Abundance and biofilm formation capability of Vibrio cholerae in aquatic environment with an emphasis on Hilsha fish (Tenualosa ilisha).

The potentially deadly and sporadic diarrhea-causing agent, Vibrio cholerae, is present in a great number in the freshwater aquatic environment and can be transmitted to humans by different aquatic organisms. In the perspective of Bangladesh, an anadromous fish species Hilsha (Tenualosa ilisha) can act as a transmission vehicle of V. cholerae from the aquatic to the household kitchen environment. The present study was carried out to investigate the presence of V. cholerae in the aquatic habitat of Bangladesh with a major emphasis on freshly caught Hilsha fish, along with river water and plankton samples from the fish capture site. The study also detected the biofilm formation capability of V. cholerae within Hilsha fish that might help the transmission and persistence of the pathogen in aquatic habitat. Twenty out of 65 freshly caught fish (30.8%) and 1 out of 15 water samples (6.67%) showed the presence of V. cholerae and none of the plankton samples were positive for V. cholerae. The isolated strains were identified as non-O1 and non-O139 serogroups of V. cholerae and contain some major toxin and virulence genes. A few strains showed cellular cytotoxicity on the HeLa cell line. All strains were able to form biofilm on the microtiter plate and the detection of three genes related to biofilm formation (vpsA, vpsL, and vpsR) were also assayed using qPCR. In this study, the in vitro biofilm formation ability of the isolated strains may indicate the long-term persistence of V. cholerae in different parts of Hilsha fish. The abundance of V. cholerae only in freshly caught Hilsha fish and the absence of the pathogen in the surrounding aquatic environment could stipulate the role of Hilsha fish as one of the major transmission routes of V. cholerae from the freshwater aquatic environment of Bangladesh to the household kitchen environment.

Individual and household exposures associated with cholera transmission in case-control studies: a systematic review.

OBJECTIVE: Uncertainty persists regarding cholera transmission routes. We conducted a structured review of case-control studies on cholera transmission and provide a qualitative summary of reported exposures in order to inform public health efforts and future cholera research. METHODS: We searched two electronic databases for published case-control studies that investigated risk factors for cholera and included any publications that did not match our exclusion criteria. From the included studies, we grouped exposures using two parameters, whether transmission domain was public or domestic, and also on the vehicle of transmission. We extracted data on study location, method of case and control inclusion, type of statistical analysis performed and which exposures were included. Additionally, two parallel subgroup analyses were performed. The first included the subgroup of all studies that used culture-confirmed cholera cases, and the second included the subgroup of all studies employing a multivariate analysis. In the second analysis, we calculated the population attributable risk (PAR). RESULTS: Our search yielded 2347 peer-reviewed publications, of which 65 did not match our exclusion criteria, comprising 69 individual studies. Water-based exposures were investigated in 97% of these studies, of which 70% found a significant association with cholera infection. Food-based exposures were investigated in 75% of studies, of which 63% found a significant association with risk of cholera infection. Close personal contact with cholera cases was investigated in 30% of studies, of which 52% found a significant association with risk of cholera infection. Hygiene-related exposures were investigated in 51% of studies, of which 63% found a significant association with cholera transmission. Among studies that examined at least one exposure related to the domestic domain, 76% found a significant association with cholera infection vs. 71% of studies investigating at least one public domain exposure. The subgroup analyses produced similar results. CONCLUSIONS: Despite strong evidence for cholera transmission via foodborne-, hygiene-, waterborne- and close personal contact-related pathways in both domestic and public domains, we found that non-waterborne-related factors are understudied. Future cholera case-control studies would benefit from investigating all transmission vehicles and transmission domains.

OBJECTIF: L'incertitude persiste quant aux voies de transmission du choléra. Nous avons mené une analyse structurée d'études cas-témoins sur la transmission du choléra et fourni un résumé qualitatif des expositions rapportées afin d'éclairer les efforts de santé publique et les recherches futures sur le choléra. MÉTHODES: Nous avons recherché dans deux bases de données électroniques, des études cas-témoins publiées, portant sur les facteurs de risque du choléra et avons inclus toutes les publications ne correspondant pas à nos critères d'exclusion. Parmi les études incluses, nous avons regroupé les expositions en utilisant deux paramètres, selon que le domaine de transmission soit public ou domestique, ainsi que sur le véhicule de transmission. Nous avons extrait les données sur le lieu de l'étude, la méthode d'inclusion des cas et des témoins, le type d'analyse statistique effectuée et les expositions incluses. En outre, deux analyses parallèles de sous-groupes ont été effectuées. La première comprenait le sous-groupe de toutes les études utilisant des cas de choléra confirmés par culture et la seconde, le sous-groupe de toutes les études utilisant une analyse multivariée. Dans la seconde analyse, nous avons calculé le risque attribuable à la population. RÉSULTATS: Notre recherche a donné 2347 publications revues par des pairs, dont 65 ne correspondaient pas à nos critères d'exclusion et comprenant 69 études individuelles. Les expositions à base d'eau ont été investiguées dans 97% de ces études, parmi lesquelles 70% ont trouvé une association significative avec l'infection par le choléra. Les expositions d'origine alimentaire ont été investiguées dans 75% des études, dont 63% ont montré une association significative avec le risque d'infection par le choléra. Le contact personnel rapproché avec des cas de choléra a été investigué dans 30% des études, parmi lesquelles 52% ont trouvé une association significative avec le risque d'infection par le choléra. Les expositions liées à l'hygiène ont été étudiées dans 51% des études, dont 63% ont montré une association significative avec la transmission du choléra. Parmi les études portant sur au moins une exposition liée au domaine domestique, 76% ont trouvé une association significative avec l'infection par le choléra, contre 71% des études portant sur au moins une exposition du domaine public. Les analyses de sous-groupes ont produit des résultats similaires. CONCLUSIONS: Malgré de solides preuves sur la transmission du choléra par des voies d'origine alimentaire, hygiénique, d'origine hydrique et de contact rapproché, dans les domaines domestique et public, nous avons constaté que les facteurs non liés à l'eau étaient peu étudiés. Les futures études cas-témoins sur le choléra gagneraient à investiguer sur tous les véhicules et tous les domaines de transmission.

