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    Frequency distribution of diarrheal episodes according to their duration in days in an urban slum of Dhaka, Bangladesh.

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    Seasonal variation of diarrheal diseases in preschool children in an urban slum of Dhaka, Bangladesh.

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EPIDEMIOLOGIC AND CLINICAL CHARACTERISTICS OF ACUTE DIARRHEA WITH EMPHASIS ON ENTAMOEBA HISTOLYTICA INFECTIONS IN PRESCHOOL CHILDREN IN AN URBAN SLUM OF DHAKA, BANGLADESH

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  • 1 Centre for Health and Population Research, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh; Department of Medicine, Unit of Infectious Disease, University of Vermont College of Medicine, Burlington, Vermont; Departments of Medicine, Microbiology and Pathology, University of Virginia, Charlottesville, Virginia; Department of International Health, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland

The epidemiology, clinical features, nutritional status, and causative agents of diarrhea were studied in 289 Bangladeshi children (147 boys and 142 girls) 2–5 years old. The use of improved diagnostic tests for amebiasis enabled for the first time analysis of the contribution of Entamoeba histolytica to total diarrheal illness in this community setting. The average incidence rate of diarrhea was 1.8/child-year, and the average number of diarrheal days was 3.7 days/child-year over an average observation period of 2.8 years/child. Seventy-five percent of the diarrheal episodes were ≤ 2 days in duration. Persistent diarrhea was relatively uncommon (0.2% of the children) and chronic diarrhea was observed in only one episode. Compared with malnourished and/or stunted children, better-nourished children experienced significantly fewer diarrheal episodes. The diarrheal incidence rate for children with blood group A was significantly less that that of the children with blood groups O and AB. The most frequent bacterial enteropathogens isolated from diarrheal stool specimens were enterotoxigenic Escherichia coli (9%) and Aeromonas species (9%), followed by Plesimonas shigelloides (4%) and Shigella flexneri (3.8%). Rotavirus was the most common viral agent isolated from diarrheal stool samples (5%). Giardia lamblia, Cryptosporidium parvum, and E. histolytica were identified in 11%, 8.4%, and 8%, respectively, of the diarrheal stool specimens. Dysentery was observed in 7.7% of all diarrheal episodes. The most common pathogens isolated from dysenteric stool were S. flexneri (11.6%), Aeromonas sp. (10%), E. histolytica (8.7%), Campylobacter jejunii (5.8%), P. shigelloides (4.3%), and A. caviae (4.3%). The overall incidence rate of E. histolytica-associated diarrhea was 0.08/child-year. Visible blood and hemoccult test-detected blood loss was found in 7% and 25%, respectively, of cases of E. histolytica-associated diarrhea. Children who had recovered from a diarrheal episode with E. histolytica, but not E. dispar, had half the chance of developing subsequent E. histolytica-associated diarrhea, consistent with the development of species-specific acquired immunity. In conclusion, the use of modern diagnostic tests demonstrated that E. histolytica contributed to overall morbidity from diarrheal illness. Understanding the etiology, frequency, and consequences of acute diarrhea in children from a developing country should aid in the design of interventions to improve child health.

INTRODUCTION

The rate of mortality from diarrheal diseases in the world has decreased, mainly because of better therapy and interventions that promote sanitary conditions and that educate inhabitants to encourage them to take part in primary health care activities.1,2 However, acute diarrheal diseases continue to be one of the major causes of morbidity and mortality in the developing world such as Bangladesh, where one in 10 children die before their fifth birthday.1,3 An epidemiologic study of an infectious disease in a community is an initial step toward the introduction of the proper interventions for controlling the disease because the features and the patterns of isolation of etiologic agents of the disease vary from place to place depending on the local meteorology, geography, and socioeconomic elements.4,5

Human infection with Entamoeba histolytica, known, as amebiasis, is prevalent worldwide and is common in children of the developing world. Entamoeba histolytica is associated with diarrhea/dysentery in endemic countries.6 Recently E. histolytica has been reclassified into pathogenic (E. histolytica sensu stricto) and nonpathogenic (E. dispar) species.7 The classic stool ova and parasite examination, whereby E. histolytica is identified by its appearance in trichrome- or iron hematoxylin-stained stool specimens, is insensitive and cannot differentiate E. histolytica from the nonpathogenic but identical-appearing parasite E. dispar. A few surveys in which the two species are distinguished have been carried out in endemic countries, and it has been found that the prevalence of E. histolytica infection varies greatly from place to place.8–11

