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Background: Household contacts of cholera patients are at a 100 times higher risk of developing cholera than the general population. The objective of this study was to examine the incidence of V. cholerae infections among household contacts of cholera patients in a rural setting in Bangladesh, to identify risk factors for V. cholerae infections among this population, and to investigate transmission pathways of V. cholerae using multilocus variable-number tandem-repeat analysis (MLVA)., Methodology/principal Findings: Stool from household contacts, source water and stored water samples were collected from cholera patient households on Day 1, 3, 5, and 7 after the presentation of the index patient at a health facility. Two hundred thirty clinical and water V. cholerae isolates were analyzed by MLVA. Thirty seven percent of households had at least one household contact with a V. cholerae infection. Thirteen percent of households had V. cholerae in their water source, and 27% had V. cholerae in stored household drinking water. Household contacts with V. cholerae in their water source had a significantly higher odds of symptomatic cholera (Odds Ratio (OR): 5.49, 95% Confidence Interval (CI): 1.07, 28.08). Contacts consuming street vended food had a significantly higher odds of a V. cholerae infection (OR: 9.45, 95% CI: 2.14, 41.72). Older age was significantly associated with a lower odds of a V. cholerae infection (OR: 0.96, 95% CI: 0.93, 0.99). Households with both water and clinical V. cholerae-positive samples all had isolates that were closely related by MLVA., Conclusions/significance: These findings emphasize the need for interventions targeting water treatment and food hygiene to reduce V. cholerae infections., Competing Interests: The authors have declared that no competing interests exist.

Vibrio cholerae , an estuarine bacterium, is the causative agent of cholera, a severe diarrheal disease that demonstrates seasonal incidence in Bangladesh. In an extensive study of V. cholerae occurrence in a natural aquatic environment, water and plankton samples were collected biweekly between December 2005 and November 2006 from Mathbaria, an estuarine village of Bangladesh near the mangrove forests of the Sundarbans. Toxigenic V. cholerae exhibited two seasonal growth peaks, one in spring (March to May) and another in autumn (September to November), corresponding to the two annual seasonal outbreaks of cholera in this region. The total numbers of bacteria determined by heterotrophic plate count (HPC), representing culturable bacteria, accounted for 1% to 2.7% of the total numbers obtained using acridine orange direct counting (AODC). The highest bacterial culture counts, including toxigenic V. cholerae , were recorded in the spring. The direct fluorescent antibody (DFA) assay was used to detect V. cholerae O1 cells throughout the year, as free-living cells, within clusters, or in association with plankton. V. cholerae O1 varied significantly in morphology, appearing as distinctly rod-shaped cells in the spring months, while small coccoid cells within thick clusters of biofilm were observed during interepidemic periods of the year, notably during the winter months. Toxigenic V. cholerae O1 was culturable in natural water during the spring when the temperature rose sharply. The results of this study confirmed biofilms to be a means of persistence for bacteria and an integral component of the annual life cycle of toxigenic V. cholerae in the estuarine environment of Bangladesh. IMPORTANCE Vibrio cholerae O1 in the estuarine environment of Mathbaria, where the bacterium is culturable when the water temperature rises and is observable predominantly as distinct rods and dividing cells. In the spring and fall, these morphological changes coincide with the two seasonal peaks of endemic cholera in Bangladesh. V. cholerae O1 in the estuarine environment of Mathbaria, where the bacterium is culturable when the water temperature rises and is observable predominantly as distinct rods and dividing cells. In the spring and fall, these morphological changes coincide with the two seasonal peaks of endemic cholera in Bangladesh. V. cholerae O1, the causative agent of cholera in Bangladesh.V. cholerae O1, the causative agent of cholera in Bangladesh., (Copyright © 2018 Sultana et al.)

Helicobacter pylori colonization is prevalent throughout the world, and is predominantly acquired during childhood. In developing countries, >70% of adult populations are colonized with H. pylori and >50% of children become colonized before the age of 10 years. However, the exact timing of acquisition is unknown. We assessed detection of H. pylori acquisition among a birth cohort of 105 children in Mirzapur, Bangladesh. Blood samples collected at time 0 (cord blood), and at 6, 12, 18, and 24 months of life were examined for the presence of IgG and IgA antibodies to whole cell H. pylori antigen and for IgG antibodies to the CagA antigen using specific ELISAs and immunoblotting. Breast milk samples were analyzed for H. pylori-specific IgA antibodies. Cord blood was used to establish maternal colonization status. H. pylori seroprevalence in the mothers was 92.8%. At the end of the two-year follow-up period, 50 (47.6%) of the 105 children were positive for H. pylori in more than one assay. Among the colonized children, CagA prevalence was 78.0%. A total of 58 children seroconverted: 50 children showed persistent colonization and 8 (7.6%) children showed transient seroconversion, but immunoblot analysis suggested that the transient seroconversion observed by ELISA may represent falsely positive results. Acquisition of H. pylori was not influenced by the mother H. pylori status in serum or breastmilk. In this population with high H. pylori prevalence, we confirmed that H. pylori in developing countries is detectable mainly after the first year of life.

