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1.  Short Communication: In Utero HIV Infection Is Associated with an Increased Risk of Nevirapine Resistance in Ugandan Infants Who Were Exposed to Perinatal Single Dose Nevirapine 
Use of single dose nevirapine (sdNVP) to prevent HIV mother-to-child transmission is associated with the emergence of NVP resistance in many infants who are HIV infected despite prophylaxis. We combined results from four clinical trials to analyze predictors of NVP resistance in sdNVP-exposed Ugandan infants. Samples were tested with the ViroSeq HIV Genotyping System and a sensitive point mutation assay (LigAmp, for detection of K103N, Y181C, and G190A). NVP resistance was detected at 6–8 weeks in 36 (45.0%) of 80 infants using ViroSeq and 33 (45.8%) of 72 infants using LigAmp. NVP resistance was more frequent among infants who were infected in utero than among infants who were diagnosed with HIV infection after birth by 6–8 weeks of age. Detection of NVP resistance at 6–8 weeks was not associated with HIV subtype (A vs. D), pre-NVP maternal viral load or CD4 cell count, infant viral load at 6–8 weeks, or infant sex. NVP resistance was still detected in some infants 6–12 months after sdNVP exposure. In this study, in utero HIV infection was the only factor associated with detection of NVP resistance in infants 6–8 weeks after sdNVP exposure.
doi:10.1089/aid.2009.0003
PMCID: PMC2752753  PMID: 19552593
2.  Short Communication: In Utero HIV Infection Is Associated with an Increased Risk of Nevirapine Resistance in Ugandan Infants Who Were Exposed to Perinatal Single Dose Nevirapine 
Abstract
Use of single dose nevirapine (sdNVP) to prevent HIV mother-to-child transmission is associated with the emergence of NVP resistance in many infants who are HIV infected despite prophylaxis. We combined results from four clinical trials to analyze predictors of NVP resistance in sdNVP-exposed Ugandan infants. Samples were tested with the ViroSeq HIV Genotyping System and a sensitive point mutation assay (LigAmp, for detection of K103N, Y181C, and G190A). NVP resistance was detected at 6–8 weeks in 36 (45.0%) of 80 infants using ViroSeq and 33 (45.8%) of 72 infants using LigAmp. NVP resistance was more frequent among infants who were infected in utero than among infants who were diagnosed with HIV infection after birth by 6–8 weeks of age. Detection of NVP resistance at 6–8 weeks was not associated with HIV subtype (A vs. D), pre-NVP maternal viral load or CD4 cell count, infant viral load at 6–8 weeks, or infant sex. NVP resistance was still detected in some infants 6–12 months after sdNVP exposure. In this study, in utero HIV infection was the only factor associated with detection of NVP resistance in infants 6–8 weeks after sdNVP exposure.
doi:10.1089/aid.2009.0003
PMCID: PMC2752753  PMID: 19552593
3.  Analysis of nevirapine (NVP) resistance in Ugandan infants who were HIV-infected despite receiving single dose (SD) nevirapine (NVP) vs. SD NVP plus daily NVP up to 6-weeks of age to prevent HIV vertical transmission 
The Journal of infectious diseases  2008;198(7):1075-1082.
Background
Single dose (SD) nevirapine (NVP) at birth plus NVP to the infant up to 6 weeks of age is superior to SD NVP alone for prevention of HIV vertical transmission through breastfeeding. We analyzed NVP resistance in HIV-infected Ugandan infants who received either SD NVP or extended NVP prophylaxis.
Methods
We tested plasma HIV using a genotyping assay (ViroSeq), a phenotypic resistance assay (PhenoSense), and sensitive point mutation assay (LigAmp, for K103N, Y181C, G190A).
Results
At 6 weeks, NVP resistance was detected by ViroSeq in a higher proportion of infants in the extended NVP arm than in the SD NVP arm (21/25=84% vs. 12/24=50%, p=0.01). Similar results were obtained with LigAmp and PhenoSense. Infants who were HIV-infected at birth had high rates of resistance in both study arms. In contrast, infants who were HIV-infected after birth were more likely to have resistance detected at 6 weeks in the extended NVP arm. Use of extended NVP prophylaxis was also associated with detection of NVP resistance by ViroSeq at 6 months (7/7=100% extended NVP arm vs. 1/6=16.7% SD NVP arm, p=0.005).
Conclusions
Use of extended NVP prophylaxis was associated with increased selection and persistence of NVP resistance in HIV-infected Ugandan infants.
