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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.

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.

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.

Abstract

We analyzed drug resistance in HIV-infected Ugandan children who received antiretroviral therapy in a prospective, observational study (2004–2006); some children had prior single-dose nevirapine (sdNVP) exposure. Children received stavudine (d4T), lamivudine (3TC), and nevirapine (NVP); treatment was continued if they were clinically and immunologically stable. Samples with >1,000 copies/ml HIV RNA were analyzed by using the ViroSeq HIV Genotyping System (ViroSeq). Subtype A and D pretreatment samples also were analyzed with the LigAmp assay (for K103N, Y181C, and G190A). ViroSeq results were obtained for 74 pretreatment samples (35 from sdNVP-exposed children (median age, 19 months) and 39 from sdNVP-unexposed children (median age, 84 months). This included 39 subtype A, 22 subtype D, 1 subtype C, and 12 inter-subtype recombinant samples. One sample had nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance, one had nucleoside reverse transcriptase inhibitor (NRTI) resistance, and three had protease inhibitor (PI) resistance. Y181C was detected by using LigAmp in five pretreatment samples [four (14.8%) of 37 samples from sdNVP-exposed children, one (4.2%) of 24 samples from children without prior sdNVP exposure; p = 0.35]. Among children who were not virally suppressed at 48 weeks of treatment, all 12 tested had NNRTI resistance, as well as resistance to 3TC and emtricitibine (FTC); three had resistance to other NRTIs. Seven of those children had a ViroSeq result at 96 weeks of treatment; four of the seven acquired resistance to additional NRTIs by 96 weeks. In Uganda, clinically and immunologically stable children receiving nonsuppressive antiretroviral treatment regimens are at risk for development of drug resistance.

Objectives

To assess whether the commercial ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plains, IL, USA) can be used in conjunction with dried blood spots (DBS) for clinical monitoring of drug resistance in patients who fail antiretroviral treatment (ART) in rural Tanzania.

Patients and methods

Patients at Haydom Lutheran Hospital with confirmed treatment failure (viral load >1000 copies/mL) of a first-line ART regimen were selected for resistance testing. DBS were stored with desiccant at −20°C for a median of 126 days (range 0–203) and shipped at ambient temperature for 20 days. After manual extraction of nucleic acids, the ViroSeq kit was used for amplification and sequencing. DBS-derived genotypes were compared with those of a plasma-based assay.

Results

Seventeen of 36 (47%) DBS specimens were successfully genotyped. Only 2 of 16 (13%) DBS with a viral load <10 000 copies/mL could be amplified, compared with 15 of 20 (75%) DBS with a viral load >10 000 copies/mL (P = 0.001). In samples that yielded a sequence, all 23 clinically significant reverse transcriptase (RT) mutations in plasma were also detected in DBS. One RT mutation was found in DBS only. In the protease region, 77 polymorphisms were found in plasma, of which 70 (91%) were also detected in DBS. Sixteen of 17 (94%) patients had identical resistance profiles to antiretroviral drugs in plasma and DBS.

Conclusions

The ViroSeq kit performed well in patients with a high viral load, but failed to genotype most DBS with a viral load <10 000 copies/mL. In DBS that yielded a genotype, there was high concordance with a plasma-based assay.

Given the diversity of human immunodeficiency virus type 1 (HIV-1) subtypes and the emergence of subtypes other than HIV-1 subtype B in the United States, genotypic assays must be capable of delivering sequence data on diverse HIV-1 subtypes. We evaluated the performance of Visible Genetics TRUGENE HIV-1 genotyping kit and Applied Biosystems ViroSeq HIV-1 genotyping system on a panel of 34 well-characterized HIV-1 viral stocks (subtypes A through H). Both assays perform well on diverse HIV-1 subtypes despite being designed for HIV-1 subtype B. The TRUGENE assay produced sequence data for 31 isolates but not for one C and two G isolates. The TRUGENE assay using prototype 1.5 RT-PCR primers and the ViroSeq assay were both successful for all variants tested, although five isolates lacked double-strand sequence coverage in the ViroSeq assay. The availability of standardized HIV-1 genotyping kits that perform reliably with all HIV subtypes will facilitate broad implementation of HIV-1 resistance testing.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Objectives

Dried blood spots (DBS) and dried plasma spots (DPS) are considered convenient alternatives to serum and plasma for HIV drug resistance testing in resource-limited settings. We sought to investigate how extreme conditions could affect the short-term ability to amplify and genotype HIV from DBS.

Methods

A panel of six matched DPS/DBS was generated using blood collected from HIV-infected donors. Replicate cards were prepared in 903 filter paper using 50 µL of blood and stored at either −20°C or at 37°C/100% humidity. Nucleic acids were extracted at baseline and after 1, 2, 8 and 16 weeks of storage and were amplified and sequenced using an in-house RT-nested PCR method or the ViroSeq assay.

