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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 ...

University of KwaZulu-Natal, Durban, South Africa

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/microl (95% confidence interval (CI); -91 to 54 cells/microl), -0.8% (95% CI; -3.6 to 2%), and -36.8 cells/microl (95% CI; -477 to 404 cells/microl) 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.

Evaluation of the Partec flow cytometer against the BD FACSCalibur system for monitoring immune responses of human immunodeficiency virus-infected patients in Zimbabwe.

To investigate the origins and evolutionary history of subtype C HIV-1 in Zimbabwe in a context of regional conflict and migration.

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

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.

Surveillance of transmitted antiretroviral drug resistance among HIV-1 infected women attending antenatal clinics in Chitungwiza, Zimbabwe.

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.

Primary drug resistance in South Africa: data from 10 years of surveys.

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.

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.

To determine the frequency and patterns of acquired antiretroviral drug resistance in a rural primary health care programme in South Africa.

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.

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.

Drug resistance in children at virological failure in a rural KwaZulu-Natal, South Africa, cohort.

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/

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

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.

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

Justen Manasa

Africa Centre for Health and Population Studies,
College of Health Sciences,
Africa Centre for Health and Population Studies, College of Health Sciences

University of KwaZulu-Natal, Durban, South Africa

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

Justen Manasa

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University of KwaZulu-Natal, Durban, South Africa

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

Africa Centre for Health and Population Studies,
College of Health Sciences,
Africa Centre for Health and Population Studies, College of Health Sciences