Related Articles

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.

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

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.

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.

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.

Background

As second-line antiretroviral treatment (ART) becomes more accessible in resource-limited settings (RLS), the need for more affordable monitoring tools such as point-of-care viral load assays and simplified genotypic HIV drug resistance (HIVDR) tests increases substantially. The prohibitive expenses of genotypic HIVDR assays could partly be addressed by focusing on a smaller region of the HIV reverse transcriptase gene (RT) that encompasses the majority of HIVDR mutations for people on ART in RLS. In this study, an in silico analysis of 125,329 RT sequences was performed to investigate the effect of submitting short RT sequences (codon 41 to 238) to the commonly used virco®TYPE and Stanford genotype interpretation tools.

Results

Pair-wise comparisons between full-length and short RT sequences were performed. Additionally, a non-inferiority approach with a concordance limit of 95% and two-sided 95% confidence intervals was used to demonstrate concordance between HIVDR calls based on full-length and short RT sequences.

The results of this analysis showed that HIVDR interpretations based on full-length versus short RT sequences, using the Stanford algorithms, had concordance significantly above 95%. When using the virco®TYPE algorithm, similar concordance was demonstrated (>95%), but some differences were observed for d4T, AZT and TDF, where predictions were affected in more than 5% of the sequences. Most differences in interpretation, however, were due to shifts from fully susceptible to reduced susceptibility (d4T) or from reduced response to minimal response (AZT, TDF) or vice versa, as compared to the predicted full RT sequence. The virco®TYPE prediction uses many more mutations outside the RT 41-238 amino acid domain, which significantly contribute to the HIVDR prediction for these 3 antiretroviral agents.

Conclusions

This study illustrates the acceptability of using a shortened RT sequences (codon 41-238) to obtain reliable genotype interpretations by virco®TYPE and Stanford algorithms. Implementation of this simplified protocol could significantly reduce the cost of both resistance testing and ARV treatment monitoring in RLS.

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.

Antiretroviral therapy (ART) scale-up in resource limited settings (RLS) has been successful, utilizing a standardized population-based approach to ART delivery. An unintended consequence of treatment scale-up is the inevitable emergence of HIV drug resistance (HIVDR) in populations even when patient adherence to ART is optimally supported. HIVDR has the potential to undermine the dramatic gains that ART has had in reducing the morbidity and mortality of HIV-infected patients in RLS. Sustaining and expanding ART coverage in RLS will depend upon the ability of ART programs to deliver ART in a way that minimizes the emergence of HIVDR. Fortunately, current evidence demonstrates that HIVDR in RLS has neither emerged nor been transmitted to the degree that had initially been feared. However, due to a lack of standardized methodologies, HIVDR data from RLS can be difficult to interpret and may not provide the programmatic evidence necessary for public health action. The World Health Organization has developed simple, standardized surveys that generate comparable results to assess acquired and transmitted HIVDR for routine public health implementation in RLS. These HIVDR surveys are designed to be implemented in conjunction with annual monitoring of program and site factors known to be associated with the emergence of HIVDR.

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.

Background

The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population.

Methods and Findings

HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma.

Conclusions

ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population.

Introduction

HIV drug resistance (HIVDR) testing is not routinely available in many resource-limited settings (RLS), therefore ART program and site factors known to be associated with HIVDR should be monitored to optimize the quality of patient care and minimize the emergence of preventable HIVDR.

Methods

In 2009, Namibia selected five World Health Organization Early Warning Indicators (EWIs) and piloted abstraction at nine antiretroviral therapy (ART) sites: ART prescribing practices, Patients lost to follow-up (LTFU) at 12 months, Patient retention on first-line ART at 12 months, On-time antiretroviral (ARV) drug pick-up, and ARV drug-supply continuity.

Results

Records supported monitoring of three of five selected EWIs. 9/9 (100%) sites met the target of 100% initiated on appropriate first-line regimens. 8/9 (89%) sites met the target of ≤20% LTFU, although 20.8% of ART starters (range 4.6%-44.6%) had a period of absence without documented ART coverage of 2.3 months (range 1.5-3.9 months). 6/9 (67%) sites met the target of 0% switched to a second-line regimen.

Conclusions

EWI monitoring directly resulted in public health action which will optimize the quality of care, specifically the strengthening of ART record systems permitting monitoring of five EWIs in future years and protocols for improved ART patient defaulter tracing.

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.

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.

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.

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.

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.

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.

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.

Objective:

To determine the predictors for early versus later (breastfeeding) transmission of HIV-1.