Results

HIV-1 pol was successfully amplified in all DBS/DPS at baseline and in those stored for up to 16 weeks at −20°C by the in-house assay. In contrast, amplification was rapidly lost during storage at 37°C/100% humidity with only 6/6 and 4/6 DBS specimens amplifiable by the in-house assay at weeks 1 and 2, respectively. Similarly, only two DPS stored at 37°C/100% humidity were amplified by the in-house assay at week 1.

Conclusions

We show that resistance testing from DBS and DPS is severely compromised after 2 and 1 weeks of storage at 37°C/100% humidity with desiccant, respectively. These findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport specimens from collection sites to locations that have appropriate storage conditions such as −20°C.

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.

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.

Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX.

Conclusions

The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.

Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy (n = 18) or were treatment naive (n = 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.

The ViroSeq HIV-1 genotyping system is used in many African countries for drug resistance testing. In this study, we used a panel of diverse HIV-1 group M isolates circulating in Cameroon to show that the performance of this assay can be altered by the sequence variation of non-B HIV-1 strains that predominate in African settings.

Background

Dried blood spots (DBSs) are an attractive alternative to plasma for HIV-1 drug resistance testing in resource-limited settings. We recently showed that HIV-1 can be efficiently genotyped from DBSs stored at −20°C for prolonged periods (0.5–4 years). Here, we evaluated the efficiency of genotyping from DBSs stored at 4°C for 1 year.

Methods

A total of 40 DBSs were prepared from residual diagnostic specimens collected from HIV subtype B-infected persons and were stored with desiccant at 4°C. Total nucleic acids were extracted after 1 year using a modification of the Nuclisens assay. Resistance testing was performed using the ViroSeq HIV-1 assay and an in-house nested RT–PCR method validated for HIV-1 subtype B that amplifies a smaller (1 kb) pol fragment.

Results

Using the ViroSeq assay, only 23 of the 40 (57.5%) DBS specimens were successfully genotyped; 22 of these specimens had plasma viraemia >10 000 RNA copies/mL. When the specimens were tested using the in-house assay, 38 of the 40 DBSs (95%) were successfully genotyped. Overall, resistance genotypes generated from the DBSs and plasma were highly concordant.

Conclusions

We show that drug resistance genotyping from DBSs stored at 4°C with desiccant is highly efficient but requires the amplification of small pol fragments and the use of an in-house nested PCR protocol with quality-controlled reagents. These findings suggest that 4°C may represent a suitable temperature for long-term storage of DBSs.

Commercial HIV-1 genotypic resistance assays are very expensive, particularly for use in resource-constrained settings like India. Hence a cost effective in-house assay for drug resistance was validated against the standard ViroSeq™ HIV-1 Genotyping System 2.0 (Celera Diagnostics, CA, USA). A total of 50 samples were used for this evaluation (21 proficiency panels and 29 clinical isolates). Known resistance positions within HIV-1 protease (PR) region (1–99 codons) and HIV-1 reverse-transcriptase (RT) region (1–240 codons) were included. The results were analysed for each codon as follows: (i) concordant; (ii) partially concordant; (iii) indeterminate and (iv) discordant. A total of 2750 codons (55 codons per patient sample × 50 samples) associated with drug resistance (1050 PR and 1700 RT) were analysed. For PR, 99% of the codon results were concordant and 1% were partially concordant. For RT, 99% of the codon results were concordant, 0.9% were partially concordant and 0.1% were discordant. No indeterminate results were observed and the results were reproducible. Overall, the in-house assay provided comparable results to those of US FDA approved ViroSeq™, which costs about a half of the commercial assay ($ 100 vs. $ 230), making it suitable for resource-limited settings.

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.

The emergence of resistance to antiretroviral drugs is a major obstacle to the successful treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients. In this work, we correlate clinical and virological trends such as viral load (VL) and CD4 counts to genotypic and phenotypic antiretroviral (ARV) resistance profiles of HIV-1 isolates from the B and non-B subtypes found in vertically infected children failing ARV therapy. Plasma samples were collected from 52 vertically HIV-1-infected children failing different ARV therapies. Samples underwent HIV-1 pol sequencing and phenotyping and were clustered into subtypes by phylogenetic analysis. Clinical data from each patient were analyzed together with the resistance (genotypic and phenotypic) data obtained. Thirty-five samples were from subtype B, 10 samples were non-B (subtypes A, C, and F), and 7 were mosaic samples. There was no significant difference concerning treatment data between B and non-B clades. Prevalence of known drug resistance mutations revealed slightly significant differences among B and non-B subtypes: L10I, 21 and 64%, K20R, 13 and 43%, M36I, 34 and 100%, L63P, 76 and 36%, A71V/T, 24 and 0%, and V77I, 32 and 0%, respectively, in the protease (0.0001 ≤ P ≤ 0.0886), and D67N, 38 and 8%, K70R, 33 and 0%, R211K, 49 and 85%, and K219Q/E, 31 and 0%, respectively, in the reverse transcriptase (0.0256 ≤ P ≤ 0.0704). Significant differences were found only in secondary resistance mutations and did not reflect significant phenotypic variation between clade B and non-B.