Evaluation of a low cost reverse transcriptase assay for plasma HIV-1 viral load monitoring.

We evaluated a low cost manual reverse transcriptase assay (ExaVir Load V.1 and V.2; Cavidi Tech AB) against commercially available HIV RNA assays that quantify viral load to assess its suitability for use in resource-constrained settings. Frozen plasma samples previously tested for RNA by RT-PCR (Roche Diagnostics) and bDNA (Bayer Diagnostics) were retested for RT activity. Text sequence obtained from HIV genotype analysis was submitted to the Stanford HIV Resistance Database V.3.9 and were examined for resistant virus. Detectable RT was present in 98% of samples (V.1; n=127) and in 95% of samples (V.2; n=69) with RNA >10,000 and >1,000 copies/ml respectively. Positive association was found between the log10 RNA copies/ml and log10 RT copies/ml equivalents variables using Pearson's correlation (V.1: r=0.89, n=189; V.2: r=0.89, n=85). The RT activity over time closely followed the trend for RNA levels in samples from 10 HIV seropositive patients with progressive disease. A strong association between RT and RNA was also found with paired samples from 19 patients taken at initiation or change of antiretroviral therapy and again within 2 months. Current (n=40) or no (n=119) exposure to efavirenz therapy had no effect on RT assay performance despite efavirenz binding tightly to the RT enzyme. Samples that demonstrated resistance to the non-nucleoside RT inhibitors (n=112) had a decrease in RT of 0.20 log10 indicating a possible decrease in RT fitness. The RT assay showed good association with current molecular assays, and V.2 is sufficiently sensitive for monitoring HIV viral load in resource-constrained settings.

Radiosensitization of colon cancer cell lines by docetaxel: mechanisms of action.

The radiation-modifying action of docetaxel in experimental systems is well established. Docetaxel is also an increasingly important drug for the treatment of cancer in concurrent radiotherapy protocols. However, the mechanisms of docetaxel radiosensitization are not fully understood. We have investigated the magnitude and mechanisms of docetaxel radiosensitization in vitro in four human colorectal cancer cell lines (SW480, SW707, SW48, and HT29) with widely differing radiosensitivities. Cell survival curves were generated for a range of docetaxel concentrations (5-20 nM) alone and for X-rays (1-5 Gy) +/- 10 or 20 nM docetaxel (for 24 h before irradiation). Cell cycle distributions and apoptotic frequencies were measured during the treatments. Sensitivity to docetaxel alone was similar in all cell lines and could be attributed to massive induction of apoptosis (60-80% by 24 h). Radiosensitivity varied widely; the surviving fractions at 2 Gy in the most resistant (HT29) and most sensitive (SW28) lines were 0.81 and 0.13, respectively. Exposure to 10 nM docetaxel induced a progressive accumulation of SW480, SW707, and SW48 cells in G2/M. After 24 h, 55-70% of the cells were in G2/M. It is likely, therefore, that accumulation in this radiosensitive phase of the cell cycle contributes significantly to radiosensitization by the drug.

Immunological and virological failure after antiretroviral therapy is associated with enhanced peripheral and thymic pathogenicity.

T cell dynamics and viral genotype were studied in human immunodeficiency virus 1-infected individuals receiving antiretroviral therapy who were viremic and had either increasing (discordant immunological responders) or decreasing (nonresponders) CD4(+) T cell counts. A comparison was made with treated individuals who were not viremic and had increasing CD4(+) T cell counts (complete responders). Nonresponders had higher CD4(+) T cell proliferation (as assessed by Ki67 expression) and immune activation (as assessed by CD38 and human leukocyte antigen-DR expression), together with a reduction in T cell receptor excision circles, compared with discordant immunological responders and complete responders, which suggests that there is enhanced viral pathogenicity in both peripheral T cells and the thymus. Although there was a high prevalence of mutations in the protease and reverse transcriptase genes in discordant immunological responders, these changes were also observed in nonresponders.

Analysis of HIV-1 viral load in seminal plasma samples.

BACKGROUND: With HIV-1-infected individuals now facing the prospect of relatively long and healthy lives, many discordant couples (where the male is HIV-1 seropositive) are seeking to have children. To assist reducing the risks of heterosexual and subsequent vertical transmission in this situation, quantification of HIV-1 viral load in seminal plasma may be effective as one of several measures to reduce the risk of infecting the mother during insemination, potentially providing a better indication of infectivity than blood plasma analysis. OBJECTIVE(S): To modify existing molecular methods for the purpose of analysing HIV-1 viral load in seminal plasma. METHODS: Two commercial assays for HIV-1 RNA quantification were used to assess their sensitivity, specificity and precision for quantification of seminal plasma samples. Seminal plasma samples were prepared with an additional centrifugation step to aid removal of inhibitors to molecular assays. RESULTS: Seminal plasma samples exhibited specificity of >95%, equivalent to that reported by the manufacturers of the commercial assays. With additional centrifugation, complete inhibition of 2/19 (10%) seminal plasma samples was observed using the RT-PCR assay, and inhibition was not apparent in the bDNA assay. Quantification of HIV-1 RNA in seminal plasma samples in both assays was equivalent to that observed in plasma samples and did not appear to be affected by the additional centrifugation step. CONCLUSION: Minor modification of the RT-PCR assay procedure by additional centrifugation of seminal plasma improved the sensitivity of the assay. Inhibition was not apparent with the bDNA assay.