Reliable epidemiologic data are essential to estimate the burden of disease due to E. histolytica and to formulate policy to control amebiasis. Amebiasis is endemic in Bangladesh. Case series of patients at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) Hospital in Dhaka show peak incidences of infection among children between two and 14 years of age and in adults > 40 years old.6 The field use of a rapid and specific antigen detection test for E. histolytica infection is providing new insights into amebiasis. The prevalence of asymptomatic infection with E. histolytica was unknown and until recently it was believed that most asymptomatic infections were due to the non-pathogen E. dispar. Currently, we are conducting a field study on amebiasis to understand the natural history of E. histolytica infection in an urban slum of Dhaka. As a part of this study, we are also conducting active diarrhea surveillance in a cohort of 2–5-year-old children at the time of enrollment. Here, we describe the epidemiology, clinical features, nutritional status, and causative agents of diarrhea, including E. histolytica, in these children with the expectation that this information may help in the ultimate control of diarrheal diseases.

MATERIALS AND METHODS

Study area and population.

The study was conducted between January 1999 and July 2002 in Mirpur, an urban slum in Dhaka as described elsewhere.12 Two hundred eighty-nine children (147 boys and 142 girls) 2–5 years old were enrolled. Half of these children had IgG antibodies against the E. histolytica galactose and N-acetyl-D-glactosamine (Gal/ GalNAc) lectin in their blood at the time of enrollment. The amebic Gal/GalNAc lectin mediates adherence to and contact-dependent cytolysis of human colonic epithelium. Serum IgG antibodies to the lectin have been detected in patients with amebiasis.8,9 All enrolled children and their family members received free primary health care services, including medications, from the project office in Mirpur. Informed consent was obtained from the parents or guardians and the human experimentation guidelines of the U.S. Department of Health and Human Services, the University of Virginia, the Johns Hopkins University Bloomberg School of Public Health, and the Center for Health and Population Research, ICDDR,B in Dhaka, Bangladesh were followed in conducting this research.

The parents and children were visited and interviewed every other day by health care workers for details about any diarrheal episodes of the child, as well as other related questions. Children with diarrhea were also detected through their parents contacting project personnel at the field clinic. When diarrheal disease was detected, the child was examined and treated with oral rehydration and/or antibiotics as appropriate and a stool sample was collected for detailed investigation of enteropathogens including E. histolytica. Stool specimens were also collected monthly from every child for detection of E. histolytica infection by antigen capture and culture.

Anthropometry.

Anthropometric measurements were taken by trained research assistants at the time of enrollment and then every four months. Each child was weighed in light clothes with an electronic weighing scale. The standing heights of children were measured to the nearest 0.1 cm using a locally constructed height stick. Nutritional status was assessed by comparing the weight and height of the study children with those of a National Center for Health Statistics (Hyattsville, MD) reference population of the same age and sex with the Epi-Info 6 version 6.04 (Centers for Disease Control and Prevention, Atlanta, GA) computer program.13

Clinical definitions.

Diarrhea was defined as having three or more unformed stools in a 24 hour period. A diarrheal episode was defined as being separated from another episode by at least three diarrhea-free days. A new episode for asymptomatic E. histolytica infection was defined as being separated from another episode by at least two negative results in monthly non-diarrheal stools for E. histolytica antigens.12 The presence of blood in the stool was defined by the gross appearance of blood in the stools, as indicated by the mother or noted by study personnel. Diarrhea was further classified as dysenteric or non-dysenteric. Dysenteric diarrhea was defined by gross blood in the stools and/or microscopic stool examination showing red blood cells ≥ 1/high-power field. Diarrheal episodes were defined as acute (< 14 days), persistent (≥ 14 days but < 30 days) and chronic (≥ 30 days) according to duration. Episodes of E. histolytica-associated diarrhea or dysentery were defined as earlier in this report, but were accompanied by the isolation of E. histolytica from diarrheal or dysenteric stools taken at the time of illness. Asymptomatic E. histolytica infection was defined as having a positive stool antigen detection test result for E. histolytica in the absence of diarrheal illness. Fever was defined according to the mother’s assessment. The project physician assessed the degree of dehydration according to World Health Organization criteria.14 The severity of each diarrheal disease episode was calculated by using a numeric scoring system.15

Stool sampling.

Stool specimens were collected within 24 hours after the reporting of a diarrheal episode. Samples were transported to the ICDDR,B laboratory within six hours after collection. From January 1999 to December 2000, stool specimens were transported to the laboratory without transport media. From January 2001 onwards, Cary-Blair and buffer glycerol saline BGS media were used for transportation of the stool samples.

Stool microbiology.

