T-Lymphocyte Subsets and Prolonged Diarrhea in Young Children from. Guinea-
Bissau. Kare Molbak,1 Ida M. Lisse,2 and Peter Aaby1. In a community-based ...
American Journal of Epidemiology Copyright O 1996 by The Johns Hopkins University School of Hygiene and PubOc Health AD rights reserved
Vol. 143, No. 1
Printed in USA
T-Lymphocyte Subsets and Prolonged Diarrhea in Young Children from Guinea-Bissau
Kare Molbak,1 Ida M. Lisse,2 and Peter Aaby1 In a community-based prospective study of 380 children conducted between 1987 and 1990, the rate of diarrhea was significantly associated with percentage of CD8 T-lymphocytes and the CD4 : CD8 ratio. After adjustment for age and previous diarrhea, the relative incidence of diarrhea with a duration of &7 days was 2.10 (95% confidence interval (CO 1.15-3.85) in children with 20-29.9% CD8 T-cells and 3.41 (95% Cl 1.29-9.01) in children with 2:30% CD8 T-cells (in comparison with children who had less than 20% CD8 cells) (p for trend = 0.004). There was a nonsignificant tendency for rates of diarrhea of >7 days to decrease according to increasing proportions of CD4 cells (p = 0.194). The authors found no significant association between T-cell subsets and diarrhea which resolved within 6 days. The association between the incidence of prolonged diarrhea and T-cell subset proportions could not be explained as a confounding effect of low weight, breastfeeding, or previous infection with measles or Cryptosporidium. However, other prior infections or micronutrient deficiencies may explain the findings, and these host factors may be significant targets in intervention against diarrheal diseases. Am J Epidemiol 1996; 143:79-84. antigens, CD4; antigens, CD8; child; diarrhea; immunity, cellular; lymphocyte subsets; risk factors; Tlymphocytes
Diarrhea in early childhood remains a major cause of morbidity and death in developing countries (1). Most episodes of diarrhea resolve within a week and may be effectively treated by oral rehydration and continued feeding (2). However, in the case of more prolonged illness, oral rehydration has little impact, the risk of developing or worsening malnutrition increases, and there is a substantial risk of death (3). Hence, persistent diarrhea, defined as diarrhea that has continued for at least 2 weeks (4), has in many areas emerged as a more important cause of serious illness and death than acute diarrhea (2, 5, 6). A series of papers (7) and reviews (2-4, 8) have recently addressed the topic of persistent diarrhea, including host risk factors for persistent diarrhea. After initial observations in Bangladesh showed that cutaneous anergy was associated with increased diarrheal morbidity (9), two prospective studies evaluated this relation further. These investigations from Bangladesh (10, 11) and
Peru (12) corroborated the finding that the risk of developing diarrhea is inversely associated with delayed-type hypersensitivity reactions to standard skin-test antigens. As part of ongoing community studies of the longterm effects of measles, we conducted serologic surveys in which the T-cell subsets of children were assessed by examination of blood smears from fingerprick blood (13, 14). A number of these children were included in prospective diarrhea studies (5, 15-17), which permitted an investigation of the association between cellular immune function, as measured by CD4 and CD8 T-lymphocyte subsets, and the incidence of acute and prolonged diarrhea. MATERIALS AND METHODS Fieldwork
The study was conducted in a peri-urban district, Bandim II, in the capital of Guinea-Bissau. The district has a population of approximately 10,000 who reside in 650 houses. From 301 randomly sampled houses, all children born after June 1, 1984, were included in the morbidity study, which started in January 1987 and ended in April 1990. Children bom in or moving to these houses were also included. Children who moved within the area were followed up in their new homes. In addition, the study included all twins and children
Received for publication August 18,1994, and in final form February 13, 1995. Abbreviation: Cl, confidence interval. 1 Epidemiology Research Unit, Danish Epidemiology Science Centre, Statens SeruminstiUit, Copenhagen, Denmark. 2 Department of Pathology, Hvidovre University Hospital, Copenhagen, Denmark. Reprint requests to Dr. Kara Molbak, Epidemiology Research Unit, Danish Epidemiology Science Centre, Statens Seruminstitirt, Artillertvej 5, DK-2300 Copenhagen S, Denmark.
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Molbak et al.