doi:10.1086/591503
PMCID: PMC2587235  PMID: 18684096
HIV-1; infant; mother-to-child transmission; nevirapine; resistance
4.  Comparison of Laboratory Methods for Analysis of Non-nucleoside Reverse Transcriptase Inhibitor Resistance in Ugandan Infants 
Detailed comparisons of HIV drug resistance assays are needed to identify the most useful assays for research studies, and to facilitate comparison of results from studies that use different methods. We analyzed nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in 40 HIV-infected Ugandan infants who had received nevirapine (NVP)-based prophylaxis using the following assays: an FDA-cleared HIV genotyping assay (the ViroSeq HIV-1 Genotyping System v2.0), a commercially available HIV genotyping assay (GeneSeq HIV), a commercially available HIV phenotyping assay (PhenoSense HIV), and a sensitive point mutation assay (LigAmp). ViroSeq and GeneSeq HIV results (NVP resistance yes/no) were similar for 38 (95%) of 40 samples. In 6 (15%) of 40 samples, GeneSeq HIV detected mutations in minor subpopulations that were not detected by ViroSeq, which identified two additional infants with NVP resistance. LigAmp detected low-level mutations in 12 samples that were not detected by ViroSeq; however, LigAmp testing identified only one additional infant with NVP resistance. GeneSeq HIV and PhenoSense HIV determinations of susceptibility differed for specific NNRTIs in 12 (31%) of the 39 samples containing mixtures at relevant mutation positions. PhenoSense HIV did not detect any infants with NVP resistance who were not identified with GeneSeq HIV testing. In this setting, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility.
doi:10.1089/aid.2008.0235
PMCID: PMC2799186  PMID: 19621988
5.  Comparison of Laboratory Methods for Analysis of Non-nucleoside Reverse Transcriptase Inhibitor Resistance in Ugandan Infants 
Abstract
Detailed comparisons of HIV drug resistance assays are needed to identify the most useful assays for research studies, and to facilitate comparison of results from studies that use different methods. We analyzed nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in 40 HIV-infected Ugandan infants who had received nevirapine (NVP)-based prophylaxis using the following assays: an FDA-cleared HIV genotyping assay (the ViroSeq HIV-1 Genotyping System v2.0), a commercially available HIV genotyping assay (GeneSeq HIV), a commercially available HIV phenotyping assay (PhenoSense HIV), and a sensitive point mutation assay (LigAmp). ViroSeq and GeneSeq HIV results (NVP resistance yes/no) were similar for 38 (95%) of 40 samples. In 6 (15%) of 40 samples, GeneSeq HIV detected mutations in minor subpopulations that were not detected by ViroSeq, which identified two additional infants with NVP resistance. LigAmp detected low-level mutations in 12 samples that were not detected by ViroSeq; however, LigAmp testing identified only one additional infant with NVP resistance. GeneSeq HIV and PhenoSense HIV determinations of susceptibility differed for specific NNRTIs in 12 (31%) of the 39 samples containing mixtures at relevant mutation positions. PhenoSense HIV did not detect any infants with NVP resistance who were not identified with GeneSeq HIV testing. In this setting, population sequencing-based methods (ViroSeq and GeneSeq HIV) were the most informative and had concordant results for 95% of the samples. LigAmp was useful for the detection and quantification of minority variants. PhenoSense HIV provided a direct and quantitative measure of NNRTI susceptibility.
doi:10.1089/aid.2008.0235
PMCID: PMC2799186  PMID: 19621988
6.  Analysis of nevirapine (NVP) resistance in HIV-infected infants who received extended NVP or NVP/zidovudine prophylaxis 
AIDS (London, England)  2011;25(7):911-917.
BACKGROUND
In the PEPI-Malawi trial, infants received up to 14 weeks of extended nevirapine (NVP) or extended NVP plus zidovudine (NVP+ZDV) to prevent postnatal HIV transmission. We examined emergence and persistence of NVP resistance in HIV-infected infants who received these regimens prior to HIV diagnosis.
METHODS
Infant plasma samples collected at 14 weeks of age were tested using the ViroSeq HIV Genotyping System and a sensitive point-mutation assay, LigAmp (for K103N and Y181C). Samples collected at 6 and 12 months of age were analyzed using LigAmp.
RESULTS
At 14 weeks of age, NVP resistance was detected in samples from 82 (75.9%) of 108 HIV-infected infants. While the frequency of NVP resistance detected by ViroSeq was lower in the extended NVP+ZDV arm than in the extended NVP arm, the difference was not statistically significant (38/55=69.1% vs. 44/53=83.0%, P=0.12). Similar results were obtained using LigAmp. Using LigAmp, the proportion of infants who still had detectable NVP resistance at 6 and 12 months was similar among infants in the two study arms (at 6 months: 17/20=85.0% for extended NVP vs. 21/26=80.8% for extended NVP+ZDV, P=1.00; at 12 months: 9/16=56.3% for extended NVP vs.10/13=76.9% for extended NVP+ZDV, P=0.43).
CONCLUSIONS
Infants exposed to extended NVP or extended NVP+ZDV had high rates of NVP resistance at 14 weeks of age, and resistant variants frequently persisted for 6–12 months. Frequency and persistence of NVP resistance did not differ significantly among infants who received extended NVP only vs. extended NVP+ZDV prophylaxis.
doi:10.1097/QAD.0b013e328344fedc
PMCID: PMC3261770  PMID: 21487249
HIV; nevirapine; resistance; infants; Malawi
7.  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.
doi:10.1016/j.jcv.2010.03.017
PMCID: PMC2909836  PMID: 20427228
HIV; minor variant; drug resistance; nevirapine; PMTCT
8.  Emergence and persistence of nevirapine (NVP) resistance in breast milk after single-dose NVP administration 
AIDS (London, England)  2010;24(4):557-561.
