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The microbiological quality of water is usually assessed by fecal coliform bacteria, and the presence of E. coli as an indicator of fecal contamination is widely recommended by international guidelines. This study aimed to assess the prevalence of diarrheagenic pathogens, in both public and personal domain water sources and examine the reliance on the WHO drinking water risk assessment guidelines. This study was conducted in a low-income urban community in Dhaka, Bangladesh between September 2014 and October 2015. Polymerase chain reaction (PCR) was used to detect the marker and virulence genes of Escherichia coli , Vibrio cholerae , Salmonella species, and Campylobacter species, and the culture method was employed for the quantitative assessment of E. coli . According to the WHO guidelines, 48% of the public domain source water and 21% of the personal domain point-of-drinking water were classified in the low-risk group, i.e., 0 CFU of E. coli /100 mL. However, when using PCR, we detected pathogens in 39% (14/36) of the point-of-drinking water samples and 65% (74/114) of the public domain water source samples classified in the low-risk group. Our study showed that relying solely on E. coli detection as a measure of water quality may overlook the presence of other pathogens in the drinking water. In addition to the culture-based method, the detection of virulence genes by PCR should also be considered to add more scrutiny to the detection of diverse types of pathogens.

The water sanitation and hygiene (WASH) sector has provided beneficiaries in low and middle-income countries with latrines and clean water for decades. However, we still need good evidence documenting the expected health impact. This paper investigates why we lack this evidence and ways to move forward. Using mTEC agar, we monitored E. coli contamination on selected "hotspot" surfaces within the kitchen environments of 32 low-income households in Dhaka, Bangladesh, every six weeks for two years. Despite being washed, the highest average contamination was found on food plates, at 253 cfu/10 cm 2 , followed by cutting knives, with 240 cfu/10 cm 2 . The drinking vessel surfaces and the latrine doorknobs had the lowest contaminations, with E. coli means of 167 and 73 cfu/10 cm 2 , respectively. These findings imply a need to measure an individual's pathogen exposure as close to the mouth as possible to estimate the true pathogen exposure. The paper proposes introducing the new "personal domain"-the point of consumption-as the physical sphere in which WASH interventions should be assessed. With this approach, we can observe and quantify the different pathogen exposure routes and, with this, further improve WASH interventions.

There is a paucity of recent research on direct water quantity measurement for personal and domestic hygiene. We aimed to measure the water quantity used for personal and domestic hygiene and to explore the reasons and determinants for variation of water usage. We conducted this study from September 2014 to June 2016 in a low-income urban community in Dhaka. In 12 households, the team conducted a day-long bimonthly ethnographic observation for one year to measure the volume of water used per activity per person. They conducted 28 in-depth interviews to explore the reasons for changes of water usage. Participants used a median of 75 L (61-100) of water per capita per day (LCPD) and of this 75 LCPD they used a median of 39 LCPD (26-58) for personal hygiene. Women used less water than men. Individual and social norms, beliefs, and weather determinants determined personal hygiene. Water availability determined domestic hygiene (e.g., washing dishes, toilets and bathrooms). This study helps to elucidate a range of determinants of water usage of the participants from the participants' perspective. The quantity of water used for domestic and personal hygiene and its relationship to fecal-oral transmitted disease can be explored in future research.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dey, Hossain, Akhter, Jensen and Begum.)

Improving hygiene practices is considered to be the single most cost-effective means of reducing the global health burden of infectious diseases. Hygiene promotion and disease prevention interventions often portray and promote "hygiene" from a biomedical perspective, which may not be optimally effective for achieving their goal of changing people's behaviors. This study aimed to educe the meaning of hygiene for the residents of a low-income community in Bangladesh and how that meaning shapes their personal hygiene practices. We conducted this study in the Tongi township in Dhaka, Bangladesh, from September 2014 to June 2016. The research team purposively selected 24 households. The team conducted day-long observations using the participant observation approach and in-depth interviews with specific members of the 24 households. The concept of "hygiene" had two separate meanings to the study participants: cleanliness and holiness. The participants reported that cleanliness was required to remove odors, grease, hot spices and dirt. The motivation for cleanliness was to feel fresh, avoid heavy feelings, feel light and feel comfortable. To maintain the holiness of the body, bathing and ablution needed to be performed following particular religious rules/rituals. The motivation of holiness was derived from their accountability to God. The participants also reported that the cleansing processes and methods for the body and the home for cleanliness reasons were also different from those for holiness reasons. The notion of "hygiene" was multidimensional for the residents of the low-income urban community in Bangladesh. Our study participants did not explicitly conceptualize a notion of hygiene that was based on the germ theory of diseases but rather a notion that was based on individual physical comfort and cultural belief systems. Future studies on the prevention of hygiene-related diseases should combine and link the biomedical aspect to religious and cultural rituals to promote improved hygiene practices.

