Nevirapine Resistance Mutations Research Articles

Erratum to: Sensitive Drug-Resistance Assays Reveal Long-Term Persistence of HIV-1 Variants with the K103N Nevirapine (NVP) Resistance Mutation in Some Women and Infants after the Administration of Single-Dose NVP: HIVNET 012.

Background. The HIV Network for Prevention Trials (HIVNET) 012 trial showed that NVP resistance (NVPR) emerged in some women and children after the administration of single-dose nevirapine (SD-NVP). We tested whether K103N-containing human immunodeficiency virus (HIV)-1 variants persisted in women and infants 1 year or more after the administration of SD-NVP. Methods. We analyzed samples from 9 women and 5 infants in HIVNET 012 who had NVPR 6-8 weeks after the administration of SD-NVP. Samples were analyzed with the ViroSeq system and with 2 sensitive resistance assays, LigAmp and TyHRT. Results. ViroSeq detected the K103N mutation in 8 of 9 women and in 2 of 5 infants. LigAmp detected the K103N mutation at low levels in 8 of 9 women and in 4 of 5 infants. K103N was not detected by ViroSeq 12-24 months after the administration of SD-NVP but was detected by LigAmp in 3 of 9 women and in 1 of 5 infants. K103N was also detected in those samples by use of the TyHRT assay. Conclusions. K103N-containing variants persist in some women and infants for 1 year or more after the administration of SD-NVP. Sensitive resistance assays may provide new insight into the impact of antiretroviral drug exposure on HIV-1 evolution.

Genetic Analyses of HIV-1 Strains Transmitted from Mother to Child in Northern Vietnam.

We previously reported mother-to-child transmission of HIV-1 in nine (6.7%) of 135 children on nevirapine prophylaxis in Vietnam. In the current study, we investigated the appearance and profile of antiretroviral drug (ARV) resistance mutations, the predicted coreceptor usage, and the genetic diversity of HIV-1 strains isolated from the eight pairs of HIV-1-infected mothers and their children, who were followed up to 12 months after birth. Portions of the pol and env C2V3 regions of the HIV-1 strains were analyzed genetically. HIV-1 CRF01_AE RNA was detected in four (50%) children at delivery. Y181C, a nevirapine resistance mutation, appeared in two (25%) children 1 and 3 months after birth, respectively. No ARV resistance mutation was detected in the mothers, though three mothers were on ARV prophylaxis. Five mothers and their children harbored CCR5-tropic (R5) viruses. Two mothers harbored both R5 and CXCR4-tropic (X4) viruses, but their children harbored only R5 viruses even though the X4 viruses were dominant in the mothers. In the remaining one mother, HIV-1 RNA was not amplified and her child harbored both R5 and X4 viruses at birth, but only X4 virus 12 months after delivery. The infants' viruses were more homogeneous than their mothers' viruses (mean distance: 0.5% vs. 1.1%, respectively). This is the first molecular epidemiological study of vertical HIV-1 infections in Vietnam. These findings may provide useful knowledge for the prevention of mother-to-child transmission of HIV-1 and the antiretroviral treatment of children in Vietnam.

Optimization of the oligonucleotide ligation assay for the detection of nevirapine resistance mutations in Zimbabwean Human Immunodeficiency Virus type-1 subtype C

An oligonucleotide ligation assay (OLA) designed to detect Human Immunodeficiency Virus type-1 (HIV)-drug-resistance to the nevirapine (NVP) selected mutations K103N, Y181C, V106M and G190A was used to evaluate 200 archived dried blood spots (DBS) from infected infants participating in the Zimbabwean Early Infant Diagnosis (EID) Program. Consensus sequencing of specimens with indeterminate OLA results was performed to identify genetic sequence polymorphisms that appeared to compromise performance of the OLA. When consistent patterns of polymorphisms were observed the probes were redesigned, and DBS specimens with indeterminate OLA results were retested with the new Zimbabwe-specific (ZW) probes. OLA results obtained in Zimbabwe were compared to repeat testing in a US reference laboratory. 188/200 (94%) DBS yielded polymerase chain reaction (PCR) amplification of HIV pol. ZW probes reduced indeterminate OLA results from 5.2% to 2.8% of codons evaluated (p=0.02), with 98.2% concordance between results obtained in the Zimbabwean and US laboratories. Optimization of OLA probes to accommodate polymorphisms in regional HIV variants improved OLA performance, and comparison to the USA results showed successful implementation of the OLA in Zimbabwe for detection of NVP resistance mutations in DBS specimens.

