Genetic relatedness of Vibrio cholerae isolates within and between households during outbreaks in Dhaka, Bangladesh.

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.

Rapid dipstick detection of Vibrio cholerae in household stored and municipal water in Dhaka, Bangladesh: CHoBI7 trial.

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.

Mouthing of Soil Contaminated Objects is Associated with Environmental Enteropathy in Young Children.

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.

Modelling stunting in LiST: the effect of applying smoothing to linear growth data.

BACKGROUND: The Lives Saved Tool (LiST) is a widely used resource for evidence-based decision-making regarding health program scale-up in low- and middle-income countries. LiST estimates the impact of specified changes in intervention coverage on mortality and stunting among children under 5 years of age. We aimed to improve the estimates of the parameters in LiST that determine the rate at which the effects of interventions to prevent stunting attenuate as children get older. METHODS: We identified datasets with serial measurements of children's lengths or heights and used random effects models and restricted cubic splines to model the growth trajectories of children with at least six serial length/height measurements. We applied WHO growth standards to both measured and modelled (smoothed) lengths/heights to determine children's stunting status at multiple ages (1, 6, 12, 24 months). We then calculated the odds ratios for the association of stunting at one age point with stunting at the next ("stunting-to-stunting ORs") using both measured and smoothed data points. We ran analyses in LiST to compare the impact on intervention effect attenuation of using smoothed rather than measured stunting-to-stunting ORs. RESULTS: A total of 21,786 children with 178,786 length/height measurements between them contributed to our analysis. The odds of stunting at a given age were strongly related to whether a child is stunted at an earlier age, using both measured and smoothed lengths/heights, although the relationship was stronger for smoothed than measured lengths/heights. Using smoothed lengths/heights, we estimated that children stunted at 1 month have 45 times the odds of being stunted at 6 months, with corresponding odds ratios of 362 for the period 6 to 12 months and 175 for the period 12 to 24 months. Using the odds ratios derived from the smoothed data in LiST resulted in a somewhat slower attenuation of intervention effects over time, but substantial attenuation was still observed in the LiST outputs. For example, in Mali the effect of effectively eliminating SGA births reduced prevalence of stunting at age 59 months from 44.4% to 43.7% when using odds ratios derived from measured lengths/heights and from 44.4% to 41.9% when using odds ratios derived from smoothed lengths/heights. CONCLUSIONS: Smoothing of children's measured lengths/heights increased the strength of the association between stunting at a given age and stunting at an earlier age. Using odds ratios based on smoothed lengths/heights in LiST resulted in a small reduction in the attenuation of intervention effects with age and thus some increase in the estimated benefits, and may better reflect the true benefits of early nutritional interventions.

Occurrence in Mexico, 1998-2008, of Vibrio cholerae CTX+ El Tor carrying an additional truncated CTX prophage.

The seventh cholera pandemic caused by Vibrio cholerae O1 El Tor (ET) has been superseded in Asia and Africa by altered ET possessing the cholera toxin (CTX) gene of classical (CL) biotype. The CL biotype of V. cholerae was isolated, along with prototypic and altered ET, during the 1991 cholera epidemic in Mexico and subsequently remained endemic until 1997. Microbiological, molecular, and phylogenetic analyses of clinical and environmental V. cholerae isolated in Mexico between 1998 and 2008 revealed important genetic events favoring predominance of ET over CL and altered ET. V. cholerae altered ET was predominant after 1991 but not after 2000. V. cholerae strains isolated between 2001 and 2003 and a majority isolated in 2004 lacked CTX prophage (Φ) genes encoding CTX subunits A and B and repeat sequence transcriptional regulators of ET and CL biotypes: i.e., CTXΦ(-). Most CTXΦ(-) V. cholerae isolated in Mexico between 2001 and 2003 also lacked toxin coregulated pili tcpA whereas some carried either tcpA(ET) or a variant tcpA with noticeable sequence dissimilarity from tcpA(CL). The tcpA variants were not detected in 2005 after CTXΦ(+) ET became dominant. All clinical and environmental V. cholerae O1 strains isolated during 2005-2008 in Mexico were CTXΦ(+) ET, carrying an additional truncated CTXΦ instead of RS1 satellite phage. Despite V. cholerae CTXΦ(-) ET exhibiting heterogeneity in pulsed-field gel electrophoresis patterns, CTXΦ(+) ET isolated during 2004-2008 displayed homogeneity and clonal relationship with V. cholerae ET N16961 and V. cholerae ET isolated in Peru.