The minimal health impact observed in large-scale water sanitation and hygiene (WASH) intervention studies motivated us to investigate the contribution of contaminated food and drinking water to the total daily Escherichia coli load ingested by the average adult in a low-income, urban area. Leftover food (food left at room temperature for more than 6 hours) from 32 households was collected eight times at 6-week intervals in 2014-2015 in the low-income area of Arichpur, Dhaka, Bangladesh. In total, 117 samples were obtained from four food types: fish, lentils, rice, and vegetables, which comprise approximately 85.2% of the average adult's personal daily food consumption. Samples were analyzed for E. coli using selective chromogenic media. For an average adult, the daily consumption of the four food types at mean contamination levels of E. coli can contribute 4.45 log colony-forming units (cfu)/day (95% confidence interval 4.06-4.84). Drinking water quality was measured 211 times at the point of drinking, with a mean, median, and maximum contamination of 1.9, 1.2, and 2.82 log E. coli cfu/100 mL, respectively. If the typical adult in Arichpur was able to drink water with 0 E. coli cfu/100 mL, it would only remove < 5.2% of the total E. coli ingested per day with a mean-contaminated diet. These approximations may suggest why insignificant effects have been observed for water quality interventions in similar, low-hygiene settings. In Arichpur, the E. coli contribution from drinking water to the total E. coli load was insufficient to exert a substantial effect.

This study collected rectal swabs from diarrheal patients and in-house environmental samples from low-income households in Dhaka City, Bangladesh, over a 4-month period and investigated these to determine the domestic transmission pathways of Escherichia coli-associated diarrhea. The environmental samples included swabs from four frequently touched surfaces, drinking water and food. Both the rectal swabs and environmental samples were examined for virulence genes characteristic of diarrheagenic E. coli pathotypes by PCR. In addition, each sample was cultured for E. coli, and the strains were analyzed for virulence profile and subjected to multilocus sequence typing (MLST). The results showed that 31% (73 of 233) of all samples including rectal swabs and household samples were positive for one or more of the diarrheagenic E. coli virulence factors. PCR analyses showed that 28% (10/36) of the rectal swabs, 43% (58/136) of household swabs, 9% (3/32) of the food, and 7% (2/29) of the water samples were positive for various virulence genes. 6 Out of the 36 rectal swab samples and associated household samples were shown to have similar E. coli pathotypic genes, and the drinking vessel surface was identified as the major source of contamination. EAEC and CTEC were the most commonly identified pathotypes in the cultured isolates. The phylogenetic tree constructed by MLST data showed that the diarrheagenic isolates were clustered in several diversified lineages. This study supports the hypothesis that there are high-risk hotspots, particularly those surfaces associated with food consumption, for diarrheagenic E. coli contamination within the household environments of Bangladesh.

Cholera, a devastating diarrheal disease that caused several global pandemics in the last centuries, may share some similarities with the new COVID-19. Cholera has affected many populations in history and still remains a significant burden in developing countries. The main transmission route was thought to be predominantly through contaminated drinking water. However, revisiting the historical data collected during the Copenhagen 1853 cholera outbreak allowed us to re-evaluate the role of drinking-water transmission in a city-wide outbreak and reconsider some critical transmission routes, which have been neglected since the time of John Snow. Recent empirical and cohort data from Bangladesh also strengthened the dynamic potentiality of other transmission routes (food, fomite, fish, flies) for transmitting cholera. Analyzing this particular nature of the cholera disease transmission, this paper will describe how the pattern of transmission routes are similar to COVID-19 and how the method of revisiting old data can be used for further exploration of new and known diseases., (© 2021 APMIS. Published by John Wiley & Sons Ltd.)

