Aletta Kliphuis

Timing of the HIV-1 subtype C epidemic in Ethiopia based on early virus strains and subsequent virus diversification.

Objective To trace the introduction of HIV-1 subtype C into Ethiopia based on virus diversification during the epidemic. Design A set of 474 serum samples obtained in Ethiopia in 1982–1985 was tested for HIV-1. HIV-1 env gp120 V3 and gag or pol regions were sequenced and analysed together with sequences from later stages of the epidemic. Results None of 98 samples from 1982–1983, one of 193 samples from 1984, and one of 183 samples from 1985 were HIV-1 positive. Phylogenetic analysis of virus sequences from positive samples revealed that they belong to the Ethiopian C, and not the C′, cluster. Analysis of 81 Ethiopian C V3 sequences from 1984–1997 revealed that the consensus sequence of the Ethiopian epidemic has been stable over time. Both the 1984 and 1985 V3 sequences, in contrast with three out of 27 (11%) of the 1988 and none out of 51 of the 1992–1997 sequences, had no synonymous substitutions compared to the reconstructed common ancestor of the Ethiopian C viruses. A highly significant correlation between sampling years of the V3 sequences and their synonymous distances to the common ancestor was demonstrated. Conclusions The increasing genetic heterogeneity together with stable consensus sequence of the Ethiopian HIV-1 C population demonstrates that evolution of the virus population is characterized by an unbiased expansion around a stationary consensus. Based on the rate of synonymous diversification of HIV-1 strains within the Ethiopian population, we were able to estimate 1983 (95% confidence interval, 1980–1984) as the year of HIV-1 C introduction into Ethiopia.

Development and evaluation of an assay for HIV-1 protease and reverse transcriptase drug resistance genotyping of all major group-M subtypes

BACKGROUND High cost and varying sensitivity for non-B HIV-1 subtypes limits application of current commercial kits for HIV-1 drug resistance genotyping of all major HIV-1 group-M subtypes. OBJECTIVES Our research aimed to develop and validate an assay specific for all major HIV-1 group-M subtypes for use as an alternative to commercial assays for HIV-1 protease (PR) and reverse transcriptase (RT) drug resistance genotyping. STUDY DESIGN A nested RT-PCR encompassing the entire PR and RT up to amino acid 321 of HIV-1 was designed to detect HIV-1 group-M subtypes. Primers compatible with group-M subtypes were defined and analytical sensitivity of the assay evaluated using a panel of reference viruses for subtypes A-H and CRF01_AE. The assay was subsequently evaluated on 246 plasma samples from HIV-1 infected individuals harboring various group-M subtypes and viral loads (VLs). RESULTS All major group-M HIV-1 subtypes were detected with an overall analytical sensitivity of 1.00E+03 RNA copies/ml. Application of the genotyping assay on 246 primarily African clinical samples comprising subtypes A (n=52; 21.7%), B (n=12; 5.0%), C (n=127; 52.9%), D (n=25; 10.4%), CRF01_AE (n=10; 4.2%), and CRF02_AG (n=10; 4.2%), and unassigned variants (n=10; 4.2%), VL range 4.32E+02-8.63E+06 (median 2.66E+04) RNA copies/ml, was ∼98% successful. CONCLUSIONS A group-M subtype-independent genotyping assay for detection of HIV-1 drug resistance was developed. The described assay can serve as an alternative to commercial assays for HIV-1 drug resistance genotyping in routine diagnostics, and for surveillance and monitoring of drug resistance in resource-limited settings (RLS).

Development and Evaluation of an Affordable Real-Time Qualitative Assay for Determining HIV-1 Virological Failure in Plasma and Dried Blood Spots

ABSTRACT Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for “open platform” applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.

Clinical Evaluation of an Affordable Qualitative Viral Failure Assay for HIV Using Dried Blood Spots in Uganda

Background WHO recommends regular viral load (VL) monitoring of patients on antiretroviral therapy (ART) for timely detection of virological failure, prevention of acquired HIV drug resistance (HIVDR) and avoiding unnecessary switching to second-line ART. However, the cost and complexity of routine VL testing remains prohibitive in most resource limited settings (RLS). We evaluated a simple, low–cost, qualitative viral–failure assay (VFA) on dried blood spots (DBS) in three clinical settings in Uganda. Methods We conducted a cross–sectional diagnostic accuracy study in three HIV/AIDS treatment centres at the Joint Clinical Research Centre in Uganda. The VFA employs semi-quantitative detection of HIV–1 RNA amplified from the LTR gene. We used paired dry blood spot (DBS) and plasma with the COBASAmpliPrep/COBASTaqMan, Roche version 2 (VLref) as the reference assay. We used the VFA at two thresholds of viral load, (>5,000 or >1,000 copies/ml). Results 496 paired VFA and VLref results were available for comparative analysis. Overall, VFA demonstrated 78.4% sensitivity, (95% CI: 69.7%–87.1%), 93% specificity (95% CI: 89.7%–96.4%), 89.3% accuracy (95% CI: 85%–92%) and an agreement kappa = 0.72 as compared to the VLref. The predictive values of positivity and negativity among patients on ART for >12 months were 72.7% and 99.3%, respectively. Conclusions VFA allowed 89% of correct classification of VF. Only 11% of the patients were misclassified with the potential of unnecessary or late switch to second–line ART. Our findings present an opportunity to roll out simple and affordable VL monitoring for HIV–1 treatment in RLS.