Stool specimens were cultured within the same day of collection using standard methods.15 The stool samples were plated on MacConkey agar, Salmonella-Shigella agar (SSA), taurocholate-tellerite-gelatin agar (TTGA) and Campy-Brucella agar plate. The specimens were also enriched in selenite F broth and bile peptone broth and were subcultured onto SSA from the former and onto TTGA from the latter. MacConkey agar and SSA were used for isolation of Escherichia coli, Salmonella sp., and Shigella sp., TTGA was used for isolation of Vibrio cholerae, and Campy-Brucella agar plate was used for isolation of Campylobacter jejuni. All microbiologic media or their ingredients were either Difco Products (Becton Dickinson Microbiology Systems, Sparks, MD) or Baltimore Biological Laboratories products (Becton Dickinson and Company, Cockeysville, MD).

Three lactose-positive colonies were picked from the Mac-Conkey agar plates for identification of E. coli. Different categories of diarrheagenic E. coli: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli, enterohemorrhagic E. coli, and enteroinvasive E. coli were detected by a hybridization technique with specific DNA probes.16 The E. coli strains positive for hybridization with EPEC adherent factor and/or the E. coli attachment and effacement gene probe were identified as EPEC.17,18

Fecal specimens were also placed in phosphate-buffered saline and kept in the freezer for later identification of rotavirus, enteric adenovirus, and astrovirus by using commercially available enzyme-linked imunosorbent assays (IDEIA™Adenovirus, IDEIA™ Rotavirus, and IDEIA™ Astrovirus; Dako, Ely, United Kingdom).

Stool parasitology.

The TechLab (Blacksburg, VA) Entamoeba test (designed to detect but not differentiate E. histolytica and E. dispar antigen in stool specimens) and the TechLab E. histolytica II test (designed to detect specifically E. histolytica antigen in stool specimens) were performed on the stool specimens according to the manufacturer’s instructions.11 Cryptosporidium was also detected by an antigen detection test (Cryptosporidium TEST; Techlab) according to the manufacturer’s instructions. Stools were examined for ova and parasites by direct microscopy, also for the identification of Giardia lamblia, Ascaris lubricoides, Trichuris trichiura, Strongyloides stercoralis, Hymenolepsis nana, and Cyclospora cayetanensis.

Stool lactoferrin was detected by a latex agglutination assay (Techlab), and fecal occult blood was detected by a hemoccult test (Beckman Coulter, Inc., Palo Alto, CA). Blood typing was done for all 235 children who remained in the study at the beginning of the third year of follow-up. ABO and Rh blood typing was done by conventional techniques.19

Statistical methods.

Basic demographic information, surveillance data, and clinical and laboratory findings of each diarrheal episode for which stool sample was collected were stored in data files using Fox-Pro® (Microsoft, Redmond, WA). Categorical data were compared by chi-square analysis with Fisher’s exact test. The relationship between numeric and categorical data was analyzed by analysis of variance or a Kruskal-Wallis test. Spearman correlation was used for correlation analysis between numeric variables. Logistic regression was used to calculate adjusted odd ratios. The percentage protection against subsequent infection was calculated as (1 − odds ratio [OR]) × 100%. The statistical package SPSS version 10.01 (SPSS, Inc., Chicago, IL) was used for data analysis.

RESULTS

Socioeconomic characteristics.

A total of 289 children from 252 households were enrolled in the study; 221 were followed for 36 months. The average follow-up period was 1,036 days (2.8 years). Baseline characteristics of the study population are shown in Table 1. The average family size was six. The percent of mothers who never attended school was 64%. Seventy-two percent of the families had very low annual incomes (< 5,000 Taka [Tk]; 1 US$ = 58 Tk). Complete information regarding sanitation could be given by 240 households. The majority of the study population provided themselves with water from a municipal supply by using plastic pipes, 18% of which were near to latrines and 27% to drains. Only 33% had direct accessibility to water from a municipal supply. Water storage in the home was found in 86% of the households. The use of food container covers was found in 95% of the households. An attached bedroom with a kitchen was found in 56%. Sixty-four percent of all adults and 26% of the study children used sanitary latrines; pit latrines were used by 2.5% of the study population. Mud floors in the house were seen in 27%. The proportion of malnourished and stunted children was more common in the younger age groups (Table 1).

Diarrheal morbidity.

Two hundred eight-nine children contributed 299,616 child-days of observation for this study. Of these, 3,046 child-days from 254 children (88%) were associated with diarrhea. The total number of episodes was 1,447. The average incidence rate of diarrhea was 1.8/child-year, and the average number of diarrheal days was 3.7 days/ child-year. Seventy-five percent of the diarrheal episodes were ≤ 2 days in duration (Figure 1). Persistent diarrhea was relatively uncommon (0.2% of the children) and chronic diarrhea was observed in only one episode. The youngest age group of children had significantly more diarrheal episodes, but not E. histolytica-associated diarrheal incidence (Table 2). Overall diarrheal morbidity was more in male children compare with female children (Table 3); however, this difference was not observed for E. histolytica-associated diarrheal illness (Table 3).