OBJECTIVE
Single-dose nevirapine (sdNVP) can reduce the risk of HIV vertical transmission. We assessed risk factors for NVP resistance in plasma and breast milk from sdNVP-exposed Ugandan women.
METHODS
Samples were analyzed using the Roche AMPLICOR HIV-1 Monitor Test Kit, v1.5, and the ViroSeq HIV-1 Genotyping System. NVP concentrations were determined by liquid chromatography with tandem mass spectroscopy.
RESULTS
HIV genotypes (plasma and breast milk) were obtained for 30 women 4 weeks after sdNVP (HIV subtypes: 15A, 1C, 12D, 2 recombinant). NVP resistance was detected in 12 (40%) of 30 breast milk samples. There was a non-significant trend between detection of NVP resistance in breast milk and plasma (p=0.06). There was no association of HIV resistance in breast milk with median maternal pre-NVP viral load or CD4 cell count, median breast milk viral load at 4 weeks, breast milk sodium >10 mmol/L, HIV subtype, or concentration of NVP in breast milk or plasma.
CONCLUSIONS
NVP resistance was frequently detected in breast milk 4 weeks after sdNVP exposure. In this study, we were unable to identify specific factors associated with breast milk NVP resistance.
doi:10.1097/QAD.0b013e3283346e60
PMCID: PMC3065236  PMID: 20057308
nevirapine; HIV-1; breast milk; Uganda; vertical transmission; nevirapine resistance
9.  Analysis of nevirapine resistance mutations in cloned HIV-1 variants from HIV-infected Ugandan infants using a single step amplification-sequencing method (AmpliSeq) 
AIDS research and human retroviruses  2008;24(9):1209-1213.
We analyzed genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV-infected despite single dose (SD) prophylaxis. Plasma samples were obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by population sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single step amplification-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 variants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealed numerous NVP resistance mutations not detected by population sequencing, genetically-linked NVP resistance mutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.
doi:10.1089/aid.2008.0109
PMCID: PMC2562759  PMID: 18788912
10.  Analysis of Nevirapine Resistance Mutations in Cloned HIV Type 1 Variants from HIV-Infected Ugandan Infants Using a Single-Step Amplification-Sequencing Method (AmpliSeq) 
AIDS Research and Human Retroviruses  2008;24(9):1209-1213.
Abstract
We analyzed the genetic linkage of nevirapine (NVP) resistance mutations and the genetic complexity of HIV-1 variants in Ugandan infants who were HIV infected despite single dose (SD) prophylaxis. Plasma samples were obtained from six HIV-infected infants who had two or more NVP resistance mutations detected by population sequencing (ViroSeq). ViroSeq PCR products were cloned and transformed, and a single-step amplification-sequencing reaction (AmpliSeq) was used to analyze NVP resistance mutations in cloned HIV-1 variants directly from bacterial colonies. Fifty clones were analyzed for each infant sample. This analysis revealed numerous NVP resistance mutations not detected by population sequencing, genetically linked NVP resistance mutations, and a high degree of genetic complexity at codons that influence NVP susceptibility.
doi:10.1089/aid.2008.0109
PMCID: PMC2562759  PMID: 18788912
11.  Addition of extended zidovudine to extended nevirapine prophylaxis reduces nevirapine resistance in infants who were HIV infected in utero 
AIDS (London, England)  2010;24(3):381-386.
BACKGROUND
In the PEPI-Malawi trial, most women received single dose nevirapine (sdNVP) at delivery, and infants in the extended study arms received sdNVP plus 1 week of daily zidovudine (ZDV), followed by either extended daily NVP or extended daily NVP+ZDV up to 14 weeks of age. While extended NVP prophylaxis reduces the risk of postnatal HIV transmission, it may increase the risk of NVP resistance among infants who are HIV-infected despite prophylaxis.
METHODS
We analyzed 88 infants in the PEPI- Malawi trial with in utero HIV infection who received prophylaxis for a median of 6 weeks prior to HIV diagnosis. HIV genotyping was performed using the ViroSeq HIV Genotyping System.
RESULTS
At 14 weeks of age, the proportion of infants with NVP resistance was lower in the extended NVP+ZDV arm than in the extended NVP arm (28/45=62.2% vs. 37/43=86.0%, p=0.015). None of the infants had ZDV resistance. Addition of extended ZDV to extended NVP was associated with reduced risk of NVP resistance at 14 weeks if prophylaxis was stopped by 6 weeks (54.5% vs. 85.7%, p=0.007), but not if prophylaxis was continued beyond 6 weeks (83.3% vs. 87.5%, p=1.00).
CONCLUSIONS
Addition of extended ZDV to extended NVP prophylaxis significantly reduced the risk of NVP resistance at 14 weeks in infants with in utero HIV infection, provided that HIV infection was diagnosed and the prophylaxis was stopped by 6 weeks of age.
doi:10.1097/QAD.0b013e3283352ef1
PMCID: PMC3063063  PMID: 19996936
HIV-1; resistance; infants; Malawi; nevirapine
12.  Impact of Maternal and Infant Antiretroviral Drug Regimens on Drug Resistance in HIV-Infected Breastfeeding Infants 
The Pediatric infectious disease journal  2013;32(4):10.1097/INF.0b013e31827f44ee.
