Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays.

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.

Absolute CD4 T-cell counting in resource-poor settings: direct volumetric measurements versus bead-based clinical flow cytometry instruments.

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in "Le Dantec" Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV- subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.

National external quality assessment scheme for lymphocyte immunophenotyping in Belgium.

In 2000, the Belgian Scientific Institute of Public Health introduced a voluntary external quality assessment scheme for lymphocyte immunophenotyping. This paper provides an analysis of the first six surveys. Specimens consisted of fresh EDTA-anticoagulated whole blood and were sent by overnight mail. The 41 participants were surveyed for methodology and were asked to report white blood cell count, percentage of lymphocytes, and percentages and absolute numbers of CD3+, CD4+, CD8+, and CD19+ cells. Median intralaboratory coefficients of variation were 1.0, 1.3, 1.7, and 3.2% for CD3+, CD4+, CD8+, and CD19+ cell percentages, respectively. Interlaboratory variability was consistently lower than 6.5% for CD3+ and CD4+CD3+ cell percentages, and lower than 9.5% for CD8+CD3+ cell percentages. Median coefficients of variation for the absolute values were higher, ranging from 10.1% for CD4+CD3+ cells to 16.5% for CD19+ cells. The percentage of CD4+CD3+ and CD8+ CD3+ cells was in several samples significantly lower than the percentage of total CD4+ and CD8+ cells. The number of laboratories measuring total CD4+ and CD8+ cells decreased by 30% during the programme. Between-laboratory variability remained stable over time. Analysis of individual laboratory performance indicated that some laboratories markedly improved their results.