Randomized Controlled Trial of Hospital-Based Hygiene and Water Treatment Intervention (CHoBI7) to Reduce Cholera.

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.

Unsafe Child Feces Disposal is Associated with Environmental Enteropathy and Impaired Growth.

OBJECTIVE: To investigate the relationship between unsafe child feces disposal, environmental enteropathy, and impaired growth, we conducted a prospective cohort study of 216 young children in rural Bangladesh. STUDY DESIGN: Using a prospective cohort study design in rural Bangladesh, unsafe child feces disposal, using the Joint Monitoring Program definition, was assessed using 5-hour structured observation by trained study personnel as well as caregiver reports. Anthropometric measurements were collected at baseline and at a 9-month follow-up. Stool was analyzed for fecal markers of environmental enteropathy: alpha-1-antitrypsin, myeloperoxidase, neopterin (combined to form an environmental enteropathy disease activity score), and calprotectin. FINDINGS: Among 216 households with young children, 84% had an unsafe child feces disposal event during structured observation and 75% had caregiver reported events. There was no significant difference in observed unsafe child feces disposal events for households with or without an improved sanitation option (82% vs 85%, P = .72) or by child's age (P = .96). Children in households where caregivers reported unsafe child feces disposal had significantly higher environmental enteropathy scores (0.82-point difference, 95% CI 0.11-1.53), and significantly greater odds of being wasted (weight-for-height z score <-2 SDs) (9% vs 0%, P = .024). In addition, children in households with observed unsafe feces disposal had significantly reduced change in weight-for-age z-score (-0.34 [95% CI -0.68, -0.01] and weight-for-height z score (-0.52 [95% CI -0.98, -0.06]). CONCLUSION: Unsafe child feces disposal was significantly associated with environmental enteropathy and impaired growth in a pediatric population in rural Bangladesh. Interventions are needed to reduce this high-risk behavior to protect the health of susceptible pediatric populations.

Geophagy Is Associated with Growth Faltering in Children in Rural Bangladesh.

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.

Shigella Infections in Household Contacts of Pediatric Shigellosis Patients in Rural Bangladesh.

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.

Genetic variation of Vibrio cholerae during outbreaks, Bangladesh, 2010-2011.

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.

Catch-up growth occurs after diarrhea in early childhood.

Diarrhea and linear growth faltering continue to burden low-income countries and are among the most important contributors to poor health during early childhood. Diarrhea is thought to adversely affect linear growth, but catch-up growth can occur if no additional insults are experienced. We sought to characterize catch-up growth in relation to diarrhea burden in a multisite dataset of 1007 children. Using longitudinal anthropometry and diarrheal surveillance data from 7 cohort studies in 4 countries, we examined the relation between diarrhea prevalence and growth in 3- to 6-mo periods using linear mixed-effect models. Growth during each period was calculated as a function of age using linear splines. We incorporated the longitudinal prevalence of diarrhea in both current and previous periods into the model. Diarrhea during the current period was associated with slower linear and ponderal growth. Faster (catch-up) growth in length was observed in children with no diarrhea in age groups immediately after an age group in which diarrhea was experienced [age group >6-12 mo: 0.03 mm/mo for each percentage diarrhea prevalence in the previous period (95% CI: 0.007, 0.06) relative to 11.3 mm/mo mean growth rate; age group >12-18 mo: 0.04 mm/mo (95% CI: 0.02, 0.06) relative to 8.9 mm/mo mean growth rate; age group >18-24 mo: 0.04 mm/mo (95% CI: 0.003, 0.09) relative to 7.9 mm/mo mean growth rate]. The associations were stronger in boys than in girls when separate models were run. Similar results were observed when weight was the outcome variable. When diarrheal episodes are followed by diarrhea-free periods in the first 2 y of life, catch-up growth is observed that may allow children to regain their original trajectories. The finding of a greater effect of diarrhea on linear growth in boys than in girls was unexpected and requires additional study. Diarrhea burdens are high throughout the first 2 y of life in these study sites, therefore reducing the likelihood of catch-up growth. Extending diarrhea-free periods may increase the likelihood of catch-up growth and decrease the prevalence of stunting.