Frequency of Antiretroviral Resistance Mutations among Infants Exposed to Single-Dose Nevirapine and Short Course Maternal Antiretroviral Regimens: ACTG A5207.

BackgroundIntrapartum single-dose nevirapine (sdNVP) reduces HIV-1 perinatal transmission but selects NVP resistance among mothers and infants. We evaluated the frequency of antiretroviral resistance among infants with intrauterine HIV-1 infection exposed to sdNVP and maternal antenatal or breastfeeding antiretroviral therapy.MethodsThis analysis included 429 infants from sub-Saharan Africa, India and Haiti whose 422 mothers received sdNVP plus maternal study treatment. At entry mothers had CD4>250/μL and were ART-naïve except for antenatal ZDV per local standard of care. Maternal study treatment started intrapartum and included ZDV/3TC, TDF/FTC or LPV/r for 7 or 21 days in a randomized factorial design. Infants received sdNVP study treatment and ZDV if local standard of care. Infant HIV RNA or DNA PCR and samples for genotype were obtained at birth and weeks 2, 4 and 12; infants who ever breast-fed were also tested at weeks 16, 24, 48 and 96. Samples from HIV-1-infected infants were tested for drug resistance by population genotype (ViroSeq). NVP or NRTI resistance mutations were assessed using the IAS-USA mutation list.ResultsPerinatal HIV-1 transmission occurred in 17 (4.0%) infants including 12 intrauterine infections. Resistance mutations were detected among 5 (42%) intrauterine-infected infants; of these, 3 had mutations conferring resistance to NVP alone, 1 had resistance to NRTI alone, and 1 had dual-class resistance mutations. Among the 2 infants with NRTI mutations, one (K70R) was likely maternally transmitted and one (K65R) occurred in the context of breastfeeding exposure to maternal antiretroviral therapy.ConclusionsInfants with intrauterine HIV infection are at risk of acquiring resistance mutations from exposure to maternal antiretroviral medications intrapartum and/or during breastfeeding. New approaches are needed to lower the risk of antiretroviral resistance in these infants.

Clonal amplification and maternal-infant transmission of nevirapine-resistant HIV-1 variants in breast milk following single-dose nevirapine prophylaxis

BackgroundIntrapartum administration of single-dose nevirapine (sdNVP) reduces perinatal HIV-1 transmission in resource-limiting settings by half. Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise de novo in the infant.FindingsWe performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation.ConclusionIn this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission.

Greater Suppression of Nevirapine Resistance With 21- vs 7-Day Antiretroviral Regimens After Intrapartum Single-Dose Nevirapine for Prevention of Mother-to-Child Transmission of HIV