below 3 years of age who had experienced measles, even if they did not reside in one of the 301 selected houses (14). Field-workers collected information weekly on episodes of diarrhea (as defined by the mother) and child feeding patterns. A sequence of days with diarrhea was regarded as one episode, provided that it was separated from previous episodes by at least 2 diarrhea-free days. Using portable spring scales and measuring boards, the field-workers measured each child's weight and length at intervals of approximately 3 months. Laboratory methods
Capillary blood was collected in a heparinized Microtainer (Becton Dickinson, Rutherford, New Jersey), and on the same day the white blood cell count was estimated by a single experienced laboratory technician using a hemocytometer. Blood films for the hematologic examination were prepared, air-dried, wrapped in aluminum foil, and stored with a desiccant at — 10°C until transfer to Denmark (13). Immunocytochemical labeling was carried out with monoclonal antibodies against CD4 and CD8 T-lymphocytes with the alkaline phosphatase technique (13). Beginning in 1988, the alkaline phosphatase was conjugated with the avidin-biotin complex (DAKO, Copenhagen, Denmark). Two hundred lymphocytes were counted for each of the immunolabeled slides. Differential counts were made visually, and the absolute values for each lymphoid subset were calculated. This method has been found to give T-cell subset values that are closely correlated with results obtained by flow cytometry (13). Data analysis
All children without diarrhea on the day of blood sampling who were included in both the serologic surveys and the diarrhea surveillance were included in the present analysis. Children with diarrhea were excluded, since they were not at risk of developing diarrhea. Thus, 380 children under 4 years of age were included, of which 317 (83.4 percent) were from the randomly selected group of 301 houses. Additionally, 50 children were included because they had had measles and 13 because they were bom a twin. There was no difference in diarrhea rates between these three groups, which allowed us to analyze the data combined. A possible confounding effect of measles was nevertheless explored in the multivariate analysis. The data were analyzed by Cox proportional hazards regression (18). In the models, "survival" time was number of days free of diarrhea from the day of
blood sampling to the day on which an episode of diarrhea was recorded (n = 305) or the child was lost to follow-up (n = 75). In the initial analysis, we categorized the results from the hematologic analysis into four groups by their quantiles and treated them as categorical variables, with the highest group serving as the referent group. In addition, age (35 months old. Table 1 shows the hematologic values and their association with diarrhea in the initial analysis, which adjusted for age and year of investigation. Total lymphocyte count, CD8 cell percentage, and CD4: CD8 ratio were associated with the rate of diarrhea. Since there was no natural trend in the association between lymphocyte count and risk of diarrhea, this finding was considered an artifact. In a following analysis, we examined possible confounders. Table 2 shows that children who had experienced diarrhea in the month before entry, had been weaned, or had had cryptosporidiosis before entry had significantly increased rates. Previous diarrhea was of biologic importance in our attempts to determine the nature and causal direction of the association between T-cell subset values and diarrhea. Hence, further analysis was limited to the 289 children for whom information could be obtained on both diarrhea in the month prior to blood sampling and T-cell subsets (table 3). After adjustment for age, year of study, and Am J Epidemiol
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Vol. 143, No. 1, 1996
T-Cell Subsets and Childhood Diantiea TABLE 1. Median hematologlc values, 25th and 75th percentiles, and p values for an association between Tlymphocyte subsets and rates of diarrhea in children from Guinea-Bissau, 1987-1990 No. Median of value cNWi'en White Wood cell countt Lymphocyte % Total no. of lymphocytest CD4 cell % Total no of CD4 cellst CD8 cell % Total no. of CDS cellst CD4:CD8 cell ratio
25th-75th percentiles
p value (or association with diantiea*
370 374
9.7 68
7.4-11.8 58-78
0.878 0.123
367 361
6.4 36
4.8-8.1 31-41
0.051 0.376
353 362
2.2 21
1.6-3.1 17-25
0.220 0.039
355 357
1.3 1.8
0.9-1.8 1.3-2.3
0.973 0.054
Likelihood ratio test from proportional hazards regression analysis, with adjustment for age and year of examination. Hematologic values were categorized at their quantiles. t No. of cells x 109/liter.
previous diarrhea, children with high proportions of CD8 cells had increased rates of diarrhea extending beyond 1 week, whereas there was no significant association between CD8 cell percentage and diarrhea of less than 7 days (p =. 0.306). Children with >30 percent CD8 cells had a relative rate of 4.52 (95 percent confidence interval (CI) 0.77-26.71) for diarrhea of at least 14 days' duration; the confidence interval was large because of a small number of persistent episodes (n = 12). Table 3 additionally shows a tendency for rates of diarrhea of £ 7 days to be lower in children with higher proportions of CD4 cells, although this relation was not statistically significant. TABLE 2.
The data were also analyzed in a Cox model with only diarrhea episodes of &7 days' duration as the endpoint. Censoring was performed after a follow-up of 180 days or at a period of ^ 7 days with missing information; episodes with a duration of less than 7 days were ignored. The results were similar: There was an increased rate of diarrhea by increasing CD8 cell percentage (p = 0.037) and a 1.99 relative rate (95 percent confidence interval 1.13-3.50; p = 0.028) in children with a CD4: CD8 ratio less than 1.0. Table 4 shows the results from a multivariate model that included diarrhea of at least 7 days as the response variable and adjusted for the confounding effect of previous diarrhea, weaning, previous infection with Cryptosporidium (15, 16), and low weight-for-age. There were no significant first-order interactions between CD8 cell percentage and the other covariates, including prior measles. The table shows that the relative incidence of diarrhea of ^ 7 days was 2.09 in children with 20-29.9 percent CD8 cells and 3.67 in children with &30 percent CD8 cells, in comparison with children who had less than 20 percent CD8 cells (p for trend = 0.004). DISCUSSION
Studies from Bangladesh (9-11) and Peru (12) indicate that cell-mediated immune deficiency is an independent risk factor for diarrhea. In the study by Koster et al. (9), anergy was associated with an 83 percent increased duration of diarrhea, and in the recent results reported by Baqui et al. (10, 11), the odds ratios were 1.55 (95 percent CI 1.26-1.92) for all types of diarrhea and 2.09 (95 percent CI 0.73-5.93) for persistent diarrhea in anergic children. The statis-
Age-adjusted rate ratios for diantiea among children from Guinea-Bissau, 1987-1990*
Determinant
Previous diarrhea}. Child weaned§ Previous cryptosporidiosis Welght-for-age z score i -21 Height-for-age z score
