Alioune Dieye

Evaluation of a flow cytometry method for CD4 T cell enumeration based on volumetric primary CD4 gating using thermoresistant reagents

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.

Relationship of Binding of Immunoglobulin G to Plasmodium falciparum-Infected Erythrocytes with Parasite Endemicity and Antibody Responses to Conserved Antigen in Immune Individuals

ABSTRACT To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.

Evaluation of PIMATM CD4 System for Decentralization of Immunological Monitoring of HIV-Infected Patients in Senegal.

Background HIV infection is a concern in the army troupes because of the risk behaviour of the military population. In order to allow regular access to CD4+ T cell enumeration of military personnel as well as their dependents and civilians living with HIV, the Senegalese Army AIDS program is implementing PIMATM Alere technology in urban and semi-urban military medical centres. Validation such device is therefore required prior their wide implementation. The purpose of this study was to compare CD4+ T cell count measurements between the PIMATM Alere to the BD FACSCountTM. Methodology We selected a total of 200 subjects including 50 patients with CD4+ T-cells below 200/mm3, 50 between 200 and 350/mm3, 50 between 351 and 500/mm3, and 50 above 500/mm3. CD4+ T-cell count was performed on venous blood using the BD FASCountTM as reference method and the PIMATM Point of Care technology. The mean biases and limits of agreement between the PIMATM Alere and BD FACSCountTM were assessed with the Bland-Altman analysis, the linear regression performed using the Passing-Bablok regression analysis, and the percent similarity calculated using the Scott method. Results Our data have shown a mean difference of 22.3 cells/mm3 [95%CI:9.1–35.5] between the BD FACSCountTM and PIMATM Alere CD4 measurements. However, the mean differences of the two methods was not significantly different to zero when CD4+ T-cell count was below 350/mm3 (P = 0.76). The Passing-Bablok regression in categorized CD4 counts has also showed concordance correlation coefficient of 0.89 for CD4+ T cell counts below 350/mm3 whilst it was 0.5 when CD4 was above 350/mm3. Conclusion Overall, our data have shown that for low CD4 counts, the results from the PIMATM Alere provided accurate CD4+ T cell counts with a good agreement compared to the FACSCountTM.

[IgG responses to candidate malaria vaccine antigens in the urban area of Dakar (Senegal): evolution according to age and parasitemia in patients with mild symptoms].

Malaria remains a major problem in African countries despite substantial decreases in morbidity and mortality due to sustained control programs. Studies for the evaluation of qualitative or quantitative Ab responses to key targets of anti-plasmodium immunity were mostly done in rural endemic setting compared to urban area. In a cohort of 200 patients with mild malaria and living in Dakar, we analyze total and subclasses IgG responses to a panel of P. falciparum blood stage antigens: MSP1p19, MSP3, EB200, GST-5 and R23. A mean age of 15 yrs (4 to 56 yrs) and parasitemia between 0.1 to 17% were found. Levels of IgG anti-MSP3 were higher in patients with low parasitemia (≤1%) and appear negatively correlated to parasite densities (Rho =. 0.54; p= 0.021). This correlation is more significant in children (≤ 15 yrs). In addition, an increase of IgG responses against MSP1p19 is highly observed in adults having a parasitemia less than 1%. In those patients, we find that IgG1 subclasses were predominant (p <0.01). Our study shows an association between Ab responses and parasitemia. This association is dependant to IgG anti-MSP3 in children and IgG anti-MSP1p19 in adults living in urban area.

Relationship between Antibody Levels, IgG Binding to Plasmodium falciparum-Infected Erythrocytes, and Disease Outcome in Hospitalized Urban Malaria Patients from Dakar, Sénégal

Background. Management of clinical malaria requires the development of reliable diagnostic methods and efficient biomarkers for follow-up of patients. Protection is partly based on IgG responses to parasite antigens exposed at the surface of infected erythrocytes (iRBCs). These IgG responses appeared low during clinical infection, particularly in severe disease. Methods. We analyzed the IgG binding capacity to the surface of live erythrocytes infected by knob positive FCR3 strain. Sera from 69 cerebral malaria (CM) and 72 mild malaria (MM) cases were analyzed by ELISA for IgG responses to five antigens from iRBC and by flow cytometry for IgG binding as expressed in labeling index ratio (LIR). The relationship between IgG levels, LIR, parasitemia, age, and the clinical outcomes was evaluated. Results. We found a significant decrease of LIR in adult CM fatal cases compared to surviving patients (p = 0.019). In MM, LIRs were correlated to IgG anti-iRBC and anti-PfEMP3/5 levels. In CM, no correlation was found between LIR, IgG levels, and parasitemia. Conclusion. The IgG binding assay was able to discriminate outcome of cerebral malaria cases and it deserves further development as a potential functional-associated assay for symptomatic malaria analysis.

