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Persistence of K103N-Containing HIV-1 Variants after Single-Dose Nevirapine for Prevention of HIV-1 Mother-to-Child Transmission
 
 
  The Journal of Infectious Diseases Jan 18, 2007
 
Tamara S. Flys,1 Deborah Donnell,2 Anthony Mwatha,2 Clemensia Nakabiito,3 Philippa Musoke,3 Francis Mmiro,4 J. Brooks Jackson,1 Laura A. Guay,1 and Susan H. Eshleman1
 
1Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; 2Statistical Center for HIV/AIDS Research and Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington; Departments of 3Paediatrics and 4Obstetrics and Gynaecology, Makerere University, Kampala, Uganda
 
ABSTRACT
K103N-containing human immunodeficiency virus (HIV)-1 variants are selected in some women who receive single-dose (SD) nevirapine (NVP) for prevention of HIV-1 mother-infant transmission. We examined the persistence of K103N in women who received SD NVP prophylaxis. K103N was detected using the LigAmp assay (assay cutoff, 0.5% K103N). K103N was detected at 6-8 weeks in 60 (41.7%) of 144 women. Fading (lack of detection) of K103N was documented in 16 women by 2 years, 43 women by 3 years, and 55 women by 4 and 5 years. Slower fading was independently associated with HIV-1 subtype (D>A) and higher pre-NVP viral load.
 
The HIV Network for Prevention Trials (HIVNET) 012 regimen of single-dose (SD) nevirapine (NVP) [1, 2] is safe, inexpensive, and effective for prevention of HIV-1 mother-to-child transmission (pMTCT). However, exposure to SD NVP is associated with selection of NVP-resistant HIV-1 variants. Using a population sequencing-based genotyping assay (ViroSeq), the portion of women with NVP resistance detected 6-8 weeks after SD NVP varied among HIV-1 subtypes (C>D>A) [3]. The most common NVP resistance mutation detected in women with all 3 subtypes was K103N. ViroSeq reliably detects the K103N mutation when it is present in >20% of the viral population [4]. Using a sensitive and quantitative point mutation assay (LigAmp), the portion of women with detectable K103N (>0.5% K103N) 6-8 weeks after SD NVP was 41.7% for subtype A, 55.3% for subtype D, and 69.8% for subtype C. The highest mean levels of K103N were found in women with subtype C (2.2% for subtype A, 5.5% for subtype D, and 11.7% for subtype C) [5]. Differences in detection of K103N in women with these 3 subtypes did not appear to reflect differences in the viral loads of the samples or differences in HIV-1 sequences at the binding sites of oligonucleotides used in the LigAmp assay [5].
 
K103N-containing variants can persist at low levels for years in the absence of antiretroviral drug exposure in patients who are infected with resistant strains [6] and in patients who discontinue treatment with a nonnucleoside reverse-transcriptase inhibitor (NNRTI) [7]. Long-term persistence of K103N-containing variants in women after SD NVP could potentially reduce the efficacy of SD NVP for pMTCT in subsequent pregnancies or the efficacy of NNRTI-based regimens for future treatment of their HIV-1 infection. In the HIVNET 012 trial, 1-2-year follow-up samples were available for 9 women who had NVP resistance mutations detected at 6-8 weeks (3 of subtype A and 6 of subtype D). Using the LigAmp assay, we detected low levels of K103N-containing variants in 3 of those women 1-2 years after SD NVP, all with subtype D [8]. In this report, we used the LigAmp assay to analyze the persistence of K103N-containing variants in women with subtype A and D HIV-1 who were enrolled in a 5-year follow-up study of the HIVNET 012 trial.
 
