Genetic attributes of blood-derived subtype-C HIV-1 tat and env in India and neurocognitive function.

Genetic elements in HIV-1 subtype B tat and env are associated with neurotoxicity yet less is known about other subtypes. HIV-1 subtype C tat and env sequences were analyzed to determine viral genetic elements associated with neurocognitive impairment in a large Indian cohort. Population-based sequences of HIV-1 tat (exon 1) and env (C2-V3 coding region) were generated from blood plasma of HIV-infected patients in Pune, India. Participants were classified as cognitively normal or impaired based on neuropsychological assessment. Tests for signature residues, positive and negative selection, entropy, and ambiguous bases were performed using tools available through Los Alamos National Laboratory (http://www.hiv.lanl.gov) and Datamonkey (http://www.datamonkey.org). HIV-1 subtype C tat and env sequences were analyzed for 155 and 160 participants, of which 34-36% were impaired. Two signature residues were unique to impaired participants in exon 1 of tat at codons 29 (arginine) and 68 (proline). Positive selection was noted at codon 29 among normal participants and at codon 68 in both groups. The signature at codon 29 was also a signature for low CD4+ (<200 cells/mm(3)) counts but remained associated with impairment after exclusion of those with low CD4+ counts. No unique genetic signatures were noted in env. In conclusion, two signature residues were identified in exon 1 of HIV-1 subtype C tat that were associated with neurocognitive impairment in India and not completely accounted for by HIV disease progression. These signatures support a linkage between diversifying selection in HIV-1 subtype C tat and neurocognitive impairment.

A combined screening platform for HIV treatment failure and resistance.

BACKGROUND: To develop a low cost method to screen for virologic failure of antiretroviral therapy (ART) and HIV-1 drug resistance, we performed a retrospective evaluation of a screening assay using serial dilutions of HIV-1 RNA-spiked blood plasma and samples from patients receiving >6 months of first-line ART. METHODS: Serial dilution testing was used to assess sensitivity of a simple PCR-based assay (targeted at ≥1,000 HIV RNA copies/mL). We created blood plasma minipools of five samples, extracted HIV RNA from the pools, PCR amplified the reverse transcriptase (RT) coding region of the HIV-1 pol gene from extracted RNA, sequenced PCR product of positive pools, and used sequences to determine drug resistance. Sensitivity, specificity, and predictive values were determined for different levels of virologic failure based on maximum viral loads of individual samples within a pool. RESULTS: Of 295 samples analyzed, 43 (15%) had virologic failure at ≥50 copies/mL (range 50-10,500 copies/mL, four at ≥1,000 copies/mL). The assay demonstrated 100% sensitivity to detect virus from these four samples, requiring only one round of PCR, and 56% and 89% sensitivity to detect samples with ≥50 and ≥500 copies/mL using two rounds. Amplified PCR products of all positive pools were successfully sequenced and 30% harbored ≥1 major resistance mutation. This method would have cost 10% of the combined costs of individual viral load and resistance testing. CONCLUSIONS: We present a novel method that can screen for both virologic failure of first-line ART and drug resistance. The method is much less expensive than current methods, which may offer sustainability in resource-limited settings.

Pooled nucleic acid testing to detect antiretroviral treatment failure in Mexico.

BACKGROUND: Similar to other resource-limited settings, cost restricts availability of viral load monitoring for most patients receiving antiretroviral therapy in Tijuana, Mexico. We evaluated if a pooling method could improve efficiency and reduce costs while maintaining accuracy. METHODS: We evaluated 700 patient blood plasma specimens at a reference laboratory in Tijuana for detectable viremia, individually and in 10 × 10 matrix pools. Thresholds for virologic failure were set at ≥500, ≥1000 and ≥1500 HIV RNA copies per milliliter. Detectable pools were deconvoluted using pre-set algorithms. Accuracy and efficiency of the pooling method were compared with individual testing. Quality assurance (QA) measures were evaluated after 1 matrix demonstrated low efficiency relative to individual testing. RESULTS: Twenty-two percent of the cohort had detectable HIV RNA (≥50 copies/mL). Pooling methods saved approximately one third of viral load assays over individual testing, while maintaining negative predictive values of >90% to detect samples with virologic failure (≥50 copies/mL). One matrix with low relative efficiency would have been detected earlier using the developed QA measures, but its exclusion would have only increased relative efficiency from 39% to 42%. These methods would have saved between $13,223 and $14,308 for monitoring this cohort. CONCLUSIONS: Despite limited clinical data, high prevalence of detectable viral loads and a contaminated matrix, pooling greatly improved efficiency of virologic monitoring while maintaining accuracy. By improving cost-effectiveness, these methods could provide sustainability of virologic monitoring in resource-limited settings, and incorporation of developed QA measures will most likely maximize pooling efficiency in future uses.