A Comparative Analysis of Vibrio cholerae Contamination in Point-of-Drinking and Source Water in a Low-Income Urban Community, Bangladesh.

Bangladesh is a cholera endemic country with a population at high risk of cholera. Toxigenic and non-toxigenic Vibrio cholerae (V. cholerae) can cause cholera and cholera-like diarrheal illness and outbreaks. Drinking water is one of the primary routes of cholera transmission in Bangladesh. The aim of this study was to conduct a comparative assessment of the presence of V. cholerae between point-of-drinking water and source water, and to investigate the variability of virulence profile using molecular methods of a densely populated low-income settlement of Dhaka, Bangladesh. Water samples were collected and tested for V. cholerae from "point-of-drinking" and "source" in 477 study households in routine visits at 6 week intervals over a period of 14 months. We studied the virulence profiles of V. cholerae positive water samples using 22 different virulence gene markers present in toxigenic O1/O139 and non-O1/O139 V. cholerae using polymerase chain reaction (PCR). A total of 1,463 water samples were collected, with 1,082 samples from point-of-drinking water in 388 households and 381 samples from 66 water sources. V. cholerae was detected in 10% of point-of-drinking water samples and in 9% of source water samples. Twenty-three percent of households and 38% of the sources were positive for V. cholerae in at least one visit. Samples collected from point-of-drinking and linked sources in a 7 day interval showed significantly higher odds (P < 0.05) of V. cholerae presence in point-of-drinking compared to source [OR = 17.24 (95% CI = 7.14-42.89)] water. Based on the 7 day interval data, 53% (17/32) of source water samples were negative for V. cholerae while linked point-of-drinking water samples were positive. There were significantly higher odds (p < 0.05) of the presence of V. cholerae O1 [OR = 9.13 (95% CI = 2.85-29.26)] and V. cholerae O139 [OR = 4.73 (95% CI = 1.19-18.79)] in source water samples than in point-of-drinking water samples. Contamination of water at the point-of-drinking is less likely to depend on the contamination at the water source. Hygiene education interventions and programs should focus and emphasize on water at the point-of-drinking, including repeated cleaning of drinking vessels, which is of paramount importance in preventing cholera.

Transmission and Toxigenic Potential of Vibrio cholerae in Hilsha Fish (Tenualosa ilisha) for Human Consumption in Bangladesh.

Fish have been considered natural reservoirs of Vibrio cholerae, the deadly diarrheal pathogen. However, little is known about the role of fish in the transmission of V. cholerae from the Bay of Bengal to the households of rural and urban Bangladesh. This study analyzes the incidence and pathogenic potential of V. cholerae in Hilsha (Tenualosa ilisha), a commonly caught and consumed fish that exhibits a life cycle in both freshwater and marine environments in Bangladesh. During the period from October 2014 to October 2015, samples from the gills, recta, intestines, and scale swabs of a total of 48 fish were analyzed. The fish were collected both at local markets in the capital city Dhaka and directly from fishermen at the river. PCR analysis by targeting V. cholerae species-specific ompW gene revealed that 39 of 48 (81%) fish were positive in at least one of the sample types. Real-time PCR analysis demonstrated that the cholera-causing ctxA gene was detected in 20% (8 of 39) of V. cholerae-positive fish. A total of 158 V. cholerae isolates were obtained which were categorized into 35 genotypic groups. Altogether, 25 O1 and 133 non-O1/O139 strains were isolated, which were negative for the cholera toxin gene. Other pathogenic genes such as stn/sto, hlyA, chxA, SXT, rtxC, and HA-P were detected. The type three secretion system gene cluster (TTSS) was present in 18% (24 of 133) of non-O1/O139 isolates. The antibiotic susceptibility test revealed that the isolates conferred high resistance to sulfamethoxazole-trimethoprim and kanamycin. Both O1 and non-O1/O139 strains were able to accumulate fluid in rabbit ileal loops and caused distinctive cell death in HeLa cell. Multilocus sequence typing (MLST) showed clonal diversity among fish isolates with pandemic clones. Our data suggest a high prevalence of V. cholerae in Hilsha fish, which indicates that this fish could serve as a potential vehicle for V. cholerae transmission. Moreover, the indigenous V. cholerae strains isolated from Hilsha fish possess considerable virulence potential despite being quite diverse from current epidemic strains. This represents the first study of the population structure of V. cholerae associated with fish in Bangladesh.