BACKGROUND
The HPTN 046 trial evaluated the efficacy of extended infant nevirapine (NVP) administration for prevention of HIV transmission through breastfeeding. Infants received daily NVP to 6 weeks of age. HIV-uninfected infants (the intent-to-treat group) received daily NVP or placebo up to 6 months of age. We analyzed emergence of NVP resistance in infants who acquired HIV-infection despite prophylaxis.
METHODS
HIV genotyping was performed using the ViroSeq HIV Genotyping System. Medians and proportions were used to summarize data. Two-sided Fisher’s exact tests were used to evaluate associations between categorical variables.
RESULTS
NVP resistance was detected in 12 (92.3%) of 13 infants who were HIV-infected by 6 weeks and in seven (28%) of 25 infants who were HIV-uninfected at 6 weeks and HIV-infected at 6 months of age (6/8=75% in the NVP arm, 1/17=5.9% in the placebo arm, P=0.001). Among those 25 infants, 4 had mothers who initiated an antiretroviral (ARV) treatment regimen by 6 months postpartum. In all 4 cases, the treatment regimen included a non-nucleoside reverse transcriptase inhibitor (NVP or efavirenz). NVP resistance was detected in all four of those infants by 6 months of age (4/4=100%). In contrast, only three (14.2%) of the remaining 21 HIV-infected infants whose mothers did not initiate ARV treatment developed NVP resistance (P=0.003).
CONCLUSIONS
Extended NVP prophylaxis significantly increased the risk of NVP resistance in infants who acquired HIV infection after 6 weeks of age. Treatment of maternal HIV infection was also associated with emergence of NVP resistance in HIV-infected, breastfed infants.
doi:10.1097/INF.0b013e31827f44ee
PMCID: PMC3826537  PMID: 23249916
Nevirapine resistance; prevention of mother-to-child transmission; extended nevirapine; HIV
13.  Performance of Applied Biosystems ViroSeq HIV-1 Genotyping System for Sequence-Based Analysis of Non-Subtype B Human Immunodeficiency Virus Type 1 from Uganda 
Journal of Clinical Microbiology  2001;39(12):4323-4327.
The Applied Biosystems ViroSeq HIV-1 Genotyping System is a commercially available, integrated system for sequence-based analysis of drug resistance mutations in human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for analysis of non-subtype B HIV-1 by analyzing plasma samples from Ugandan women and infants. Plasma samples were obtained from 105 women and 25 infants enrolled in a Ugandan clinical trial. HIV-1 analysis was performed with the ViroSeq system according to the manufacturer's instructions, except that the volume of plasma used for analysis was less than the recommended 0.5 ml for some samples. Viral loads ranged from 2,313 to 2,336,400 copies/ml. PCR products suitable for sequencing were amplified from all samples tested. Complete sequences for protease (amino acids 1 to 99) and RT (amino acids 1 to 320) were obtained for 102 of 105 (97%) of the maternal samples tested and all 25 of the infant samples tested. Complete double-stranded sequences were obtained for 90 of 105 (86%) of the maternal samples tested and 22 of 25 (88%) of the infant samples tested. The sequences obtained with this system were used for HIV-1 subtyping. The subtypes identified were A, C, D, and A/D recombinant HIV-1. The performances of the seven sequencing primers were similar for the subtypes examined. The ViroSeq system performs well for analysis of Ugandan plasma samples with subtypes A, C, D, and A/D recombinant HIV-1. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in countries where these subtypes are prevalent.
doi:10.1128/JCM.39.12.4323-4327.2001
PMCID: PMC88543  PMID: 11724839
14.  Detection of HIV-1 Drug Resistance in Women Following Administration of a Single Dose of Nevirapine: Comparison of Plasma RNA to Cellular DNA by Consensus Sequencing and by Oligonucleotide Ligation Assay▿  
Journal of Clinical Microbiology  2010;48(5):1555-1561.
A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%, P < 0.01). When resistance was detected only by OLA (n = 45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (n = 51) (P < 0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.
doi:10.1128/JCM.02062-09
PMCID: PMC2863880  PMID: 20181911
15.  Ultrasensitive Detection of Minor Drug-Resistant Variants for HIV After Nevirapine Exposure Using Allele-Specific PCR: Clinical Significance 
Abstract
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.
doi:10.1089/aid.2009.0082
PMCID: PMC2864062  PMID: 20334564
16.  Viremia and HIV-1 Drug Resistance Mutations Among Patients Receiving Second-Line Highly Active Antiretroviral Therapy in Chennai, Southern India 
Analysis of human immunodeficiency virus type 1 pol gene sequences from 107 patients receiving second-line antiretroviral therapy (ART) revealed that a high prevalence of resistance mutations among second-line ART-experienced patients limits the ART-sequencing options, suggesting darunavir as the third-line drug in India.
