI resistance by standard genotype resembled that of the genotype-positive NNRTI-experienced subgroup, which was inferior to that of the NNRTI-naive subgroup. This observation led to the hypothesis that minor populations of NNRTI-resistant variants compromised the response to efavirenz-containing multidrug therapy in NNRTI-experienced patients with negative standard genotypes for NNRTI resistance. To test this hypothesis, we used 2 more-sensitive methods, single-genome sequencing and allele-specific PCR, to detectminor NNRTI-resistant variants in plasma samples from study entry and to assess their impact on virologic response.

Figure 1.
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Figure 1.

Median change in human immunodeficiency virus type 1 RNA (log10 copies/mL), by nonnucleoside reverse-transcriptase inhibitor (NNRTI) experience and baseline NNRTI resistance. NNRTI resistance was assessed by standard genotype (ViroSeq v2.0). Subgroups shown are (1) NNRTI naive, standard genotype negative for NNRTI resistance, (2) NNRTI experienced, standard genotype positive for NNRTI resistance, and (3) NNRTI experienced, standard genotype negative for NNRTI resistance. Vertical bars represent 95% confidence intervals.

NNRTI resistance mutations detected by single-genome sequencing. For single-genome sequencing analyses, 27 baseline and virologic failure sample pairs were randomly selected as described in the Methods. For NNRTI-experienced patients, the median duration after discontinuation of prior NNRTI therapy was 366 days (range, 0–555 days). Sequences were generated and analyzed at entry and at virologic failure, except for participant 11E for whom no sample was available at virologic failure.

Single-genome sequencing results are summarized in Table 1. A total of 1566 sequences from study entry were analyzed (a mean of 46 sequences/sample), allowing the detection of variants present at a frequency of ⩾5% with 90% confidence. Only ∼12 sequences/sample were analyzed at the time of virologic failure because NNRTI-resistant variants were dominant, as determined by standard genotype analysis.

The number and types of NNRTI resistance mutations detected at study entry were significantly different between NNRTI-naive and -experienced groups (Table 1). Specifically, ⩾1 sequences encoding an NNRTI resistance mutation were detected in 8 of 12 NNRTI-experienced patients, comparedwith 3 of 15 NNRTI-naive patients (P = .022). Furthermore, the fraction of sequences encoding NNRTI resistance mutations was higher in the NNRTI-experienced group (31 of 468 sequences) than in the NNRTI-naive group (3 of 773 sequences; P < .001).

In this small subset of 27 participants analyzed by singlegenome sequencing, the HIV-1 RNA response from entry to virologic failure was inferior in the NNRTI-experienced group (change ± standard error, +0.13 ± 0.19 log10 copies/mL), compared with the NNRTI-naive group (change ± standard error, −.35 ± 0.13 log10 copies/mL; P = .032).

Phylogenetic analyses. Phylogenetic analyses were performed to assess the relatedness between sequences with NNRTI resistance mutations at entry and virologic failure. Close clustering between sequences obtained at baseline and at virologic failure that contain NNRTI resistance mutations, as evidenced by high bootstrap values, was observed in 1 of 3 NNRTI-naive subjects and 1 of 8 NNRTI-experienced subjects, with the suggestion of clustering in a second NNRTI-experienced subject. Figure 2 illustrates these phylogenetic relationships. In subject 7N from the NNRTI-naive group (Figure 2A), a baseline sequence containing K103N clustered closely (bootstrap value, 96) with sequences at the time of virologic failure containing K103N linked to Y188C or M230L. By contrast, baseline sequences from NNRTI-naive subject 5N and 12N containing P225H and L100I, respectively, did not cluster with the predominant resistant variants at virologic failure (Figure 2B and 2C).

Figure 2.
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Figure 2.

Phylogenetic analysis comparing relatedness between single-genome sequences at baseline and virologic failure. Nonnucleoside reverse-transcriptase inhibitor (NNRTI)-naive subjects are shown in panels A-C, and NNRTI-experienced subjects are shown in panels D-F. Solid green circles represent baseline sequences with NNRTI resistance mutations. Open circles represent baseline sequences without NNRTI resistance mutations. Solid red squares represent sequences obtained at virologic failure with NNRTI resistance mutations. Open squares represent sequences obtained at virologic failure without NNRTI resistance mutations. Bootstrap values >60 are shown.

NNRTI-experienced subject 5E had 2 distinct populations of sequences at entry, 1 wild-type and 1 containing several linked NNRTI resistance mutations (K101E, Y181C, and G190A). The mutant sequences at entry intermingled in multiple clusters (bootstrap values, >60) with the mutant sequences at virologic failure (Figure 2D). For NNRTI-experienced subject 3E, entry sequences containing K101E showed a trend toward clustering (bootstrap value, 46) with sequences obtained at virologic failure containing L100I-K101E-Y188L or L100I-K101E-G190A (Figure 2E). The observed clustering of mutant sequences at entry and at virologic failure for these subjects was not altered by changing drug-resistance mutations to wild-type (data not shown).