Background: Household contacts of cholera patients have a 100 times higher risk of developing a cholera infection than the general population. To compare the genetic relatedness of clinical and water source Vibrio cholerae isolates from cholera patients' households across three outbreaks, we analyzed these isolates using whole-genome-sequencing (WGS) and multilocus variable-number tandem-repeat analysis (MLVA)., Results: The WGS analyses revealed that 80% of households had source water isolates that were more closely related to clinical isolates from the same household than to any other isolates. While in another 20% of households an isolate from a person was more closely related to clinical isolates from another household than to source water isolates from their own household. The mean pairwise differences in single nucleotide-variant (SNV) counts for isolates from the same household were significantly lower than those for different households (2.4 vs. 7.7 p < 0.0001), and isolates from the same outbreak had significantly fewer mean pairwise differences compared to isolates from different outbreaks (mean: 6.2 vs. 8.0, p < 0.0001). Based on MLVA in outbreak 1, we observed that the majority of households had clinical isolates with MLVA genotypes related to other clinical isolates and unrelated to water source isolates from the same household. While in outbreak 3, there were different MLVA genotypes between households, however within the majority of households, the clinical and water source isolates had the same MLVA genotypes. The beginning of outbreak 2 resembled outbreak 1 and the latter part resembled outbreak 3. We validated our use of MLVA by comparing it to WGS. Isolates with the identical MLVA genotype had significantly fewer mean pairwise SNV differences than those isolates with different MLVA genotypes (mean: 4.8 vs. 7.7, p < 0.0001). Furthermore, consistent with WGS results, the number of pairwise differences in the five MLVA loci for isolates within the same household was significantly lower than isolates from different households (mean: 1.6 vs. 3.0, p < 0.0001)., Conclusion: These results suggest that transmission patterns for cholera are a combination of person-to-person and water-to-person cholera transmission with the proportions of the two modes varying within and between outbreaks.

Objective: To characterise childhood mouthing behaviours and to investigate the association between object-to-mouth and food-to-mouth contacts, diarrhoea prevalence and environmental enteropathy., Methods: A prospective cohort study was conducted of 216 children ≤30 months of age in rural Bangladesh. Mouthing contacts with soil and food and objects with visible soil were assessed by 5-h structured observation. Stool was analysed for four faecal markers of intestinal inflammation: alpha-1-antitrypsin, myeloperoxidase, neopterin and calprotectin., Results: Overall 82% of children were observed mouthing soil, objects with visible soil, or food with visible soil during the structured observation period. Sixty two percent of children were observed mouthing objects with visible soil, 63% were observed mouthing food with visible soil, and 18% were observed mouthing soil only. Children observed mouthing objects with visible soil had significantly elevated faecal calprotectin concentrations (206.81 μg/g, 95% confidence interval [CI]: 6.27, 407.36). There was also a marginally significant association between Escherichia coli counts in soil from a child's play space and the prevalence rate of diarrhoea (diarrhoea prevalence ratio: 2.03, 95% CI 0.97, 4.25)., Conclusion: These findings provide further evidence to support the hypothesis that childhood mouthing behaviour in environments with faecal contamination can lead to environmental enteropathy in susceptible paediatric populations. Furthermore, these findings suggest that young children mouthing objects with soil, which occurred more frequently than soil directly (60% vs. 18%), was an important exposure route to faecal pathogens and a risk factor for environmental enteropathy., (© 2017 John Wiley & Sons Ltd.)

Objective: In urban Dhaka, Bangladesh, 30% of source water samples collected from the households of patients with cholera had detectable Vibrio cholerae. These findings indicate an urgent need for a public health intervention for this population. The Crystal VC ® dipstick test is a rapid method for detecting V. cholerae in stool and water. However, to date no study has investigated the use of the rapid dipstick test for household surveillance of stored drinking water., Methods: The efficacy of the Crystal VC ® dipstick test for detecting V. cholerae in the Dhaka city municipal water supply and stored household drinking water sources after enrichment for 18 h in alkaline peptone water (APW) was compared to bacterial culture as the gold standard., Results: A total of 1648 water samples (824 stored household drinking water samples and 824 municipal water supply samples) were collected from households of patients with cholera. The overall specificity and sensitivity of the dipstick test compared to bacterial culture was 99.6% (95% confidence interval (CI): 99.2%, 99.9%) and 65.6% (95% CI: 55.2%, 75%), respectively. The specificities for stored household drinking water and Dhaka city municipal supply water compared to bacterial culture were 99.8% (95% CI: 99.1%, 100%) and 99.5% (95% CI: 98.6%, 99.9%), respectively (P = 0.138), and the sensitivities were 66.7% (95% CI: 43.0%, 85.4%) and 65.3% (95% CI: 53.5%, 76.0%), respectively (P = 0.891)., Conclusion: The Crystal VC ® dipstick is a promising screening tool for cholera outbreak surveillance in resource-limited settings where elimination of false-positive results is critical. The lower than expected sensitivity should be further investigated in future studies., (© 2016 John Wiley & Sons Ltd.)

Household members of cholera patients are at a 100 times higher risk of cholera infections than the general population because of shared contaminated drinking water sources and secondary transmission through poor household hygiene practices. In this study, we investigated the bactericidal concentration of free chlorine required to inactivate Vibrio cholerae in household drinking water in Dhaka, Bangladesh. In laboratory experiments, we found that the concentrations of free chlorine required to inactivate 10 5 colony-forming units (CFU)/mL of V. cholerae serogroups O1 and O139 were 0.1 mg/L and 0.2 mg/L, respectively. The concentration of free chlorine generated by a single chlorine tablet (sodium dichloroisocyanurate [33 mg]) after a 30-minute reaction time in a 10-L sealed vessel containing Dhaka city municipal supply water was 1.8 mg/L; and the concentration declined to 0.26 mg/L after 24 hours. In field measurements, water collected from 165 households enrolled in a randomized controlled trial (RCT) of a chlorine and handwashing with soap intervention (Cholera-Hospital-Based-Intervention-for-7-Days [CHoBI7]), we observed significantly higher free chlorine concentrations in the 82 intervention arm households (mean = 1.12 mg/L, standard deviation [SD] = 0.52, range = 0.07-2.6 mg/L) compared with the 83 control households (0.017 mg/L, SD = 0.01, range = 0-0.06 mg/L) (P < 0.001) during spot check visits. These findings suggest that point-of-use chlorine tablets present an effective approach to inactivate V. cholerae from drinking water in households of cholera patients in Dhaka city. This result is consistent with the findings from the RCT of CHoBI7 which found that this intervention led to a significant reduction in symptomatic cholera infections among household members of cholera patients and no stored drinking water samples with detectable V. cholerae., (© The American Society of Tropical Medicine and Hygiene.)

