Novel Cholera Toxin Variant and ToxT Regulon in Environmental Vibrio mimicus Isolates: Potential Resources for the Evolution of Vibrio cholerae Hybrid Strains.

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in V. mimicusctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) isolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence-related genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains.IMPORTANCE Natural diversification of CTXФ and ctxAB genes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains of V. cholerae This report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Hygiene intervention reduces contamination of weaning food in Bangladesh.

OBJECTIVE: This study was conducted to measure the impact of a hygiene intervention on the contamination of weaning food in Bangladesh. METHODS: Sixty households were selected: 30 study and 30 control households. Samples of weaning food were collected from all the 60 households at baseline and examined for faecal coliforms (FC), faecal streptococci (FS) and Clostridium perfringens (CP) following standard procedures. After cooking, food samples were collected on three occasions before feeding. Following Hazard Analysis Critical Control Point (HACCP) procedures, critical control points were determined. The mothers in the 30 study households were then trained for 4 weeks in how to attain the control point conditions. Then, again the food samples were collected and analysed. RESULTS: At baseline, weaning foods from study and control households were heavily contaminated with FC and FS. The FC and FS counts were 1.84 log(10) and 1.92 log(10) colony-forming unit (cfu)/g, respectively, in the study households, and 0.86 log(10) and 1.33 log(10)  cfu/g, respectively, in the control households in the first feeding. After the intervention, the FC and FS counts in study households had dropped to 0.10 log(10) and 0.09 log(10)  cfu/g, respectively, a statistically significant reduction (P < 0.001). Monitoring the sustainability of the behaviour change after 3 months showed that the mothers were maintaining food hygiene. CONCLUSIONS: A hygiene intervention following the HACCP approach reduced the weaning food contamination significantly. Awareness building among mothers about weaning food hygiene could be an important intervention for preventing weaning food-related diarrhoea in Bangladesh.

Protocol for a parallel group, two-arm, superiority cluster randomised trial to evaluate a community-level complementary-food safety and hygiene and nutrition intervention in Mali: the MaaCiwara study (version 1.3; 10 November 2022).

BACKGROUND: Diarrhoeal disease remains a significant cause of morbidity and mortality among the under-fives in many low- and middle-income countries. Changes to food safety practices and feeding methods around the weaning period, alongside improved nutrition, may significantly reduce the risk of disease and improve development for infants. We describe a protocol for a cluster randomised trial to evaluate the effectiveness of a multi-faceted community-based educational intervention that aims to improve food safety and hygiene behaviours and enhance child nutrition. METHODS: We describe a mixed-methods, parallel group, two-arm, superiority cluster randomised controlled trial with baseline measures. One hundred twenty clusters comprising small urban and rural communities will be recruited in equal numbers and randomly allocated in a 1:1 ratio to either treatment or control arms. The community intervention will be focussed around an ideal mother concept involving all community members during campaign days with dramatic arts and pledging, and follow-up home visits. Participants will be mother-child dyads (27 per cluster period) with children aged 6 to 36 months. Data collection will comprise a day of observation and interviews with each participating mother-child pair and will take place at baseline and 4 and 15 months post-intervention. The primary analysis will estimate the effectiveness of the intervention on changes to complementary-food safety and preparation behaviours, food and water contamination, and diarrhoea. Secondary outcomes include maternal autonomy, enteric infection, nutrition, child anthropometry, and development scores. A additional structural equation analysis will be conducted to examine the causal relationships between the different outcomes. Qualitative and health economic analyses including process evaluation will be done. CONCLUSIONS: The trial will provide evidence on the effectiveness of community-based behavioural change interventions designed to reduce the burden of diarrhoeal disease in the under-fives and how effectiveness varies across different contexts. TRIAL REGISTRATION: ISRCTN14390796. Registration date December 13, 2021.

A comparison of traditional diarrhoea measurement methods with microbiological and biochemical indicators: A cross-sectional observational study in the Cox's Bazar displaced persons camp.