Nevirapine (NVP) resistance emerges in up to 70% of women exposed to single-dose (sd) NVP for prevention of mother-to-child transmission of human immunodeficiency virus (HIV). HIV-infected pregnant women were randomized to receive sdNVP and either zidovudine/lamivudine (3TC), tenofovir/emtricitabine (FTC), or lopinavir/ritonavir for either 7 or 21 days. The primary endpoint was the emergence of new NVP resistance mutations as detected by standard population genotype at 2 and 6 weeks after treatment. Low-frequency NVP- or 3TC/FTC-resistant mutants at codons 103, 181, and 184 were sought using allele-specific polymerase chain reaction (ASP). Among 484 women randomized, 422 (87%) received study treatment. Four hundred twelve (98%) women had primary endpoint results available; of these, 5 (1.2%) had new NVP resistance detected by population genotype: 4 of 215 in the 7-day arms (1.9%; K103N in 4 women with Y181C, Y188C, or G190A in 3 of 4) and 1 of 197 (0.5%; V108I) in the 21-day arms (P = .37). Among women with ASP results, new NVP resistance mutations emerged significantly more often in the 7-day arms (13/74 [18%]) than in the 21-day arms (3/66 [5%], P = .019). 3TC/FTC-resistant mutants (M184V/I) emerged infrequently (7/134 [5%]), and their occurrence did not differ by arm. Three short-term antiretroviral strategies, begun simultaneously with the administration of sdNVP, resulted in a low rate (1.2%) of new NVP-resistance mutations when assessed at 2 and 6 weeks following completion of study treatment by standard genotype. ASP revealed that 21-day regimens were significantly better than 7-day regimens at preventing the emergence of minor NVP resistance variants. Clinical Trials Registration.NCT00099632.

Low-Frequency Nevirapine Resistance at Multiple Sites May Predict Treatment Failure in Infants on Nevirapine-Based Treatment

Resistance commonly arises in infants exposed to single-dose nevirapine (sdNVP) for prevention of mother to child transmission. Although K103N and Y181C are common following sdNVP, multiple other mutations also confer NVP resistance. It remains unclear whether specific NVP resistance mutations or combinations of mutations predict virologic failure in infants when present at low frequencies before NVP-based treatment. Twenty sdNVP-exposed infants who were subsequently treated with NVP-based highly active antiretroviral therapy were examined. Pretreatment plasma samples were tested for the presence of NVP resistance mutations by allele-specific polymerase chain reaction for K103N and Y181C and ultradeep pyrosequencing (UDPS) for all primary NVP mutations. Viral levels were determined every 3 months for up to 24 months on NVP-highly active antiretroviral therapy. Cox proportional hazard models were used to determine correlates of viral failure. The NVP resistance mutations K103N or Y181C were detected in pretreatment plasma samples in 6 infants by allele-specific polymerase chain reaction. NVP resistance at these or other sites was detectable by UDPS in 10 of 20 infants tested. Virologic failure occurred in 50% of infants with any NVP resistance mutations detected, whereas only 20% of infants without resistance experienced viral failure, but the difference was not significant (P = 0.19). An increase in the number of NVP resistance mutations detectable by UDPS in an infant was significantly associated with an increased risk of virologic failure [HR = 1.79 (95% confidence interval: 1.07 to 2.99), P = 0.027]. Low frequencies of multiple NVP resistance mutations, in addition to K103N and Y181C, present in infants before NVP-based treatment may predict treatment outcome.

Effects of Short-Course Zidovudine on the Selection of Nevirapine-Resistant HIV-1 in Women Taking Single-Dose Nevirapine

Single-dose nevirapine (sdNVP) given to prevent mother-to-child-transmission of HIV-1 selects NVP-resistance. Short-course zidovudine (ZDV) was hypothesized to lower rates of NVP-resistance. HIV-1 infected pregnant women administered sdNVP with or without short-course ZDV were assessed for HIV-1 mutations (K103N, Y181C, G190A, and V106M) prior to delivery and postpartum. Postpartum NVP-resistance was lower among 31 taking ZDV+sdNVP compared to 33 taking only sdNVP (35.5% vs. 72.7%; χ2 P = .003). NVP mutants decayed to <2% in 24/35 (68.6%) at a median 6 months postpartum, with no differences based on ZDV use (logrank P = .99). Short-course ZDV was associated with reduced NVP-resistance mutations among women taking sdNVP.