The illness cost borne by households, known as out-of-pocket expenditure, was 74% of the total health expenditure in Bangladesh in 2017. Calculating economic burden of diarrhea of low-income urban community is important to identify potential cost savings strategies and prioritize policy decision to improve the quality of life of this population. This study aimed to estimate cost of illness and monthly percent expenditure borne by households due diarrhea in a low-income urban settlement of Dhaka, Bangladesh. We conducted this study in East Arichpur area of Tongi township in Dhaka, Bangladesh from September 17, 2015 to July 26, 2016. We used the World Health Organization (WHO) definition of three or more loose stool in 24 hours to enroll patients and enrolled 106 severe patients and 158 non-severe patients from Tongi General Hospital, local pharmacy and study community. The team enrolled patients between the first to third day of the illness (≤ 72 hours) and continued daily follow-up by phone until recovery. We considered direct and indirect costs to calculate cost-per-episode. We applied the published incidence rate to estimate the annual cost of diarrhea. The estimated average cost of illness for patient with severe diarrhea was US$ 27.39 [95% CI: 24.55, 30.23] (2,147 BDT), 17% of the average monthly income of the households. The average cost of illness for patient with non-severe diarrhea was US$ 6.36 [95% CI: 5.19, 7.55] (499 BDT), 4% of the average monthly income of households. A single diarrheal episode substantially affects financial condition of low-income urban community residents: a severe episode can cost almost equivalent to 4.35 days (17%) and a non-severe episode can cost almost equivalent to 1 day (4%) of household's income. Preventing diarrhea preserves health and supports financial livelihoods., Competing Interests: The authors have declared that no competing interests exist.

Cholera, an acute diarrheal disease spread by lack of hygiene and contaminated water, is a major public health risk in many countries. As cholera is triggered by environmental conditions influenced by climatic variables, establishing a correlation between cholera incidence and climatic variables would provide an opportunity to develop a cholera forecasting model. Considering the auto-regressive nature and the seasonal behavioral patterns of cholera, a seasonal-auto-regressive-integrated-moving-average (SARIMA) model was used for time-series analysis during 2000-2013. As both rainfall (r = 0.43) and maximum temperature (r = 0.56) have the strongest influence on the occurrence of cholera incidence, single-variable (SVMs) and multi-variable SARIMA models (MVMs) were developed, compared and tested for evaluating their relationship with cholera incidence. A low relationship was found with relative humidity (r = 0.28), ENSO (r = 0.21) and SOI (r = -0.23). Using SVM for a 1 °C increase in maximum temperature at one-month lead time showed a 7% increase of cholera incidence (p < 0.001). However, MVM (AIC = 15, BIC = 36) showed better performance than SVM (AIC = 21, BIC = 39). An MVM using rainfall and monthly mean daily maximum temperature with a one-month lead time showed a better fit (RMSE = 14.7, MAE = 11) than the MVM with no lead time (RMSE = 16.2, MAE = 13.2) in forecasting. This result will assist in predicting cholera risks and better preparedness for public health management in the future.

Epidemiological studies considered water use and hygiene practices as central risk factors for diarrhea. Few studies focused on independent association of water quantity with diarrheal diseases. This study aimed to describe the methodological protocol that adapted multidisciplinary and mixed-method research approach to assess how water usage including water quantity influences the attributable risk for diarrhea in a low-income urban community in Bangladesh. The quantitative, anthropological and microbiological approaches were threaded together to provide a greater understanding of the infrastructural, behavioral and microbial interactions to fathom the dimensions of fecal oral transmission pathways within the households. The use of the 'Choleraphone' (i.e. a mobile phone based real time diarrheal reporting system) was a contemporary approach intended to cut down on resources, reduce research fatigue and provide more accurate data compared to the 'gold standard' (i.e. visiting a household of diarrhea cases within 48 hours) for measuring diarrhea incidence. Development of methods to measure water quantity using qualitative and quantitative approach within a setting where meter water connection is rare was another unique feature of this protocol. This protocol provided guidance and insight on how multiple methods of different disciplines can be combined to enrich understanding of waterborne diseases., Competing Interests: Authors have no conflict of interest, (© 2019 The Authors.)

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., (© 2019 John Wiley & Sons Ltd.)