Background. A cross-sectional study among individuals receiving second-line antiretroviral treatment was conducted to report on the level of detectable viremia and the types of drug resistance mutations among those with detectable human immunodeficiency virus (HIV) type 1 plasma viral loads (PVLs).
Methods. PVLs were measured using Abbott m2000rt real-time polymerase chain reaction, and genotyping was performed with the ViroSeq genotyping system, version 2.0, and ViroSeq analysis software, version 2.8.
Results. Of 107 patient plasma specimens consecutively analyzed, 30 (28%) had undetectable PVLs (<150 copies/mL), and 77 (72%) were viremic with a median PVL of 5450 copies/mL (interquartile range, 169–1 997 967). Sequencing was done for 107 samples with PVLs >2000 copies/mL: 33 patients (73%) had 1 of the protease (PR) inhibitor mutations; 41 (91%) had nucleoside reverse-transcriptase inhibitor (NRTI) mutations; 33 (73%) had non-NRTI (NNRTI) mutations; and 30 (66.7%) had both NRTI and NNRTI mutations. Triple-class resistance to NRTIs, NNRTIs, and PR inhibitors was observed in 24 (53%) patients. Based on the mutational profiles observed, all 45 sequences were susceptible to darunavir and tipranavir, whereas 47% showed resistance to lopinavir, 58% showed resistance to atazanavir, and >60% showed resistance to saquinavir, indinavir, nelfinavir, and fosamprenavir.
Conclusions. The results of the study showed that the majority of patients receiving second-line antiretroviral therapy started to accumulate PR resistance mutations, and the mutation profiles suggest that darunavir might be the drug of choice for third-line regimens in India.
doi:10.1093/cid/cir967
PMCID: PMC3571716  PMID: 22323567
17.  Performance of the Applied Biosystems ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Sequence-Based Analysis of HIV-1 in Pediatric Plasma Samples 
Journal of Clinical Microbiology  2001;39(4):1254-1257.
The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.
doi:10.1128/JCM.39.4.1254-1257.2001
PMCID: PMC87920  PMID: 11283037
18.  One-, Two-, and Three-Class Resistance among HIV-Infected Patients on Antiretroviral Therapy in Private Care Clinics: Mumbai, India 
Abstract
HIV-infected patients receiving antiretroviral (ARV) therapy (ART) in India are not all adequately virally suppressed. We analyzed ARV drug resistance in adults receiving ART in three private clinics in Mumbai, India. HIV viral load was measured in 200 patients with the Roche AMPLICOR HIV-1 Monitor Test, v1.5. HIV genotyping was performed with the ViroSeq HIV-1 Genotyping System for 61 participants who had HIV-1 RNA >1000 copies/ml. Genotyping results were obtained for 51 samples. The participants with resistance results were on ART for a median of 24 months and were on their current regimen for a median of 12 months (median CD4 cell count: 217 cells/mm3; median HIV viral load: 28,200 copies/ml). ARV regimens included nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens (n = 27), dual nucleoside reverse transcriptase inhibitors (NRTIs, n = 19), protease inhibitor (PI)-based regimens (n = 3), and other regimens (n = 2). Twenty-six participants (51.0%) were on their first ARV regimen and 24 (47%) reported >95% adherence. Forty-nine participants (96.1%) had resistance to at least one ARV drug; 47 (92.2%) had NRTI resistance, 32 (62.7%) had NNRTI resistance, and four (7.8%) had PI resistance. Thirty (58.8%) had two-class resistance and three (5.9%) had three-class resistance. Four (8%) had three or more resistance mutations associated with etravirine resistance and two (4%) had two mutations associated with reduced darunavir susceptibility. Almost all patients with HIV-1 RNA >1000 copies/ml had NRTI resistance and nearly two-thirds had NNRTI resistance; PI resistance was uncommon. Nearly 60% and 6% had two- and three-class resistance, respectively. This emphasizes the need for greater viral load and resistance monitoring, use of optimal ART combinations, and increased availability of second- and third-line agents for patients with ARV resistance.
doi:10.1089/aid.2009.0102
PMCID: PMC2858895  PMID: 20063995
19.  Detection of HIV-1 DNA resistance mutations by a sensitive assay at initiation of antiretroviral therapy is associated with virologic failure 
Background
Antiretroviral therapy (ART) has become more available throughout the developing world during the past five years. The World Health Organization recommends nonnucleoside reverse transcriptase inhibitor-based regimens as initial ART. However, their efficacy may be compromised by resistance mutations selected by single-dose nevirapine (sdNVP) used to prevent mother-to-child-transmission of HIV-1 (PMTCT). There is no simple and efficient method to detect such mutations at initiation of ART.
Methods
181 women participating in a PMTCT clinical trial who started NVP-ART after they had received sdNVP or placebo were tested for nevirapine-resistance point-mutations (K103N, Y181C, and G190A) using 100 copies of HIV-1 DNA with a sensitive oligonucleotide ligation assay (OLA) able to detect mutants at low concentrations (≥5% of the viral population). Virologic failure was defined as plasma HIV-1 RNA confirmed >50 copies/mL between 6–18 months of NVP-ART.
