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1.  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
2.  Implementing antiretroviral resistance testing in a primary health care HIV treatment programme in rural KwaZulu-Natal, South Africa: early experiences, achievements and challenges 
Background
Antiretroviral drug resistance is becoming increasingly common with the expansion of human immunodeficiency virus (HIV) treatment programmes in high prevalence settings. Genotypic resistance testing could have benefit in guiding individual-level treatment decisions but successful models for delivering resistance testing in low- and middle-income countries have not been reported.
Methods
An HIV Treatment Failure Clinic model was implemented within a large primary health care HIV treatment programme in northern KwaZulu-Natal, South Africa. Genotypic resistance testing was offered to adults (≥16 years) with virological failure on first-line antiretroviral therapy (one viral load >1000 copies/ml after at least 12 months on a standard first-line regimen). A genotypic resistance test report was generated with treatment recommendations from a specialist HIV clinician and sent to medical officers at the clinics who were responsible for patient management. A quantitative process evaluation was conducted to determine how the model was implemented and to provide feedback regarding barriers and challenges to delivery.
Results
A total of 508 specimens were submitted for genotyping between 8 April 2011 and 31 January 2013; in 438 cases (86.2%) a complete genotype report with recommendations from the specialist clinician was sent to the medical officer. The median turnaround time from specimen collection to receipt of final report was 18 days (interquartile range (IQR) 13–29). In 114 (26.0%) cases the recommended treatment differed from what would be given in the absence of drug resistance testing. In the majority of cases (n = 315, 71.9%), the subsequent treatment prescribed was in line with the recommendations of the report.
Conclusions
Genotypic resistance testing was successfully implemented in this large primary health care HIV programme and the system functioned well enough for the results to influence clinical management decisions in real time. Further research will explore the impact and cost-effectiveness of different implementation models in different settings.
doi:10.1186/1472-6963-14-116
PMCID: PMC3973961  PMID: 24606875
HIV-1; Drug resistance; Anti-retroviral agents; Primary health care; Treatment failure; Process assessment (health care); Capacity building
3.  Drug resistance in children at virological failure in a rural KwaZulu-Natal, South Africa, cohort 
Background
Better understanding of drug resistance patterns in HIV-infected children on antiretroviral therapy (ART) is required to inform public health policies in high prevalence settings. The aim of this study was to characterise the acquired drug resistance in HIV-infected children failing first-line ART in a decentralised rural HIV programme.
Methods
Plasma samples were collected from 101 paediatric patients (≤15 yrs of age) identified as failing ART. RNA was extracted from the plasma, reverse transcribed and a 1.3 kb region of the pol gene was amplified and sequenced using Sanger sequencing protocols. Sequences were edited in Geneious and drug resistance mutations were identified using the RegaDB and the Stanford resistance algorithms. The prevalence and frequency of mutations were analysed together with selected clinical and demographic data in STATA v11.
Results
A total of 101 children were enrolled and 89 (88%) were successfully genotyped; 73 on a non-nucleoside reverse-transcriptase inhibitor (NNRTI)-based regimen and 16 on a protease inhibitor (PI)-based regimen at the time of genotyping. The majority of patients on an NNRTI regimen (80%) had both nucleoside reverse-transcriptase inhibitor (NRTI) and NNRTI resistance mutations. M184V and K103N were the most common mutations amongst children on NNRTI-based and M184V among children on PI-based regimens. 30.1% had one or more thymidine analogue mutation (TAM) and 6% had ≥3 TAMs. Only one child on a PI-based regimen harboured a major PI resistance mutation.
Conclusions
Whilst the patterns of resistance were largely predictable, the few complex resistance patterns seen with NNRTI-based regimens and the absence of major PI mutations in children failing PI-based regimens suggest the need for wider access to genotypic resistance testing in this setting.
doi:10.1186/1742-6405-11-3
PMCID: PMC3922737  PMID: 24444369
4.  High-Levels of Acquired Drug Resistance in Adult Patients Failing First-Line Antiretroviral Therapy in a Rural HIV Treatment Programme in KwaZulu-Natal, South Africa 
PLoS ONE  2013;8(8):e72152.
Objective
To determine the frequency and patterns of acquired antiretroviral drug resistance in a rural primary health care programme in South Africa.
Design
Cross-sectional study nested within HIV treatment programme.