[Profiles of IgG responses against CSP, GLURP and LSA-3NR2 in urban malaria (Dakar): relations with haemoglobin levels and parasite densities].

Malaria remains a major health problem in sub- Saharan African countries despite substantial decreases in morbidity and mortality due to sustained control programs. Vaccines candidates were mainly tested in rural endemic setting; however increasing proportion of the population is living in urban area. Evaluation of the qualitative or quantitative immune responses to key targets of anti-Plasmodium immunity requires further investigation in urban area. In a cohort of 144 patients with mild malaria living in Dakar, we analyzed IgG responses against target antigens of P. falciparum: CSP, LSA-3NR2 and GLURP by ELISA. A mean age of 15 yrs (4-65 yrs) was found and patients were separated in 59 adults (<15yrs) and 85 children (≤15 yrs). Parasites densities (0,01-15%) did not differ between the two age groups. In contrast, haemoglobin levels appeared lower in children (4.5-16.6 g/dl) (p<0.01). For the immune results, the most recognized antigens were GLURP and CSP compared to LSA-3NR2. Levels of IgG against these antigens were significantly different between the two age groups and they were positively correlated (rho = 0.32; p<0.001). In addition, levels of IgG anti-GLURP were associated with low parasitemia (≤1%) and absence of anemia (≥11g/dl), particularly in adults (p<0.001). In a multiple regression analysis, no significant relationship was found between parasite densities and IgG responses against all the tested antigens. Our study shows the implication of IgG anti-GLURP in humoral immune response against the parasite. The present work contributes to determine IgG levels that can be used as relevant immunologic biomarkers in urban clinical malaria.

Evolution of autoantibodies profile in systemic lupus erythematosus according to age and clinical manifestations

Clinical features and auto-antibodies profile of 35 Senegalese patients diagnosed systemic lupus erythematosus (SLE) were analyzed after measurement of antinuclear antibodies (ANA) by IFI, detection of Abs anti-DNA native by ELISA and evaluation of antibodies anti-Sm, anti-RNP, anti-SSA anti-SSB, anti-CCP2, anti-J0, and anti-Scl70 levels by immunodot. Mean age of 33 yrs (18-50 yrs) and sex ratio (F/M) of 16 were found. The most frequent clinical features were rheumatic (88.7%) and cutaneous (79.4%) disorders. ANA and anti-DNAn Abs were detected in 85.7% and 62.5% of the patients respectively. Abs anti-RNP, anti-Sm, anti-SSA, anti-SSB and anti-CCP2 were detected in 30 to 70% of patients. In young patients, the levels of anti-DNAn and anti-Sm Abs were higher than in patients older than 40 yrs (P<0.05). In addition, associations of cutaneous and rheumatic symptoms were characterized by high levels of anti-DNAn, anti-SSA and anti-SSB Abs. Our study shows the interest of a measurement of anti-DNAn, anti-SSA and anti-SSB Abs during the follow of SLE patients particularly in those presenting both rheumatic and cutaneous symptoms.

[Decentralization of the immunological monitoring of people living with HIV/AIDS in a limiting setting with a low HIV seroprevalence: the experience of Senegal]

Our work aimed to propose a manual method of counting CD4 T lymphocytes which is an alternative magnetic immunoseparation followed by a reading with a fluorescence microscope as an alternative to the automated flow cytometry. This alternative technique is easier for use, less expensive and could answer the difficulties encountered for the monitoring CD4 T cells count in developing countries. The specific objectives were: 1) to train the technicians of the peripheral sites in order to make the numeration of the CD4 T lymphocytes more accessible at the peripheral level; 2) to equip the sites with necessary facilities for the T lymphocytes CD4 count; 3) to put in place a system of quality control permitting the reliability of the results. A hundred and fifty patients have been enrolled in three care services for people living with HIV/AIDS in Dakar. This population was constituted of 119 seropositive and 31 seronegative patients acting as control group to have some patients with high rates of T lymphocytes CD4. For the follow-up at peripheral level, the patients were constituted of the active line of the patients living with HIV/AIDS supported in the targeted sites. The measurements allowed studying concordances for different rates of lymphocytes: 0 to 199, 200 to 499 and over 500 cells by mm3. The results showed also a very good correlation (r = 0.97 or r = 0.98 according to the operator) between the two methods for CD4 rates inferior to 500 cells by mm3 among both the negative group and the HIV positive patients. We also discussed the profit of decentralization for the program and the patient, as well as the setting up of an external quality control to validate the alternative technique. According to the results, the Dynabeads is well correlated with the Facscount. It is a technique that can be used as an alternative in the zones with limited resources, low prevalence and for a small number of samples.