Intrapartum Single-Dose Carbamazepine Reduces Nevirapine Levels Faster and May Decrease Resistance After a Single Dose of Nevirapine for Perinatal HIV Prevention

World Health Organization guidelines recommend zidovudine + lamivudine for 7 days from labor onset in HIV-infected women receiving single-dose nevirapine (sdNVP) to cover prolonged subtherapeutic nevirapine concentrations. Although effective, this is complicated and does not eliminate resistance; alternative strategies could add benefit. Antiretroviral-naive HIV-infected pregnant women aged 18-40 years, with CD4 >200 cells per cubic millimeter, able to regularly attend the antenatal clinics in Moshi, Tanzania, were enrolled 1:1 by alternate allocation to receive 200 mg sdNVP alone or in combination with open-label 400-mg single-dose carbamazepine (sdNVP/CBZ) at delivery (ClinicalTrials.gov NCT00294892). The coprimary outcomes were nevirapine plasma concentrations 1 week and nevirapine resistance mutations 6 weeks postpartum. Analyses were based on those still eligible at delivery. Ninety-seven women were assigned to sdNVP and 95 to sdNVP/CBZ during pregnancy, of whom 75 sdNVP and 83 sdNVP/CBZ were still eligible at delivery at study sites. The median (interquartile range) nevirapine plasma concentration was 1.55 (0.88-1.84) mg/L in sdNVP (n = 61) and 1.40 (0.93-1.97) mg/L in sdNVP/CBZ (n = 72) at delivery (P = 0.91), but 1 week later was significantly lower in sdNVP/CBZ [n = 63; 0.09 (0.05-0.20) mg/L] than in sdNVP [n = 52; 0.20 (0.09-0.31) mg/L; rank-sum: P = 0.004] (geometric mean ratio: 0.64, 95% confidence interval: 0.43 to 0.96; P = 0.03). Six weeks postpartum, nevirapine mutations were observed in 11 of 52 (21%) in sdNVP and 6 of 55 (11%) in sdNVP/CBZ (odds ratio = 0.46, 95% confidence interval: 0.16 to 1.34; P = 0.15). Addition of single-dose carbamazepine to sdNVP at labor onset in HIV-infected, pregnant women did not affect nevirapine plasma concentration at delivery, but significantly reduced it 1 week postpartum, with a trend toward fewer nevirapine resistance mutations.

Monitoring of HIV Type 1 DNA Load and Drug Resistance in Peripheral Blood Mononuclear Cells During Suppressive Antiretroviral Therapy Does Not Predict Virologic Failure

Our objective was to determine whether monitoring HIV-1 DNA concentration or new resistance mutations in peripheral blood mononuclear cells (PBMCs) during effective antiretroviral therapy (ART) predicts virologic failure. A retrospective analysis used blood specimens and clinical data from three nevirapine containing arms of a four-arm, open-label, randomized trial comparing ART regimens in HIV-1-infected children who had failed mono- or dual-nucleoside therapy. Sensitive assays compared cell-associated HIV-1 DNA concentrations and nevirapine (NVP) and lamivudine (3TC) resistance mutations in children with plasma HIV-1 RNA <400 copies(c)/ml who did or did not experience subsequent virologic failure. Forty-six children were analyzed through the last available follow-up specimen, collected at 48 (n=16) or 96 (n=30) weeks of ART. Thirty-five (76%) had sustained viral suppression and 11 (24%) had plasma viral rebound to ≥ 400 c/ml (virologic failure detected at a median of 36 weeks). HIV-1 DNA levels at baseline, 24, 48, and 96 weeks of ART were similar in children who did vs. did not experience virologic failure (p=0.82). HIV-1 DNA levels did not increase prior to viral rebound. NVP resistance mutations were detected in 91% of subjects in the failure group vs. 3% in the suppressed group (p <0.0001). Among nine evaluable children, NVP mutations were first detected prior to virologic failure in two (22%), at viral rebound in five (56%), and after failure in two (22%) children. HIV-1 DNA concentrations did not predict virologic failure in this cohort. New drug resistance mutations were detected in the PBMCs of a minority of virologically suppressed children who subsequently failed ART.