Existing methodologies to record diarrheal disease incidence in households have limitations due to a high-episode recall error outside a 48-hour window. Our objective was to use mobile phones for reporting diarrheal episodes in households to provide real-time incidence data with minimum resource consumption and low recall error. From June 2014 to June 2015, we enrolled 417 low-income households in Dhaka, Bangladesh, and asked them to report diarrheal episodes to a call center. A team of data collectors then visited persons reporting the episode to collect data. In addition, each month, the team conducted in-home surveys on diarrhea incidence for a preceding 48-hour period. The mobile phone surveillance reported an incidence of 0.16 cases per person-year (95% CI: 0.13-0.19), with 117 reported diarrhea cases, and the routine in-home survey detected an incidence of 0.33 cases per person-year (95% CI: 0.18-0.60), the incidence rate ratio was 2.11 (95% CI: 1.08-3.78). During focus group discussions, participants reported a lack in motivation to report diarrhea by phone because of the absence of provision of intervening treatment following reporting. Mobile phone technology can provide a unique tool for real-time disease reporting. The phone surveillance in this study reported a lower incidence of diarrhea than an in-home survey, possibly because of the absence of intervention and, therefore, a perceived lack of incentive to report. However, this study reports the untapped potential of mobile phones in monitoring infectious disease incidence in a low-income setting.

Whole genome sequencing was utilized to investigate the genomic profile of Vibrio cholerae O1 strains, isolated from symptomatic patients in a low-income urban area of Dhaka, Bangladesh. Comparative genomics using bioinformatics tools were applied to identify major virulence factors, biotype and antimicrobial resistance genes in three V. cholerae O1 strains (VC-1, 2 and 3) isolated from two case patients. A phylogenetic SNP (single nucleotide polymorphism)-based analysis was conducted to infer the relatedness to V. cholerae O1 strains isolated elsewhere. The V. cholerae strains were the El Tor variant carrying ctxB1 (standard classical genotype). SNP-based global phylogeny revealed that the three isolates were strictly clonal and the closest neighbouring genomes were epidemic clones of V. cholerae O1 isolated in 2010 from cholera patients in Pakistan. All strains harboured the integrase gene of the SXT element (int SXT ), antimicrobial resistance genes for aminoglycosides, phenicol, sulphonamide and trimethoprim except VC-1 that lacked sulphonamide resistance genes. The multilocus sequence typing (MLST) revealed that the strains belonged to sequence type, ST69. The study provides knowledge on current genetic traits of clinical V. cholerae O1 circulating in urban household clusters of Bangladesh which may help in predicting emergence of new pandemic strains in Bangladesh., Significance and Impact of the Study: Vibrio cholerae has frequently experienced genetic changes with rapid evolution of pandemic clones in the Ganges Delta region. Whole genome sequencing can reveal genetic information of current pathogenic V. cholerae in Bangladesh which includes cefotaxime genotypes, virulence factors, altered antimicrobial resistance pattern as well as mobile genetic element compared to global pandemic strains. This study data could be used in planning future surveillance strategies in Ganges Delta region by informing new epidemiology of current outbreak strains., (© 2018 The Society for Applied Microbiology.)

Cholera outbreaks in Africa have been attributed to both droughts and floods, but whether the risk of a cholera outbreak is elevated during droughts is unknown. We estimated the risk of cholera outbreaks during droughts and floods compared with drought- and flood-free periods in 40 sub-Saharan African countries during 1990-2010 based on data from Emergency Events Database: the Office of Foreign Disaster Assistance /Centre for Research on the Epidemiology of Disasters International Disaster Database (www.emdat.be). A cholera outbreak was registered in one of every three droughts and one of every 15 floods. We observed an increased incidence rate of cholera outbreaks during drought periods (incidence rate ratio [IRR] = 4.3, 95% confidence interval [CI] = 2.9-7.2) and during flood periods (IRR = 144, 95% CI = 101-208) when compared with drought/flood-free periods. Floods are more strongly associated with cholera outbreaks, yet the prevalence of cholera outbreaks is higher during droughts because of droughts' long durations. The results suggest that droughts in addition to floods call for increased cholera preparedness.

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.

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.

