T Lymphocyte Subset Values Among Healthy Cameroonians

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To the Editor: In human immunodeficiency virus (HIV) infected individuals, progressive clinical and immunological deterioration is associated with CD4+ T-lymphocyte decline (1,2). Abrupt decline in CD4+ cell counts to very low levels with an inverted CD4/CD8 ratio were shown to be predictors of AIDS (3). CD4+ T-lymphocyte counts are currently one of the best parameters for monitoring disease progression among HIV-infected individuals (4); they are also used to stratify HIV-1 positive subjects into various treatment protocols and vaccine trials. In many developed countries, CD4+ cell counts have been documented among healthy HIV-negative adults to serve as reference values in determining the level that is indicative of AIDS in HIV-positive subjects (5,6). Similar information is currently lacking from most developing countries. The objective of our study was to establish baseline means of the CD4+, CD8+, CD3+ T lymphocyte and the CD4/CD8 ratio among healthy HIV-seronegative Cameroonian adults living in Yaoundé (the administrative headquarters). This was carried out from June to November 1995, and the values obtained were compared with those of AIDS patients referred to us for routine CD4+ cell counts. The study group consisted of randomly selected visitors and guardians of patients admitted in the four largest hospitals in Yaoundé. A verbal informed consent was obtained from all subjects, followed by completion of a questionnaire and a thorough physical examination. Subjects were considered ineligible for the study if they had taken any prescribed medication other than oral contraceptives 48 h before enrollment, or had had any acute illness, malignancy, or hospitalization within 12 months of enrollment. Subjects vaccinated within 12 months of study entry, smokers, and pregnant women were also excluded from the study. Five milliliters of whole blood was anonymously collected in K3 (liquid) EDTA Vacutainer tubes between 7:30 and 9:30 a.m. local time and immediately taken to the central laboratory, where it was analyzed within 6 h. The CD4+, CD8+, T-lymphocyte absolute counts, CD3+ T-lymphocyte total count, as well as the CD4/CD8 ratio were obtained by a FACScount cytometer (Becton Dickinson, San Jose, CA, U.S.A.) according to the manufacturer's instructions. The leukocyte and the differential counts were determined using standard automated techniques (Coulter Counter S plus Junior, Margency, France). HIV antibody status was assessed by enzyme-linked immunosorbent assay (ELISA) (Enzygnost Anti-HIV1/2 plus, Behringwerke Marburg, Germany); ELISA-reactive specimens were confirmed using HIV-1 group M Western blot (Novapath HIV-1 Immunoblot, Biorad, Munich, Germany) and an “inhouse” HIV-1 group O Western blot (Prof. Gurtler, Max Von Pettenkofer Institut, Munich, Germany). HIV-confirmed individuals were excluded. Descriptive statistics (means, standard deviation, etc.), χ2, and Z test were used. Of the 868 individuals contacted, 348 (40%) refused to be enrolled in the study, the major objection being the HIV testing. After completion of questionnaires and physical examination, 307 (35%) were excluded. Of the 307 individuals excluded, 12% were smokers, 4% were pregnant women, 25% were taking prescribed medication, 49% had had an acute illness (malaria, typhoid fever, etc.) or hospitalization within 12 months of enrollment and 10% had one or more of the following symptoms: fever anemia, diarrhoea, hypertension, persistent lymph nodes, and abscess. Blood was collected from 213 subjects and after HIV testing, 10 (4.7%) were found to be HIV positive. The final study group of 203 healthy HIV-seronegative subjects consisted of 142 men (70%) and 61 women (30%) with a mean age of 28 years (SD: ±7). The control group was made of 38 AIDS patients (1 in stage III and 37 in stage IV according to the CDC classification). Table 1 shows the mean values of CD4+, CD8+, CD3+ T lymphocytes, absolute number of lymphocytes, and the CD4/CD8 ratio among healthy HIV-negative Cameroonians. These values appeared to be higher than those described among Whites (5) being respectively (per μl): 830 (p < 0.0001); 560 (p = 0.0027); 1,450 (p < 0.0001); 1,900 (p < 0.0001) and 1.51 (p = 0.007). The mean values (per μl) for CD4+ T cells were significantly greater among females than among males (1,048 versus 951 p = 0.035); no age variation was found. These findings are consistent with other reports (6-8). The analysis of CD8+ T cells (per μl) showed no difference between sexes (606 among women versus 625 among men, p = 0.61) and age (χ2 = 5.3, p = 0.81). No difference was observed with the distribution of the CD3+ T cells among women (1,729 per μl) and men (1,666 per μl) (p = 0.36). With the CD4/CD8 ratio, a significant difference was found between gender (1.87 in women versus 1.63 in men p = 0.006); no correlation was found with age (χ2 = 13.3, p = 0.15). Seven of our 203 HIV-negative subjects (3.4%) had a CD4+ T lymphopenia (<400 per μl) (4 of 7 without having an inverted CD4/CD8 ratio); a similar value (2.8%) was reported among healthy individuals (5). Factors such as time of sampling, seasonal and day-to-day variation (9), as well as heterogeneity of CD4 epitopes to some monoclonal antibodies among different ethnic groups could account for CD4+ T-lymphopenia among healthy individuals. An inverted CD4/CD8 ratio (<1) was found among 18 of 203 HIV-negative subjects (9%); similar results ranging from 10 to 27% were reported in HIV-negative controls (5,6). When CD4+ T lymphopenia and inverted CD4/CD8 ratio were associated, only 3 of 203 (1.4%) exhibited abnormalities in both parameters. Among AIDS patients, infection with HIV-1 group M was predominant (37 of 38), with only 1 case of HIV-1 group O. Comparison of HIV-negative subjects and AIDS patients revealed significant differences among CD4+ (per μl) (980 versus 190, p < 0.00001), CD8+ (619 versus 730, p < 0.00001), CD3+ (1,685 versus 1,070 p < 0.00001) cell counts and the CD4/CD8 ratio (1.7 versus 0.25 p < 0.00001). Twelve of 38 (31%) AIDS patients had CD4+ T-cell counts (per μl) ranging from 201 to 540; this did not meet the proposed Centers for Disease Control definition for AIDS based on CD4+ T-cell counts ≤200 per μl (10). In our study, we have reported the distribution of lymphocyte populations bearing CD4, CD8, and CD3 antigens as well as the CD4/CD8 ratio among healthy HIV-negative Cameroonians living in Yaoundé; mean values were compared with those of HIV-positive controls. They provide baseline values for comparing results with those of different countries in following up HIV-positive individuals. Our results are preliminary, and further investigations involving a larger sample size should be carried out nationwide. However, our findings differ from those reported for populations outside Africa. Acknowledgment: We thank Dr. B. Schmidt-Ehry, GTZ Cameroon, for his constant support, and Dr. J. Shang, St. Thomas Hospital, London, England, for proofreading and advice. This work was made possible with the support of Boehringer Mannheim GmbH, Germany. L. Zekeng; A. Sadjo; J. Meli; L. Kaptué Haematology and Immunology Laboratory; University Teaching Hospital; Yaoundé, Cameroon E. Mpoudi Ngole Military Hospital; Yaoundé, Cameroon G. Hess; R. Babiel Boehringer Mannheim GmbH; Mannheim, Germany

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