BACKGROUND: Water, Sanitation, and Hygiene (WASH) systems aim to reduce the spread of enteric pathogens, particularly amongst children under five years old. The most common primary outcome of WASH trials is carer-reported diarrhoea. We evaluate different diarrhoea survey instruments as proxy markers of enteric pathogen presence in stool. METHODS: We recruited 800 community-based participants from the Cox's Bazar Displaced Person's Camp in Bangladesh, split evenly between the rainy (July/August 2020) and dry (November/December 2020) periods. Participants were randomized evenly into either a standard survey asking carers if their child under five years old has had diarrhoea in the past fortnight, or a pictorial survey asking carers to pick from a pictorial chart which stools their child under five years old has had in the past fortnight. We collected stools from a random sub-sample of 120. Stools were examined visually, and tested for proteins associated with enteric infection and 16 enteric pathogens. We calculated sensitivities and specificities for each survey type, visual examination, and proteins with respect to enteric pathogen presence. FINDINGS: The sensitivity of the standard survey for enteric pathogen presence was 0.49[95%CI:0.32,0.66] and the specificity was 0.65[0.41,0.85]. Similar sensitivities and specificities were observed for pictorial survey, visual inspection, and proteins. INTERPRETATION: While diarrhoea is an important sign in clinical practice it appears that it is a poor proxy for enteric pathogen presence in stool in epidemiological surveys. When enteric infection is of interest, this should be measured directly. FUNDING: The project was funded by the National Institutes for Health Research Global Health Research Unit on Improving Health in Slums (16/136/87) and by the University of Warwick.

An atypical cause of myocardial infarction: case report of an obstructing papillary fibroelastoma of the aortic valve.

BACKGROUND: Papillary fibroelastomas are rare primary cardiac tumours with a prevalence of 0.01% at autopsy. They are histologically benign tumours but have been demonstrated through case series to confer an increased risk of thrombo-embolism resulting in: transient ischaemic attack, stroke, myocardial infarction, and pulmonary and systemic embolization. CASE SUMMARY: A 54-year-old woman presented with central chest pain radiating to her left arm. At presentation there was a significant troponin rise; initial high-sensitivity troponin-I (hsTn-I) 660 pg/mL increased to 3340 pg/mL at 6 h. Coronary angiogram did not reveal any obstructing coronary artery disease. Echocardiography revealed a rounded, mobile mass on the left coronary cusp of the aortic valve suspicious for papillary fibroelastoma. The patient underwent shave excision of the lesion. Intra-operatively it was noted that the mass intermittently sat within the ostium of the left main resulting in its occlusion. Histology confirmed a papillary fibroelastoma. DISCUSSION: Primary cardiac tumours are rare but can cause life-threatening complications such as stroke, myocardial infarction, and cardiac arrest. In the literature, the mechanism of these complications is mainly attributed to thrombo-embolism. This case demonstrates the utility of echocardiogram in investigating and diagnosing a rare cause of myocardial infarction and highlights an unusual mechanism, that is tumour causing obstruction of the coronary ostium.

Development and validation of BLRI Mastitis Test Kit at Bangladesh Livestock Research Institute Regional Station, Sirajganj.

OBJECTIVE: The objective of this study was to develop a low-cost kit for the detection of subclinical mastitis (SCM) and to check its validity, reproducibility, and efficacy at the field level. MATERIALS AND METHODS: A total of 550 quarter milk samples from crossbred dairy cows were collected, of which 400 milk samples were used to validate the newly developed BLRI mastitis test (BMT) kit to justify its efficacy as an individual test kit in detecting SCM based on somatic cell count (SCC) by direct microscopic count (DMC). The efficacy of the newly developed BMT was compared with the California Mastitis Test (CMT) kit. Another 150 milk samples were subjected to SCC determined by DMC and DCC (De Laval cell counter®) categorized by CMT and BMT scores. RESULTS: A SCM test kit, namely, BMT kit was successfully developed in this study. The percentage accuracy of CMT and BMT were 76.75% and 75.75%; sensitivity 69.36% and 67.56%; specificity 85.95% and 85.85%; positive predictive value 86.03% and 85.71%; negative predictive value 69.23% and 68%, respectively. A p value of 0.001 was found for both CMT and BMT. However, CMT and BMT had no significant difference in sensitivity (p = 0.778). Average SCCs (cells/ml) determined by DCC and DMC, respectively, were mostly corresponded to the SCC ranges of both CMT and BMT scores. CONCLUSION: The newly developed BMT kit is an independent, cheap, farmer-friendly, first country made, and reliable SCM diagnostic test kit that can be used at field condition.