Objective: To determine the relationship between geophagy (mouthing of dirt, sand, clay, or mud) and growth faltering in young children., Study Design: We examined linear growth as height and weight standardized by age and sex, and weight standardized by height, in a cohort of children aged 6-36 months in rural Mirzapur, Bangladesh. We determined geophagy behavior at baseline through caregiver report. Anthropometric measurements were assessed at baseline and at a 1-year follow-up., Results: We found that among children not stunted at baseline, those with caregiver-reported geophagy at baseline grew less over 1 year compared with their peers, with a difference in the change of standardized height for age and sex of -0.31 (95% CI, -0.61 to -0.01)., Conclusion: These findings show that caregiver-reported geophagy was associated with growth faltering in a pediatric population in rural Bangladesh. Future studies are needed to learn more about this exposure pathway and its relevance to child growth., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Diarrhea is the second leading cause of death in children under 5 years of age globally. The time patients and caregivers spend at a health facility for severe diarrhea presents the opportunity to deliver water, sanitation, and hygiene (WASH) interventions. We recently developed Cholera-Hospital-Based Intervention for 7 days (CHoBI7), a 1-week hospital-based handwashing with soap and water treatment intervention, for household members of cholera patients. To investigate if this intervention could lead to sustained WASH practices, we conducted a follow-up evaluation of 196 intervention household members and 205 control household members enrolled in a randomized controlled trial of the CHoBI7 intervention 6 to 12 months post-intervention. Compared with the control arm, the intervention arm had four times higher odds of household members' handwashing with soap at a key time during 5-hour structured observation (odds ratio [OR]: 4.71, 95% confidence interval [CI]: 2.61, 8.49) (18% versus 50%) and a 41% reduction in households in the World Health Organization very high-risk category for stored drinking water (OR: 0.38, 95% CI: 0.15, 0.96) (58% versus 34%) 6 to 12 months post-intervention. Furthemore, 71% of observed handwashing with soap events in the intervention arm involved the preparation and use of soapy water, which was promoted during the intervention, compared to 9% of control households. These findings demonstrate that the hospital-based CHoBI7 intervention can lead to significant increases in handwashing with soap practices and improved stored drinking water quality 6 to 12 months post-intervention., (© The American Society of Tropical Medicine and Hygiene.)

The risk for cholera infection is >100 times higher for household contacts of cholera patients during the week after the index patient seeks hospital care than it is for the general population. To initiate a standard of care for this high-risk population, we developed Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7), which promotes hand washing with soap and treatment of water. To test CHoBI7, we conducted a randomized controlled trial among 219 intervention household contacts of 82 cholera patients and 220 control contacts of 83 cholera patients in Dhaka, Bangladesh, during 2013-2014. Intervention contacts had significantly fewer symptomatic Vibrio cholerae infections than did control contacts and 47% fewer overall V. cholerae infections. Intervention households had no stored drinking water with V. cholerae and 14 times higher odds of hand washing with soap at key events during structured observation on surveillance days 5, 6, or 7. CHoBI7 presents a promising approach for controlling cholera among highly susceptible household contacts of cholera patients.

To examine rates of Shigella infections in household contacts of pediatric shigellosis patients, we followed contacts and controls prospectively for 1 week after the index patient obtained care. Household contacts of patients were 44 times more likely to develop a Shigella infection than were control contacts (odds ratio 44.7, 95% CI 5.5-361.6); 29 (94%) household contacts of shigellosis patients were infected with the same species and serotype as the index patient's. Pulsed-field gel electrophoresis showed that 14 (88%) of 16 with infected contacts had strains that were indistinguishable from or closely related to the index patient's strain. Latrine area fly counts were higher in patient households compared with control households, and 2 patient household water samples were positive for Shigella. We show high susceptibility of household contacts of shigellosis patients to Shigella infections and found environmental risk factors to be targeted in future interventions.

There is a growing body of literature indicating an association between stunting and environmental enteropathy (EE), a disorder thought to be caused by repeated exposures to enteric pathogens. To investigate the relationship between exposure to enteric pathogens through geophagy, consumption of soil, EE, and stunting, we conducted a prospective cohort study of 216 children under 5 years of age in rural Bangladesh. Geophagy was assessed at baseline using 5 hour structured observation and caregiver reports. Stool was analyzed for fecal markers of intestinal inflammation: alpha-1-antitrypsin, myeloperoxidase, neopterin (all three combined to form an EE disease activity score), and calprotectin. Eighteen percent of children had observed geophagy events by structured observation and 28% had caregiver reported events in the past week. Nearly all households had Escherichia coli (97%) in soil, and 14% had diarrheagenic E. coli. Children with caregiver-reported geophagy had significantly higher EE scores (0.72 point difference, 95% confidence interval [CI]: 0.01, 1.42) and calprotectin concentrations (237.38 μg/g, 95% CI: 12.77, 462.00). Furthermore, at the 9-month follow-up the odds of being stunted (height-for-age z-score < -2) was double for children with caregiver-reported geophagy (odds ratio [OR]: 2.27, 95% CI: 1.14, 4.51). These findings suggest that geophagy in young children may be an important unrecognized risk factor for EE and stunting., (© The American Society of Tropical Medicine and Hygiene.)

Cholera remains a major public health problem. To compare the relative contribution of strains from the environment with strains isolated from patients during outbreaks, we performed multilocus variable tandem repeat analyses on samples collected during the 2010 and 2011 outbreak seasons in 2 geographically distinct areas of Bangladesh. A total of 222 environmental and clinical isolates of V. cholerae O1 were systematically collected from Chhatak and Mathbaria. In Chhatak, 75 of 79 isolates were from the same clonal complex, in which extensive differentiation was found in a temporally consistent pattern of successive mutations at single loci. A total of 59 isolates were collected from 6 persons; most isolates from 1 person differed by sequential single-locus mutations. In Mathbaria, 60 of 84 isolates represented 2 separate clonal complexes. The small number of genetic lineages in isolates from patients, compared with those from the environment, is consistent with accelerated transmission of some strains among humans during an outbreak.