Methods
Adult (≥18 years) HIV-infected individuals initially treated with a first-line stavudine- or zidovudine-based antiretroviral therapy (ART) regimen and with evidence of virological failure (one viral load >1000 copies/ml) were enrolled from 17 rural primary health care clinics. Genotypic resistance testing was performed using the in-house SATuRN/Life Technologies system. Sequences were analysed and genotypic susceptibility scores (GSS) for standard second-line regimens were calculated using the Stanford HIVDB 6.0.5 algorithms.
Results
A total of 222 adults were successfully genotyped for HIV drug resistance between December 2010 and March 2012. The most common regimens at time of genotype were stavudine, lamivudine and efavirenz (51%); and stavudine, lamivudine and nevirapine (24%). Median duration of ART was 42 months (interquartile range (IQR) 32–53) and median duration of antiretroviral failure was 27 months (IQR 17–40). One hundred and ninety one (86%) had at least one drug resistance mutation. For 34 individuals (15%), the GSS for the standard second-line regimen was <2, suggesting a significantly compromised regimen. In univariate analysis, individuals with a prior nucleoside reverse-transcriptase inhibitor (NRTI) substitution were more likely to have a GSS <2 than those on the same NRTIs throughout (odds ratio (OR) 5.70, 95% confidence interval (CI) 2.60–12.49).
Conclusions
There are high levels of drug resistance in adults with failure of first-line antiretroviral therapy in this rural primary health care programme. Standard second-line regimens could potentially have had reduced efficacy in about one in seven adults involved.
doi:10.1371/journal.pone.0072152
PMCID: PMC3749184  PMID: 23991055
5.  Primary Drug Resistance in South Africa: Data from 10 Years of Surveys 
Abstract
HIV-1 transmitted drug resistance (TDR) could reverse the gains of antiretroviral rollout. To ensure that current first-line therapies remain effective, TDR levels in recently infected treatment-naive patients need to be monitored. A literature review and data mining exercise was carried out to determine the temporal trends in TDR in South Africa. In addition, 72 sequences from seroconvertors identified from Africa Centre's 2010 HIV surveillance round were also examined for TDR. Publicly available data on TDR were retrieved from GenBank, curated in RegaDB, and analyzed using the Calibrated Population Resistance Program. There was no evidence of TDR from the 2010 rural KwaZulu Natal samples. Ten datasets with a total of 1618 sequences collected between 2000 and 2010 were pooled to provide a temporal analysis of TDR. The year with the highest TDR rate was 2002 [6.67%, 95% confidence interval (CI): 3.09–13.79%; n=6/90]. After 2002, TDR levels returned to <5% (WHO low-level threshold) and showed no statistically significant increase in the interval between 2002 and 2010. The most common mutations were associated with NNRTI resistance, K103N, followed by Y181C and Y188C/L. Five sequences had multiple resistance mutations associated with NNRTI resistance. There is no evidence of TDR in rural KwaZulu-Natal. TDR levels in South Africa have remained low following a downward trend since 2003. Continuous vigilance in monitoring of TDR is needed as more patients are initiated and maintained onto antiretroviral therapy.
doi:10.1089/aid.2011.0284
PMCID: PMC3358100  PMID: 22251009
6.  Surveillance of Transmitted Antiretroviral Drug Resistance among HIV-1 Infected Women Attending Antenatal Clinics in Chitungwiza, Zimbabwe 
PLoS ONE  2011;6(6):e21241.
The rapid scale-up of highly active antiretroviral therapy (HAART) and use of single dose Nevirapine (SD NVP) for prevention of mother-to-child transmission (pMTCT) have raised fears about the emergence of resistance to the first line antiretroviral drug regimens. A cross-sectional study was conducted to determine the prevalence of primary drug resistance (PDR) in a cohort of young (<25 yrs) HAART-naïve HIV pregnant women attending antenatal clinics in Chitungwiza, Zimbabwe. Whole blood was collected in EDTA for CD4 counts, viral load, serological estimation of duration of infection using the BED Calypte assay and genotyping for drug resistance. Four hundred and seventy-one women, mean age 21 years; SD: 2.1 were enrolled into the study between 2006 and 2007. Their median CD4 count was 371cells/µL; IQR: 255–511 cells/µL. Two hundred and thirty-six samples were genotyped for drug resistance. Based on the BED assay, 27% were recently infected (RI) whilst 73% had long-term infection (LTI). Median CD4 count was higher (p<0.05) in RI than in women with LTI. Only 2 women had drug resistance mutations; protease I85V and reverse transcriptase Y181C. Prevalence of PDR in Chitungwiza, 4 years after commencement of the national ART program remained below WHO threshold limit (5%). Frequency of recent infection BED testing is consistent with high HIV acquisition during pregnancy. With the scale-up of long-term ART programs, maintenance of proper prescribing practices, continuous monitoring of patients and reinforcement of adherence may prevent the acquisition and transmission of PDR.
doi:10.1371/journal.pone.0021241
PMCID: PMC3116901  PMID: 21698125
7.  Evolution and molecular epidemiology of subtype C HIV-1 in Zimbabwe 
AIDS (London, England)  2009;23(18):2523-2532.
