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1.  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
2.  Optimization of a Low Cost and Broadly Sensitive Genotyping Assay for HIV-1 Drug Resistance Surveillance and Monitoring in Resource-Limited Settings 
PLoS ONE  2011;6(11):e28184.
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.
doi:10.1371/journal.pone.0028184
PMCID: PMC3223235  PMID: 22132237
3.  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
4.  Use of Dried-Blood-Spot Samples and In-House Assays To Identify Antiretroviral Drug Resistance in HIV-Infected Children in Resource-Constrained Settings ▿  
Journal of Clinical Microbiology  2011;49(12):4077-4082.
Monitoring HIV drug resistance is an important component of the World Health Organization's global HIV program. HIV drug resistance testing is optimal with commercially available clinically validated test kits using plasma; however, that type of testing may not be feasible or affordable in resource-constrained settings. HIV genotyping from dried blood spots (DBS) with noncommercial (in-house) assays may facilitate the capture of HIV drug resistance outcomes in resource-constrained settings but has had varying rates of success. With in-house assays for HIV reverse transcriptase, we evaluated the yield of genotyping DBS samples collected from HIV-infected children who were enrolled in two clinical trials conducted in sub-Saharan Africa (median HIV viral load, 5.88 log10 HIV RNA copies/ml; range, 4.04 to 6.99). Overall, HIV genotypes were obtained for 94 (89.5%) of 105 samples tested (95% and 84% from clinical trials #1 and #2, respectively); however, successful analysis of 15 (16.1%) of the 94 samples required repeat testing using a different set of primers on previously synthesized cDNA. The yield of genotyping was lower on the DBS that were stored suboptimally from clinical trial #2 (56% versus 88% for optimally stored). Concordance with plasma genotypes derived using a clinically validated, commercial kit-based assay (ViroSeq HIV-1 genotyping system) was also assessed in a subset of children with paired testing. For 34 samples with paired DBS and plasma genotypes, there was 100% concordance for major drug resistance mutations. DBS genotyping using in-house assays provides an alternative for antiretroviral drug resistance testing in children in resource-constrained regions but may require region-specific optimization before widespread use.
doi:10.1128/JCM.01004-11
PMCID: PMC3232965  PMID: 21956987
5.  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
6.  Development, Validation and Clinical Evaluation of a Low Cost In-House HIV-1 Drug Resistance Genotyping Assay for Indian Patients 
PLoS ONE  2014;9(8):e105790.
Human Immunodeficiency Virus-1 (HIV-1) drug resistance genotyping assay is a part of clinical management of HIV-1 positive individuals under treatment with highly active antiretroviral therapy (HAART). Routine monitoring of drug resistance mutations in resource limited settings like India is not possible due to high cost of commercial drug resistance assays. In this study we developed an in-house, cost effective HIV-1 drug resistance genotyping assay for Indian patients and validated it against the US-FDA-approved ViroSeq HIV-1 drug resistance testing system. A reference panel of 20 clinical samples was used to develop and validate the assay against ViroSeq HIV-1 drug resistance testing system which was subsequently used to genotype a clinical panel of 225 samples. The Stanford HIV database was used to identify drug resistant mutations. The analytical sensitivity of the assay was 1000 HIV-1 RNA copies/ml of plasma sample while precision and reproducibility was 99.68±0.16% and 99.76±0.18% respectively. One hundred and one drug resistant mutations were detected by the in-house assay compared to 104 by ViroSeq system in the reference panel. The assay had 91.55% success rate in genotyping the clinical panel samples and was able to detect drug resistant mutations related to nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse-transcriptase inhibitor (NNRTI) as well as protease inhibitor (PI) classes of antiretroviral drugs. It was found to be around 71.9% more cost effective compared to ViroSeq genotyping system. This evaluation of the assay on the clinical panel demonstrates its potential for monitoring clinical HIV-1 drug resistance mutations and population-based surveillance in resource limited settings like India.
doi:10.1371/journal.pone.0105790
PMCID: PMC4144911  PMID: 25157501
7.  Evaluation of a Cost Effective In-House Method for HIV-1 Drug Resistance Genotyping Using Plasma Samples 
PLoS ONE  2014;9(2):e87441.
Objectives
Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples.
Design
The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity.
Methods
The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000–1.8 million RNA copies/ml.
Results
Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$), thus making it cost effective.
Conclusions
The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients.
doi:10.1371/journal.pone.0087441
PMCID: PMC3922725  PMID: 24533056
8.  Affordable in-house antiretroviral drug resistance assay with good performance in non-subtype B HIV-1 
Journal of virological methods  2009;163(2):505-508.
