1 Epidemiology Branch, National Institute of Child Health and Human Development, Bethesda, MD.
2 US Naval Medical Research Unit-3, Cairo, Egypt.
3 International Vaccine Institute, Seoul, South Korea.
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ABSTRACT |
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Campylobacter; cohort studies; convalescence; diarrhea
Abbreviations: CI, confidence interval; OR, odds ratio; RR, relative rate
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INTRODUCTION |
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Poor hygiene and suboptimal water sanitation are often linked to childhood diarrheal illness in less developed settings. Environmental conditions generally vary with season and climate. Additionally, maternal hygiene behaviors relating to child-care practices, such as feeding, hand washing, and cleaning, are influenced by the age of the child and generally change as the child ages. These time-varying behaviors and changing environmental conditions influence the child's risk of diarrhea. Most of the past reports investigating the epidemiolology of Campylobacter were based on prevalence studies (1114
). Such studies do not take into consideration the influence of past exposure on current susceptibility and the seasonality of the disease. Another weakness in the literature on Campylobacter diarrhea is that the few published community-based cohort studies have used only the baseline characteristics of the population to identify risk factors for the disease rather than re-collecting data on the hygiene conditions in the households at regular intervals throughout the study (8
, 10
).
Between February 1995 and February 1998, we conducted a community-based longitudinal study of diarrhea in rural Egyptian children. Using this resource, the present study was undertaken to study the epidemiology of Campylobacter and to identify potential risk factors for Campylobacter diarrhea. In addition, we evaluated whether there is an association between excretion of Campylobacter and the occurrence of diarrheal symptoms (pathogenicity) and whether there is prolonged shedding of Campylobacter after a symptomatic infection.
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MATERIALS AND METHODS |
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Cohort assembly
After mapping and numbering the houses of the two villages, we conducted a door-to-door census in which demographic characteristics of the families and baseline socio-economic data were collected. Beginning in February 1995, children under the age of 24 months living in either of the two study villages were eligible for study enrollment. Thereafter, only new births were enrolled into the study until September 1997. Subjects were followed until they were aged 36 months or until February 1998, whichever came first. Written informed consent was obtained from the parent or guardian of each child before enrollment. Human subject guidelines of the US Departments of Defense and of Health and Human Services were followed throughout the study.
Epidemiologic surveillance
Beginning in February 1995, all study subjects were visited in their homes twice weekly by trained fieldworkers. At each visit, a history of gastrointestinal symptoms since the last scheduled visit was obtained. If loose or liquid stools were reported, a rectal swab and a stool specimen were obtained and a physician examined the child. For children aged under 1 year, a detailed dietary history with reference to breastfeeding and introduction of supplementary liquids and solids was obtained at each visit. If a visit was missed because of the absence of the child or the caretaker, a revisit was attempted the following day. The surveillance enabled monitoring of deaths and out-migrations, which were censoring events for follow-up. Of the scheduled twice-weekly visits, 95 percent were successfully completed, and rectal swab specimens were obtained from all children who reported having loose or liquid stools at the time of the visit.
Cross-sectional surveys of the cohort were conducted at 2-month intervals over the 3-year period. In these surveys dietary information was collected for all children under surveillance at that point of time. In addition, rectal swabs and stool specimens were collected from the children irrespective of their diarrheal histories. Of the scheduled survey visits, 99 percent were successfully completed, and rectal swabs were obtained from all completed interviews.
We conducted hygiene surveys every March and September, corresponding to periods just before the warm and cool seasons, respectively. During these surveys, detailed household information was collected from observations of toilet areas (including type of facility, presence of feces on the floor, and puddling of water in and around the facility) in addition to the cooking, eating, and sleeping areas of the houses. Additional information collected during the surveys included observations within the house relating to the presence of animals, whether feces were visible, storage conditions of food and water, and whether there were barriers to prevent animals from entering the house. Since hygiene surveys were not started until the second year of surveillance, the first year diarrheal surveillance was excluded from the analysis relating to the hygiene surveys. All the scheduled hygiene surveys were successfully completed.