The highly populated floodplains of the Bengal Delta have a long history of endemic and epidemic cholera outbreaks, both coastal and inland. Previous studies have not addressed the spatio-temporal dynamics of population vulnerability related to the influence of underlying large-scale processes. We analyzed spatial and temporal variability of cholera incidence across six surveillance sites in the Bengal Delta and their association with regional hydroclimatic and environmental drivers. More specifically, we use salinity and flood inundation modeling across the vulnerable districts of Bangladesh to test earlier proposed hypotheses on the role of these environmental variables. Our results show strong influence of seasonal and interannual variability in estuarine salinity on spring outbreaks and inland flooding on fall outbreaks. A large segment of the population in the Bengal Delta floodplains remain vulnerable to these biannual cholera transmission mechanisms that provide ecologic and environmental conditions for outbreaks over large geographic regions.

Vibrio cholerae, an environmental organism, is a facultative human pathogen. Here, we report the virulence profiles, comprising 18 genetic markers, of 102 clinical and 692 environmental V. cholerae strains isolated in Bangladesh between March 2004 and January 2006, showing the variability of virulence determinants within the context of public health.

Vibrio cholerae O1 biotype El Tor (ET), causing the seventh cholera pandemic, was recently replaced in Bangladesh by an altered ET possessing ctxB of the Classical (CL) biotype, which caused the first six cholera pandemics. In the present study, V. cholerae O1 strains associated with endemic cholera in Dhaka between 2006 and 2011 were analysed for major phenotypic and genetic characteristics. Of 54 representative V. cholerae isolates tested, all were phenotypically ET and showed uniform resistance to trimethoprim/sulfamethoxazole (SXT) and furazolidone (FR). Resistance to tetracycline (TE) and erythromycin (E) showed temporal fluctuation, varying from year to year, while all isolates were susceptible to gentamicin (CN) and ciprofloxacin (CIP). Year-wise data revealed erythromycin resistance to be 33.3 % in 2006 and 11 % in 2011, while tetracycline resistance accounted for 33, 78, 0, 100 and 27 % in 2006, 2007, 2008, 2009 and 2010, respectively; interestingly, all isolates tested were sensitive to TE in 2011, as observed in 2008. All V. cholerae isolates tested possessed genetic elements such as SXT, ctxAB, tcpA(ET), rstR(ET) and rtxC; none had IntlI (Integron I). Double mismatch amplification mutation assay (DMAMA)-PCR followed by DNA sequencing and analysis of the ctxB gene revealed a point mutation at position 58 (C→A), which has resulted in an amino acid substitution from histidine (H) to asparagine (N) at position 20 (genotype 7) since 2008. Although the multi-resistant strains having tetracycline resistance showed minor genetic divergence, V. cholerae strains were clonal, as determined by a PFGE (NotI)-based dendrogram. This study shows 2008-2010 to be the time of transition from ctxB genotype 1 to genotype 7 in V. cholerae ET causing endemic cholera in Dhaka, Bangladesh.

Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a severe watery, life-threatening diarrheal disease occurring predominantly in developing countries. V. cholerae, including both serogroups O1 and O139, is found in association with crustacean zooplankton, mainly copepods, and notably in ponds, rivers, and estuarine systems globally. The incidence of cholera and occurrence of pathogenic V. cholerae strains with zooplankton were studied in two areas of Bangladesh: Bakerganj and Mathbaria. Chitinous zooplankton communities of several bodies of water were analyzed in order to understand the interaction of the zooplankton population composition with the population dynamics of pathogenic V. cholerae and incidence of cholera. Two dominant zooplankton groups were found to be consistently associated with detection of V. cholerae and/or occurrence of cholera cases, namely, rotifers and cladocerans, in addition to copepods. Local differences indicate there are subtle ecological factors that can influence interactions between V. cholerae, its plankton hosts, and the incidence of cholera.

Despite the known presence of rotavirus-associated diarrhoea in Bangladesh, its prevalence, including records of hospitalization in rural health facilities, is largely unknown. In a systematic surveillance undertaken in two government-run rural health facilities, 457 children, aged less than five years, having acute watery diarrhoea, were studied between August 2005 and July 2007 to determine the prevalence of rotavirus. Due to limited financial support, the surveillance of rotavirus was included as an addendum to an ongoing study for cholera in the same area. Rotavirus infection was detected in 114 (25%) and Vibrio cholerae in 63 (14%) children. Neither rotavirus nor V cholerae was detected in 280 (61%) samples; these were termed 'non-rotavirus and non-cholera' diarrhoea. Both rotavirus and cholera were detected in all groups of patients (<5 years). The highest proportion (41%; 47/114) of rotavirus was in the age-group of 6-11 months. In children aged less than 18 months, the proportion (67%; 76/114) of rotavirus was significantly (p < 0.001) higher than that of cholera (16%; 10/63). By contrast, the proportion (84%; 53/63) of cholera was significantly (p < 0.001) higher than that of rotavirus (33%; 38/114) in the age-group of 18-59 months. During the study period, 528 children were hospitalized for various illnesses. Thirty-eight percent (202/528) of the hospitalizations were due to acute watery diarrhoea, and 62% were due to non-diarrhoeal illnesses. Rotavirus accounted for 34% of hospitalizations due to diarrhoea. Severe dehydration was detected in 16% (74/457) of the children. The proportion (51%; 32/63) of severe dehydration among V cholerae-infected children was significantly higher (p < 0.001) compared to the proportion (16%; 18/114) of rotavirus-infected children. The study revealed that 12-14% of the hospitalizations in rural Bangladesh in this age-group were due to rotavirus infection, which has not been previously documented.