A Comparison of 3 Regimens to Prevent Nevirapine Resistance Mutations in HIV-Infected Pregnant Women Receiving a Single Intrapartum Dose of Nevirapine

Intrapartum single-dose (SD) nevirapine (NVP) reduces perinatal transmission of human immunodeficiency virus (HIV) infection but selects for NVP-resistant virus, which compromises subsequent NVP-based therapy. A 1-week "tail" of lamivudine and zidovudine after SD-NVP decreases the risk of resistance. We hypothesized that increasing the duration or potency of the tail would further reduce this risk to <10%, using a sensitive assay to measure resistance. HIV-infected pregnant Thai women with a CD4 cell count >250 cells/μL, most receiving zidovudine, were randomized at 28-38 weeks gestation to receive 1 of 3 intrapartum and postpartum regimens: (A) zidovudine plus enteric-coated didanosine plus lopinavir and ritonavir for 7 days, (B) zidovudine plus enteric-coated didanosine for 30 days, or (C) regimen 1 for 30 days. The incidence of NVP resistance mutations at day 10 or week 6 post partum in each arm was compared with that of a historical comparison group who received prenatal zidovudine and SD-NVP. NVP resistance was identified by consensus sequencing and a sensitive oligonucleotide ligation assay (OLA). At entry, the 169 participants had a median CD4 cell count of 456 cells/μL and an HIV load of 3.49 log(10) copies/mL. The incidence of mutations in each of the 3 P1032 arms was 0% by sequencing and 1.8%, 7.1%, and 5.3% by OLA in arms A, B, and C, respectively, compared with 13.4% by sequencing and 29.4% by OLA in the comparison group (P < .001 for each study arm vs comparison group). Grade 4 anemia developed in 1 woman. A 7-day tail of highly active combination therapy or 1 month of dual therapy after SD-NVP prevents most NVP resistance to minimal toxicity. The IMPAACT P1032 Clinical Trial is NCT00109590, and the PHPT-2 Clinical Trial is NCT00398684.

Docking Analysis and Resistance Evaluation of Clinically Relevant Mutations Associated with the HIV‐1 Non‐nucleoside Reverse Transcriptase Inhibitors Nevirapine, Efavirenz and Etravirine

An integrated computational and statistical approach was used to determine the association of non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine, efavirenz and etravirine with resistance mutations that cause therapeutic failure and their impact on NNRTI resistance. Mutations detected for nevirapine virological failure with a prevalence greater than 10% in the used patient set were: K103N, Y181C, G190A, and K101E. A support vector regression model, based on matched genotypic/phenotypic data (n=850), showed that among 6365 analyzed mutations, K103N, Y181C and G190A have the first, third, and sixth greatest significance for nevirapine resistance, respectively. The most common indicator of treatment failure for efavirenz was K103N mutation present in 56.7% of the patients where the drug failed, followed by V108I, L100I, and G190A. For efavirenz resistance, K103N, G190, and L100I have the first, fourth, and eighth greatest significance, respectively, as determined in support vector regression model. No positive interactions were observed among nevirapine resistance mutations, while a more complex situation was observed with treatment failure of efavirenz and etravirine, characterized by the accumulation of multiple mutations. Docking simulations and free energy analysis based on docking scores of mutated human immunodeficiency virus (HIV) RT complexes were used to evaluate the influence of selected mutations on drug recognition. Results from support vector regression were confirmed by docking analysis. In particular, for nevirapine and efavirenz, a single mutation K103N was associated with the most unfavorable energetic profile compared to the wild-type sequence. This is in line with recent clinical data reporting that diarylpyrimidine etravirine, a very potent third generation drug effective against a wide range of drug-resistant HIV-1 variants, shows increased affinity towards K103N/S mutants due to its high conformational flexibility.

