Improved Sensitivity of a Dual-Target HIV-1 Qualitative Test for Plasma and Dried Blood Spots.

The use of nucleic acid detection for HIV type 1 (HIV-1) detection is strongly recommended in infants <18 months of age, in whom serology is unreliable. This study evaluated the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test v2.0 (TaqMan HIV-1 Qual Test, v2.0), a dual-target total nucleic acid real-time PCR assay. The limit of detection (LOD) of the new test in plasma and dried blood spots (DBS) was determined with the 2nd International HIV-1 RNA WHO standard. The specificity of the assay was tested with EDTA plasma (n = 1,301) and DBS from HIV-negative adults (n = 1,000). The sensitivity was determined using HIV-1-positive samples (n = 169 adult EDTA plasma, n = 172 adult DBS, and n = 100 infant DBS) that included group M, subtypes A to H, CRF01_AE, CRF02_AG, and groups O and N. All positive specimens and a subset of the negative specimens were also tested with the Abbott RealTime HIV-1 Qual assay (RealTime). The LOD of the TaqMan assay was 20 copies/ml in plasma and 300 copies/ml in DBS, with specificities of 99.8% in plasma and 99.9% in DBS. The TaqMan assay results were 100% concordant with RealTime results in EDTA plasma samples and in 100 HIV-1-negative adult DBS. Among 172 HIV-1-positive DBS from adults, the TaqMan assay showed positive results for all DBS while RealTime missed five DBS with low target concentrations. Infant DBS results were 100% concordant. The improved sensitivity of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Qualitative Test, v2.0, compared to current commercially available assays may enable earlier diagnosis and treatment in adults and infants. The dual-target test may ensure HIV-1 detection even if a mutation is present in one of the two target regions. The DBS sample matrix facilitates virological testing in remote areas.

The reversibility of dissimilatory sulphate reduction and the cell-internal multi-step reduction of sulphite to sulphide: insights from the oxygen isotope composition of sulphate.

Dissimilatory sulphate reduction (DSR) leads to an overprint of the oxygen isotope composition of sulphate by the oxygen isotope composition of water. This overprint is assumed to occur via cell-internally formed sulphuroxy intermediates in the sulphate reduction pathway. Unlike sulphate, the sulphuroxy intermediates can readily exchange oxygen isotopes with water. Subsequent to the oxygen isotope exchange, these intermediates, e.g. sulphite, are re-oxidised by reversible enzymatic reactions to sulphate, thereby incorporating the oxygen used for the re-oxidation of the sulphur intermediates. Consequently, the rate and expression of DSR-mediated oxygen isotope exchange between sulphate and water depend not only on the oxygen isotope exchange between sulphuroxy intermediates and water, but also on cell-internal forward and backward reactions. The latter are the very same processes that control the extent of sulphur isotope fractionation expressed by DSR. Recently, the measurement of multiple sulphur isotope fractionation has successfully been applied to obtain information on the reversibility of individual enzymatically catalysed steps in DSR. Similarly, the oxygen isotope signature of sulphate has the potential to reveal complementary information on the reversibility of DSR. The aim of this work is to assess this potential. We derived a mathematical model that links sulphur and oxygen isotope effects by DSR, assuming that oxygen isotope effects observed in the oxygen isotopic composition of ambient sulphate are controlled by the oxygen isotope exchange between sulphite and water and the successive cell-internal oxidation of sulphite back to sulphate. Our model predicts rapid DSR-mediated oxygen isotope exchange for cases where the sulphur isotope fractionation is large and slow exchange for cases where the sulphur isotope fractionation is small. Our model also demonstrates that different DSR-mediated oxygen isotope equilibrium values are observed, depending on the importance of oxygen isotope exchange between sulphite and water relative to the re-oxidation of sulphite. Comparison of model results to experimental data further leads to the conclusion that sulphur isotope fractionation in the reduction of sulphite to sulphide is not a single-step process.