Subjects, materials, and methods.
Two-hundred thirty-two (75.8%) of 306 antiretroviral drug-naive Ugandan women who received SD NVP in HIVNET 012 were subsequently enrolled in a follow-up study with annual study visits 2-5 years after their original SD NVP exposure. Eighty-eight of the 232 women were excluded from analysis for the following reasons: (1) the receipt of SD NVP in a subsequent pregnancy during the follow-up period (n = 39), (2) the presence of a subtype other than A or D (3 of subtype C, 23 recombinant, and 5 of undetermined subtypes), (3) the receipt of treatment with combination antiretroviral therapy during the follow-up period (n = 2), (4) the lack of a 6-8-week sample available for analysis (n = 7), (5) the failure of the 6-8-week sample to amplify (n = 4), (6) the occurrence of the 6-8 week-study visit >100 days after receiving SD NVP (n = 4), and (7) the presence of an unusual K103Q polymorphism in HIV-1 reverse transcriptase (n = 1). In the woman with the K103Q polymorphism, 3%-6% K103N was detected in samples both before and up to 5 years after SD NVP exposure. Analysis of HIV-1 clones from the pre-NVP sample confirmed that detection of K103N represented a false-positive test result. In contrast, analysis of other samples with 0.5%-1% K103N with an independent assay confirmed the presence of low levels of K103N-containing variants [8].
 
The LigAmp assay involves a mutation-specific oligonucleotide ligation step followed by a universal real-time polymerase chain reaction (PCR) detection step [4]. The LigAmp assay was performed using PCR products remaining after HIV-1 genotyping with ViroSeq (assay cutoff, 0.5% K103N). Samples were analyzed in duplicate, and the results for the percentage of K103N were averaged.
 
HIV-1 subtyping was performed previously by phylogenetic analysis of HIV-1 pol region sequences [3]. Baseline HIV-1 load and CD4 cell count were measured before NVP administration in the HIVNET 012 trial, as described elsewhere [1, 2]. Informed consent was obtained from all subjects for participation in HIVNET 012 and the follow-up study. HIVNET 012 and the follow-up study were approved by the local institutional review boards in Uganda and at Johns Hopkins University School of Medicine. Guidelines of the US Department of Health and Human Services and the authors' institutions were followed in the conduct of this research.
 
Results.
Among 144 women in this study, 60 (41.7%) had K103N detected at the 6-8-week visit (>0.5% K103N). We analyzed persistence of K103N among those 60 women by use of samples collected 2, 3, 4, and 5 years after SD NVP administration. For each woman, after fading of K103N was documented (<0.5% K103N detected in a sample), samples from subsequent study visits were not tested. Many of the 60 women did not have 2-year samples because approval of the amendment for the follow-up study and their consent for the follow-up study were obtained >2 years after they received SD NVP in HIVNET 012. The level of K103N detected at 6-8 weeks was similar among women with 2-year samples (median, 2.2%) to that of those without 2-year samples (mean, 2.3%; P = .44); however, there is some imbalance in subtype. Women with subtype A compose a higher proportion of those with samples at 2 years (14/20 of those who were tested at 2 years were subtype A vs. 16/40 of those who were not tested; P = .055).
 
Among all 144 women, we documented undetectable K103N in a total of 100 women by 2 years, 127 by 3 years, and 139 by 4 and 5 years (figure 1). The remaining women either had K103N detected or had no sample available for testing. Figure 1 shows the number of women tested at each study visit. Among the 60 women who had K103N detected at 6-8 weeks, we documented undetectable K103N in 16 women by 2 years, 43 by 3 years, and 55 by 4 and 5 years. Whenever K103N was detected in samples after the 6-8-week visit, the level of K103N in the viral population was low. The mean percentage of K103N among women with detectable K103N was 1.2% at 2 years (n = 4 women; range, 0.9%-1.8%) and 0.8% at 3 years (n = 9 women; range, 0.5%-1.2% K103N).
 
A statistical model was used to estimate the cumulative rate of fading of K103N. In this study, in which many women did not have 2-year results but did have 3-year results (interval censored), standard Kaplan-Meier estimates could not be used to estimate the cumulative probability of fading at each study visit. The Turnbull estimate (a Kaplan-Meier-type estimate modified for interval censored data) uses the intuitive assumption that the distribution of missing test data at each study visit can be estimated from available test data from the same visit. This approach provides estimates and corresponding confidence intervals (CIs) of the cumulative percentage of women with undetectable K103N at each study visit (cumulative rate of fading). The estimated cumulative rates of fading for all 144 women at 2, 3, 4, and 5 years by use of this model were 87.6%, 91.9%, 99.1%, and 99.1%, respectively (table 1). When the analysis was limited to the 60 women with K103N detected at 6-8 weeks, the estimated cumulative rates of fading at 2, 3, 4, and 5 years were 63.0%, 80.7%, 97.8%, and 97.8%, respectively (table 1). The estimated rates of fading were consistently lower in subtype D than A, both among all 144 women and among the subset of 60 women who had K103N detected at 6-8 weeks (30 women with subtype A and 30 women with subtype D) (table 1). Among those 60 women, the mean number of years to the first available follow-up sample was similar for women with subtype A, compared with those with D (2.9 years for both subtypes; P = .94, t test).
 