Objective
To investigate the origins and evolutionary history of subtype C HIV-1 in Zimbabwe in a context of regional conflict and migration.
Design
HIV-1C pol sequence datasets were generated from four sequential cohorts of antenatal women in Harare, Zimbabwe sampled over 15 years (1991–2006).
Methods
One hundred and seventy-seven HIV-1C pol sequences were obtained from four successive cohorts in Zimbabwe. Maximum-likelihood methods were used to explore phylogenetic relationships between Zimbabwean HIV-1C sequences and subtype C strains from other regions. A Bayesian coalescent-based framework was used to estimate evolutionary parameters for HIV-1C in Zimbabwe, including origin and demographic growth patterns.
Results
Zimbabwe HIV-1C pol demonstrated increasing sequence divergence over the 15-year period. Nearly all Zimbabwe sequences clustered phylogenetically with subtype C strains from neighboring countries. Bayesian evolutionary analysis indicated a most recent common ancestor date of 1973 with three epidemic growth phases: an initial slow phase (1970s) followed by exponential growth (1980s), and a linearly expanding epidemic to the present. Bayesian trees provided evidence for multiple HIV-1C introductions into Zimbabwe during 1979–1981, corresponding with Zimbabwean national independence following a period of socio-political instability.
Conclusion
The Zimbabwean HIV-1C epidemic likely originated from multiple introductions in the late 1970s and grew exponentially during the 1980s, corresponding to changing political boundaries and rapid population influx from neighboring countries. The timing and phylogenetic clustering of the Zimbabwean sequences is consistent with an origin in southern Africa and subsequent expansion. HIV-1 sequence data contain important epidemiological information, which can help focus treatment and prevention strategies in light of more recent political volatility in Zimbabwe.
doi:10.1097/QAD.0b013e3283320ef3
PMCID: PMC2923658  PMID: 19770693
epidemiology; evolutionary history; HIV-1; origin; subtype C; Zimbabwe
8.  Evaluation of the Partec Flow Cytometer against the BD FACSCalibur System for Monitoring Immune Responses of Human Immunodeficiency Virus-Infected Patients in Zimbabwe▿  
Clinical and Vaccine Immunology  2007;14(3):293-298.
A single-platform volumetric flow cytometer, the Partec Cyflow SL_3, was evaluated against a BD FACSCalibur/Sysmex XT1800i dual platform for measuring CD4+ lymphocytes, total lymphocytes, and the percentage of CD4 lymphocytes in whole-blood samples for monitoring the immune systems of human immunodeficiency virus (HIV)/AIDS patients. Statistical analyses for precision, correlation, and agreement were performed. Coefficients of variation (CV) of 5.8, 4.6, and 3.9% were obtained for low, medium, and high CD4+ cell counts, respectively, using the SL_3, and CV of 3.7, 4.0, and 0.94 were obtained for the same categories, using the BD FACSCalibur. Significant correlations (P < 0.005) between the two assays for CD4 counts, total lymphocyte counts, and percentages of CD4 were obtained, with correlation coefficients of 0.99, 0.96, and 0.99, respectively (n = 229). Using the Bland-Altman plot, mean biases of −18 cell/μl (95% confidence interval (CI); −91 to 54 cells/μl), −0.8% (95% CI; −3.6 to 2%), and −36.8 cells/μl (95% CI; −477 to 404 cells/μl) were obtained for comparisons of CD4 counts, percentages of CD4 cells, and total lymphocyte counts, respectively. The effects of the age of the samples on the three parameters were also analyzed by comparing results from the same samples analyzed at 6, 24, and 48 h after collection. The correlation coefficients for comparisons among different time points for the same machine and among all the time points for the two different machines were greater than 0.90. These data showed that the Partec Cyflow SL_3 assay is comparable to the BD FACSCalibur/Sysmex XT1800i dual-platform method for measuring the amount of CD4+ cells and total lymphocytes and the percentages of CD4 cells in blood samples for the purpose of monitoring HIV/AIDS patients.
doi:10.1128/CVI.00416-06
PMCID: PMC1828850  PMID: 17267593

Results 1-8 (8)