Relationship between diarrheal diseases and baseline nutritional status of the children.

Compared with malnourished children, better-nourished children experienced significantly fewer diarrheal episodes (Table 4). Similarly, children not stunted at baseline had significantly less diarrheal morbidity (Table 4). Entamoeba histolytica-associated diarrheal incidence was also significantly less in better-nourished and not-stunted children compared with malnourished and stunted children (Table 4). However, asymptomatic E. histolytica infection and E. histolytica-associated dysenteric incidence were not related to the base line nutritional status of the children (Table 4).

Effect of breast-feeding on diarrheal incidence.

A complete history of breast-feeding was available for 285 children. Forty-seven children had a history of breast-feeding for less than the first 12 months of life and had a mean diarrheal incidence rate of 2.32 episodes/child-year. Two hundred thirty-eight children were breast-fed for a mean of 12 months and had an average diarrheal incidence of 1.68/child-year (P < 0.05) (Table 4). Although not statistically significant, the incidence rates of E. histolytica-associated diarrhea, dysentery, and asymptomatic E. histolytica infection were higher in children who were breast-fed less than 12 months (Table 4).

Relationship between blood group and diarrheal diseases.

Overall diarrheal illness showed a significant relation with blood type. Children with blood group A experienced significantly fewer episodes of diarrhea compare with the children with blood group O and blood group AB (Table 5). However, such a relationship was not observed for E. histolytica-associated diarrheal morbidity (Table 5).

Season and diarrheal incidence.

The incidence of diarrhea showed a marked seasonality, with significantly higher rates being recorded in the months of March–August than in the months of September–February; P < 0.0001) (Figure 2). Entamoeba histolytica–associated diarrheal incidence did not show such a relationship with season.

Enteropathogens isolated from diarrheal stools.

Of 1,447 diarrheal episodes, diarrheal stool samples from 893 episodes (62%) were available for analysis. All were examined for routine bacterial enteropathogens (Salmonella sp., Shigella sp., V. cholerae, Campylobacter sp., Plesiomonas sp., Aeromonas sp.) and parasitic agents. Of these 893 samples, a randomly selected subset of 210 were also examined for diarrheagenic E. coli and selected viral enteropathogens. Analysis of these 210 stool samples that received a complete bacteriologic, parasitologic, and virologic work-up resulted in an isolation rate for enteropathogens of 59%. Mixed infection with two or more pathogens was found in 19%. The prevalence of enteropathogens isolated from all 893 diarrheal stools is shown in Table 6.

The most frequent bacterial enteropathoges isolated from diarrheal stool specimens were ETEC (9%) and Aeromonas sp. (9%), followed by P. shigelloides (4%) and S. flexneri (3.8%). Rotavirus was the most common viral agent isolated from diarrheal stool samples (5%). Giardia lamblia, C. parvum, and E. histolytica were identified in 11%, 10%, and 8%, respectively, of the diarrheal stool specimens. Co-infection was most frequent in 63% (12 of 19) of the cases with ETEC and 44% (11 of 25) of the cases with C. jejuni. Co-infection of E. histolytica and other bacterial enteropathogens was found in 32% (22 of 69). Re-infection (isolation of a given pathogen from different diarrheal stools of different diarrheal episodes of the same child) was comparatively more common with E. histolytica (14 of 43, 32%), C. parvum (6 of 16, 37%), Aeromonas sp. (11 of 65, 16%), and A. hydrophilia (3 of 17, 17%).

Dysenteric and non-dysenteric episodes were observed in 7.7% and 92.3% of the specimens, respectively. The most common pathogens isolated from dysenteric stool were S. flexneri (11.6%), Aeromonas sp. (10%), E. histolytica (8.7%), C. jejuni (5.8%), P. shigelloides (4.3%), and A. caviae (4.3%).

Entamoeba histolytica-associated diarrhea incidence and association with diarrhea and dysentery.

