Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.

OBJECTIVES: To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries. MATERIAL AND METHOD: An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR. RESULTS: IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR. CONCLUSION: A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.

The development of HIV research laboratories in the Royal Thai Army Medical Department.

The development of HIV research laboratories at the Armed Forces Research Institute of Medical Sciences (AFRIMS), Royal Thai Army Medical Department in supporting of HIV-1 vaccine trials in Thailand was implemented in 1991. The collaboration between AFRIMS, Royal Thai Army Medical Department, and the US Military HIV Research Program with the ultimate goal to conduct the HIV-1 vaccine trial phase III. The HIV serology lab was set up for surveillance program in military recruits. Then, there was a need to strengthen more on the existing laboratories by training personnel to cope with the confidentiality of the lab results, specimen processing and data management which are critical. Later on, the necessary laboratory for measuring of vaccine immunogenicity was developed, such as lymphoproliferation assay. Additionally, a molecular biology lab was also developed. The HIV research laboratory management must include an ability to deal with some problems, such as late specimen receiving, fluctuating of power supply, technical staffs maintained. Good laboratory practices and safety must be strictly implemented. Communication network among facilities also played an important role in HIV laboratory strengthening at AFRIMS.

A new circulating recombinant form, CRF15_01B, reinforces the linkage between IDU and heterosexual epidemics in Thailand.

HIV-1 subtype B and CRF01_AE have been in circulation in Thailand and Southeast Asia for more than a decade. Initially separated by risk group, the two strains are increasingly intermixed, and two recombinant strains of essentially reciprocal structure have been recently reported. Here we identify additional CRF_01B recombinants and provide the evidence that HIV-1 strains now pass freely between the two high-risk populations. HIV isolates that showed discordance between CRF01_AE and subtype B in multi-region genotyping assays were selected for the study. They were drawn from 3 different cohorts in Thailand representing different risk behaviors and demographic characteristics: a drug user cohort in the north, a family planning clinic attendee cohort in the southeast, and a cohort study of the mucosal virology and immunology of HIV-1 infection in Thailand. The DNA from these isolates was PCR amplified to recover the full HIV-1 genome and subjected to sequencing and phylogenetic analysis. We establish that one particular CRF_01B recombinant, with the external envelope of subtype B and the rest of the genome from CRF01_AE, is circulating widely in Thailand. Termed CRF15_01B (also referred to as CRF15), the strain was primarily heterosexually transmitted, although injecting drug use (IDU) also played a role. In aggregate data from the studies, CRF15 constituted 1.7% of all HIV-1 infections (95% confidence interval 0.5-4.4%) and was dispersed widely in the country. The previously separate heterosexual and IDU epidemics have apparently been bridged by a new CRF. The entry of CRF15 into the mainstream of the epidemic signals new complexity in the long stable molecular picture in Thailand. These recombinants must be considered in ongoing or projected efficacy evaluations of HIV-1 vaccines and antiviral therapies.