Dried blood spot specimens are a suitable alternative sample type for HIV-1 viral load measurement and drug resistance genotyping in patients receiving first-line antiretroviral therapy.

BACKGROUND: Antiretroviral therapy (ART) is being administered in developing nations at unprecedented numbers following the World Health Organization's (WHO) development of standardized first-line drug regimens. To ensure continued efficacy of these drug regimens, WHO recommends monitoring virological responses and development of human immunodeficiency virus (HIV) drug resistance (HIVDR) in HIV-infected patients in a prospective cohort. The current study compared dried fluid spot specimens with the reference standard plasma specimens as a practical tool for viral load (VL) and HIVDR genotyping in resource-limited settings. METHODS: Dried blood spot (DBS), dried plasma spot (DPS), and plasma specimens were collected from 173 -patients receiving ART at 2 hospital sites in Abuja, Nigeria. HIV-1 VL analysis was performed using NucliSENS EasyQ HIV-1 v1.1 RUO test kits. Genotyping of the HIV-1 pol gene was performed using a broadly sensitive in-house assay. RESULTS: Direct comparison of VL levels showed that DBS specimens, and not DPS specimens, gave results comparable to those of plasma specimens (P = .0619 and .0007, respectively); however, both DBS and DPS specimens had excellent correlation with plasma specimens in predicting virological failure (VL, ≥1000 copies/mL) in patients (κ = 0.78 and 0.83, respectively). Of the 18 specimens with a plasma VL ≥1000 copies/mL, HIVDR genotyping rates were 100% in DBS and 38.9% in DPS specimens, and DBS specimens identified 61 of 65 HIVDR mutations (93.8%) identified in plasma specimens. CONCLUSIONS: Our results indicate that DBS specimens could be used for surveys to monitor HIVDR prevention failure in resource-limited settings.

Virological response and HIV drug resistance 12 months after antiretroviral therapy initiation at 2 clinics in Nigeria.

This report describes a pilot study, conducted in Nigeria, of the World Health Organization protocol for monitoring human immunodeficiency virus (HIV) drug resistance (HIVDR) and associated program factors among patients receiving first-line antiretroviral therapy (ART). In 2008, 283 HIV-infected patients starting ART were consecutively enrolled at 2 ART clinics in Abuja. Twelve months after ART initiation, 62% were alive and on first-line ART, 3% had died, 1% had transferred out of the program, and 34% were lost to follow-up. Among patients on first-line ART at 12 months, 90% had viral suppression. However, in view of the high loss to follow-up rate (34%), strategies for patient retention and tracking are critical to minimize possible HIVDR and optimize treatment outcomes.

Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.

UNLABELLED: Commercially available HIV-1 drug resistance (HIVDR) genotyping assays are expensive and have limitations in detecting non-B subtypes and circulating recombinant forms that are co-circulating in resource-limited settings (RLS). This study aimed to optimize a low cost and broadly sensitive in-house assay in detecting HIVDR mutations in the protease (PR) and reverse transcriptase (RT) regions of pol gene. The overall plasma genotyping sensitivity was 95.8% (N = 96). Compared to the original in-house assay and two commercially available genotyping systems, TRUGENE® and ViroSeq®, the optimized in-house assay showed a nucleotide sequence concordance of 99.3%, 99.6% and 99.1%, respectively. The optimized in-house assay was more sensitive in detecting mixture bases than the original in-house (N = 87, P<0.001) and TRUGENE® and ViroSeq® assays. When the optimized in-house assay was applied to genotype samples collected for HIVDR surveys (N = 230), all 72 (100%) plasma and 69 (95.8%) of the matched dried blood spots (DBS) in the Vietnam transmitted HIVDR survey were genotyped and nucleotide sequence concordance was 98.8%; Testing of treatment-experienced patient plasmas with viral load (VL) ≥ and <3 log10 copies/ml from the Nigeria and Malawi surveys yielded 100% (N = 46) and 78.6% (N = 14) genotyping rates, respectively. Furthermore, all 18 matched DBS stored at room temperature from the Nigeria survey were genotyped. Phylogenetic analysis of the 236 sequences revealed that 43.6% were CRF01_AE, 25.9% subtype C, 13.1% CRF02_AG, 5.1% subtype G, 4.2% subtype B, 2.5% subtype A, 2.1% each subtype F and unclassifiable, 0.4% each CRF06_CPX, CRF07_BC and CRF09_CPX. CONCLUSIONS: The optimized in-house assay is broadly sensitive in genotyping HIV-1 group M viral strains and more sensitive than the original in-house, TRUGENE® and ViroSeq® in detecting mixed viral populations. The broad sensitivity and substantial reagent cost saving make this assay more accessible for RLS where HIVDR surveillance is recommended to minimize the development and transmission of HIVDR.