Stability of HIV RNA in plasma specimens stored at different temperatures.

OBJECTIVES: In resource-limited countries, HIV-1 RNA quantification is usually performed in reference laboratories. Samples from remote areas are transported under suboptimal conditions. Here we evaluated HIV-1 RNA stability in plasma stored at different temperatures for 1 week. METHODS: Blood samples collected in ethylenediaminetetraacetic acid (EDTA) and processed within 6 h of collection were tested by HIV-1 RNA quantification using Roche Cobas Ampliprep-Cobas TaqMan (Roche Diagnostics). The results were compared with matched HIV-1 RNA concentrations determined from plasma stored for 1 week at 4, 22, 30 or 37 degrees C. RESULTS: A total of 51 samples were evaluated: 10 stored at 4 degrees C, 15 at 22 degrees C, 16 at 30 degrees C and 10 at 37 degrees C. Keeping plasma at 4, 22 or 30 degrees C for 1 week did not affect HIV RNA measurement. Compared with HIV-1 RNA concentrations determined from fresh plasma, the correlation was significant for each of the three temperatures with no RNA decay. In contrast, HIV-1 RNA levels decreased significantly when plasma was stored at 37 degrees C. The 10 samples submitted at this temperature showed a weaker correlation (rho=0.84; P=0.012) and a significantly reduced median HIV-1 RNA concentration (-0.92 log(10) HIV-1 RNA copies/mL; P=0.005). CONCLUSION: Plasma can be saved for up to 1 week at 30 degrees C before shipping to a reference laboratory for HIV-1 RNA quantification.

Viral efficacy maintained and safety parameters improved with a reduced dose of stavudine: a pilot study.

OBJECTIVES: Stavudine (d4T) is a potent but potentially toxic nucleoside reverse transcriptase inhibitor that is still widely used in developing countries. This study's aim was to determine the efficacy and safety profile of lower-dose d4T. METHODS: Multi-centre, open-label, single-arm, pilot, 48-week study in French patients weighing >60 kg with viral load <400 HIV-1 RNA copies/mL who were receiving d4T 40 mg twice daily and then switched to 30 mg twice daily. The primary endpoint was the proportion with plasma viral load <400 copies/mL at week 24. Secondary endpoints included the proportion with <50 copies/mL at weeks 24 and 48, changes in mitochondrial DNA, CD4 cell count and pharmacokinetics, and clinical and laboratory safety. RESULTS: Fifty-seven patients enrolled. Baseline CD4 count was 584 cells/microL; viral loads were <400 copies/mL and <50 copies/mL in 100% and 89%, respectively. Prior antiretroviral drug exposure was 6.9 years, d4T exposure was 6.3 years. Fifty-six out of 57 (98%) patients had viral load <400 copies/mL and 51 (89%) had viral load <50 copies/mL at week 24. Median CD4 count increased by 63 cells/microL at week 48 (P=0.006). At 48 weeks, total cholesterol decreased by 0.24 mmol (P=0.02), high-density lipoprotein cholesterol by 0.15 mmol (P=0.0001) and alanine aminotransferase by 5.74 mg/dL (P=0.01). Paired baseline DNA and week 24 RNA mutations were unchanged. Mitochondrial DNA (copies/cell) content increased from 672+/-254 to 682+/-269. d4T area under the plasma concentration time curve (AUC) decreased by 31% (P=0.003) and C(max) by 44% (P=0.004). Clinical and laboratory parameters improved or were unchanged. CONCLUSIONS: Reduced-dose d4T is effective with improved safety parameters.

Recovery of fat following a switch to nucleoside reverse transcriptase inhibitor-sparing therapy in patients with lipoatrophy: results from the 96-week randomized ANRS 108 NoNuke Trial.

OBJECTIVES: To evaluate the impact on peripheral fat tissue of a nucleoside reverse transcriptase inhibitor (NRTI)-sparing regimen in lipoatrophic HIV-1 infected patients. METHODS: This 96-week prospective, randomized study compared lipoatrophic patients switched to an NRTI-sparing regimen with patients remaining on an NRTI-containing regimen. The primary endpoint was the change in thigh subcutaneous fat tissue volume between baseline and week 48, as assessed by computerized tomography. RESULTS: One hundred patients were included, 50 in each arm. At baseline, patients had been on highly active antiretroviral therapy (HAART) for a median time of 6.6 years (4.9-9.7); 71% of the patients had received thymidine analogues [stavudine (37%), zidovudine (34%)]. The mean change in fat volume between baseline and week 48 significantly favoured the NRTI-sparing arm over the NRTI-maintaining arm in the intent-to-treat analysis, with a last-observation-carried-forward approach [+34 cm(3); 95% confidence interval (CI) 5-63 cm(3); P=0.002]. This was confirmed in the intent-to-treat analysis of available data, with a mean difference of +109 cm(3) (95% CI 34-185 cm(3)) at week 96 (n=53; P=0.001). This corresponded to increases of 12 and 30% in fat volume at weeks 48 and 96, respectively, in the NRTI-sparing arm. CONCLUSIONS: Switching from an effective NRTI-containing regimen to an NRTI-sparing regimen preserves immunovirological status and increases subcutaneous fat volume at weeks 48 and 96.