The overall incidence rate of E. histolytica-associated diarrhea was 0.08/child-year for the sampling rate of 62%. Of the 651 E. histolytica antigen-positive monthly stool specimens, 25 (3.84%) were followed by diarrhea and 6 (0.92%) with dysentery. Only 44 (0.92%) and 13 (0.15%) of 8,842 E. histolytica antigen-negative monthly stool samples were followed by diarrhea and dysentery, respectively. The age-adjusted OR for the association between diarrhea and E. histolytica was 4.7 (95% confidence interval [CI] = 2.9−7.6). The age-adjusted OR between dysentery and E. histolytica was 2.9 (95% CI = 1.2−7.1). Of 43 children who contributed 69 E. histolytica-associated diarrhea specimens, only 14 had a subsequent E. histolytica-associated diarrheal episode. The crude OR for developing a subsequent episode of E. histolytica-associated diarrhea was 0.48 (95% CI = 0.26−0.91).

Clinical findings of the first E. histolytica-associated diarrhea/dysentery episodes.

The clinical data and some laboratory findings of E. histolytica-associated diarrhea are shown in Table 7. The mean weight-for-age Z-score was −2.01 and 47% of the affected children were malnourished. Abdominal pain and mild-to-moderate dehydration were the most frequent findings. The average duration of diarrheal episodes was three days. Visible blood was found in 7% of the cases, whereas blood loss was detected by the hemoccult test in 25% of the cases. A fecal lactoferrin test result was positive in 20% of the cases.

Asymptomatic E. histolytica infection incidence.

To determine the incidence rate of asymptomatic E. histolytica infection and its relationship with other variables, including age, sex, and nutritional status of the study children, we tested 9,493 non-diarrheal monthly stool specimens for E. histolytica antigen. Six hundred seventy specimens were positive. Nineteen samples were positive at baseline and the remaining 651 were positive during follow-up. One hundred ninety-nine (69%) children were positive at least once by the E. histolytica antigen detection test. The total number of new episodes was 359. The incidence rate of asymptomatic infection with E. histolytica was 0.44 episodes per child-year (95% CI = 0.39−0.49). The incidence rate was not related to baseline nutritional status, age category, and duration of breast-feeding of the children (Tables 2 and 4). It was also not related to the sex of the children or their blood group (Tables 3 and 5). However, it was significantly related to the baseline E. histolytica anti-lectin IgG status of the children, as previously reported.11 The incidence rate was 0.51 episodes/child-year (95% CI = 0.44−0.58) in children positive for IgG antibody to lectin and 0.34 episodes/child-year (95% CI = 0.28−0.40) (P = 0.001) in children negative for IgG antibody to lectin. The average duration of asymptomatic infection was 1.96 months.

Asymptomatic E. histolytica infection and season.

During the surveillance of this cohort, one or more children had a positive antigen detection test result for E. histolytica during all months (100%). This demonstrated that the reservoir of E. histolytica was present within the pediatric population in Mirpur during all the calendar year, with two peaks of incidence rate during April and October (Figure 2).

DISCUSSION

The most important outcome of the present study is the description of the epidemiology, clinical features, nutritional status, and causative agents of diarrhea, with special emphasis on E. histolytica-associated diarrhea. This study of 289 Bangladeshi children 2–5 years old at Mirpur, an urban slum of Dhaka, used modern diagnostic techniques to allow for the first time a more accurate picture of the contribution of amebiasis to the overall burden of diarrheal disease. It additionally provided additional insight into the natural history of amebiasis, including evidence of species-specific immunity to re-infection.

Our study demonstrated that diarrheal disease is a major health concern for the children of this community. Overall diarrheal illness rates ranged from 1.37 to 2.57/child-year depending on age groups. The rate was highest for those 2–3 years old and lowest for children 4–5 years old. Our results are similar with those reported by Ferrecio and others,5 but different from the results of other studies in Bangladesh.20,21 These studies, which showed incidence rates of diarrhea ranging from 1.94 to 4.41/child-year, were conducted in a rural setting of Bangladesh in which 78% of the children were malnourished.20,21 Average diarrheal days ranged from 2.72 to 6 days/child-year depending on age and were less than those of children in northeastern Brazil.22 Male children had a higher diarrheal incidence than female children. Similar results have been found in other studies, although the explanation for this difference is not known.23–25 Entamoeba histolytica-associated diarrhea did not show any relationship with the sex of the children.

Children in this community were better nourished compared with children of the same age from other studies conducted in Bangladesh.19,20 In the present study 39% and 32% of the children, respectively, were malnourished and stunted, which is still very high and proved to be major risk factors for diarrheal morbidity. Most episodes of diarrhea were of short duration; 87% of episodes were ≤ 3 days in duration. This may explain the comparatively low collection rate of diarrheal stool samples for etiologic diagnosis (62%). The percentage of children having persistent diarrhea was only 0.4%, which was considerably less than that observed in another field study.21 The low incidence of persistent diarrhea could be explained by the average better nutritional status of these children.