Laboratory evaluation
After collection, rectal swabs were immediately placed in Cary-Blair medium and stored on ice packs along with the stool specimens and transported to the Abu Homos field laboratory where they were refrigerated. The specimens were then transported to Cairo twice each week to the US Naval Medical Research Unit-3 for microbiologic evaluation. Standard microbiologic methods were used to isolate Salmonella, Shigella, and Vibrionaceae (24). In addition, the swabs were plated on MacConkey's medium, and five lactose-positive colonies were evaluated for both heat-labile enterotoxin and heat-stable enterotoxin Escherichia coli, using GM1 ganglioside enzyme-linked immunosorbent assays (25
, 26
). For isolation of Campylobacter, fecal specimens were inoculated onto modified Skirrow's medium and incubated at 42°C in a microaerophilic environment (5 percent O2/10 percent CO2) for 48 hours (27
). Suspect colonies were evaluated for Gram's stain morphology, motility, oxidase and catalase reactivity, and sensitivity to nalidixic acid (28
). Campylobacter isolates were differentiated between Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) by hippurate hydrolysis (29
). Commercial enzyme-linked immunosorbent assay kits were used to detect rotavirus in the stool specimens (Rotaclone; Meridian Diagnostics, Inc., Cincinnati, Ohio).
Definition of events
A "diarrheal day" was defined as passage of three or more loose or liquid stools in any 24-hour period (in addition, for breastfed infants, the mother had to state that the stools were less formed or more liquid than usual) or having at least one loose or liquid stool with the presence of visible blood. A diarrheal episode was defined to begin on the first day of loose or liquid stools after at least 3 consecutive nondiarrheal days. The episode was defined as completed when there were 3 consecutive nondiarrheal days after a diarrheal day. An episode of diarrhea was classified as Campylobacter diarrhea, if Campylobacter was isolated at any time in the entire duration of the episode. Based on the clinical examination, dehydration was classified as "none," "some," or "severe" according to World Health Organization criteria (30). A child was considered to be breastfed if breast milk constituted any part of the child's diet.
Analytical methods
To determine incidence rates we divided the number of episodes by the total person-time at risk. To identify risk factors that were time dependent, we linked all the 2-month and hygiene surveys to the twice-weekly surveillance. The survey information was assumed to be time invariant for the interval between two surveys. Crude relative rates were computed as a ratio of the incidence rates in the presence and absence of the factor under consideration. Incidence rates were compared using the Mantel-Haenszel test statistic for density follow-up studies (31).
Because the follow-up visits for a child are not statistically independent entities, it is necessary to account for the correlation between repeated observations. Generalized estimating equations were used to adjust for nonindependence of the diarrheal episodes for a child (32). We fit Poisson regression models to obtain relative rates adjusted for time-varying covariates and other potential confounding variables. Because the risk of Campylobacter diarrhea increased with age during infancy and subsequently decreased in the second and third years of life, the confounding effect of age was adjusted using a linear spline with knots at 12 and 24 months of age. Follow-up was aggregated over 1-month periods for each child to fit these models. Adjusted relative rates of diarrheal incidence were obtained by exponentiation of the parameter estimates of the independent variables. Empirical standard error estimates from the generalized estimating equation models were used to calculate the 95 percent confidence intervals of the estimated relative rates.
Case-control studies
To evaluate pathogenicity, a case-control analysis was performed on a subset of the cohort to examine the association between the occurrence of diarrheal symptoms and excretion of Campylobacter. Because a previous infection with Campylobacter alters the risk of acquiring a subsequent infection with the organism, it was necessary to restrict this analysis to newborns enrolled within the first 28 days of birth. Cases were defined as study subjects with diarrhea detected during the twice-weekly surveillance. Controls were defined as children without diarrhea who had cultures performed on fecal samples obtained during the routine 2-month survey visits. Both cases and controls with a prior history of a Campylobacter infection were excluded (either as a case or a control). Because the controls had a single fecal specimen collected at each visit for microbiologic workup, to avoid a bias of selectively detecting Campylobacter more frequently among cases as compared with the controls, only the microbiologic result of the first fecal specimen from each episode was included in the analysis.