Evaluation of the use of dried spots and of different storage conditions of plasma for HIV-1 RNA quantification.

OBJECTIVES: The aim of the study was to evaluate the use of dried plasma spots to determine HIV-1 RNA viral loads. METHODS: The viral loads of 30 liquid plasma samples were compared with those of corresponding dried plasma spots on filter paper (DPS-FP) and in tubes (DPS-T), both of which were left for 7 days at 22 degrees C. Also, 10 liquid plasma samples with detectable viral load were stored at 4, 22 or 37 degrees C for 7 days and five further liquid plasma samples were air-dried for up to 54 h to assess the effects of temperature and the drying step on HIV-1 viral load. RESULTS: The viral loads of the 30 liquid plasma samples correlated significantly with those of the paired dried spots DPS-FP and DPS-T, but with median losses of 0.64 and 0.69 log(10) HIV-1 RNA copies/mL, respectively, and a limit of detection of 3 log(10) copies/mL. The 10 liquid plasma samples stored for 1 week at 37 degrees C showed a weaker correlation and had a significantly reduced median viral load (-0.92 log(10); P=0.005) when compared with the viral load of the matched plasma stored at - 80 degrees C. Most of the loss happened during the drying step. CONCLUSIONS: Reliable measurement of HIV-1 RNA viral load requires good plasma storage conditions. HIV RNA stability was affected by desiccation and 1 week of storage at 37 degrees C. However, our findings suggest that liquid plasma can be kept at 4 or 22 degrees C for a week with no effect on viral load.

Constant mitochondrial DNA levels in blood leukocytes of patients enrolled in a NRTI-free therapeutic trial (BIKS-2 study).

OBJECTIVES: Determine if a nucleoside reverse transcriptase inhibitors (NRTI)-free regimen affected mitochondrial DNA (mtDNA) levels in peripheral blood mononuclear cells (PBMCs) of patients enrolled in BIKS-2 trial. METHODS: Antiretroviral (ARV) naïve (N=13) and NRTI experienced (N=7) patients, received lopinavir/ritonavir, a boosted protease inhibitor, and efavirenz, a non-nucleoside reverse transcriptase inhibitor from Month (M) 0 to M12 (1-year BIKS trial) and from M12 to M36 (2-year BIKS-2 trial). MtDNA was quantified at M12, M24 and M36 via real-time PCR assay. RESULTS: From M12 to M36, the 20 patients have maintained undetectable plasma HIV-1 RNA, gained CD4 cells and had no side effects attributable to these drugs. Median mtDNA contents were constant: 478.6 at M12, 478.6 at M24 and 324.4 copies/cell at M36 (pM12-M36=0.5). Because M0 data is missing, these results were compared to those of two groups of age matched individuals: healthy donors and HIV-infected patients before and after exposure to NRTIs. Healthy donors have higher contents (871), followed by patients never treated (602), than by BIKS patients where 7 had toxic NRTIs (478.6) and at last by patients exposed for six months to the most toxic combination (ddI-d4T) (85 copies/cell). CONCLUSION: Lopinavir/ritonavir+efavirenz did not affect mtDNA contents in PBMCs.

Quantitative analysis of human mitochondrial DNA using a real-time PCR assay.

OBJECTIVES: Known for their ability to inhibit the human DNA polymerase-gamma, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-gamma ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria. METHODS: Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors. RESULTS: The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited. CONCLUSIONS: The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL.

The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L.

Several expression systems were used in studies aimed at characterizing the equine herpesvirus 1 (EHV-1) glycoprotein H and L homologues of HSV-1 (EHV-1 gH and gL) and the products were compared to the authentic proteins synthesized in virus infected cells. Using an in vitro transcription/translation system two gH species were detected (an unprocessed 89 kDa and a processed 116 kDa product). Three low molecular weight proteins were found in the case of gL (21.8 kDa, 22.9 kDa and 26.9 kDa) and these showed a slight reduction in mobility on the addition of microsomal membranes to the reactions. A gL fusion protein was produced in pGEX-2T, expression being confirmed by Western blotting using a gL-specific antiserum raised against a peptide incorporating the 13 carboxyl terminal amino acids of the protein. A gH specific peptide antiserum precipitated both gH and two smaller proteins from EHV-1 infected cells thought to be two forms of gL. Insect cells infected with gH or gL baculovirus recombinants were used to vaccinate C3H (H-2k) mice. Some protection against EHV-1 infection was conferred to the gH inoculated mice. The results will enable further studies on the importance of the gH and gL interaction in the pathogenesis of EHV-1 to be evaluated and their potential in contributing to a subunit vaccine to be assessed.