ABSTRACT
In Thailand, the cost of antiretrovirals has recently been reduced more than 10 fold. Likewise strategies for a cost reduction in laboratory monitoring are warranted. This study was designed to explore if the most expensive reagent in flow cytometry based CD4+ cell monitoring, the CD4+/CD8+ monoclonal antibodies, can be reduced without a loss of accuracy. Blood samples from 55 HIV seronegative (HIV-) and 76 HIV+ subjects were analyzed for %CD4+ and %CD8+ T cells using a two color monoclonal antibody panel (BD Biosciences, CA, USA) with 3 different amounts of the recommended reagents for staining: 1) standard, 2) half, and 3) one-fourth. A significant Spearman correlation of 0.987 was shown for the % CD4+ T cell test results for one half as well as one-fourth of the recommended amount compared to the standard staining according to the manufacturer's instruction (p < 0.0001). For the % CD8+ T cell test results, the correlation between the standard and the half or one-fourth reduced staining was 0.972 (p < 0.0001). Bland-Altman analysis showed no significant bias between the results from one half or one-fourth of the recommended amount versus the standard. The sensitivity and specificity of the two methods at the CD4+ T cell count cut-off of 200 cells/microl were 93% and 100%; and 96% and 99%, respectively. Our study indicates that a reduction of the reagents to half or one-fourth of the amount recommended by the manufacturer was still able to generate reliable results for CD4+ and CD8+ T cell counts. Such an approach will significantly reduce the cost of CD4+ monitoring for resource limited settings where a flow cytometer is available.
Subject(s)
CD4 Lymphocyte Count/economics , Cost Savings , Flow Cytometry/economics , HIV Infections/immunology , Humans , Sensitivity and Specificity , ThailandABSTRACT
We report a 7-year HIV-1 clade A/E-infected child untreated with antiretroviral therapy who had positive HIV antibody testing but undetectable plasma HIV-1 RNA by Roche Amplicor version 1.5 and bDNA version 3.0. DNA PCR was positive by methods using gag/pol primers but not env/pol primers. The patient had strong HIV-1-specific cytotoxic T lymphocyte responses, which likely contributed to her low viral burden and undetectable plasma HIV-1 RNA.
Subject(s)
Anti-Retroviral Agents , Blotting, Western , Child , DNA Primers , DNA, Viral/blood , Enzyme-Linked Immunosorbent Assay , Female , Fusion Proteins, gag-pol/blood , HIV Infections/blood , HIV-1/genetics , Humans , Interferon-gamma/blood , Polymerase Chain Reaction , RNA, Viral/blood , Viral LoadABSTRACT
DNA immunization represents one of the promising HIV-1 vaccine approaches. To overcome the obstacle of genetic variation, we used the last common ancestor (LCA) or "center-of-the-tree" approach to study a DNA fragment of the HIV-1 envelope surrounding the V3 region. A humanized codon of the 297-bp consensus ancestral sequence of the HIV-1 envelope (codons 291-391) was derived from the 80 most recent HIV-1 isolates from the 8 circulating HIV-1 subtypes worldwide. This 297-bp humanized "multi-clade" V3 DNA was amplified by a PCR-based technique. The PCR product was well expressed in vitro whereas the corresponding non-humanized V3 DNA (subtype A/E) could not be expressed. However, both V3 DNA constructs as well as the full-length HIV-1 envelope construct (A/E) were found to be immunogenic in mice by the footpad-swelling assay. Moreover, intracellular and extracellular interferon-gamma could be detected upon in vitro stimulation of spleen cells although the response was relatively weak. Further improvement of our humanized V3 DNA is needed.