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.

Development and evaluation of an affordable real-time qualitative assay for determining HIV-1 virological failure in plasma and dried blood spots.

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.

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).

Surveillance technology for HIV-1 subtype C in Ethiopia: an env-based NASBA molecular beacon assay to discriminate between subcluster C and C'.

Forty-nine samples with known C2V3 sequences were used for the evaluation of an env-based molecular beacon assay to distinguish between the two genetic subclusters C and C' which characterize the HIV-1 epidemic in Ethiopia. Two subcluster C and two subcluster C' beacons targeting two different loci in the C2V3 region were developed. Using a three beacon-based (2C and 1C'=C prime), isothermal amplification assay, concordance with DNA sequencing was achieved for 43 (87.8%) samples. Sensitivity was 81.8% and specificity 97.4% for subcluster C beacons. For the subcluster C' beacon, a sensitivity of 97% and a specificity of 87.5% was achieved. Five samples were ambiguous by sequencing of which two samples were subcluster C' by the beacon assay and one subcluster C. Two of the samples remained ambiguous with different beacon-pair combinations as well. From samples with a clear C or C' phylogeny by sequencing, three were undetected by the first-line beacon genotyping assay. Genotype ambiguity was resolved in the three samples using beacon pair combinations restricted to each targeted locus. The beacons were evaluated further in a panel including all HIV-1 subtypes. Four of five subtype C isolates were identified correctly, and no cross-reactivity was observed with other subtypes.

Phenotypic and genotypic comparisons of CCR5- and CXCR4-tropic human immunodeficiency virus type 1 biological clones isolated from subtype C-infected individuals.

Individuals infected with human immunodeficiency virus type 1 (HIV-1) subtype C infrequently harbour X4 viruses. We studied R5 and X4 biological clones generated from HIV-1 subtype C-infected individuals. All subtype C R5 viruses demonstrated slower profiles of replication on CD4(+) lymphocytes in comparison to subtype B viruses, whereas subtype C X4 viruses replicated with comparable efficiency to subtype B X4 viruses. No differences were identified in CC or CXC chemokine inhibitions (RANTES and SDF-1alpha, respectively) between subtype C and subtype B viruses. Immature dendritic cells were shown in coculture experiments to similarly enhance the infection of subtype C and subtype B R5 as well as X4 viruses. By amino acid sequence analysis, we showed that the R5 and X4 subtype C gp120 envelope gene alterations were similar to those for a switching subtype B virus, specifically with respect to the V3 charge and envelope N-linked glycosylation patterns. By phylogenetic analysis, we showed that one patient was infected with HIV-1 C' and the other was infected with HIV-1 C" and that one of the patients harbored a virus that was a recombinant in the gp120 env gene between an R5 and an X4 virus, with the resultant virus being R5. No differences were identified between the long terminal repeat regions of the subtype C R5 and X4 biological clones. These results indicate that even though R5 subtype C viruses are restrictive for virus replication, the R5-to-X4 phenotype switch can occur and does so in a manner similar to that of subtype B viruses.

Recombination of HIV type 1C (C'/C") in Ethiopia: possible link of EthHIV-1C' to subtype C sequences from the high-prevalence epidemics in India and Southern Africa.

The magnitude and complexity of the HIV-1 genetic diversity are major challenges for vaccine development. Investigation of the genotypes circulating in areas of high incidence, as well as their interactions, will be a milestone in the development of an efficacious vaccine. Because HIV-1 subtype C (HIV-1C) is responsible for most of the 36 million infections worldwide we investigated the HIV-1C strains circulating in Ethiopia in a retrospective, cross-sectional study. Serum samples from HIV-1-positive individuals were collected in seven Ethiopian cities and towns. Nucleotide sequences of the gag, pol, and env genes were analyzed. We performed phylogenetic analysis by the neighbor-joining and maximum-likelihood methods with sequences from 30 isolates, and we determined recombination by the bootscanning method as implemented in the SIMPLOT program. Sequence analyses of a 2600-nucleotide fragment (including the gag gene, the protease, and the 5' half of reverse transcriptase of the pol gene) and the corresponding V1V2/C2V3 envelope regions confirmed that two distinct HIV-1C genotypes (C' and C") are cocirculating in Ethiopia, as shown previously by the analysis of the C2V3 envelope region. We have identified intrasubtype recombination between the two HIV-1C genotypes, C' and C", with 6 of the 30 (20%) analyzed viruses being recombinants. The C' sequences were phylogenetically linked to the fast spreading viruses in India and southern Africa. Furthermore, all the recombinant viruses shared the C' V1V3 region of the envelope, suggesting that the prevalence of viruses with the C' envelope is increasing compared to the C" envelope. The possibility that viruses with a C' envelope have a biological advantage over the viruses with a C" envelope should be further investigated in biological and epidemiological studies.

HIV type 1 C and C' subclusters based on long terminal repeat sequences in the Ethiopian type 1 subtype C epidemic.

While the Ethiopian HIV-1 epidemic is dominated by subtype C, two distinguishable cocirculating C genotypes have been identified based on sequences of the C2V3 envelope region. In this study we sequenced and analyzed the long terminal repeat (LTR) sequence from 22 Ethiopian HIV-1-positive individuals. The two phylogenetically distinguishable genotypes C (n = 13) and C' (n = 4) are separated by significant bootstrap values. Nucleotide differences between the two groups were identified in the NF-AT, TCF-1alpha, and SP1 transcription factor binding sites, whereas the NF-kappaB and NRE-core sequences were identical between the two groups. Five isolates that could not be classified C or C' were found to be recombinants within the LTR sequence upon boots can analysis. Comparison of all the LTR sequences with their corresponding C2V3 envelope sequence revealed four intersubtype C/C' recombinant isolates. Thus, the prevalence of C/C' recombinant viruses is well over 40%. Interestingly, the C2V3 envelope sequences of all recombinant viruses belonged to the genotype C', whereas every LTR sequence belonged to the genotype C. This result indicates that recombination between the two genotypes is unidirectional, possibly as the result of evolutionary pressure on the respective biological functions of the LTR promoter and the envelope protein.

Primary HIV-1 subtype C infection in Ethiopia.

Between 1997 and 2001, 1624 Ethiopian factory workers were enrolled in prospective HIV-1 cohorts in Ethiopia, at Akaki and Wonji towns. HIV-1 seroprevalence at intake was 11.8% (Akaki) and 7.1% (Wonji). HIV-1 incidence was .75 per 100 person-years (Akaki) and .35 per 100 person-years (Wonji). During follow up, CD4 T-cell counts remained significantly lower and CD8 T-cell counts significantly higher in Ethiopian seroconverters compared with Dutch seroconverters. Viral loads were lower in Ethiopian seroconverters versus Dutch seroconverters in the first months after seroconversion, subsequently increasing to similar levels. All 20 Ethiopian seroconverters were infected with HIV-1 subtype C (15 with sub-cluster C' and 5 with sub-cluster C). Viral loads were higher in sub-cluster C'-infected Ethiopian seroconverters. One subject demonstrated a window period of at least 204 days, combined with a high preseroconversion viral load and no decline of CD4 T cells over a follow-up period of at least 3 years.