Results
At initiation of NVP-ART, resistance mutations were identified in 26% of 148 participants given sdNVP (K103N-13%, Y181C-5%, G190A-19%; ≥2 mutations-10%) at a median 9.3 months after sdNVP. The risk of virologic failure was .62 (95% confidence interval (CI), 0.46–0.77) in women with ≥1 resistance mutation, compared to 0.25 (95% CI, 0.17–0.35) in those without detectable resistance mutations (P<.0001). Failure was independently associated with resistance, an interval of <6 months between sdNVP and NVP-ART initiation, and a viral load above the median at NVP-ART initiation.
Conclusions
Access to simple and inexpensive assays to detect low-concentrations of NVP-resistant HIV-1 DNA prior to the initiation of ART could help improve the outcome of first-line antiretroviral therapy.
doi:10.1086/652148
PMCID: PMC2856716  PMID: 20377404
HIV-1; resistance mutations; nevirapine; HAART; oligonucleotide ligation assay; developing countries
20.  Low-frequency nevirapine resistance at multiple sites may predict treatment failure in infants on nevirapine-based treatment 
Background
Resistance commonly arises in infants exposed to single-dose nevirapine (sdNVP) for prevention of mother to child transmission (PMTCT). While 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 prior to NVP-based treatment.
Methods
Twenty sdNVP-exposed infants who were subsequently treated with NVP-based highly active antiretroviral therapy (HAART) were examined. Pre-treatment plasma samples were tested for the presence of NVP-resistance mutations by allele-specific PCR (ASPCR) for K103N and Y181C and ultra-deep pyrosequencing (UDPS) for all primary NVP mutations. Viral levels were determined every 3 months for up to 24months on NVP-HAART. Cox proportional hazard models were used to determine correlates of viral failure.
Results
The NVP resistance mutations K103N or Y181C were detected in pre-treatment plasma samples in 6 infants by ASPCR. NVP resistance at these or other sites was detectable by UDPS in 10 out of 20 infants tested. Virologic failure occurred in 50% of infants with any NVP resistance mutations detected, while 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%CI: 1.07, 2.99), p=0.027).
Conclusions
Low frequencies of multiple NVP resistance mutations, in addition to K103N and Y181C, present in infants before NVP-based treatment may predict treatment outcome.
doi:10.1097/QAI.0b013e3182515730
PMCID: PMC3383885  PMID: 22395670
HIV; infants; nevirapine; resistance; HAART; treatment failure
21.  Performance of the Celera Diagnostics ViroSeq HIV-1 Genotyping System for Sequence-Based Analysis of Diverse Human Immunodeficiency Virus Type 1 Strains 
Journal of Clinical Microbiology  2004;42(6):2711-2717.
The Celera Diagnostics ViroSeq HIV-1 Genotyping System is a Food and Drug Administration-cleared, integrated system for sequence-based analysis of drug resistance mutations in subtype B human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase (RT). We evaluated the performance of this system for the analysis of diverse HIV-1 strains. Plasma samples were obtained from 126 individuals from Uganda, Cameroon, South Africa, Argentina, Brazil, and Thailand with viral loads ranging from 2.92 to >6.0 log10 copies/ml. HIV-1 genotyping was performed with the ViroSeq system. HIV-1 subtyping was performed by using phylogenetic methods. PCR products suitable for sequencing were obtained for 125 (99%) of the 126 samples. Genotypes including protease (amino acids 1 to 99) and RT (amino acids 1 to 321) were obtained for 124 (98%) of the samples. Full bidirectional sequence data were obtained for 95 of those samples. The sequences were categorized into the following subtypes: A1/A2 (16 samples), B (12 samples), C (13 samples), D (11 samples), CRF01_AE (9 samples), F/F2 (9 samples), G (7 samples), CRF02_AG (32 samples), H (1 sample), and intersubtype recombinant (14 samples). The performances of the individual sequencing primers were examined. Genotyping of duplicate samples in a second laboratory was successful for 124 of the 126 samples. The identity level for the sequence data from two laboratories ranged from 98 to 100% (median, 99.8%). The ViroSeq system performs well for the analysis of plasma samples with diverse non-B subtypes. The availability of this genotyping system should facilitate studies of HIV-1 drug resistance in non-subtype B strains of HIV-1.
doi:10.1128/JCM.42.6.2711-2717.2004
PMCID: PMC427844  PMID: 15184457
22.  Field Evaluation of a Broadly Sensitive HIV-1 In-House Genotyping Assay for Use with both Plasma and Dried Blood Spot Specimens in a Resource-Limited Country 
Journal of Clinical Microbiology  2013;51(2):529-539.