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 Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Objective: To investigate the transmission of faecal bacteria by flies to food under natural settings., Methods: Over a period of 2 months, paired (exposed and non-exposed) containers with cooked rice were placed on the ground in kitchen areas in an urban slum area in Dhaka, Bangladesh, and the numbers of flies landing on the exposed rice were counted. Following exposure, the surface of the rice was microbiologically and molecularly analysed for the presence of Escherichia coli and genes of diarrhoeagenic E. coli and Shigella strains., Results: Rice was at greater risk (P < 0·001) of being contaminated with E. coli if flies landed on the rice than if no flies landed on the rice (odds ratio 5·4 (P < 0·001, 95% CI: 2·5-11·7). Mean contamination in exposed rice samples (n = 60) was 3·1 × 103 CFU/g (95% CI: 2·2 × 103-4·0 × 103). Furthermore, for approximately half of the observed fly landings, the average CFU per fly landing was >0·6 × 103 CFU. Genes of diarrhoeagenic E. coli and Shigella species were detected in 39 of 60 (65%) of exposed rice samples. Two fly species were identified: the common housefly (Musca domestica) and the oriental latrine fly (Chrysomya megacephala)., Conclusion: Flies may transmit large quantities of E. coli to food under field settings. The findings highlight the importance of implementing control measures to minimise exposure of food to flies to ensure food safety. Fly control measures should be considered for the prevention of diarrhoeal diseases caused by E. coli., (© 2017 John Wiley & Sons Ltd.)

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.

Vibrio cholerae O1 and O139 has been known for its ability to cause epidemics. These strains produce cholera toxin which is the main cause of secretory diarrhea. V. cholerae non-O1 and non-O139 strains are also capable of causing gastroenteritis as well as septicemia and peritonitis. It has been proven that virulence factors such as T6SS, hapA, rtxA , and hlyA are present in almost all V. cholerae strains. It is imperative that viable but non-culturable cells of V. cholerae are also detected since they are also known to cause diarrhea. Thus, the aim of this study was to develop an assay that detects all V. cholerae regardless of their serotype, culturable state, and virulence genes present, by targeting the species specific conserved ompW sequence. The developed assay meets these goals with 100% specificity and is capable of detecting as low as 5.46 copy number of V. cholerae . Detection is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24-1.32) and remarkable reproducibility (CV%: 1.08-3.7). Amplification efficiencies in the 89-100% range were observed. The assay is more economical than Taqman-based multiplex real-time PCR assays. Compared to other real-time assays, the ompW assay is specific and sensitive, has better repeatability and reproducibility, and is more economical.

Quantitative real-time PCR (qPCR) is a dynamic and cogent assay for the detection and quantification of specified nucleic acid sequences and is more accurate compared to both traditional culture based techniques and 'end point' conventional PCR. Serial dilution of bacterial cell culture provides information on colony forming unit (CFU) counts. This is crucial for obtaining optimal standard curves representative of DNA concentration. This approach eliminates variation in the standard curves caused by loss of DNA by serial dilution of nucleic acid elute. In this study, an assay was developed to detect and quantify DNA by real-time PCR for two pathogenic species, Escherichia coli (E. coli) and Vibrio cholerae (V. cholerae). In order to generate a standard curve, total bacterial DNA was diluted in a 10-fold series and each sample was adjusted to an estimated cell count. The starting bacterial DNA concentration was 11ng/µL. An individual E. coli cell has approximately 5.16 femtograms of DNA. Therefore, 11 ng/µL of DNA would indicate 2.48×10 7 cells. Both SYBR Green and TaqMan assays were validated for uidA region in E. coli and ctxA region in V. cholerae, respectively and was based on previously published assays for this standard curve experiment. PCR efficiency for uidA gene and ctxA gene were obtained 103.8% and 99.21%, respectively. Analysis of Variance (ANOVA) and coefficient of variation (CV %) indicated that standard curve generated by genomic DNA dilution had higher repeatability. Although not statistically significant, low F ratios indicated that there was some variation in CT values when genomic DNA dilution was compared to dilution of cell suspension in media. Different water samples spiked with pure cultures of E. coli and V. cholerae were used as unknown samples. The standard curve constructed by the serial dilution of genomic DNA exhibited greater efficiency when compared to that of the standard curve obtained from serial dilution of cell suspension since in the former method DNA is not lost during extraction from culture dilutions.