A second case-control analysis was conducted to determine whether Campylobacter diarrhea is associated with prolonged convalescent excretion of Campylobacter. To study convalescent excretion, follow-up of Campylobacter diarrheal episodes alone is not sufficient, because it is possible that persistent detection of Campylobacter in the stool may result from intercurrent asymptomatic reinfections with the organism. In this case-control analysis, cases and controls were selected from the 2-month surveys (i.e., both cases and controls were asymptomatic). Cases were children who had no diarrhea but excreted Campylobacter, and controls were children who had no diarrhea and were not excreting Campylobacter. We postulate that, if there is prolonged excretion of Campylobacter after an episode of Campylobacter diarrhea, more cases (asymptomatic and Campylobacter excretors) in the 2-month surveys will have a history of Campylobacter diarrhea compared with the controls (asymptomatic and not excreting Campylobacter). On the other hand, if there was no chronic excretion but significant intercurrent asymptomatic infections after episodes of Campylobacter diarrhea, the odds of having a history of Campylobacter diarrhea for controls would be expected to be the same as the odds of a positive history of Campylobacter diarrhea in cases. A recent history of Campylobacter diarrhea was defined as a diarrheal episode within the past 30 days in which Campylobacter was isolated. Duration since exposure is defined as the duration between the end of the Campylobacter diarrheal episode and the date when the cases and controls were visited. For multiple episodes of Campylobacter diarrhea within the past month, the most recent episode is used to classify the duration since exposure. To eliminate prevalent and incubating infections that could be the cause for the excretion of Campylobacter identified in the 2-month surveys, persons having diarrheal symptoms on the survey day or having a diarrheal episode that started within 3 days following the survey visit were excluded from both the cases and the controls.
For case-control analyses, odds ratios were calculated to assess the association between exposure and case-control status. Proportions were statistically compared using the chi-square test or Fisher's exact test when the data were sparsely distributed. To estimate adjusted odds ratios we used multiple logistic regression to control for the simultaneous confounding effect of several variables. Because the repeated visits for a child are not independent, we used generalized estimating equations to adjust for the correlation between repeated observations. To test whether pathogenicity or convalescent excretion of Campylobacter varied by age, we examined the statistical significance of an interaction term between exposure and age, entered into the model along with the main effects and confounding variables. Adjusted odd ratios were obtained by exponentiating the parameter estimate for exposure, and 95 percent confidence intervals were calculated from the standard error of the parameter estimate.
All statistical tests were interpreted in a two-tailed manner to estimate p values and confidence intervals. A p value of <0.05 was considered to be statistically significant.
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RESULTS |
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Risk factors
As shown in table 1, village 830 experienced a higher incidence of Campylobacter diarrhea compared with village 820 (relative rate (RR) = 1.29; 95 percent confidence interval (CI): 1.01, 1.67; p < 0.05), and the incidence rates between May and August were significantly higher than those of the remaining months (RR = 2.34; 95 percent CI: 1.90, 2.89; p < 0.001). Breastfeeding did not appear to alter the risk of Campylobacter diarrhea (RR = 0.87; 95 percent CI: 0.64, 1.20), but having a flush toilet in the house was associated with a lower risk of Campylobacter illness (RR = 0.38; 95 percent CI: 0.15, 0.96; p < 0.05). Mere ownership of livestock or fowl was not associated with an increased risk of Campylobacter diarrhea. The presence of animals in the cooking area was, however, associated with a higher risk of developing Campylobacter diarrhea (RR = 1.50; 95 percent CI: 1.02, 2.17; p < 0.05). Houses where uncovered garbage was present in the cooking areas also had higher rates of Campylobacter diarrhea (RR = 2.02; 95 percent CI: 1.27, 3.20; p < 0.01) (table 1).