Occurrence, seasonality and genetic diversity of Vibrio vulnificus in coastal seaweeds and water along the Kii Channel, Japan.

Vibrio vulnificus is a ubiquitous toxigenic bacterium found in a coastal environment but little is known about its occurrence and seasonality among seaweeds, which are widely consumed as seafood in Japan. Therefore, we have observed the bacterium's abundance in seawater and seaweed samples from three areas of the Kii Channel, Japan, during June 2003 to May 2004. A total of 192 samples were collected: 24 from each source in summer, autumn, winter and spring. The samples were selectively cultivated following the most probable number (MPN) technique. Vibrio vulnificus population ranged from 0 to 10(3) MPN 100 mL(-1) seawater or 10 g seaweeds; higher counts were observed during summer. The optimum temperature, salinity and pH for the bacterium were 20-24 degrees C, 24-28 p.p.t. and 7.95-8.15, respectively. However, seaweeds always contained higher V. vulnificus than seawater. Among 280 V. vulnificus strains, detected by species-specific colony hybridization and PCR, 78, 74, 11 and 16 were from seaweeds and 46, 42, 2 and 11 were from seawater during summer, autumn, winter and spring, respectively. Ribotyping of 160 selected strains revealed a higher genotypic diversity (18 patterns) among strains from seaweeds than from seawater (10 patterns). Seaweeds can thus act as a potential habitat for V. vulnificus and are more unsafe for consumption during summer.

A Cross Sectional Study of the Association between Sanitation Type and Fecal Contamination of the Household Environment in Rural Bangladesh.

We conducted a cross sectional study to assess 1) the association between access to basic sanitation and fecal contamination of sentinel toy balls and 2) if other sanitation factors such as shared use and cleanliness are associated with fecal contamination of sentinel toy balls. We assessed sanitation facilities in 454 households with a child aged 6-24 months in rural Bangladesh. We defined "basic" sanitation as access to improved sanitation facilities (pit latrine with a slab or better) not shared with other households. In each household, an identical toy ball was given to the target child. After 24 hours, the balls were rinsed to enumerate fecal coliforms as an indicator of household fecal contamination. Households with basic sanitation had lower fecal coliform contamination than households with no access to basic sanitation (adjusted difference in means: -0.31 log10 colony forming units [CFU]/toy ball; 95% confidence interval [CI]: -0.61, -0.01). Shared sanitation facilities of otherwise improved type were more likely to have visible feces on the latrine slab compared with private facilities. Among households with access to improved sanitation, households with no visible feces on the latrine slab had less toy ball contamination than households with visible feces on the latrine slab (adjusted difference in means: -0.38 log10 CFU/toy ball; 95% CI: -0.77, 0.02). Access to basic sanitation may prevent fecal contamination of the household environment. An Improved sanitation facility used by an individual household may be better in preventing household fecal contamination compared with improved facilities shared with other households.

Diversity and distribution of cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae.

Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains.

Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh.

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.

Organization of the CTX prophage in environmental isolates of Vibrio mimicus.

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment.

Virulence properties of rough and smooth strains of Vibrio cholerae O1.

A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains. A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme-linked immunosorbent assay (ELISA) in Richardson's and AKI media. All the smooth and rough strains, except one, showed a titre of 1: 10 and 1: 100 in Richardson's and AKI media, respectively. Both types of strains produced enterotoxin in rabbit ileal loop (RIL). The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P <0.05). However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P >0.05) between these properties. This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains.

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.