Justen Manasa

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

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.

Evolution and molecular epidemiology of subtype C HIV-1 in Zimbabwe.

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.

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.

High level of HIV-2 false positivity in KwaZulu-Natal province: A region of South Africa with a very high HIV-1 subtype C prevalence

Human immunodeficiency virus 2 (HIV‐2) is found predominantly in West Africa. It is not unlikely, however, that HIV‐2 may also be found in South Africa, due to the influx of immigrants into this country. It is important to distinguish between HIV‐1 and HIV‐2 since the clinical courses and treatment responses of these viruses are different. Routine serological methods for diagnosing HIV do not differentiate between HIV‐1 and ‐2 infections, while rapid tests, viral load quantification and PCR are HIV‐type—specific. The objective of this study was to describe the seroprevalence and molecular epidemiology of HIV‐2 in KwaZulu‐Natal, one of the regions with the highest HIV prevalence in the world and home of the two largest harbors in South Africa. HIV‐1 positive samples were screened for antibodies against HIV‐2, using a rapid test. The confirmation of HIV‐2 positive samples was done by PCR. Of the 2,123 samples screened, 319 (15%) were identified as positive by the rapid test. None of these samples were confirmed positive by PCR. To explore this discrepancy in the results, a subset (n = 52) of the rapid HIV‐2 positive samples was subjected to Western blotting. Thirty‐seven (71%) of these were positive, yielding an overall HIV‐2 seroprevalence of 10.6%. Three out of 28 (10.7%) Western blot positive samples were positive by a Pepti‐LAV assay. This discrepancy between serological and molecular confirmation may be attributed to non‐specific or cross‐reacting antibodies. The use of rapid tests and Western blots for HIV‐2 diagnosis in South Africa should be interpreted with caution. J. Med. Virol. 85:2065–2071, 2013.

Southern African Treatment Resistance Network (SATuRN) RegaDB HIV drug resistance and clinical management database: supporting patient management, surveillance and research in southern Africa

Abstract Substantial amounts of data have been generated from patient management and academic exercises designed to better understand the human immunodeficiency virus (HIV) epidemic and design interventions to control it. A number of specialized databases have been designed to manage huge data sets from HIV cohort, vaccine, host genomic and drug resistance studies. Besides databases from cohort studies, most of the online databases contain limited curated data and are thus sequence repositories. HIV drug resistance has been shown to have a great potential to derail the progress made thus far through antiretroviral therapy. Thus, a lot of resources have been invested in generating drug resistance data for patient management and surveillance purposes. Unfortunately, most of the data currently available relate to subtype B even though >60% of the epidemic is caused by HIV-1 subtype C. A consortium of clinicians, scientists, public health experts and policy markers working in southern Africa came together and formed a network, the Southern African Treatment and Resistance Network (SATuRN), with the aim of increasing curated HIV-1 subtype C and tuberculosis drug resistance data. This article describes the HIV-1 data curation process using the SATuRN Rega database. The data curation is a manual and time-consuming process done by clinical, laboratory and data curation specialists. Access to the highly curated data sets is through applications that are reviewed by the SATuRN executive committee. Examples of research outputs from the analysis of the curated data include trends in the level of transmitted drug resistance in South Africa, analysis of the levels of acquired resistance among patients failing therapy and factors associated with the absence of genotypic evidence of drug resistance among patients failing therapy. All these studies have been important for informing first- and second-line therapy. This database is a free password-protected open source database available on www.bioafrica.net. Database URL: http://www.bioafrica.net/regadb/

Implementing antiretroviral resistance testing in a primary health care HIV treatment programme in rural KwaZulu-Natal, South Africa: early experiences, achievements and challenges

BackgroundAntiretroviral 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.MethodsAn 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.ResultsA 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.ConclusionsGenotypic 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.