Slower Clearance of Nevirapine Resistant Virus in Infants Failing Extended Nevirapine Prophylaxis for Prevention of Mother-to-Child HIV Transmission

Nevirapine resistance mutations arise commonly following single or extended-dose nevirapine (ED-NVP) prophylaxis to prevent mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV), but decay within 6-12 months of single-dose exposure. Use of ED-NVP prophylaxis in infants is expected to rise, but data on decay of nevirapine resistance mutations in infants in whom ED-NVP failed remain limited. We assessed, in Ethiopian infants participating in the Six-Week Extended Nevirapine (SWEN) Trial, the prevalence and persistence of nevirapine resistance mutations at 6 and 12 months following single-dose or up to 6 weeks of ED-NVP, and correlated their presence with the timing of infection and the type of resistance mutations. Standard population genotyping followed by high-throughput cloning were done on dried blood spot samples collected during the trial. More infants who received ED-NVP had nevirapine resistance detected by standard population genotyping (high frequencies) at age 6 months compared with those who received single-dose nevirapine (SD-NVP) (58% of 24 vs. 26% of 19, respectively; p = 0.06). Moreover, 56% of ED-NVP-exposed infants with nevirapine resistance at age 6 months still had nevirapine resistance mutations present at high frequencies at age 1 year. Infants infected before 6 weeks of age who received either SD- or ED-NVP were more likely to have Y181C or K103N; these mutations were also more likely to persist at high frequencies through 1 year of age. HIV-infected infants in whom ED-NVP prophylaxis fails are likely to experience delayed clearance of nevirapine-resistant virus in the first year of life, which in turn places them at risk for early selection of multidrug-resistant HIV after initial therapy with nonnucleoside reverse transcriptase inhibitor-based regimens.

Detection of HIV drug resistance mutations in pregnant women receiving single dose Nevirapine in south India

Single dose of Nevirapine to prevent mother to child transmission of HIV is the commonest preventive regimen in resource-limited countries. The objective of this study was to detect drug-resistant virus after single dose of Nevirapine (sdNVP) provided to delivering HIV seropositive (HIV+ve) women and to evaluate the time taken for its decay. Of the 36 consenting HIV+ve pregnant women enrolled into the study, the mean hemoglobin and total lymphocyte counts were 10.8 g/dl and 1843 cells/mm 3 , respectively. Mean CD4 counts in 64% of women was 363 cells/mm 3 and mean viral load for 16/36 women was 28,143 copies/ml of plasma. Nevirapine-resistance mutations were detected in 28% of women at delivery; using OLA (Oligonucleotide Ligation Assay). K103N mutations were seen in 19.4% of women while the Y181C mutation was seen in 5%. Both the mutations were detected in 2.7% of women. Sequential blood samples collected at delivery, 7-10 days, 6 weeks, 4 months, 6 months and one year postpartum showed that 81% of K103N mutations and 66.7% of Y181C mutations were detected at 6 weeks postpartum . Wild-type virus had replaced the mutants by one year postpartum in all women except one. These observations are relevant for future treatment with antiretroviral therapy in these women for their HIV disease.

Estimating Frequencies of Minority Nevirapine-Resistant Strains in Chronically HIV-1-Infected Individuals Naïve to Nevirapine by Using Stochastic Simulations and a Mathematical Model

Nevirapine forms the mainstay of our efforts to curtail the pediatric AIDS epidemic through prevention of mother-to-child transmission of HIV-1. A key limitation, however, is the rapid selection of HIV-1 strains resistant to nevirapine following the administration of a single dose. This rapid selection of resistance suggests that nevirapine-resistant strains preexist in HIV-1 patients and may adversely affect outcomes of treatment. The frequencies of nevirapine-resistant strains in vivo, however, remain poorly estimated, possibly because they exist as a minority below current assay detection limits. Here, we employ stochastic simulations and a mathematical model to estimate the frequencies of strains carrying different combinations of the common nevirapine resistance mutations K103N, V106A, Y181C, Y188C, and G190A in chronically infected HIV-1 patients naïve to nevirapine. We estimate the relative fitness of mutant strains from an independent analysis of previous competitive growth assays. We predict that single mutants are likely to preexist in patients at frequencies ( approximately 0.01% to 0.001%) near or below current assay detection limits (>0.01%), emphasizing the need for more-sensitive assays. The existence of double mutants is subject to large stochastic variations. Triple and higher mutants are predicted not to exist. Our estimates are robust to variations in the recombination rate, cellular superinfection frequency, and the effective population size. Thus, with 10(7) to 10(8) infected cells in HIV-1 patients, even when undetected, nevirapine-resistant genomes may exist in substantial numbers and compromise efforts to prevent mother-to-child transmission of HIV-1, accelerate the failure of subsequent antiretroviral treatments, and facilitate the transmission of drug resistance.

