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ABSTRACT Between 2018 and 2024, we conducted systematic whole-genome sequencing and phylogenomic analysis on 263 V. cholerae O1 isolates from cholera patients across four provinces in the Democratic Republic of Congo (North-Kivu, South-Kivu, Tanganyika, and Kasai Oriental). These isolates were classified into the AFR10d and AFR10e sublineages of AFR10 lineage, originating from the third wave of the seventh El Tor cholera pandemic (7PET). Compared to the strains analysed between 2014 and 2017, both sublineages had few genetic changes in the core genome but recent isolates (2022-2024) had significant CTX prophage rearrangement. AFR10e spread across all four provinces, while AFR10d appeared to be extinct by the end of 2020. Since 2022, most V. cholerae O1 isolates exhibited significant CTX prophage rearrangements, including a tandem repeat of an environmental satellite phage RS1 downstream the ctxB t oxin gene of the CTX-Φ-3 prophage on the large chromosome, as well as two or more arrayed copies of an environmental pre-CTX-Φ prophage precursor on the small chromosome. We used Illumina data for mapping and coverage estimation to identify isolates with unique CTX-Φ genomic features. Gene localization was then determined on MinION-derived assemblies, revealing an organization similar to that of non-O1  V. cholerae isolates found in Asia (O139 VC1374, and environmental O4 VCE232), but never described in V. cholerae O1 El Tor from the third wave. In conclusion, while the core genome of AFR10d and AFR10e showed minimal changes, significant alterations in the CTX-Φ and pre-CTX-Φ prophage content and organization were identified in AFR10e from 2022 onwards.

Analysis of clinical and environmental Vibrio cholerae O1 strains obtained during 2008-2015 in Nigeria showed that lineages Afr9 and Afr12 carrying cholera toxin and Vibrio pathogenicity island 1 can be isolated from water. Our findings raise concerns about the role of the environment in maintenance and emergence of cholera outbreaks in Nigeria.

In Bangladesh, Vibrio cholerae lineages are undergoing genomic evolution, with increased virulence and spreading ability. However, our understanding of the genomic determinants influencing lineage transmission and disease severity remains incomplete. Here, we developed a computational framework using machine-learning, genome scale metabolic modelling (GSSM) and 3D structural analysis, to identify V. cholerae genomic traits linked to lineage transmission and disease severity. We analysed in-patients isolates from six Bangladeshi regions (2015-2021), and uncovered accessory genes and core SNPs unique to the most recent dominant lineage, with virulence, motility and bacteriophage resistance functions. We also found a strong correlation between V. cholerae genomic traits and disease severity, with some traits overlapping those driving lineage transmission. GSMM and 3D structure analysis unveiled a complex interplay between transcription regulation, protein interaction and stability, and metabolic networks, associated to lifestyle adaptation, intestinal colonization, acid tolerance and symptom severity. Our findings support advancing therapeutics and targeted interventions to mitigate cholera spread., (© 2024. The Author(s).)

BackgroundThe number of cholera cases reported to the World Health Organization (WHO) in 2022 was more than double that of 2021. Nine countries of the WHO European Region reported 51 cases of cholera in 2022 vs five reported cases in 2021.AimWe aimed to confirm that the Vibrio cholerae O1 isolates reported by WHO European Region countries in 2022 belonged to the seventh pandemic El Tor lineage (7PET). We also studied their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships.MethodsWe used microbial genomics to study the 49 V. cholerae O1 isolates recovered from the 51 European cases. We also used > 1,450 publicly available 7PET genomes to provide a global phylogenetic context for these 49 isolates.ResultsAll 46 good-quality genomes obtained belonged to the 7PET lineage. All but two isolates belonged to genomic Wave 3 and were grouped within three sub-lineages, one of which, Pre-AFR15, predominated (34/44). This sub-lineage, corresponding to isolates from several countries in Southern Asia, the Middle East and Eastern or Southern Africa, was probably a major contributor to the global upsurge of cholera cases in 2022. No unusual AMR profiles were inferred from analysis of the AMR gene content of the 46 genomes.ConclusionReference laboratories in high-income countries should use whole genome sequencing to assign V. cholerae O1 isolates formally to the 7PET or non-epidemic lineages. Periodic collaborative genomic studies based on isolates from travellers can provide useful information on the circulating strains and their evolution, particularly as concerns AMR.