HIV-1 drug resistance (HIVDR) assays are important tools in clinical management of HIV-infected patients on antiretroviral therapy (ART) and surveillance of drug-resistant variants at population levels. The high cost associated with commercial assays hinders their use in resource-limited settings. We adopted and validated a low-cost in-house assay using 68 matched plasma and dried blood spot (DBS) samples with a median viral load (VL) of 58,187 copies/ml, ranging from 253 to 3,264,850 against the commercial assay ViroSeq. Results indicated that the in-house assay not only had a higher plasma genotyping rate than did ViroSeq (94% versus 78%) but also was able to genotype 89.5% (51/57) of the matched DBS samples with VLs of ≥1,000 copies/ml. The sensitivity in detecting DR mutations by the in-house assay was 98.29% (95% confidence interval [CI], 97.86 to 98.72) on plasma and 96.54 (95% CI, 95.93 to 97.15) on DBS, and the specificity was 99.97% (95% CI, 99.91 to 100.00) for both sample types compared to ViroSeq. The minor DR mutation differences detected by the in-house assay against ViroSeq did not result in clinical significance. In addition, cost analysis showed that the in-house assay could reduce the genotyping cost by about 60% for both plasma and DBS compared to ViroSeq. This field condition evaluation highlights the potential utility of a cost-effective, subtype-independent, in-house genotyping assay using both plasma and DBS specimens for HIVDR clinical monitoring and population-based surveillance in resource-limited settings.
doi:10.1128/JCM.02347-12
PMCID: PMC3553877  PMID: 23224100
23.  Sensitivity and Specificity of the ViroSeq Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping System for Detection of HIV-1 Drug Resistance Mutations by Use of an ABI PRISM 3100 Genetic Analyzer 
Journal of Clinical Microbiology  2005;43(2):813-817.
The ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system is an integrated system for identification of drug resistance mutations in HIV-1 protease and reverse transcriptase (RT). Reagents are included for sample preparation, reverse transcription, PCR amplification, and sequencing. Software is provided to assemble and edit sequence data and to generate a drug resistance report. We determined the sensitivity and specificity of the ViroSeq system for mutation detection using an ABI PRISM 3100 genetic analyzer with a set of clinical samples and recombinant viruses. Twenty clinical plasma samples (viral loads, 1,800 to 10,500 copies/ml) were characterized by cloning and sequencing individual viral variants. Twelve recombinant-virus samples (viral loads, approximately 2,000 to 5,000 copies/ml) were also prepared. Eleven recombinant-virus samples contained drug resistance mutations as 40% mixtures. One recombinant-virus sample contained an insertion at codon 69 in RT (100% mutant). Plasma and recombinant-virus samples were analyzed using the ViroSeq system. Each sample was analyzed on three consecutive days at each of three testing laboratories. The sensitivity of mutation detection was 99.65% for the clinical plasma samples and 99.7% for the recombinant-virus preparations. The specificity of mutation detection was 99.95% for the clinical samples and 100% for the recombinant-virus mixtures. The base calling accuracy of the 3100 instrument was 99.91%. Mutations in clinical plasma samples and recombinant-virus samples were detected with high sensitivity and specificity, including mutations present as mixtures. This report supports the use of the ViroSeq system for identification of drug resistance mutations in HIV-1 protease and RT genes.
doi:10.1128/JCM.43.2.813-817.2005
PMCID: PMC548107  PMID: 15695685
24.  Efficacy of Short-Course AZT Plus 3TC to Reduce Nevirapine Resistance in the Prevention of Mother-to-Child HIV Transmission: A Randomized Clinical Trial 
PLoS Medicine  2009;6(10):e1000172.
Neil Martinson and colleagues report a randomized trial of adding short-course zidovudine+lamivudine to reduce drug resistance from single-dose nevirapine used to prevent mother-to-child transmission of HIV.
Background
Single-dose nevirapine (sdNVP)—which prevents mother-to-child transmission of HIV—selects non-nucleoside reverse-transcriptase inhibitor (NNRTI) resistance mutations in the majority of women and HIV-infected infants receiving it. This open-label, randomised trial examined the efficacy of short-course zidovudine (AZT) and lamivudine (3TC) with sdNVP in reducing NNRTI resistance in mothers, and as a secondary objective, in infants, in a setting where sdNVP was standard-of-care.
Methods and Findings
sdNVP alone, administered at the onset of labour and to the infant, was compared to sdNVP with AZT plus 3TC, given as combivir (CBV) for 4 (NVP/CBV4) or 7 (NVP/CBV7) days, initiated simultaneously with sdNVP in labour; their newborns received the same regimens. Women were randomised 1∶1∶1. HIV-1 resistance was assessed by population sequencing at: baseline, 2, and 6 wk after birth. An unplanned interim analysis resulted in early stopping of the sdNVP arm. 406 pregnant women were randomised and took study medication (sdNVP 74, NVP/CBV4 164, and NVP/CBV7 168). HIV-1 resistance mutations emerged in 59.2%, 11.7%, and 7.3% of women in the sdNVP, NVP/CBV4, and NVP/CBV7 arms by 6 wk postpartum; differences between NVP-only and both NVP/CBV arms were significant (p<0.0001), but the difference between NVP/CBV4 and NVP/CBV7 was not (p = 0.27). Estimated efficacy comparing combined CBV arms with sdNVP was 85.6%. Similar resistance reductions were seen in infants who were HIV-infected by their 6-wk visit.
Conclusions
A short course of AZT plus 3TC, supplementing maternal and infant sdNVP, reduces emergent NNRTI resistance mutations in both mothers and their infants. However, this trial was not powered to detect small differences between the CBV arms.