Minor resistant variants in nevirapine-exposed infants may predict virologic failure on nevirapine-containing ART

Background Single-dose nevirapine (sdNVP) is widely used to prevent mother-to-child transmission (PMTCT) of HIV-1. This may result in NVP resistance in both mother and infant. The significance of low levels of NVP resistance mutations in infants treated with NVP-containing antiretroviral treatment (ART) is unknown. Objectives To determine the presence of pre-treatment NVP resistance in HIV-infected infants with and without prior NVP exposure. Study design 33 HIV-1-infected infants in a PMTCT trial received NVP-containing ART (26 infants with prior NVP exposure). Plasma and buffy coat samples obtained prior to ART initiation were evaluated for drug resistance by bulk sequencing and allele-specific PCR (ASPCR). Results ViroSeq™ identified NVP resistance in 3 of 33 infants; all failed first-line therapy. Pre-ART plasma NVP resistance by ASPCR was detected in 9 of 16 children experiencing virologic failure compared to 4 of 17 children without virologic failure (risk ratio 2.4, CI 0.94–7.8, p = 0.08). Proviral resistance was not associated with virologic failure (risk ratio 1.2, CI 0.8–2.0, p = 0.40). In the nevirapine-exposed infants, those who started ART before 7 months had higher risk of virologic failure (RR 2.3, CI 0.96–9.2, p = 0.11). Conclusions Low level drug resistance detected in plasma after NVP exposure prior to ART initiation may be associated with virologic failure on ART, while resistance in the DNA reservoir was not predictive of treatment outcome.

Ultrasensitive Detection of Minor Drug-Resistant Variants for HIV After Nevirapine Exposure Using Allele-Specific PCR: Clinical Significance

HIV-1 drug resistance mutations have been detected at low frequencies after single-dose nevirapine (sdNVP) for prevention of mother-to-child transmission (PMTCT). We investigated the relationship between these "minor variant" NVP-resistant viruses and clinical outcome with NVP-containing antiretroviral therapy (ART). An allele-specific quantitative PCR (ASPCR) assay was used to quantify the pre-ART frequency of K103N and Y181C in 26 women who had received sdNVP. The cohort was composed of 7 patients who experienced virologic failure and 19 control patients who maintained virologic suppression on NVP-containing ART; all were negative for resistance by standard genotyping. NVP resistance mutations were found in 17 of 26 (65%) patients using ASPCR. The frequency of NVP-resistant viruses ranged from 0.1% to 4.11%. Receiver operating characteristics (ROC) analysis identified a clinical threshold frequency of 0.19% for the ASPCR assay. Application of this threshold demonstrated minor variant resistance in 6 of 7 patients (86%) who failed treatment compared to 6 of 19 patients (32%) who were successful (OR = 13; 95% CI 1.27-133). ASPCR provides a means of detecting minor variant drug-resistant viruses that may impact subsequent treatment response. These data suggest a clinical role for highly sensitive assays to detect and quantify resistant viruses at low frequencies.