Cholera is a life-threatening gastrointestinal infection caused by a toxigenic bacterium, Vibrio cholerae. After a lull of almost 30 years, a first case of cholera was detected in Lebanon in October 2022. The outbreak lasted three months, with 8007 suspected cases (671 laboratory-confirmed) and 23 deaths. In this study, we use phenotypic methods and microbial genomics to study 34 clinical and environmental Vibrio cholerae isolates collected throughout this outbreak. All isolates are identified as V. cholerae O1, serotype Ogawa strains from wave 3 of the seventh pandemic El Tor (7PET) lineage. Phylogenomic analysis unexpectedly reveals the presence of two different strains of the seventh pandemic El Tor (7PET) lineage. The dominant strain has a narrow antibiotic resistance profile and is phylogenetically related to South Asian V. cholerae isolates and derived African isolates from the AFR15 sublineage. The second strain is geographically restricted and extensively drug-resistant. It belongs to the AFR13 sublineage and clusters with V. cholerae isolates collected in Yemen. In conclusion, the 2022-2023 Lebanese cholera outbreak is caused by the simultaneous introduction of two different 7PET strains. Genomic surveillance with cross-border collaboration is therefore crucial for the identification of new introductions and routes of circulation of cholera, improving our understanding of cholera epidemiology., (© 2024. The Author(s).)

Vibrio cholerae O1 causes the diarrheal disease cholera, and the small intestine is the site of active infection. During cholera, cholera toxin is secreted from V. cholerae and induces a massive fluid influx into the small intestine, which causes vomiting and diarrhea. Typically, V. cholerae genomes are sequenced from bacteria passed in stool, but rarely from vomit, a fluid that may more closely represents the site of active infection. We hypothesized that V. cholerae O1 population bottlenecks along the gastrointestinal tract would result in reduced genetic variation in stool compared to vomit. To test this, we sequenced V. cholerae genomes from 10 cholera patients with paired vomit and stool samples. Genetic diversity was low in both vomit and stool, consistent with a single infecting population rather than coinfection with divergent V. cholerae O1 lineages. The amount of single-nucleotide variation decreased from vomit to stool in four patients, increased in two, and remained unchanged in four. The variation in gene presence/absence decreased between vomit and stool in eight patients and increased in two. Pangenome analysis of assembled short-read sequencing demonstrated that the toxin-coregulated pilus operon more frequently contained deletions in genomes from vomit compared to stool. However, these deletions were not detected by PCR or long-read sequencing, indicating that interpreting gene presence or absence patterns from short-read data alone may be incomplete. Overall, we found that V. cholerae O1 isolated from stool is genetically similar to V. cholerae recovered from the upper intestinal tract., Importance: Vibrio cholerae O1, the bacterium that causes cholera, is ingested in contaminated food or water and then colonizes the upper small intestine and is excreted in stool. Shed V. cholerae genomes from stool are usually studied, but V. cholerae isolated from vomit may be more representative of where V. cholerae colonizes in the upper intestinal epithelium. V. cholerae may experience bottlenecks, or large reductions in bacterial population sizes and genetic diversity, as it passes through the gut. Passage through the gut may select for distinct V. cholerae mutants that are adapted for survival and gut colonization. We did not find strong evidence for such adaptive mutations, and instead observed that passage through the gut results in modest reductions in V. cholerae genetic diversity, and only in some patients. These results fill a gap in our understanding of the V. cholerae life cycle, transmission, and evolution., Competing Interests: The authors declare no conflict of interest.