The NNRTI-resistant mutants detected at entry for the 5 other NNRTI-experienced subjects (Table 1) did not cluster with the mutant sequences obtained at virologic failure. To illustrate, patient 8E had sequences at entry and at virologic failure that contained K103N, but these sequences were not closely related (Figure 2F). NNRTI resistance mutations detected by allele-specific PCR.

Table 1.
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Table 1.

Nonnucleoside Reverse-Transcriptase Inhibitor (NNRTI) Resistance Detected by Single-Genome Sequencing

To further assess whether minor populations of NNRTI-resistant variants were more frequent in the NNRTI-experienced patients and associated with virologic failure, baseline samples from 103 subjects (76 NNRTI naive and 27 NNRTI experienced) with negative standard genotype for NNRTI resistance were tested using allele-specific PCR for mutants K103N and Y181C. Of the 103 subjects selected, 5 subjects (2 NNRTI naive and 3 NNRTI experienced) were excluded because follow-up HIV-1 RNA measurements were not available. In addition, allele-specific PCR results could not be obtained in 4 subjects for Y181C and 3 for K103N, leaving 95 subjects evaluated for K103N and 94 for Y181C (Table 2). A significant association was found between NNRTI experience and Y181C variants at mutant cutoff frequencies >0.5% (5 of 22 versus 3 of 72 subjects; P = .016) and >1% (5 of 22 versus 3 of 72 subjects; P = .016). A trend for association was found between NNRTI experience and K103N at frequencies >0.5% (4 of 24 versus 4 of 71 subjects; P = .11) and >1% (3 of 24 versus 2 of 7 subjects; P = .10).

Table 2.
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Table 2.

Impact of K103N and Y181C Variants on Human Immunodeficiency Virus Type 1 (HIV-1) RNA Response

K103N at frequencies >1.0% was strongly associated with inferior HIV-1 RNA response from baseline to week 24 by intent-to-treat analysis (P < .001; Table 2). Specifically, K103N at frequencies >1.0% was associated with an increase in HIV-1 RNA of 0.5 log10 copies/mL, whereas K103N ⩽1.0% was associated with a decrease in HIV-1 RNA of 1.1 log10 copies/mL. K103N at a >0.5% frequency was also significantly associated with inferior HIV-1 RNA response, although the strength of association was lower (P = .006 ). K103N at frequencies >1% was also associated with protocol-defined virologic failure in both on-treatment (P = .036) and intent-to-treat analyses (P = .053). In contrast, Y181C variants were not significantly associated with either protocol-defined virologic failure or change in HIV-1 RNA from entry to week 24 (Table 2), although the average reduction in HIV-1 level was lower for those with >1% Y181C (−0.4 log10 copies/mL) versus those with ⩽1.0% mutant (−1.1 log10 copies/mL).

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Discussion

This work provides several lines of evidence that minor populations of NNRTI-resistant variants not detected by currently recommended resistance assays can contribute to failure of antiretroviral regimens containing efavirenz, especially among patients with prior exposure to nevirapine or delavirdine. This evidence includes (1) more-frequent detection of minor NNRTI-resistant variants in NNRTI-experienced than NNRTI-naive patients by 2 different methods (single-genome sequencing and allele-specific PCR), (2) strong phylogenetic relatedness in several subjects between single-genome sequences containing NNRTI resistance mutations detected before the initiation of efavirenz-containing therapy and those present at the time of virologic failure, and (3) a significant association between K103N variants at frequencies >0.5 or >1% and reduced HIV-1 RNA response to efavirenz-containing regimens. These findings contribute to the growing body of evidence that the current generation of resistance assays used in clinical practice may miss minor but clinically important populations of drug-resistant variants [9, 10].

Although the regimens compared in ACTG 398 included adefovir and single or dual unboosted protease inhibitors, which are no longer relevant for management of HIV-1 infection, the study provided an important opportunity to examine the potential role of minor drug-resistant variants on treatment response to multidrug regimens. We focused on NNRTI-resistant variants because 44% of study subjects had a history of prior exposure to nevirapine or delavirdine, and all subjects were randomized to a new regimen containing efavirenz. Because of the limited potency of the other regimen components—adefovir, abacavir, and single or dual unboosted protease inhibitors—in nucleoside reverse-transcriptase inhibitor- and protease inhibitor-experienced patients, efavirenz activity is a key driver of treatment response [13]. In addition, it is known that single point mutations in HIV-1 reverse transcriptase (eg, K103N) can confer high-level cross-resistance to other NNRTIs and that such mutations can be detected by several methods, including single-genome sequencing and allele-specific PCR [7]. As such, ACTG 398 provided key opportunities to investigate the influence of major and minor populations of NNRTI-resistant variants on response to efavirenz-containing regimens. The results of these investigations provide strong evidence that both major (detected by standard genotype) and minor populations of NNRTI-resistant variants can compromise virologic response to efavirenz-containing regimens [13, 14].

