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1.  Early HIV-1 diagnosis using in-house real time PCR amplification on dried blood spots for infants in remote and resource limited settings 
Background
In resource-limited settings, most perinatally HIV-1-infected infants do not receive timely antiretroviral therapy because early HIV-1 diagnosis is not available or affordable.
Objective
To assess the performance of a low cost in-house real-time PCR assay to detect HIV-1 DNA in infant dried blood spots (DBS).
Methods
1319 DBS collected throughout Thailand from non-breastfed infants born to HIV-1-infected mothers were shipped at room temperature to a central laboratory. In-house real-time DNA-PCR results were compared to Roche Amplicor® HIV-1 DNA test (Version 1.5) results. In addition, we verified the Roche test performance on DBS sampled from 1218 other infants using as reference HIV serology result at 18 months of age.
Results
Real-time DNA-PCR and Roche DNA-PCR results were 100% concordant. Compared to HIV-serology results, the Roche test sensitivity was 98.6% (95% CI: 92.6 to 100.0%) and its specificity at 4 months of age was 99.7% (95% CI: 99.2 to 99.9%).
Conclusions
In-house real-time PCR performed as well as the Roche test in detecting HIV-1 DNA on DBS in Thailand. Combined use of DBS and real-time PCR assays is a reliable and affordable tool to expand access to early HIV-1 diagnosis in remote and resource-limited settings, enabling timely treatment for HIV-1-infected infants.
doi:10.1097/QAI.0b013e31818e2531
PMCID: PMC3111749  PMID: 18989220
HIV-1 real-time DNA-PCR; Dried Blood Spots; early diagnosis; infant; access to treatment; remote and resource limited-settings
2.  Detection of HIV-1 DNA resistance mutations by a sensitive assay at initiation of antiretroviral therapy is associated with virologic failure 
Background
Antiretroviral therapy (ART) has become more available throughout the developing world during the past five years. The World Health Organization recommends nonnucleoside reverse transcriptase inhibitor-based regimens as initial ART. However, their efficacy may be compromised by resistance mutations selected by single-dose nevirapine (sdNVP) used to prevent mother-to-child-transmission of HIV-1 (PMTCT). There is no simple and efficient method to detect such mutations at initiation of ART.
Methods
181 women participating in a PMTCT clinical trial who started NVP-ART after they had received sdNVP or placebo were tested for nevirapine-resistance point-mutations (K103N, Y181C, and G190A) using 100 copies of HIV-1 DNA with a sensitive oligonucleotide ligation assay (OLA) able to detect mutants at low concentrations (≥5% of the viral population). Virologic failure was defined as plasma HIV-1 RNA confirmed >50 copies/mL between 6–18 months of NVP-ART.
Results
At initiation of NVP-ART, resistance mutations were identified in 26% of 148 participants given sdNVP (K103N-13%, Y181C-5%, G190A-19%; ≥2 mutations-10%) at a median 9.3 months after sdNVP. The risk of virologic failure was .62 (95% confidence interval (CI), 0.46–0.77) in women with ≥1 resistance mutation, compared to 0.25 (95% CI, 0.17–0.35) in those without detectable resistance mutations (P<.0001). Failure was independently associated with resistance, an interval of <6 months between sdNVP and NVP-ART initiation, and a viral load above the median at NVP-ART initiation.
Conclusions
Access to simple and inexpensive assays to detect low-concentrations of NVP-resistant HIV-1 DNA prior to the initiation of ART could help improve the outcome of first-line antiretroviral therapy.
doi:10.1086/652148
PMCID: PMC2856716  PMID: 20377404
HIV-1; resistance mutations; nevirapine; HAART; oligonucleotide ligation assay; developing countries

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