Abstract: Acute gastroenteritis is the most common clinical manifestation of Vibrio cholerae infection. Cases of non-O1 V. cholerae infections in cancer patients have been previously reported in the literature. To our best knowledge, this is a unique case of V. cholerae classical biotype, serovar Ogawa infection in a young female patient with gastric malignancy., (Copyright © 2023 Copyright: © 2023 Journal of Cancer Research and Therapeutics.)

Objective: Globally, there are estimated to be 2.9 million cholera cases annually. Early detection of cholera outbreaks is crucial for resource allocation for case management and for targeted interventions to be delivered to stop the spread of cholera. In resource limited settings such as Eastern Democratic Republic of the Congo (DRC), there is often limited laboratory capacity for analysing stool samples for cholera by bacterial culture. Therefore, rapid diagnostic tests (RDTs) for cholera present a promising tool to rapidly test stool samples in a health facility setting for cholera. Our objective is to evaluate the Crystal VC O1 RDT for cholera detection compared with bacterial culture and polymerase chain reaction (PCR) for Vibrio cholerae., Methods: From March 2020 to December 2022, stool samples were collected from 644 diarrhoea patients admitted to 94 health facilities in Bukavu in Eastern DRC. Patient stool samples were analysed by Crystal VC O1 RDT for cholera and by bacterial culture and PCR for V. cholerae O1., Results: Twenty six percent of diarrhoea patients (166/644) had stool samples positive for cholera by RDT, and 24% (152/644) had stool samples positive for V. cholerae O1 by bacterial culture or PCR. The overall specificity and sensitivity of the Crystal VC O1 RDT by direct testing was 94% (95% confidence interval [CI]: 92%-96%) and 90% (95% CI, 84%-94%), respectively, when compared with either a positive result by bacterial culture or PCR., Conclusion: Our findings suggest that the Crystal VC O1 RDT presents a promising tool for cholera surveillance in this cholera endemic setting in sub-Saharan Africa., (© 2024 John Wiley & Sons Ltd.)

Background: In Addis Ababa, Ethiopia, open ditches along innner roads in residential areas serve to convey domestic wastewater and rainwater away from residences. Contamination of drinking water by wastewater through faulty distribution lines could expose households to waterborne illnesses. This prompted the study to assess the microbiological safety of wastewater and drinking water in Addis Ababa, identify the pathogens therein, and determine their antibiotic resistance patterns., Results Vibrio Cholerae: O1, mainly Hikojima serotype, was isolated from 23 wastewater and 16 drinking water samples. Similarly, 19 wastewater and 10 drinking water samples yielded Escherichia coli O157:H7. V. cholerae O1 were 100% resistant to the penicillins (Amoxacillin and Ampicillin), and 51-82% were resistant to the cephalosporins. About 44% of the V. cholerae O1 isolates in this study were Extended Spectrum Beta-Lactamase (ESBL) producers. Moreover, 26% were resistant to Meropenem. Peperacillin/Tazobactam was the only effective β-lactam antibiotic against V. cholerae O1. V. cholerae O1 isolates showed 37 different patterns of multiple resistance ranging from a minimum of three to a maximum of ten antimicrobials. Of the E. coli O157:H7 isolates, 71% were ESBL producers. About 96% were resistant to Ampicillin. Amikacin and Gentamicin were very effective against E. coli O157:H7 isolates. The isolates from wastewater and drinking water showed multiple antibiotic resistance against three to eight antibiotic drugs., Conclusions: Open ditches for wastewater conveyance along innner roads in residence areas and underground faulty municipal water distribution lines could be possible sources for V. cholerae O1 and E. coli O157:H7 infections to surrounding households and for dissemination of multiple drug resistance in humans and, potentially, the environment., (© 2024. The Author(s).)