The Burden of HIV Associated Drug Resistance Mutations in an Early Infant Diagnosis Program: A Glance through the Paediatric Window of Zimbabwe

There is paucity of information on the prevalence of human immunodeficiency virus type 1 drug-resistance mutations in infants infected despite prevention of mother to child transmission in Zimbabwe. This study examined 32 dried blood spot specimens from HIV- 1 C infected infants born alive to women receiving antiretroviral therapy, who were part of the WHO/ResNet early infant diagnosis program between January 2010 and January 2011. The objective was to determine the patterns and levels of HIV drug resistance and inform on policy formulation. Overall HIV prevalence of the infants in the retrospect study period was 2.4% (32/1356). Half of the samples (16/32) analyzed had HIV associated drug resistance mutations, however excluding polymorphic mutations the HIV drug resistant mutations were 25% (8/32). Frequencies of the mutations were (E138A, n=6; G190A, n=1; M230ML, n=1; K103KN, n=1; Y181C, n=5; V90VI, n=1; E138G, n=1; E138EA, n=1). One patient had two mutations the K103KN and the Y181C. Of the sixteen patients with HIV drug resistance mutations, 5 had nevirapine only resistance, none had lamivudine resistance and 8 had both etravirine and rilpivirine resistance, 2 had both nevirapine, efavirenz and 1 had rilpivirine only. The study shows an apparent non-nucleoside reverse transcriptase inhibitor (NNRTI) drug resistance predominance and suggests a judicious use of NNRTI regimens and a prudent strategy to minimize the selection of drug resistance mutations.

Evolution of gag and gp41 in Patients Receiving Ritonavir-Boosted Protease Inhibitors

Several groups have proposed that genotypic determinants in gag and the gp41 cytoplasmic domain (gp41-CD) reduce protease inhibitor (PI) susceptibility without PI-resistance mutations in protease. However, no gag and gp41-CD mutations definitively responsible for reduced PI susceptibility have been identified in individuals with virological failure (VF) while receiving a boosted PI (PI/r)-containing regimen. To identify gag and gp41 mutations under selective PI pressure, we sequenced gag and/or gp41 in 61 individuals with VF on a PI/r (n = 40) or NNRTI (n = 20) containing regimen. We quantified nonsynonymous and synonymous changes in both genes and identified sites exhibiting signal for directional or diversifying selection. We also used published gag and gp41 polymorphism data to highlight mutations displaying a high selection index, defined as changing from a conserved to an uncommon amino acid. Many amino acid mutations developed in gag and in gp41-CD in both the PI- and NNRTI-treated groups. However, in neither gene, were there discernable differences between the two groups in overall numbers of mutations, mutations displaying evidence of diversifying or directional selection, or mutations with a high selection index. If gag and/or gp41 encode PI-resistance mutations, they may not be confined to consistent mutations at a few sites.

HIV drug resistance levels in adults failing first-line antiretroviral therapy in an urban and a rural setting in South Africa

Urban and rural HIV treatment programmes face different challenges in the long‐term management of patients. There are few studies comparing drug resistance profiles in patients accessing treatment through these programmes. The aim of this study was to perform such a comparison.

HIV Drug Resistance Patterns at the Epicentre of the HIV-1 Epidemic in Kwazulu-Natal, South Africa 2003-2013

South Africa has the highest number of individuals with HIV-1 infection worldwide with the epicentre of the epidemic in the province of KwaZulu-Natal (KZN). Currently South Africa has the largest antiretroviral treatment (ART) programme with KZN and Gauteng provinces accounting for 56% of all patients on ART. In order to understand transmission dynamics and temporal trends in drug resistance, we performed a comprehensive literature review of primary and acquired drug resistance in adults and children in KZN over a ten year period, 2003-2013. Transmitted drug resistance (TDR) to non-nucleoside reverse transcriptase inhibitors (NNRTIs) in adults is increasing to moderate levels (5-15%). Acquired drug resistance to first line drug regimens in adults was associated with at least one mutation to nucleoside reverse transcriptase inhibitors (NRTI) or NNRTIs or both, with M184V and K103N the most common mutations. Prevalence of thymidine analogues (TAMs) was low to moderate. There was no published data on acquired drug resistance to second line agents. Children failing first line drug regimens harboured drug resistance mutations to NRTI (M184V and TAMs), NNRTI (V106M and K103N) and protease inhibitors (PIs) (V82A). Concerns have been raised about the high levels of the K65R mutation associated with tenofovir regimens in adults and children. Continued vigilance is needed for ART failure in both adults and children, so as not to compromise second line treatment options. Drug resistance genotyping is now cost effective and should be considered for inclusion in regional surveillance studies and during routine care.