Cholera outbreaks (2012) in three districts of Nepal reveal clonal transmission of multi-drug resistant Vibrio cholerae O1.

BACKGROUND: Although endemic cholera causes significant morbidity and mortality each year in Nepal, lack of information about the causal bacterium often hinders cholera intervention and prevention. In 2012, diarrheal outbreaks affected three districts of Nepal with confirmed cases of mortality. This study was designed to understand the drug response patterns, source, and transmission of Vibrio cholerae associated with 2012 cholera outbreaks in Nepal. METHODS: V. cholerae (n = 28) isolated from 2012 diarrhea outbreaks {n = 22; Kathmandu (n = 12), Doti (n = 9), Bajhang (n = 1)}, and surface water (n = 6; Kathmandu) were tested for antimicrobial response. Virulence properties and DNA fingerprinting of the strains were determined by multi-locus genetic screening employing polymerase chain reaction, DNA sequencing, and pulsed-field gel electrophoresis (PFGE). RESULTS: All V. cholerae strains isolated from patients and surface water were confirmed to be toxigenic, belonging to serogroup O1, Ogawa serotype, biotype El Tor, and possessed classical biotype cholera toxin (CTX). Double-mismatch amplification mutation assay (DMAMA)-PCR revealed the V. cholerae strains to possess the B-7 allele of ctx subunit B. DNA sequencing of tcpA revealed a point mutation at amino acid position 64 (N → S) while the ctxAB promoter revealed four copies of the tandem heptamer repeat sequence 5'-TTTTGAT-3'. V. cholerae possessed all the ORFs of the Vibrio seventh pandemic island (VSP)-I but lacked the ORFs 498-511 of VSP-II. All strains were multidrug resistant with resistance to trimethoprim-sulfamethoxazole (SXT), nalidixic acid (NA), and streptomycin (S); all carried the SXT genetic element. DNA sequencing and deduced amino acid sequence of gyrA and parC of the NAR strains (n = 4) revealed point mutations at amino acid positions 83 (S → I), and 85 (S → L), respectively. Similar PFGE (NotI) pattern revealed the Nepalese V. cholerae to be clonal, and related closely with V. cholerae associated with cholera in Bangladesh and Haiti. CONCLUSIONS: In 2012, diarrhea outbreaks in three districts of Nepal were due to transmission of multidrug resistant V. cholerae El Tor possessing cholera toxin (ctx) B-7 allele, which is clonal and related closely with V. cholerae associated with cholera in Bangladesh and Haiti.

Diarrhea in early childhood: short-term association with weight and long-term association with length.

The short-term association between diarrhea and weight is well-accepted, but the long-term association between diarrhea and growth is less clear. Using data from 7 cohort studies (Peru, 1985-1987; Peru, 1989-1991; Peru, 1995-1998; Brazil, 1989-1998; Guinea-Bissau, 1987-1990; Guinea-Bissau, 1996-1997; and Bangladesh, 1993-1996), we evaluated the lagged relationship between diarrhea and growth in the first 2 years of life. Our analysis included 1,007 children with 597,638 child-days of diarrhea surveillance and 15,629 anthropometric measurements. We calculated the associations between varying diarrhea burdens during lagged 30-day periods and length at 24 months of age. The cumulative association between the average diarrhea burden and length at age 24 months was -0.38 cm (95% confidence interval: -0.59, -0.17). Diarrhea during the 30 days prior to anthropometric measurement was consistently associated with lower weight at most ages, but there was little indication of a short-term association with length. Diarrhea was associated with a small but measurable decrease in linear growth over the long term. These findings support a focus on prevention of diarrhea as part of an overall public health strategy for improving child health and nutrition; however, more research is needed to explore catch-up growth and potential confounders.