The association of diarrheal illness with seasons is a well-established fact.23 In this study, we have found the most pronounced seasonality with a large number of diarrheal episodes in the warm months of the year (March–August).

One of the interesting findings of our study was the association between ABO blood group types of the children with overall diarrheal illness. The association between different enteropathogens, especially ETEC and V. cholerae O1, and ABO blood groups has been well studied.26,27 Our data suggests that overall diarrheal illness is significantly higher in children with blood group O and AB, compared with children with blood group A and blood group B. This is the first report of a relationship between blood group and overall diarrheal incidence. In other studies, investigators have not looked for an association of blood groups with overall diarrheal diseases. Children with blood groups O and AB also were more prone to E. histolytica-associated diarrhea, but this association was not statistically significant.

Our study demonstrated that the etiologic agents of diarrhea in the Mirpur community were essentially identical to those found in other studies,18,24 except for E. histolytica and Aeromonas, which were isolated more frequently with diarrheal stools. The status of Aeromonas sp. as a human enteric pathogen is controversial.28,29 A case-control study in this community in the future may explain this controversy. The isolation rate of Cryptosporidium from diarrheal stool was higher (8.4%) when compared with the clinical study conducted at the ICDDR,B Hospital in Dhaka which isolated Cryptosporidium from only 3.5% of the diarrheal stools.30 This difference may be caused by the different methods used for the diagnosis of Cryptosporidium infection. In our study, we used an antigen detection test for diagnosis, whereas the study at the ICDDR,B Hospital used microscopy. The present study has shown that along with Aeromonas sp., ETEC, and Cryptosporidium parvum, E. histolytica is also a major enteropathogen in the community children of Mirpur, Dhaka. The isolation rate of E. histolytica was 8%, which was higher than that found in another study.31 The low isolation rate of E. histolytica reported by Zaki and others31 may be due to the exclusive use of stool microscopy, which has a low sensitivity and is no longer recommended. In Egypt, a recent survey using antigen detection tests showed an even higher isolation rate of E. histolytica in individuals presenting with acute diarrhea to an outpatient clinic.32 Additional studies in other communities will be required to derive the overall contribution of amebiasis to diarrheal disease.

Our present study has demonstrated that the asymptomatic carrier state of E. histolytica infection increased by five-fold the risk of E. histolytica-associated diarrhea and by three-fold the risk of E. histolytica-associated dysentery. Our results are in contrast with the results of the case-control study of the ICDDR, B, Hospital in Dhaka that failed to demonstrate an association between E. histolytica and diarrhea.18 The lack of an association between E. histolytica and diarrhea, as observed by Albert MJ and others,18 might have been caused by sampling bias because 84% (679 of 814) of their cases were less than 24 months old or by their failure to use an E. histolytica-specific diagnostic test. A previous study at the ICDDR, B Hospital demonstrated that E. histolytica infection was very low in children less than two years old.6

Of all clinical symptoms, abdominal pain and mild-to-moderate dehydration were the most frequent findings in E. histolytica-associated diarrheal episodes. These data support the findings reported by Wanke and others.6 Stool examination showed that E. histolytica-associated dysentery was underestimated when dysentery was defined by the gross appearance of blood in stool, which was found in only 5% of the cases of E. histolytica-associated diarrhea. Dysentery was demonstrated in 12–25% of cases of E. histolytica-associated diarrhea if stool samples were examined microscopically for red blood cells or examined for occult blood. Test results for fecal lactoferrin, another indicator of invasive infection, were positive in 20% of cases of E. histolytica-associated diarrhea. Thus, our study demonstrates that 12–25% of E. histolytica-associated diarrhea illnesses are invasive.

Of 43 children with a first episode, only 14 had a subsequent episode of E. histolytica-associated diarrhea. The OR was 0.48, indicating that children after a diarrheal episode with E. histolytica had half the chance of developing subsequent E. histolytica-associated diarrhea. This also supports our previous observations from this cohort that immunity exists after natural E. histolytica infection.12

Table 1

Baseline characteristics of the study population in Mirpur, Dhaka, Bangladesh*

No. (Male:Female)% malnourished (WAZ <-2) (male:female)% stunted (HAZ <-2) (male:female)
* WAZ = weight-for-age Z-score; HAZ = height-for-age Z-score; Tk = Taka.
Age (months)
    24–3656 (28:28)51.79 (42.8:60.7)41.07 (32.1:32.1)
    37–4883 (40:43)43.37 (40:46.5)43.37 (32.5:53.4)
    49–60150 (79:71)32.00 (39.2:23.9)22.67 (22.7:22.5)
    Total289 (147:142)39.10 (40.1:38.0)32.18 (27.2:37.3)
Household information (n = 252)
    Average family size (minimum, maximum)6 (2, 17)
    Maternal schooling, no. (%)
        Never attended school162 (64)
        Attended 1–5 years59 (23)
        Attended 6–10 years19 (7.5)
        Attended >10 years4 (1.5)
    Annual income in Tk, no. (%)
        <5,000 (<$U.S. 68)183 (72.6)
        5,000–10,000 ($US 68–172)60 (23.8)
        >10,000 (>$US 172)9 (3.5)
Table 2