Trial registration
www.ClinicalTrials.gov NCT 00144183
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Currently, about 33 million people are infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV can be treated with combination antiretroviral therapy (ART), commonly three individual antiretroviral drugs that together efficiently suppress the replication of the virus. HIV infection of a child by an HIV-positive mother during pregnancy, labor, delivery, or breastfeeding is called mother-to-child transmission (MTCT). In 2007, an estimated 420,000 children were newly infected with HIV, the majority through MTCT. Most of these mothers and children live in sub-Saharan Africa where child and maternal mortality rates are high and mortality in HIV-infected children is extremely high. MTCT is preventable and there is a global commitment, agreed at the UN General Assembly Session on HIV/AIDS in 2001, to reduce the proportion of infants infected with HIV by 50% by 2010.
Why Was This Study Done?
In many resource-limited settings, MTCT is prevented by giving a single dose of nevirapine (an antiretroviral drug which has a long duration in the body and protects the fetus during labor and delivery only) to HIV-infected women in labor and also to a baby within 72 hours of birth. However, nevirapine, a non-nucleoside reverse-transcriptase inhibitor (NNRTI), which suppresses the replication of the virus, is associated with increased resistance of HIV, in mother and child, to NNRTI. This resistance reduces the effectiveness of future treatments of both mother and child with combination ART that includes an NNRTI; such regimens are the mainstay for long-term treatment of HIV in developing countries. The researchers investigated whether giving other antiretroviral drugs with nevirapine, during labor and delivery, to both mother and her newborn reduced the chances of them developing resistance to NNRTIs.
What Did the Researchers Do and Find?
The researchers selected 406 HIV-positive pregnant women for study across five sites in South Africa between February 2003 and May 2007. The women and their newborn babies were randomly assigned to receive, either (i) a single dose of nevirapine, (ii) a single dose of nevirapine plus combivir (zidovudine combined with lamivudine) for four days, or (iii) a single dose of nevirapine plus combivir for seven days. At two days, two weeks, and six weeks after delivery blood was collected from mothers and babies. HIV virus from blood samples was analyzed for resistance mutations, and mothers and children with resistance mutations were monitored for a further 96 weeks until no resistance was detected or combination ART (also called “HAART”) was started. Enrollment into the single-dose nevirapine arm was stopped early because a very high rate of NNRTI resistance mutations was found and other investigators reported long-term bad consequences of NNRTI-resistance on subsequent ART. The two nevirapine plus combivir arms were continued. The researchers found that selection of resistance mutations by single-dose nevirapine was reduced in mother and child by the addition of zidovudine and lamivudine for a short period; resistance mutations were found in 59.2% of women who got nevirapine only but only 11.7%, and 7.3% of women treated nevirapine plus four days combivir, and nevirapine plus seven days combivir respectively. A reduction was also seen in new NNRTI resistant mutations in the HIV-infected infants that received combivir. The study did not have enough women to show that there was a real difference between the resistance in the four-day and seven-day combivir regimens.
What Do These Findings Mean?
These findings show that a short-course treatment of zidovudine and lamivudine in addition to a single dose of nevirapine during labor and birth reduces the selection of NNRTI resistance mutations in both mother and child. The drug regimens appeared safe, and easy to provide and adhere to. Preliminary results from this study contributed to a change in clinical practice for the care of pregnant women with HIV; in 2004 the World Health Organisation guidelines introduced a short course of combivir with nevirapine for the management of pregnant HIV-infected women. However, the study had some limitations. It used HIV-positive women who were mainly infected with a subtype of HIV called HIV-1 clade C and who had a lot of virus in their blood. NNRTI resistance after treatment with nevirapine is more common in clade C than in others and this study does not address the effect of these combinations for preventing NNRTI resistance in other HIV subtypes. Also, World Health Organization, national, and international guidelines recommend combination ART during pregnancy, as it decreases HIV transmission from mother to child in the uterus to <2% in resource-limited settings. Although long-term combination treatment may not be available in all locations, this study does not tell us how the short-term combinations during and after delivery tested would compare to longer-term combinations given to pregnant women in reducing both HIV transmission and HIV drug resistance.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000172.
This study is further discussed in a PLoS Medicine Perspective by Lehman et al.
The US Centers for Disease Control and Prevention provide information for HIV treatment and prevention
MedlinePlus provides extensive information on symptoms and treatment for HIV/AIDS as well as access to related clinical trials and medical literature
aidsmap, a nonprofit, nongovernmental organization provides information on HIV and supporting those living with HIV
The World Health Organization gives information on the prevention of mother-to-child transmission of HIV
doi:10.1371/journal.pmed.1000172
PMCID: PMC2760761  PMID: 19859531
25.  Effects of Short-Course Zidovudine on the Selection of Nevirapine-Resistant HIV-1 in Women Taking Single-Dose Nevirapine 
The Journal of Infectious Diseases  2012;205(12):1811-1815.
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.
doi:10.1093/infdis/jis282
PMCID: PMC3415891  PMID: 22492850

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