Predictors of Early and Late Mother-to-Child Transmission of HIV in a Breastfeeding Population: HIV Network for Prevention Trials 012 Experience, Kampala, Uganda

To determine the predictors for early versus later (breastfeeding) transmission of HIV-1. Secondary data analysis was performed on HIV Network for Prevention Trials 012, a completed randomized clinical trial assessing the relative efficacy of nevirapine (NVP) versus zidovudine in reducing mother-to-child transmission (MTCT) of HIV-1. We used Cox regression analysis to assess risk factors for MTCT. The ViroSeq HIV genotyping and a sensitive point mutation assay were used to detect NVP resistance mutations. In this subset analyses, 122 of 610 infants were HIV infected, of whom 99 (81.1%) were infected early (first positive polymerase chain reaction < or =56 days). Incidence of MTCT after 56 days was low [0.7% per month (95% confidence interval, CI: 0.4 to 1.0)], but continued through 18 months. In multivariate analyses, early MTCT "factors" included NVP versus zidovudine (hazard ratio (HR) = 0.57, 95% CI: 0.38 to 0.86), pre-entry maternal viral load (VL, HR = 1.76, 95% CI: 1.28 to 2.41), and CD4 cell count (HR = 1.16, 95% CI: 1.05 to 1.28). Maternal VL (6-8 weeks) was associated with late MTCT (HR = 3.66, 95% CI: 1.78 to 7.50), whereas maternal NVP resistance (6-8 weeks) was not. Maternal VL was the best predictor of both early and late transmission. Maternal NVP resistance at 6-8 weeks did not predict late transmission.

Identification of Nevirapine‐Resistant HIV‐1 in the Latent Reservoir after Single‐Dose Nevirapine to Prevent Mother‐to‐Child Transmission of HIV‐1

Intrapartum single-dose nevirapine decreases mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) but promotes nevirapine resistance. Although resistant viruses fade to undetectable levels in plasma, they may persist as stably integrated proviruses within the latent reservoir in resting CD4(+) T cells, potentially complicating future treatment. Blood samples were collected from 60 women from South Africa and Uganda >6 months after they had received single-dose nevirapine. To selectively analyze the stable latent form of HIV-1, resting CD4(+) T cells were isolated and activated in the presence of reverse-transcriptase inhibitors and integrase inhibitors, which allows for the specific isolation of viruses produced by cells with stably integrated proviral DNA. These viruses were then analyzed for nevirapine resistance. Although only a small number of latently infected cells were present in each blood sample (mean, 162 cells), nevirapine resistance mutations (K103N and G190A) were detected in the latent reservoir of 4 (8%) of 50 evaluable women. A single dose of nevirapine can establish antiretroviral resistance within the latent reservoir. This results in a potentially lifelong risk of reemergence of nevirapine-resistant virus and highlights the need for strategies to prevent transmission that do not compromise successful future treatment.

Women exposed to single-dose nevirapine in successive pregnancies: effectiveness and nonnucleoside reverse transcriptase inhibitor resistance

To assess the impact of prior exposure to single-dose nevirapine (sdNVP) on mother-to-child transmission and genotypic resistance in HIV-infected women. Prospective study of 120 women exposed to the HIVNET 012 sdNVP regimen in two successive pregnancies and 240 antiretroviral (ARV)-naïve, multiparous women who received sdNVP for the first time. One hundred and eight of 120 and 193 of 240 women returned for a postpartum visit by 6 weeks. HIV-1 was detected in 11.1% (95% confidence interval = 5.9-18.6) of the infants of women previously exposed to sdNVP and 4.2% (95% confidence interval = 1.3-7.0) of those exposed for the first time (P = 0.028). Rates of maternal HIV-1 genotypic resistance at 6 weeks postdelivery were 37.5% and 46.4%, respectively (P = 0.119). Sensitive mutation-specific real-time PCR testing found three of 12 previously exposed women who transmitted HIV-1 to their infants had either K103N or Y181C at baseline compared with one of eight ARV-naïve, transmitting women who had Y181C. None of 40 randomly selected nontransmitting women from either group had detectable NVP resistance mutations prior to sdNVP exposure. This study shows that effectiveness of sdNVP may be compromised by prior exposure to sdNVP, although the increase in transmission rate after prior exposure could not be explained by the detection of NVP resistance mutations prior to re-exposure as measured both by standard genotyping and highly sensitive allele-specific PCR assays. Furthermore, transmission rates of women with prior exposure were not higher than those reported elsewhere.