Diarrheal morbidity by age of the children at Mirpur, Dhaka, Bangladesh

Age group (months)Number of ChildrenDays observedNumber of diarrheal episodes (child year)Number of Entamoeba histolytica–associated diarrheal episodes (child year)Number of E. histolytica–associated asymptomatic episodes (child/year)
* P < 0.001 versus age group 24–36 months.
24–365659,898410 (2.50)14 (0.08)73 (0.44)
37–488384,496320 (1.38)*14 (0.06)90 (0.39)
49–60150155,222717 (1.69)*41 (0.10)196 (0.46)
All289299,6161,447 (1.76)69 (0.08)359 (0.44)
Table 3

Diarrheal morbidity by sex of the children at Mirpur, Dhaka, Bangladesh

SexNumber of childrenDays observedNumber of diarrheal episodes (child/year)Number of E. histolytica–associateddiarrheal episodes (child/year)Number of E. histolytica–associated asymptomatic episodes (child/year)
* P < 0.001.
Male147149,241784 (1.92)*33 (0.08)170 (0.42)
Female142150,375663 (1.61)*36 (0.09)189 (0.46)
All289299,6161,447 (1.76)69 (0.08)359 (0.44)
Table 4

Relationship between diarrheal morbidity, nutritional status, and duration of breastfeeding at baseline of the study children*

IndicatorsNo.Days observedNumber of diarrheal episodes (child/year)Number of Entamoebahistolytica–associated diarrheal episodes (child/year)Number ofE. histolytica–associated asymptomatic episodes (child/year)Number ofE. histolytica–associated dysenteric episodes (child/year)
* WAZ = weight-for-age Z-score; HAZ = height-for-age Z-score.
P < 0.01.
P < 0.038.
Malnourished (WAZ <-2)113113,814653 (2.1)†37 (0.12)†138 (0.44)8 (0.025)
Better-nourished (WAZ ≥-2)176185,802794 (1.6)†32 (0.06)†221 (0.43)11 (0.022)
Stunted (HAZ <-2)9399,307545 (2.0)†31 (0.11)‡125 (0.46)7 (0.03)
Not-stunted (HAZ ≥-2)196200,309902 (1.64)†38 (0.07)‡234 (0.43)12 (0.02)
Breastfed
    <12 months4740,972255 (2.3)†13 (1.2)58 (0.52)4 (0.04)
    ≥12 months242258,6441,192 (1.68)†56 (0.08)301 (0.42)15 (0.02)
Table 5

Diarrheal morbidity by blood group of the children at Mirpur, Dhaka, Bangladesh

Blood groupNumber of childrenDays observedNumber of diarrheal episodes (child/year)Number of Entamoeba histolytica–associated diarrheal episodes (child/year)Number of E. histolytica–associated asymptomatic episodes (child/year)
a,b,c Indicate p = <0.05 between groups.
P < 0.05 versus group A.
A7994,059380 (1.47)18 (0.07)96 (0.37)
B7992,390433 (1.71)20 (0.08)105 (0.41)
O6677,581432 (2.03)*21 (0.10)106 (0.50)
AB1112,96278 (2.20)*4 (0.11)13 (0.037)
Table 6

Organisms identified in diarrheal stools of children in Mirpur, Dhaka, Bangladesh