The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.

Morbidity and mortality data are important for planning and implementing healthcare strategies of a country. To understand the major causes for hospitalizations in rural Bangladesh, demographic and clinical data were collected from the hospital-records of five government-run rural health facilities (upazila health complexes) situated at different geographical regions of the country from January 1997 to December 2001. During this period, 75,598 hospital admissions in total were recorded, of which 54% were for male, and 46% were for female. Of all the admissions, diarrhoeal disease was the leading cause for hospitalization (25.1%), followed by injuries (17.7%), respiratory tract diseases (12.6%), diseases of the gastrointestinal tract (10.5%), obstetric and gynaecological causes (8.5%), and febrile illnesses (6.7%). A considerable proportion (8.3%) of the hospitalized patients remained undiagnosed. Despite the limitations of hospital-based data, this paper gives a reasonable insight of the important causes for hospitalizations in upazila health complexes that may guide the policy-makers in strengthening and prioritizing the healthcare needs at the upazila level in Bangladesh.

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., "clonal cluster," as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.

We examined whether malnutrition (underweight [WAZ] < -2) increased the risk of diarrhea equally for all enteropathogens. The study was conducted prospectively between January 1999 and July 2002 in Mirpur, an urban slum in Dhaka. Two hundred eighty-nine Bangladeshi children (147 male and 142 female) 2-5 years of age were included in the study. Malnutrition was present in 39% of the children at the time of enrollment. The parents and children were visited and interviewed every other day by health care workers for details about any diarrheal episodes. Stool samples were successfully collected from 62% of episodes of diarrhea. Of the identified enteropathogens, only enterotoxigenic Escherichia coli (ETEC), Cryptosporidium sp., and Entamoeba histolytica were significantly more prevalent in malnourished children. We concluded that the malnutrition attributed risk is not equal for enteric pathogens associated with diarrheal illness.

The causative agent of cholera, Vibrio cholerae, has been shown to be autochthonous to riverine, estuarine, and coastal waters along with its host, the copepod, a significant member of the zooplankton community. Temperature, salinity, rainfall and plankton have proven to be important factors in the ecology of V. cholerae, influencing the transmission of the disease in those regions of the world where the human population relies on untreated water as a source of drinking water. In this study, the pattern of cholera outbreaks during 1998-2006 in Kolkata, India, and Matlab, Bangladesh, and the earth observation data were analyzed with the objective of developing a prediction model for cholera. Satellite sensors were used to measure chlorophyll a concentration (CHL) and sea surface temperature (SST). In addition, rainfall data were obtained from both satellite and in situ gauge measurements. From the analyses, a statistically significant relationship between the time series for cholera in Kolkata, India, and CHL and rainfall anomalies was determined. A statistically significant one month lag was observed between CHL anomaly and number of cholera cases in Matlab, Bangladesh. From the results of the study, it is concluded that ocean and climate patterns are useful predictors of cholera epidemics, with the dynamics of endemic cholera being related to climate and/or changes in the aquatic ecosystem. When the ecology of V. cholerae is considered in predictive models, a robust early warning system for cholera in endemic regions of the world can be developed for public health planning and decision making.

Clinical and environmental Vibrio cholerae organisms collected from February 2004 through April 2005 were systematically isolated from 2 rural Bangladeshi locales. Their genetic relatedness was evaluated at 5 loci that contained a variable number of tandem repeats (VNTR). The observed minimal overlap in VNTR patterns between the 2 communities was consistent with sequential, small outbreaks from local sources.

Several species of the genus Vibrio, including Vibrio cholerae, are bioluminescent or contain bioluminescent strains. Previous studies have reported that only 10% of V. cholerae strains are luminescent. Analysis of 224 isolates of non-O1/non-O139 V. cholerae collected from Chesapeake Bay, MD, revealed that 52% (116/224) were luminescent when an improved assay method was employed and 58% (130/224) of isolates harbored the luxA gene. In contrast, 334 non-O1/non-O139 V. cholerae strains isolated from two rural provinces in Bangladesh yielded only 21 (6.3%) luminescent and 35 (10.5%) luxA+ isolates. An additional 270 clinical and environmental isolates of V. cholerae serogroups O1 and O139 were tested, and none were luminescent or harbored luxA. These results indicate that bioluminescence may be a trait specific for non-O1/non-O139 V. cholerae strains that frequently occur in certain environments. Luminescence expression patterns of V. cholerae were also investigated, and isolates could be grouped based on expression level. Several strains with defective expression of the lux operon, including natural K variants, were identified.

The study investigated the burden of acute otitis media (AOM) during the first two years of life in a cohort of 252 newborns in rural Bangladesh using data collected on occurrences of AOM. Trained community health workers (CHWs) conducted household surveillance and picked up cases of AOM using the study algorithm. The incidence rate was 0.9 episodes per child-year observed. Forty-six percent (n=115) of the 252 subjects developed AOM: 36% (n=91) during the first year of life and 10% (n=24) during the second year of life (p<0.001). The age-specific incidence rates of AOM varied; peaks occurred in the 6-12-month age-group and the lowest in the first three months of life. In total, 20% (n=49) of the study subjects had single, 26% (n=66) recurrent, and 54% (n=137) no episode of AOM. Perforation with discharge developed in 85% (n=322) of 375 episodes. The duration of discharge from the ears was < or =6 weeks in 95% of the episodes, but in 5% of the episodes, discharge from the ears continued for >6 weeks. The incidence of AOM was higher in the monsoon season compared to the summer season (p<0.003). The study documented AOM as an important cause of morbidity among rural children up to two years of age in Bangladesh and should be addressed with strategies to overcome the burden of disease.