Distribution of virulence genes in clinical and environmental Vibrio cholerae strains in Bangladesh.

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.

Wasting is associated with stunting in early childhood.

The longitudinal relationship between stunting and wasting in children is poorly characterized. Instances of wasting or poor weight gain may precede linear growth retardation. We analyzed longitudinal anthropometric data for 1599 children from 8 cohort studies to determine the effect of wasting [weight-for-length Z-score (WLZ) < -2] and variability in WLZ in the first 17 mo on length-for-age Z-score (LAZ) at 18-24 mo of age. In addition, we considered the effects of change in WLZ during the previous 6-mo period on length at 18 and 24 mo. Wasting at 6-11 or 12-17 mo was associated with decreased LAZ; however, children who experienced wasting only at 0-5 mo did not suffer any long-term growth deficits compared with children with no wasting during any period. Children with greater WLZ variability (≥0.5 SD) in the first 17 mo of life were shorter [LAZ = -0.51 SD (95% CI: -0.67, -0.36 SD)] at 18-24 mo of age than children with WLZ variability <0.5. Change in WLZ in the previous 6-mo period was directly associated with greater attained length at 18 mo [0.33 cm (95% CI: 0.11, 0.54 cm)] and 24 mo [0.72 cm (95% CI: 0.52, 0.92 cm)]. Children with wasting, highly variable WLZ, or negative changes in WLZ are at a higher risk for linear growth retardation, although instances of wasting may not be the primary cause of stunting in developing countries.

Role of zooplankton diversity in Vibrio cholerae population dynamics and in the incidence of cholera in the Bangladesh Sundarbans.

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.

The discovery of cholera - like enterotoxins produced by Escherichia coli causing secretory diarrhoea in humans.

Non-vibrio cholera has been recognized as a clinical entity for as long as cholera was known to be caused by Vibrio cholerae. Until 1968, the aetiologic agent of this syndrome was not known. Following a series of studies in patients with non-vibrio cholera it was found that these patients had large concentrations of Escherichia coli in the small bowel and stools which produced cholera toxin-like enterotoxins, and had fluid and electrolyte transport abnormalities in the small bowel similar to patients with documented cholera. Furthermore, these patients developed antibodies to the cholera-like enterotoxin. Later studies showed that these strains, when fed to volunteers produced a cholera-like disease and that two enterotoxins were found to be produced by these organisms: a heat-labile enterotoxin (LT) which is nearly identical to cholera toxin, and a heat-stable enterotoxin (ST), a small molecular weight polypeptide. E. coli that produced one or both of these enterotoxins were designated enterotoxigenic E. coli (ETEC). ETEC are now known not only to cause a severe cholera-like illness, but to be the most common bacterial cause of acute diarrhoea in children in the developing world, and to be the most common cause of travellers' diarrhoea in persons who visit the developing world.

Environmental signatures associated with cholera epidemics.

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.

Risk factors and case management of acute diarrhoea in North Gondar Zone, Ethiopia.

In Ethiopia, evidence is lacking about maternal care-taking and environmental risk factors that contribute to acute diarrhoea and the case management of diarrhoea. The aim of this study was to identify the risk factors and to understand the management of acute diarrhoea. A pretested structured questionnaire was used for interviewing mothers of 440 children in a prospective, matched, case-control study at the University of Gondar Referral and Teaching Hospital in Gondar, Ethiopia. Results of multivariate analysis demonstrated that children who were breastfed and not completely weaned and mothers who were farmers were protective factors; risk factors for diarrhoea included sharing drinking-water and introducing supplemental foods. Children presented with acute diarrhoea for 3.9 days with 4.3 stools per day. Mothers usually did not increase breastmilk and other fluids during diarrhoea episodes and generally did not take children with diarrhoea to traditional healers. Incorporating messages about the prevention and treatment of acute diarrhoea into child-health interventions will help reduce morbidity and mortality associated with this disease.

Serogroup, virulence, and genetic traits of Vibrio parahaemolyticus in the estuarine ecosystem of 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.