Cholera Epidemics of the Past Offer New Insights Into an Old Enemy.

Background: Although cholera is considered the quintessential long-cycle waterborne disease, studies have emphasized the existence of short-cycle (food, household) transmission. We investigated singular Danish cholera epidemics (in 1853) to elucidate epidemiological parameters and modes of spread. Methods: Using time series data from cities with different water systems, we estimated the intrinsic transmissibility (R0). Accessing cause-specific mortality data, we studied clinical severity and age-specific impact. From physicians' narratives we established transmission chains and estimated serial intervals. Results: Epidemics were seeded by travelers from cholera-affected cities; initial transmission chains involving household members and caretakers ensued. Cholera killed 3.4%-8.9% of the populations, with highest mortality among seniors (16%) and lowest in children (2.7%). Transmissibility (R0) was 1.7-2.6 and the serial interval was estimated at 3.7 days (95% confidence interval, 2.9-4.7 days). The case fatality ratio (CFR) was high (54%-68%); using R0 we computed an adjusted CFR of 4%-5%. Conclusions: Short-cycle transmission was likely critical to early secondary transmission in historic Danish towns. The outbreaks resembled the contemporary Haiti outbreak with respect to transmissibility, age patterns, and CFR, suggesting a role for broader hygiene/sanitation interventions to control contemporary outbreaks.

The importance of thinking beyond the water-supply in cholera epidemics: A historical urban case-study.

BACKGROUND: Planning interventions to respond to cholera epidemics requires an understanding of the major transmission routes. Interrupting short-cycle (household, foodborne) transmission may require different approaches as compared long-cycle (environmentally-mediated/waterborne) transmission. However, differentiating the relative contribution of short- and long-cycle routes has remained difficult, and most cholera outbreak control efforts focus on interrupting long-cycle transmission. Here we use high-resolution epidemiological and municipal infrastructure data from a cholera outbreak in 1853 Copenhagen to explore the relative contribution of short- and long-cycle transmission routes during a major urban epidemic. METHODOLOGY/PRINCIPAL FINDINGS: We fit a spatially explicit time-series meta-population model to 6,552 physician-reported cholera cases from Copenhagen in 1853. We estimated the contribution of long-cycle waterborne transmission between neighborhoods using historical municipal water infrastructure data, fitting the force of infection from hydraulic flow, then comparing model performance. We found the epidemic was characterized by considerable transmission heterogeneity. Some neighborhoods acted as localized transmission hotspots, while other neighborhoods were less affected or important in driving the epidemic. We found little evidence to support long-cycle transmission between hydrologically-connected neighborhoods. Collectively, these findings suggest short-cycle transmission was significant. CONCLUSIONS/SIGNIFICANCE: Spatially targeted cholera interventions, such as reactive vaccination or sanitation/hygiene campaigns in hotspot neighborhoods, would likely have been more effective in this epidemic than control measures aimed at interrupting long-cycle transmission, such as improving municipal water quality. We recommend public health planners consider programs aimed at interrupting short-cycle transmission as essential tools in the cholera control arsenal.

Survival of Ascaris eggs and hygienic quality of human excreta in Vietnamese composting latrines.

BACKGROUND: For centuries farmers in Vietnam have fertilized their fields with human excreta collected directly from their household latrines. Contrary to the official guideline of six-month storage, the households usually only store human excreta for three to four months before use, since this is the length of time that farmers have available to produce fertilizer between two cropping seasons. This study aimed to investigate whether hygienically safe fertilizer could be produced in the latrines within this period of time. METHODS: By inoculating eggs of the helminth parasite indicator Ascaris suum into heaps of human excreta, a die-off experiment was conducted under conditions similar to those commonly used in Vietnamese latrines. Half a ton of human excreta was divided into five heaps containing increasing concentrations of lime from 0% to 11%. RESULTS: Regardless of the starting pH, which varied from 9.4 to 11.6, a >99% die-off of eggs was obtained after 105 to 117 days of storage for all lime concentrations and 97% of eggs were non-viable after 88 days of storage. The most critical parameter found to determine the die-off process was the amount of ammonia (urine) in the excreta which indicates that longer storage periods are needed for parasite egg die-off if urine is separated from the excreta. CONCLUSION: By inactivating >99% of all A. suum eggs in human excreta during a storage period of only three months the commonly used Double Vault Composting (DVC) latrine, in which urine is not separated, could therefore potentially provide a hygienic acceptable fertilizer.