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DISCUSSION |
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Similar to other studies from developing areas, our study observed an age-specific decline in the incidence of Campylobacter diarrhea, suggesting that there is an age-related acquisition of immunity (9, 14
, 19
, 23
, 33
). However, we have shown that first-time infections with Campylobacter are pathogenic regardless of the age of the child, suggesting that prior exposure and immunity influence the risk of Campylobacter diarrhea for a child. This association was consistent even when we excluded Campylobacter infections with bacterial copathogens from the cases and controls, suggesting that pathogenicity observed in older age children is not explained by a co-infection. The observation that Campylobacter remains highly pathogenic in the first 6 months of life even in those with a prior symptomatic infection suggests that early infections may not be immunogenic enough to confer protection against subsequent disease. This observation is corroborated by the finding that children aged under 6 months have very poor serologic responses to natural Campylobacter infection (33
). However, consistent with observations from most developing countries, our study found that infections beyond the first 6 months of life that occur subsequent to a symptomatic infection are not pathogenic. These findings have implications for the use of new Campylobacter vaccines in children from less developed settings where the risk of disease is greatest in early life. The vaccine will need to confer immunity in the very young in order to protect children during the period of greatest risk.
We found evidence suggesting that prolonged convalescent excretion of Campylobacter lasted for a month after the diarrheal episode. Our results are similar to findings in Thailand, but they differ from the shorter convalescent excretion periods noted in Mexico and Chile (9, 21
, 23
). Although our study was not designed to examine the role of prolonged convalescent Campylobacter excretion in secondary transmission, this is an important area for future investigation.
In vitro studies have demonstrated that C. coli are more readily phagocytosed and killed by peritoneal macrophages than C. jejuni (34). These findings have raised the possibility that infections due to C. coli are not as severe as those caused by C. jejuni. Compared with C. jejuni infections, C. coli infections have less often been found to be associated with bloody diarrhea and symptomatic disease (23
). In our study population, we found no difference in the proportion of severe disease in infections resulting from C. coli compared with C. jejuni. This observation remained consistent even when Campylobacter episodes with other copathogens were excluded from the analysis.
Studies that have shown Campylobacter diarrhea to be associated with the consumption of contaminated water or foods, ownership of livestock or poultry, or contact with animals have not factored in the time-dependent nature of these environmental conditions (8, 15
, 18
, 22
, 35
39
). After incorporating these changing conditions into our analyses, we found that the presence of animals and uncovered garbage in the cooking area is associated with an increased risk of Campylobacter diarrhea. In our observations, we did not record the species of animals in the house, so this increased risk could not be associated with specific types of animals. However, we know from the census that more than 80 percent of the enrolled children resided in houses that owned barnyard animals. An observational study conducted in a shantytown in Peru showed that toddlers frequently come into contact with poultry feces that lie within the homes and have an average of 3.9 feces-to-mouth episodes in a 12-hour period (40
). This observation, coupled with the fact that the feces from poultry when cultured yielded viable C. jejuni for up to 48 hours after deposition, suggests that there is a high risk of Campylobacter transmission in environments where there may be frequent human-animal contact. Our findings support the suggestion that it is the presence of the animals themselves and the poor hygienic conditions within the house, as opposed to the mere ownership of animals, that place the child at an increased risk of Campylobacter disease.
In conclusion, the high burden of disease resulting from Campylobacter infection in this population highlights the need for improved methods for disease control. Improving hygiene-related behaviors and minimizing contact with animals may be recommended, and consideration must be given to the development of safe, effective candidate vaccines that can confer protection in early infancy.
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ACKNOWLEDGMENTS |
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The authors thank Sahar Abd El Samad, Manal El Sayed, and the staff of the Abu Homos Field Research Center for their contributions to field and laboratory work and Dr. Mahmoud Abu El Nasr and Dr. Badriya Z. Morsy of the Egyptian Ministry of Health and Population for their advice and support. In addition, the authors would like to thank Dr. Lawrence Moulton, Johns Hopkins University Bloomberg School of Public Health, for his comments and suggestions.
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NOTES |
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REFERENCES |
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