Methods:

Secondary data analysis was performed on HIV Network for Prevention Trials 012, a completed randomized clinical trial assessing the relative efficacy of nevirapine (NVP) versus zidovudine in reducing mother-to-child transmission (MTCT) of HIV-1. We used Cox regression analysis to assess risk factors for MTCT. The ViroSeq HIV genotyping and a sensitive point mutation assay were used to detect NVP resistance mutations.

Results:

In this subset analyses, 122 of 610 infants were HIV infected, of whom 99 (81.1%) were infected early (first positive polymerase chain reaction ≤56 days). Incidence of MTCT after 56 days was low [0.7% per month (95% confidence interval, CI: 0.4 to 1.0)], but continued through 18 months. In multivariate analyses, early MTCT “factors” included NVP versus zidovudine (hazard ratio (HR) = 0.57, 95% CI: 0.38 to 0.86), pre-entry maternal viral load (VL, HR = 1.76, 95% CI: 1.28 to 2.41), and CD4 cell count (HR = 1.16, 95% CI: 1.05 to 1.28). Maternal VL (6–8 weeks) was associated with late MTCT (HR = 3.66, 95% CI: 1.78 to 7.50), whereas maternal NVP resistance (6–8 weeks) was not.

Conclusions:

Maternal VL was the best predictor of both early and late transmission. Maternal NVP resistance at 6–8 weeks did not predict late transmission.

Background

One of the challenges facing the tuberculosis (TB) control programmes in resource-limited settings is lack of rapid techniques for detection of drug resistant TB, particularly multi drug resistant tuberculosis (MDR TB). Results obtained with the conventional indirect susceptibility testing methods come too late to influence a timely decision on patient management. More rapid tests directly applied on sputum samples are needed. This study compared the sensitivity, specificity and time to results of four direct drug susceptibility testing tests with the conventional indirect testing for detection of resistance to rifampicin and isoniazid in M. tuberculosis. The four direct tests included two in-house phenotypic assays – Nitrate Reductase Assay (NRA) and Microscopic Observation Drug Susceptibility (MODS), and two commercially available tests – Genotype® MTBDR and Genotype® MTBDRplus (Hain Life Sciences, Nehren, Germany).

Methods

A literature review and meta-analysis of study reports was performed. The Meta-Disc software was used to analyse the reports and tests for sensitivity, specificity, and area under the summary receiver operating characteristic (sROC) curves. Heterogeneity in accuracy estimates was tested with the Spearman correlation coefficient and Chi-square.

Results

Eighteen direct DST reports were analysed: NRA – 4, MODS- 6, Genotype MTBDR® – 3 and Genotype® MTBDRplus – 5. The pooled sensitivity and specificity for detection of resistance to rifampicin were 99% and 100% with NRA, 96% and 96% with MODS, 99% and 98% with Genotype® MTBDR, and 99% and 99% with the new Genotype® MTBDRplus, respectively. For isoniazid it was 94% and 100% for NRA, 92% and 96% for MODS, 71% and 100% for Genotype® MTBDR, and 96% and 100% with the Genotype® MTBDRplus, respectively. The area under the summary receiver operating characteristic (sROC) curves was in ranges of 0.98 to 1.00 for all the four tests. Molecular tests were completed in 1 – 2 days and also the phenotypic assays were much more rapid than conventional testing.

Conclusion

Direct testing of rifampicin and isoniazid resistance in M. tuberculosis was found to be highly sensitive and specific, and allows prompt detection of MDR TB.

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.

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.

Objective. The increased use of antiretroviral therapy (ART) has reduced the morbidity and mortality associated with HIV, adversely leading to the emergence of HIV drug resistance (HIVDR). In this study we aim to evaluate the prevalence of HIVDR mutations in ART-naive HIV-1 infected patients from northern India. Design. Analysis was performed using Viroseq genotyping system based on sequencing of entire protease and two-thirds of the Reverse Transcriptase (RT) region of pol gene. Results. Seventy three chronic HIV-1 infected ART naïve patients eligible for first line ART were enrolled from April 2006 to August 2008. In 68 patients DNA was successfully amplified and sequencing was done. 97% of HIV-1 strains belonged to subtype C, and one each to subtype A1 and subtype B. The overall prevalence of primary DRMs was 2.9% [2/68, 95% confidence interval (CI), 0.3%–10.2%]. One patient had a major RT mutation M184V, known to confer resistance to lamivudine, and another had a major protease inhibitor (PI) mutation D30N that imparts resistance to nelfinavir. Conclusion. Our study shows that primary HIVDR mutations have a prevalence of 2.9% among ART-naive chronic HIV-1 infected individuals.