[show abstract][hide abstract] ABSTRACT: Laboratory follow-up of HIV patients in resource-limited settings requires appropriate instruments for CD4 T cell enumeration. In this study, we evaluated the application of a simplified, mobile and robust flow cytometry system, the Apogee Auto 40 analyzer (Auto40) using thermoresistant reagents, for CD4 T cell enumeration. We measured the absolute CD4 counts in fresh whole blood samples from 170 Senegalese subjects, including 129 HIV-positive (HIV+) patients and 41 HIV-negative (HIV-) controls. Based on volumetric primary CD4 gating, cells were stained with commercially available reagents (Easy MoAb CD4;Bio-D, Valenzano, Italy) and analyzed on the Auto40. The results were compared with those from the FACSCount system (Becton Dickinson, San Jose, USA). Repeatability analysis was performed on duplicate testing of 49 samples on both FACSCount and Auto40. The intra-run precision was measured by 10 replicates using 3 clinical blood samples with low, intermediate and high CD4 concentrations. The results from the two instruments were in good agreement. The percent similarity between the results of both instruments was 99%±relative standard deviation of 12.7%. The concordance correlation coefficient was 0.99. The absolute bias and limits of agreement (LOA) between the two instruments, calculated by Bland-Altman analysis, were clinically acceptable (bias: +4 cells/μl; LOA: -111 to +120 cells/μl). The clinical agreement between the two instruments at a cutoff of 200 CD4 cells/μl was 94%. The repeatability of measurements on the Auto40 was also similar to that observed with FACSCount system (bias +0.1 cells/μl, coefficient of variation 2.5% vs bias -1.1cells/μl, coefficient of variation 2.9% respectively). In conclusion, our results indicate that the Auto 40 system, using thermoresistant reagents, is suitable for CD4 T cell enumeration and will be a helpful tool to improve HIV laboratory monitoring in resource-limited settings.
Journal of immunological methods 07/2011; 372(1-2):7-13. · 2.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Immune activation has been suggested to increase susceptibility to human immunodeficiency virus type 1 (HIV-1) transmission, while at the same time it could be deemed essential for mounting an effective antiviral immune response. In this study, we compared levels of T cell activation between exposed seronegative (ESN) partners in HIV-1 discordant couples and HIV-unexposed control subjects in Dakar, Senegal. ESN subjects showed lower levels of CD38 expression on CD4(+) T cells than did control subjects. However, this was found to be associated with concurrent differences in the use of condoms: ESN subjects reported a higher degree of condom use than did control subjects, which correlated inversely with CD38 expression. In addition, we observed markedly higher levels of T cell activation in women compared with men, irrespective of sexual behavior. These findings question the relevance of low-level CD4(+) T cell activation in resistance to HIV-1 infection and underscore the need to take gender and sexual behavior characteristics of high-risk populations into account when analyzing correlates of protective immunity.
The Journal of Infectious Diseases 02/2010; 201(6):835-42. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: The measurement of CD4 counts and viral loads on a single instrument such as an affordable flow cytometer could considerably reduce the cost related to the follow-up of antiretroviral therapy in resource-poor settings. The aim of this study was to assess whether the HIV-1 p24 antigen could be measured using a microsphere-based flow cytometric (FC) assay and the experimental conditions necessary for processing plasma samples. A commercial anti-p24 antibody pair from Biomaric was used to develop a p24 microsphere immunoassay (MIA) using HIV culture supernatant as the source of antigen. The ultrasensitive Perkin Elmer enzyme immunoassay (EIA) served as a reference assay. Quantification of HIV p24 using the heat-mediated immune complex disruption format described for plasma samples was feasible using the Biomaric MIA and applicable to a broad range of HIV-1 Group M subtypes. The inclusion of a tyramide amplification step was successful and increased the fluorescence signal up to 3 logs as compared with the MIA without amplification. The analytical sensitivity of this ultrasensitive Biomaric assay reached 1 pg/mL, whereas the ultrasensitive Perkin Elmer EIA was sensitive to less than 0.17 pg/mL. Our data indicate, for the first time, that the principle of p24 detection using the heat-denatured ultrasensitive format can be applied to FC.
Cytometry Part B Clinical Cytometry 12/2008; 76(3):231-6. · 2.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alternative, affordable, and simple assays to monitor antiretroviral therapy (ART) in resource-poor settings are needed. We have evaluated and compared a heat-denatured (HD) HIV p24 amplified enzyme-linked immunosorbent assay from Perkin-Elmer and CD38CD8 T-cell levels, determined by flow cytometry, for their capacity to predict viral load (VL) in HIV-1-infected patients from Senegal. Median fluorescence intensity (MFI) of CD38 expression on memory (CD45RO) CD8 T cells correlated better with RNA VL than HD p24 antigenemia (R = 0.576, P < 0.0001 vs R = 0.548, P < 0.0001). MFI of CD38 expression on memory CD8 T cells could predict detectable RNA VL (VL = 2.6 log10) with a sensitivity of 87% and a specificity of 74%. A comparable sensitivity (89%) could be reached for HD p24 assay, but only to predict RNA VL of more than 5 logs, which might lead to unacceptable delays in clinical decision making. The clinical use of the HD p24 assay to monitor ART in Senegal would require more comparative data about the kinetics of p24 antigen and HIV RNA in peripheral blood as well as further evaluation regarding its sensitivity toward subtype A and CRF02. MFI of CD38 expression on memory CD8 T cells appeared to be a better alternative to monitor ART in HIV-infected patients from Senegal.
[show abstract][hide abstract] ABSTRACT: 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.