Vibrio cholerae persists in aquatic environments predominantly in a nonculturable state. In this study coccoid, nonculturable V. cholerae O1 in biofilms maintained for 495 days in Mathbaria, Bangladesh, pond water became culturable upon animal passage. Culturability, biofilm formation, and the wbe, ctxA, and rstR2 genes were monitored by culture, direct fluorescent antibody (DFA), and multiplex PCR. DFA counts were not possible after formation of biofilm. Furthermore, wbe, but not ctxA, were amplifiable, even after incubation for 54 and 68 days at room temperature ( approximately 25 degrees C) and 4 degrees C, respectively, when no growth was detectable. Slower biofilm formation and extended culturability were observed for cultures incubated at 4 degrees C, compared with approximately 25 degrees C, suggesting biofilm production to be temperature dependent and linked to loss of culturability. Small colonies appearing after incubation in microcosms for 54 and 68 days at 25 degrees C and 4 degrees C, respectively, were wbe positive and ctxA and rstR2 negative, indicating loss of bacteriophage CTXphi. The coccoid V. cholerae O1 observed as free cells in microcosms incubated for 495 days could not be cultured, but biofilms in the same microcosms yielded culturable cells. It is concluded that biofilms can act as a reservoir for V. cholerae O1 between epidemics because of its long-term viability in biofilms. In contrast to biofilms produced in Mathbaria pond water, V. cholerae O1 in biofilms present in cholera stools and incubated under identical conditions as the Mathbaria pond water biofilms could not be cultured after 2 months, indicating that those V. cholerae cells freshly discharged into the environment are significantly less robust than cells adapted to environmental conditions.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of bacterial infection leading to acute watery diarrhea in infants and young children. Although the prevalence of ETEC is high in Bangladesh and infections can be spread through food and contaminated water, limited information is available about ETEC in the surface water. We carried out studies to isolate ETEC from surface water samples from ponds, rivers, and a lake from a site close to field areas known to have a high incidence of diarrhea in Dhaka, Bangladesh, and Matlab, Bangladesh. ETEC strains isolated from the water sources were compared with ETEC strains isolated from patients with diarrhea at two hospitals in these areas. ETEC were isolated from 30% (45 of 150) of the samples from the surface water sources and 19% (518 of 2700) of the clinical specimens. One hundred ETEC strains isolated from patients with similar phenotypes as the environmental strains were compared for phenotypic and genotypic properties. The most common O serogroups on ETEC were O6, O25, O78, O115, and O126 in both types of strains. Pulsed-field gel electrophoresis analyses of the ETEC strains showed that multiple clones of ETEC were present within each colonization factor type and that some clones detected in the environment were also isolated from the stools of patients. The strains showed multiple and similar antibiotic resistance patterns. This study shows that ETEC is prevalent in surface water sources in Bangladesh suggesting a possible reason for the endemicity of this pathogen in Bangladesh.

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.

Predicting cholera epidemics through monitoring the environment for the presence of pathogenic Vibrio cholerae is complicated by the presence in water of a large number of mostly nonpathogenic V. cholerae strains. V. cholerae strains causing recent cholera epidemics in Bangladesh carry the sulfamethoxazole-trimethoprim (SXT) element, which encodes resistance to several antibiotics. Here, we show that the use of a culture medium containing streptomycin, sulfamethoxazole, and trimethoprim (the antibiotic selection technique [AST]) can significantly enhance the isolation of environmental V. cholerae O1 with epidemic potential (P<.001). The AST was also used to monitor the recent emergence and spread of a new multiple-antibiotic-resistant strain of V. cholerae in Bangladesh. The results of this study support the hypothesis that pre-epidemic amplification of pathogenic V. cholerae occurs in the human host and leads to the start of an epidemic cycle dominated by a single clone of V. cholerae that spreads rapidly through environmental waters.

The incidence of aetiology-specific diarrhoea and the pathogenicity of infectious agents in a birth cohort (n=252) in rural Bangladesh were determined. Stool specimens or rectal swabs were collected from diarrhoeal cases over two years and routinely on a monthly basis. Stool samples from children with diarrhoea were compared with stool samples from children without diarrhoea to calculate rates of isolation and pathogenicity of agents. In total, 1750 stool specimens from diarrhoea patients and 5679 stool specimens from children without diarrhoea were tested. An infectious agent was identified in 58% of the stool specimens from diarrhoea patients and 21.6% of the stool specimens from children without diarrhoea. The most commonly-isolated pathogens from all specimens were enterotoxigenic Escherichia coli (ETEC), enteroadherent E. coli, Shigella, Campylobacter jejuni, Giardia, and rotavirus. ETEC (ST and LT-ST toxin), enterotoxigenic Bacteroides fragilis, Shigella, and rotavirus were associated more with disease than with asymptomatic infections. Aetiology-specific infections were associated with acute episodes. The isolated enteropathogens were essentially the same as those found in other tropical rural settings. Enterotoxigenic B. fragilis was also identified as a pathogen. Ongoing vaccine efforts focusing on Shigella, rotavirus, and ETEC would be useful.

The contribution of amebiasis to the burden of diarrheal disease in children and the degree to which immunity is acquired from natural infection were assessed in a 4-year prospective observational study of 289 preschool children in an urban slum in Dhaka, Bangladesh. Entamoeba histolytica infection was detected at least once in 80%, and repeat infection in 53%, of the children who completed 4 years of observation. Annually there were 0.09 episodes/child of E. histolytica-associated diarrhea and 0.03 episodes/child of E. histolytica-associated dysentery. Fecal immunoglobulin A (IgA) anti-parasite Gal/GalNAc lectin carbohydrate recognition domain (anti-CRD) was detected in 91% (183/202) of the children at least once and was associated with a lower incidence of infection and disease. We concluded that amebiasis was a substantial burden on the overall health of the cohort children. Protection from amebiasis was associated with a stool anti-CRD IgA response. The challenge of producing an effective vaccine will be to improve upon naturally acquired immunity, which does not provide absolute protection from reinfection.