Allele-specific PCR revealed that prior exposure to delavirdine or nevirapine was more strongly associated with the presence of Y181C variants at frequencies >0.5 and >1.0% in entry samples than with K103N variants at similar frequencies. This difference is likely explained by the preferential selection of Y181C over K103N by nevirapine and to a lesser extent by delavirdine [26, 27, 28]. By contrast, K103N at frequencies >0.5 and >1% was was strongly associated with decreased virologic response to efavirenz-containing regimens, whereas Y181C variants were not significantly associated with response. This finding is consistent with greater fitness and higher level resistance of K103N variants to efavirenz than Y181C variants [29–31].

Phylogenetic analyses were performed on single-genome sequences to assess the genetic relatedness between NNRTI-resistant variants at entry and virologic failure. These analyses revealed several important findings. In 1 of 15 NNRTI-naive subjects (subject 7N; Figure 2A), an entry sequence containing K103N clustered closely with all the mutant sequences obtained at virologic failure that contained K103N with M230L or Y181C. This observation suggests that the virus population at virologic failure evolved from a K103N mutant population detected at entry. Although anecdotal, this example is consistent with other reports of major and minor NNRTI-resistant mutants in treatment-naive individuals that arise spontaneously or are acquired through transmission of resistant virus and can compromise response to NNRTI-containing regimens [9, 10, 32]. In 2 other NNRTI-naive patients (Figure 2B and 2C), sequences at entry had NNRTI resistance mutations, but completely different NNRTI-resistant mutant sequences were present at virologic failure. These examples illustrate the complexity of assigning significance to a specific variant that is identified using moresensitive methods of detection. In 1 of 8 NNRTI-experienced patients, entry sequences containing NNRTI resistance mutations clustered closely with mutant sequences at virologic failure, again suggesting that the virus population at virologic failure evolved from the mutant population at entry. Identifying such associations is noteworthy given the small number of sequences (N = 45) examined at entry relative to the total population of virus. Indeed, in the 5 other NNRTI-experienced patients, there was no obvious clustering of mutant sequences from entry and virologic failure time points (eg, patient 8E; Figure 2F). The lack of clustering may be attributable to recombination and the large potential impact of few nucleotide substitutions on phylogenetic relationships between closely related sequences from the same individual. In addition, the phylogenetic analyses highlight the limited sampling possible by single-genome sequencing and the advantage of allele-specific PCR for detecting specific variants at low frequency in virus populations as well as relating their frequency to treatment response as described above (Table 1 vs Table 2).

In summary, our findings support the general concept that minor populations of NNRTI-resistant variants that are missed by current clinical testing can persist after NNRTI exposure and contribute to failure of multidrug regimens containing an NNRTI. This concept is particularly relevant for women in developing countries who have received single-dose nevirapine for prevention of mother-to-child transmission of HIV-1 and who begin therapy with an NNRTI-containing regimen [33]. Although 1 report suggests that levels of NNRTI-resistant variants decrease to clinically insignificant levels in most women 6 months after receipt of single-dose nevirapine [34], others have reported long-term persistence of variants and poor response to NNRTI-containing regimens [24, 35, 36]. Additional studies are thus needed to define the clinical significance and optimal management of minor variants that are resistant to NNRTI or other drug classes.

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Acknowledgments

We acknowledge the ACTG 398 Protocol Team, the pharmaceutical cosponsors (Agouron, DuPont-Merck, Gilead Sciences, GlaxoSmithKline, Merck, Roche, and ViroLogic), volunteers, and research staff. We would like to thank Bob Stephen and Beena Neelam of the Advanced Biomedical Computing Center, SAIC-Frederick, National Cancer Institute, National Institutes of Health, Frederick, Maryland, for the help in assembling singlegenome sequences.

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Footnotes

  • Potential conflicts of interest: J.W.M. is a consultant to Gilead Sciences, Merck, and Chimerix; has received grant support from Merck; and is a shareholder for RFS Pharma. S.M.H. is a scientific advisor to Merck and Progenics. All other authors: none reported.

  • Financial support: NIAID, Division of AIDS (University of Pittsburgh CTU Grant 1U01 AI069494-01), and a Virology Support Laboratory subcontract (204VC009) of the ACTG Central Group Grant (1U01AI068636-01).

  • Received May 15, 2009.
  • Accepted September 2, 2009.
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