The introduction of antiretroviral therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. This has led to a demand for affordable HIV-1 drug resistance assays, since treatment failure due to development of drug resistance is common. This study developed and evaluated an affordable “in–house” genotyping assay to monitor HIV-1 drug resistance in Africa, particularly South Africa. An “in-house” assay using automated RNA extraction, and subtype C specific PCR and sequencing primers was developed and successfully evaluated 396 patient samples (viral load ranges 1,000->1.6million RNA copies/ml). The “in-house” assay was validated by comparing sequence data and drug resistance profiles from 90 patient and 10 external quality control samples to data from the ViroSeqTM HIV-1 Genotyping kit. The “in-house” assay was more efficient, amplifying all 100 samples, compared to 91 samples using Viroseq. The “in house” sequences were 99.2%) homologous to the ViroSeq sequences, and identical drug resistance mutation profiles were observed in 96 samples. Furthermore, the “in-house” assay genotyped 260 of 295 samples from seven African sites, where 47% were non-subtype C. Overall, the newly validated “in-house” drug resistance assay is suited for use in Africa as it overcomes the obstacle of subtype diversity.
doi:10.1016/j.jviromet.2009.11.011
PMCID: PMC2932961  PMID: 19917318
HIV-1 subtype C; antiretroviral drug resistance; mutation profile; affordable
9.  Analysis of Drug Resistance in Children Receiving Antiretroviral Therapy for Treatment of HIV-1 Infection in Uganda 
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.
doi:10.1089/aid.2009.0164
PMCID: PMC2875950  PMID: 20455758
10.  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
11.  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
12.  HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4°C 
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.
doi:10.1093/jac/dkn100
PMCID: PMC2386080  PMID: 18344550
resistance testing; ViroSeq assay; 903 filter paper
13.  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
14.  Characterization of HIV-1 antiretroviral drug resistance after second-line treatment failure in Mali, a limited-resources setting 
Journal of Antimicrobial Chemotherapy  2012;67(12):2943-2948.
Objectives
We describe the outcomes of second-line drug resistance profiles and predict the efficacy of drugs for third-line therapy in patients monitored without the benefit of plasma HIV-1 RNA viral load (VL) or resistance testing.
Methods
We recruited 106 HIV-1-infected patients after second-line treatment failure in Mali. VL was determined by the Abbott RealTime system and the resistance by the ViroSeq HIV-1 genotyping system. The resistance testing was interpreted using the latest version of the Stanford algorithm.
Results
Among the 106 patients, 93 had isolates successfully sequenced. The median age, VL and CD4 cells were respectively 35 years, 72 000 copies/mL and 146 cells/mm3. Patients were exposed to a median of 4 years of treatment and to six antiretrovirals. We found 20% of wild-type viruses. Resistance to etravirine was noted in 38%, to lopinavir in 25% and to darunavir in 12%. The duration of prior nucleos(t)ide reverse transcriptase inhibitor exposure was associated with resistance to abacavir (P < 0.0001) and tenofovir (P = 0.0001), and duration of prior protease inhibitor treatment with resistance to lopinavir (P < 0.0001) and darunavir (P = 0.06).
Conclusion
Long duration of therapy prior to failure was associated with high levels of resistance and is directly related to limited access to VL monitoring and delayed switches to second-line treatment, precluding efficacy of drugs for third-line therapy. This study underlines the need for governments and public health organizations to recommend the use of VL monitoring and also the availability of darunavir and raltegravir for third-line therapies in the context of limited-resource settings.
doi:10.1093/jac/dks310
PMCID: PMC3584968  PMID: 22888273
resistance; third-line; Africa
15.  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
16.  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
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.  Population-Based Monitoring of Emerging HIV-1 Drug Resistance on Antiretroviral Therapy and Associated Factors in a Sentinel Site in Cameroon: Low Levels of Resistance but Poor Programmatic Performance 
PLoS ONE  2013;8(8):e72680.
Background
Scale-up of antiretroviral therapy (ART) in resource-limited settings has drastically reduced HIV-related morbidity and mortality. However, challenges in long-term ART, adherence and HIV drug resistance (HIVDR) itself, require monitoring to limit HIVDR emergence among ART-experienced populations, in order to ensure regimen efficacy.
Methods
A longitudinal study was conducted from 2009–2011 in a cohort of 141 HIV-infected adult patients (aged >21) at the national social insurance centre hospital in Yaounde, Cameroon. As per-WHO HIVDR protocol, HIV-1 protease-reverse transcriptase genotyping was performed at baseline and at endpoint (12 months) on first-line ART using ViroSeq™ Genotyping kit.