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype Vibrio cholerae O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic Vibrio cholerae serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a "reemerging" disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis. 187:96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably V. cholerae). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of V. cholerae is proving useful in developing environmental models for the prediction of cholera epidemics.

The burden of enterotoxigenic Bacteroides fragilis (ETBF)-related diarrhea was determined in a birth cohort of 252 children in rural Bangladesh. Isolation rates of ETBF in stool and risk factors for acquisition of ETBF and disease were established. Of 382 B. fragilis-positive specimens, 14.4% of the strains found in them produced enterotoxin, as determined by a tissue-culture assay. The overall isolation rate of ETBF was 2.3% (40/1750) from diarrheal specimens and 0.3% (15/5679) from nondiarrheal specimens collected throughout the 2 years of the study (P < .001). ETBF was isolated from 20.3% (40/197) of the B. fragilis-positive diarrheal specimens and from 8.1% (15/185) of the B. fragilis-positive nondiarrheal specimens (P < .001) and was significantly associated with acute diarrheal disease in children > or = 1 year of age (P = .0001). The diarrheal illness was mild in nature. In conditional multivariate analyses that examined environmental and host risk factors, the presence of livestock in the household area was linked to the acquisition of ETBF (chickens, P < .05; cows, P = .06). ETBF was found to be a small but significant contributor to diarrheal disease in this rural community. Improved management of livestock may be useful for the prevention of ETBF infection.

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.

Background: The association of antibody responses with both innate and acquired immunity to amebiasis indicate that CD4+ T cells play a role in protection against Entamoeba histolytica infection. To test this hypothesis, we compared the genotype frequencies of human leukocyte antigen (HLA) class II alleles in a cohort of Bangladeshi children intensively monitored for E. histolytica infection for a 3-year period., Methods: Using logistic regression, we calculated the odds of disease by genotype and by haplotype., Results: The DQB1*0601 heterozygous and homozygous genotypes were found in 55% of E. histolytica-negative children but in only 34% of E. histolytica-positive children (overall odds ratio, 2.39; 95% confidence interval [CI], 1.26-4.54). Children who were heterozygous for the DQB1*0601/DRB1*1501 haplotype were 10.1 times (95% CI, 2.02-50.6) more likely to be both E. histolytica negative and serum anti-lectin immunoglobulin G negative at baseline. Other DQB1 and DRB1 alleles (DQB1*0202, DQB1*0301, and DRB1*0701) were not associated with any of the clinical outcomes related to amebiasis., Conclusion: A potential protective association was observed with the HLA class II allele DQB1*0601 and the heterozygous haplotype DQB1*0601/DRB1*1501. This association may explain why amebiasis does not occur in some children who are exposed to the parasite and implicates HLA class II-restricted immune responses in protection against E. histolytica infection.

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.

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 < or = 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.

Based on results of ecological studies demonstrating that Vibrio cholerae, the etiological agent of epidemic cholera, is commensal to zooplankton, notably copepods, a simple filtration procedure was developed whereby zooplankton, most phytoplankton, and particulates >20 microm were removed from water before use. Effective deployment of this filtration procedure, from September 1999 through July 2002 in 65 villages of rural Bangladesh, of which the total population for the entire study comprised approximately 133,000 individuals, yielded a 48% reduction in cholera (P < 0.005) compared with the control.

The emergence of Vibrio cholerae O139 Bengal during 1992-1993 was associated with large epidemics of cholera in India and Bangladesh and, initially, with a total displacement of the existing V. cholerae O1 strains. However, the O1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains. Since the initial emergence of the O139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized. The clinical and epidemiological characteristics of these strains have been studied. Rapid genetic reassortment in O139 strains appears to be a response to the changing epidemiology of V. cholerae O1 and also a strategy for persistence in competition with strains of the O1 serogroup. The emergence of V. cholerae O139 has provided a unique opportunity to witness genetic changes in V. cholerae that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera. The genetic changes and natural selection involving both environmental and host factors are likely to influence profoundly the genetics, epidemiology, and evolution of toxigenic V. cholerae, not only in the Ganges Delta region of India and Bangladesh, but also in other areas of endemic and epidemic cholera.

How Vibrio cholerae spreads around the world and what determines its seasonal peaks in endemic areas are not known. These features of cholera have been hypothesized to be primarily the result of environmental factors associated with aquatic habitats that can now be identified. Since 1997, fortnightly surveillance in 4 widely separated geographic locations in Bangladesh has been performed to identify patients with cholera and to collect environmental data. A total of 5670 patients (53% <5 years of age) have been studied; 14.3% had cholera (10.4% due to V. cholerae O1 El Tor, 3.8% due to O139). Both serogroups were found in all locations; outbreaks were seasonal and often occurred simultaneously. Water-use patterns showed that bathing and washing clothes in tube-well water was significantly protective in two of the sites. These data will be correlated with environmental factors, to develop a model for prediction of cholera outbreaks.

Diversity, relatedness, and ecological interactions of toxigenic Vibrio cholerae O1 populations in two distinctive habitats, the human intestine and the aquatic environment, were analyzed. Twenty environmental isolates and 42 clinical isolates were selected for study by matching serotype, geographic location of isolation in Bangladesh, and season of isolation. Genetic profiling was done by enterobacterial repetitive intergenic consensus sequence-PCR, optimized for profiling by using the fully sequenced V. cholerae El Tor N16961 genome. Five significant clonal clusters of haplotypes were found from 57 electrophoretic types. Isolates from different areas or habitats intermingled in two of the five significant clusters. Frequencies of haplotypes differed significantly only between the environmental populations (exact test; P < 0.05). Analysis of molecular variance yielded a population genetic structure reflecting the differentiating effects of geographic area, habitat, and sampling time. Although a parameter confounding the latter differences explained 9% of the total molecular variance in the entire population (P < 0.01), the net effect of habitat and time could not be separated because of the small number of environmental isolates included in the study. Five subpopulations from a single area were determined, and from these we were able to estimate a relative differentiating effect of habitat, which was small compared with the effect of temporal change. In conclusion, the resulting population structure supports the hypothesis that spatial and temporal fluctuations in the composition of toxigenic V. cholerae populations in the aquatic environment can cause shifts in the dynamics of the disease.