Etiologic agentDiarrheal stool % (no. with agent/no. tested)
Bacterial agent39.04 (82/210)
    Enterotoxigenic Escherichia coli9.05 (19/210)
        Heat-stable toxin (+)3.33 (7/210)
        Heat-labile toxin (+)6.19 (13/210)
        Both (+)0.48 (1/210)
    Enteropathogenic E. coli3.33 (7/210)
    Enteroinvasive E. coli1.9 (4/210)
    Enteroaggregative E. coli2.86 (6/210)
    Enterohemorrhagic E. coli0 (0/210)
    Aeromonas species (non-typable)9.29 (83/893)
    A. hydrophilia2.24 (20/893)
    A. sobria2.24 (20/893)
    A. caviae2.35 (21/893)
    Shigella flexneri3.81 (34/893)
    S. sonnei0.34 (3/893)
    S. dysenteriae0.67 (6/893)
    S. boydii1.57 (14/893)
    Shigella spices0.22 (2/893)
    Campylobacter jejuni2.80 (25/893)
    Plesiomonas shigelloides4.03 (36/893)
    Salmonella sp.0.78 (7/893)
    Vibrio cholerae 01 El tor inaba0.11 (1/893)
    V. cholerae O1390.11 (1/893)
    Vibrio sp.1.0 (9/893)
Viral agent8.09 (17/210)
    Rotavirus5.24 (11/210)
    Adenovirus2.86 (6/210)
    Astrovirus0 (0/210)
Parasitic agent82.47 (607/736)
    Entamoeba histolytica7.75 (69/893)
    E. dispar11.98 (107/893)
    Cryptosporidium parvum8.4 (75/893)
    Giardia lamblia11.08 (99/893)
    Ascaris lumbricoides58.90 (526/893)
    Trichuris trichiura51.06 (456/893)
    Hookworm0.56 (5/893)
    Hymenolepis nana1.23 (11/893)
    Strongyloides stercoralis0.22 (2/893)
Table 7

Clinical and laboratory findings of Entamoeba histolytica–associated diarrhea in 43 children*

Findings
* WAZ = weight-for-age Z-score; RBCs = red blood cells; hpf = high-power field.
Age in months, mean (SD)62 (16)
Mean WAZ (SD)−2.01 (1.04)
Children with WAZ <−2, no. (%)20 (47)
Fever present, no. (%)17 (40)
Abdominal pain, no. (%)27 (63)
Vomiting present, no. (%)5 (12)
Average peak stool frequency/24 hours (SD)6 (4)
Stool consistency (loose-liquid not watery), no. (%)42 (98)
Other family member with diarrhea, no. (%)8 (19)
Mild-moderate dehydration present, no. (%)28 (65)
Average duration in days (SD)3 (2)
Average diarrheal severity score (SD)6 (2)
Visible blood in stool, no. (%)3 (7)
Stool RBCs ≥1/hpf, no. (%)5 (12)
Fecal hemoccult positive, number positive/number tested (%)17/69 (25)
Fecal lactoferrin positive, number positive/number tested (%)8/41 (20)
Figure 1.
Figure 1.

Frequency distribution of diarrheal episodes according to their duration in days in an urban slum of Dhaka, Bangladesh.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.398

Figure 2.
Figure 2.

Seasonal variation of diarrheal diseases in preschool children in an urban slum of Dhaka, Bangladesh.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 4; 10.4269/ajtmh.2003.69.398

Authors’ addresses: Rashidul Haque, Dinesh Mondal, and Selim Akther, Laboratory Sciences Division, Center for Health and Population Resaerch, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B), GPO Box 128, Dhaka 1000, Bangladesh. Beth D. Kirkpatrick, Unit of Infectious Diseases, University of Vermont/Fletcher Allen Health Care, Burgess 303, MCHV Campus, Burlington, VT 05401. Barry M. Farr and William A. Petri, Jr., Department of Medicine, University of Virginia Health System, Charlottesville, VA 22908. R. Bradley Sack, Department of International Health, Johns Hopkins University Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205-2179.

Acknowledgments: We thank Dr. Lyerly of TechLab Inc. for providing parasite fecal antigen detection tests. The ICCRD,B acknowledges the commitment of the National Institutes of Health (NIH) (Bethesda, MD) and the University of Virginia to its effort. We thank the parents and children of Mirpur for their participation, and the field team, including the study supervisor Lutfar Rahaman; field assistants Janata Rani Shaha, Salma Akther, Sahina Parveen, Nurjahan Akther Baby, and Dulari Begum; the Data Management Officer Mahbubur Rahman; and Dr. Hamidur Rahman from the laboratory staffs for their contributions to this study.

Financial support: The study was conducted at the ICDDR, B Centre for Health and Population Research with the support of a grant (AI-43596) from the NIH. This work was also supported by NIH grant AI-43596. Rashidul Haque is a Howard Hughes Medical Institute International Research Scholar and William A. Petri, Jr. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology.

Disclosure: The authors wish to disclose that the University of Virginia has a license agreement with Techlab, Inc. for diagnostic tests for amebiasis. Dr. Petri donates all of his royalties from this agreement to the American Society of Tropical Medicine and Hygiene

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Author Notes

Reprint requests: Rashidul Haque, Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), GPO Box 128, Dhaka1000, Bangladesh, Telephone: 880-2-881-751, extension 2411, Fax: 880-2-8812529 or 880-2-8823116, E-mail: rhaque@icddrb.org.
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