Results
At baseline, a prevalence of 3.6% (5/139) HIVDR was observed [protease inhibitors M46I (1/5), G73A (1/5), L90LM (1/5); nucleoside reverse transcriptase inhibitors: M184V (1/5), T215F (1/5); non-nucleoside reverse transcriptase inhibitors: K103N (1/5), Y181Y/C (2/5), M230ML (1/5)]. At endpoint, 54.0% (76) patients were followed-up, 9.2% (13) died, and 3.5% (5) transferred, 38.5% (47) lost to follow-up (LTFU). 69.7% (53/76) of those followed-up had viremia <40 copies/ml and 90.8% (69/76) <1000 copies/ml. 4/7 patients with viremia ≥1000 copies/ml harbored HIVDR (prevalence: 5.3%; 4/76), with M184V/I (4/4) and K103K/N (3/4) being the most prevalent mutations. LTFU was favored by costs for consultation/laboratory tests, drug shortages, workload (physician/patient ratio: 1/180) and community disengagement.
Conclusions
Low levels of HIVDR at baseline and at endpoint suggest a probable effectiveness of ART regimens used in Cameroon. However the possible high rate of HIVDR among LTFUs limited the strengths of our findings. Evaluating HIVDR among LTFU, improving adherence, task shifting, subsidizing/harmonizing costs for routine follow-up, are urgent measures to ensure an improved success of the country ART performance.
doi:10.1371/journal.pone.0072680
PMCID: PMC3753336  PMID: 23991142
19.  Performance Characteristics of Human Immunodeficiency Virus Type 1 (HIV-1) Genotyping Systems in Sequence-Based Analysis of Subtypes Other than HIV-1 Subtype B 
Journal of Clinical Microbiology  2003;41(3):998-1003.
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.
doi:10.1128/JCM.41.3.998-1003.2003
PMCID: PMC150292  PMID: 12624021
20.  An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings 
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
doi:10.3791/51242
PMCID: PMC4024245  PMID: 24747156
Medicine; Issue 85; Biomedical Technology; HIV-1; HIV Infections; Viremia; Nucleic Acids; genetics; antiretroviral therapy; drug resistance; genotyping; affordable
21.  Virological efficacy and emergence of drug resistance in adults on antiretroviral treatment in rural Tanzania 
Background
Virological response to antiretroviral treatment (ART) in rural Africa is poorly described. We examined virological efficacy and emergence of drug resistance in adults receiving first-line ART for up to 4 years in rural Tanzania.
Methods
Haydom Lutheran Hospital has provided ART to HIV-infected patients since October 2003. A combination of stavudine or zidovudine with lamivudine and either nevirapine or efavirenz is the standard first-line regimen. Nested in a longitudinal cohort study of patients consecutively starting ART, we carried out a cross-sectional virological efficacy survey between November 2007 and June 2008. HIV viral load was measured in all adults who had completed at least 6 months first-line ART, and genotypic resistance was determined in patients with viral load >1000 copies/mL.
Results
Virological response was measured in 212 patients, of whom 158 (74.5%) were women, and median age was 35 years (interquartile range [IQR] 29–43). Median follow-up time was 22.3 months (IQR 14.0–29.9). Virological suppression, defined as <400 copies/mL, was observed in 187 patients (88.2%). Overall, prevalence of ≥1 clinically significant resistance mutation was 3.9, 8.4, 16.7 and 12.5% in patients receiving ART for 1, 2, 3 and 4 years, respectively. Among those successfully genotyped, the most frequent mutations were M184I/V (64%), conferring resistance to lamivudine, and K103N (27%), Y181C (27%) and G190A (27%), conferring resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs), whereas 23% had thymidine analogue mutations (TAMs), associated with cross-resistance to all nucleoside reverse transcriptase inhibitors (NRTIs). Dual-class resistance, i.e. resistance to both NRTIs and NNRTIs, was found in 64%.
Conclusion
Virological suppression rates were good up to 4 years after initiating ART in a rural Tanzanian hospital. However, drug resistance increased with time, and dual-class resistance was common, raising concerns about exhaustion of future antiretroviral drug options. This study might provide a useful forecast of drug resistance and demand for second-line antiretroviral drugs in rural Africa in the coming years.
doi:10.1186/1471-2334-9-108
PMCID: PMC2713244  PMID: 19583845
22.  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
23.  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
24.  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
25.  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

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