This paper defines high-risk areas of cholera based on environmental risk factors of the disease in an endemic area of Bangladesh. The risk factors include proximity to surface water, high population density, and low educational status, which were identified in an earlier study by the authors. Cholera data were analyzed by spatially referenced extended household units for two time periods, 1983-1987 and 1992-1996. These periods were chosen because they had different dominant cholera agents. From 1983-1987 classical cholera was dominant and from 1992-1996 El Tor was dominant. By defining high-risk areas based on risk factors, this study builds a spatial risk model for cholera. The model is then evaluated based on the locations of observed cholera cases. The study also identifies the determinants of death due to cholera for the two different time periods dominated by the different cholera agents. The modeled risk areas that were based on the risk factors were found to correspond with actual distributions of cholera morbidity and mortality. The high-risk areas of the dominant cholera agents are relatively stable over time. However, from 1983-1987 El Tor cholera, which was not the dominant agent during that period, was not associated with high-risk areas, suggesting that the El Tor habitat may have changed over time. The case fatality rate for cholera was related to proximity to a diarrhea treatment hospital in the study area.

The bacteria that cause cholera are known to be normal inhabitants of surface water, however, the environmental risk factors for different biotypes of cholera are not well understood. This study identifies environmental risk factors for cholera in an endemic area of Bangladesh using a geographic information systems (GIS) approach. The study data were collected from a longitudinal health and demographic surveillance system and the data were integrated within a geographic information system database of the research area. Two study periods were chosen because they had different dominant biotypes of the disease. From 1992 to 1996 El Tor cholera was dominant and from 1983 to 1987 classical cholera was dominant. The study found the same three risk factors for the two biotypes of cholera including proximity to surface water, high population density, and poor educational level. The GIS database was used to measure the risk factors and spatial filtering techniques were employed. These robust spatial methods are offered as an example for future epidemiological research efforts that define environmental risk factors for infectious diseases.

Entamoeba histolytica infection and colitis occurred in 55% and 4%, respectively, of a cohort of Bangladeshi preschool children observed for 2 years. DNA typing demonstrated that infecting E. histolytica isolates were genetically diverse. Innate resistance to infection in children was linked to the absence of serum anti-trophozoite IgG. Most children who lacked serum anti-trophozoite IgG failed to develop it in response to a new infection. The serum anti-trophozoite IgG response clustered in families, which is consistent with genetic inheritance. Acquired resistance to infection was linked to intestinal IgA against the carbohydrate-recognition domain of the E. histolytica galactose N-acetyl-d-galactosamine lectin. This was associated with an 86% reduction in new infection over 1 year. Amebiasis is a common and potentially serious infection in children from Dhaka, and both innate and acquired immune responses limit infection.

Despite nearly 200 years of study, the mechanisms contributing to the maintenance of endemic cholera and the causes of periodic epidemics remain poorly understood. To investigate these patterns, cholera data collected over 33 years (1966-1998) in Matlab, Bangladesh, were analyzed. Time-lagged autocorrelations were stratified by Vibrio cholerae serogroup, serotype, and biotype. Both classical and El Tor biotypes alternated and persisted between 1966 and 1988; the classical biotype disappeared by 1988, and the O139 serogroup first appeared in 1993. Both the Ogawa and Inaba serotypes circulated the entire time. The autocorrelations revealed that both Inaba and Ogawa epidemics were followed 12 months later by epidemics of the same serotype. Ogawa epidemics, however, were also followed by further Ogawa epidemics only 6 months later. Thus, epidemics of Inaba may selectively confer short-term population-level immunity for a longer period than those of Ogawa. These observations suggest that the Inaba antigen should be maximized in cholera vaccine designs.

Infections due to non-typhoid Salmonella, resistant to antibiotics, have recently emerged as an important health problem worldwide. Antibiotic resistance was studied by the disc-diffusion method among 3,876 (2.78%) non-typhoid Salmonella isolates cultured from 139,279 faecal samples in a diarrhoea treatment centre in Dhaka, Bangladesh, during 1989-1996. Of 499 salmonellae isolated in 1989, serogroup C (1.12%) was the most common, followed by Salmonella Typhi (0.72%) and serogroup B (0.71%). Isolation rate of serogroup B increased significantly to 2.18% (p < 0.01) in 1992 compared to 0.56% in 1991, 2.86% in 1995, and 2.48% in 1996. Serotyping of 194 serogroup B isolates revealed Salmonella Typhimurium (52%) and Salmonella Gloucester (45%) as predominant serotypes. Resistance to ampicillin (A), chloramphenicol (C), and trimethoprim-sulphamethoxazole (Sxt) (R type-ACSxt) increased to 89-100% during 1992-1996 from 20-28% during 1989-1991 (p < 0.01) among S. Typhimurium and S. Gloucester isolates. In 1993, 8-10% of the strains of both the serotypes, resistant to ampicillin, chloramphenicol, and trimethoprim-sulphamethoxazole, acquired resistance to ceftriaxone (Cr) (R type-ACSxtCr), which increased to 85-92% in 1996 (p < 0.01). All were susceptible to ciprofloxacin. A 157-kb conjugative plasmid transferred R type-ACSxt from both the serotypes to Escherichia coli K-12. The findings of the study suggest the emergence of multidrug-resistant S. Gloucester and S. Typhimurium for the first time as a significant health problem in Bangladesh, and surveillance is essential to monitor the resistant non-typhoid Salmonella and identify its sources and modes of transmission.