In univariate models examining predictors of fading among all 144 women, subtype (D>A), higher baseline viral load, and lower baseline CD4 cell count were significant predictors for a longer time to fading of K103N. In a multivariate model, subtype (hazard ratio [HR] for D vs. A, 0.50 [95% CI, 0.33-0.77]; P = .0007) and baseline viral load (HR per log increase in baseline viral load, 0.63 [95% CI, 0.48-0.83]; P = .0006) were independently associated with time to fading, but baseline CD4 cell count was not (HR per decrease of 100 cells, 0.97 [95% CI, 0.90-1.05]; P = .43). The time to fading was approximately twice as long for women with subtype D, compared with women with subtype A, and a log increase in baseline viral RNA increased time to fading by a factor of 1.57. Among the subset of 60 women who had K103N-containing variants detected at 6-8 weeks, time to fading was predicted by the level of K103N at the 6-8-week visit in a univariate model but not in a multivariate model. In a multivariate model, results for this subset of 60 women were similar to those obtained for all 144 women; only subtype and viral load predicted time to fading (HR for subtype, 0.34 [95% CI, 0.17-0.68] [P = .005]; HR for viral load, 0.45 [95% CI, 0.22-0.90] [P = .003]).
 
Discussion.
This report demonstrates that HIV-1 variants with K103N can persist at very low levels for years in some women following SD NVP exposure and that HIV-1 subtype (D>A) and higher baseline viral load are both associated with slower fading of those variants. Since we first described selection of K103N-containing variants after SD NVP [9], concerns have been raised about whether persistence of NVP-resistant HIV-1 would lower the efficacy of SD NVP for pMTCT in subsequent pregnancies. Two preliminary studies found no significant difference in the efficacy of the HIVNET 012 regimen for pMTCT with first versus second use [10, 11]. Early reports of NVP resistance after SD NVP also raised concerns that prior exposure to SD NVP would adversely affect the response of women to future treatment with NNRTI-containing regimens. Available studies suggest that NVP-resistant variants that emerge after SD NVP exposure are unlikely to compromise subsequent treatment of women with NNRTI-containing regimens, provided that there is sufficient time between the initial NVP exposure and treatment initiation to allow fading of NVP-resistant strains [12-15]. Data in this report suggest that the rate of fading of NVP-resistant strains may vary from one geographic region to another, depending on which subtypes are prevalent. Further studies are needed to evaluate whether subtype-based differences in the fading of NVP-resistant HIV-1 variants influence clinical or virologic outcome in NVP-exposed women who are subsequently treated with NNRTIs.
 
Many countries now offer pregnant women potent antiretroviral regimens for pMTCT and for treatment of their own HIV-1 infection. Women with higher HIV-1 loads are more likely to transmit HIV-1 to their infants and are also more likely to have NVP-resistant strains detected after SD NVP exposure. We now show that K103N-containing variants are also more likely to persist in the years following delivery among women with higher HIV-1 loads. These findings emphasize the importance of evaluating HIV-1 disease status in pregnant HIV-1-positive women and the importance of providing eligible women with highly active antiretroviral therapy (HAART) during pregnancy, if it is available.
 
Unfortunately, HAART is still not available to pregnant women in many countries that shoulder the greatest burden of the HIV-1 epidemic, because of cost, lack of needed infrastructure, and other factors
. Additionally, many women in resource-limited settings do not know their HIV-1 infection status and do not present for medical care until close to or at the time of delivery; in such situations, initiation of HAART during pregnancy, even if available, would not be possible. Our findings demonstrate fading of NVP resistance to very low levels after SD NVP exposure, and recent clinical studies suggest that the response to NNRTI-based HAART is not compromised by prior SD NVP exposure if treatment initiation occurs at a time distant from the exposure. These observations support continued use of SD NVP as an important option for pMTCT in settings where more complex regimens are not available.
 
 
 
 
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