We investigated the evolution, phylogeny and antimicrobial resistance of Vibrio cholerae O1 isolates (VCO1) from Ghana. Outbreak and environmental sources of VCO1 were characterized, whole-genome sequenced and compared to globally available seventh pandemic (7P) strains of V. cholerae at SNP resolution. Final analyses included 636 isolates. Novel Ghanaian isolates clustered into three distinct clades (clades 1, 2 and 3) in wave 3 of the 7P lineage. The closest relatives of our novel Ghanaian isolates were from Benin, Cameroon, Togo, Niger and Nigeria. All novel Ghanaian isolates were multi-drug resistant. Environmental isolates clustered into clade 2, despite being isolated years later, showing the possibility of persistence and re-emergence of older clades. A lag phase of several years from estimated introduction to reported cases suggests pathogen persistence in the absence of reported cholera cases. These results highlight the importance of deeper surveillance for understanding transmission routes between bordering countries and planning tailored vaccination campaigns in an effort to eradicate cholera.

Background: Cholera, an acute diarrheal disease is a major public health problem in many developing countries. Several rapid diagnostic tests (RDT) are available for the detection of cholera, but their efficacies are not compared in an endemic setting. In this study, we have compared the specificity and sensitivity of three RDT kits for the detection of Vibrio cholerae O1 and compared their efficiency with culture and polymerase chain reaction (PCR) methods., Methods: Five hundred six diarrheal stool samples collected from patients from two different hospitals in Kolkata, India were tested using SD Bioline Cholera, SMART-II Cholera O1 and Crystal-VC RDT kits. All the stool samples were screened for the presence of V. cholerae by direct and enrichment culture methods. Stool DNA-based PCR assay was made to target the cholera toxin (ctxAB) and O1 somatic antigen (rfb) encoding genes. Statistical evaluation of the RDTs has been made using STATA software with stool culture and PCR results as the gold standards. The Bayesian latent class model (LCM) was used to evaluate the diagnostic tests in the absence of the gold standard., Results: Involving culture technique as gold standard, the sensitivity and specificity of the cholera RDT kits in the direct testing of stools was highest with SAMRT-II (86.1%) and SD-Cholera (94.4%), respectively. The DNA based PCR assays gave very high sensitivity (98.4%) but the specificity was comparatively low (75.3%). After enrichment, the high sensitivity and specificity was detected with SAMRT-II (78.8%) and SD-Cholera (99.1%), respectively. Considering PCR as the gold standard, the sensitivity and specificity of the RDTs remained between 52.3-58.2% and 92.3-96.8%, respectively. In the LCM, the sensitivity of direct and enrichment testing was high in SAMRT-II (88% and 92%, respectively), but the specificity was high in SD cholera for both the methods (97% and 100%, respectively). The sensitivity/specificity of RDTs and direct culture have also been analyzed considering the age, gender and diarrheal disease severity of the patients., Conclusion: Overall, the performance of the RDT kits remained almost similar in terms of specificity and sensitivity. Performance of PCR was superior to the antibody-based RDTs. The RTDs are very useful in identifying cholera cases during outbreak/epidemic situations and for making them as a point-of-care (POC) testing tool needs more improvement., Competing Interests: The authors have declared that no competing interests exist.

Background: Cholera has been present and recurring in Zambia since 1977. However, there is a paucity of data on genetic relatedness and diversity of the Vibrio cholerae isolates responsible for these outbreaks. Understanding whether the outbreaks are seeded from existing local isolates or if the outbreaks represent separate transmission events can inform public health decisions., Results: Seventy-two V. cholerae isolates from outbreaks in 2009/2010, 2016, and 2017/2018 in Zambia were characterized using multilocus variable number tandem repeat analysis (MLVA) and whole genome sequencing (WGS). The isolates had eight distinct MLVA genotypes that clustered into three MLVA clonal complexes (CCs). Each CC contained isolates from only one outbreak. The results from WGS revealed both clustered and dispersed single nucleotide variants. The genetic relatedness of isolates based on WGS was consistent with the MLVA, each CC was a distinct genetic lineage and had nearest neighbors from other East African countries. In Lusaka, isolates from the same outbreak were more closely related to themselves and isolates from other countries than to isolates from other outbreaks in other years., Conclusions: Our observations are consistent with i) the presence of random mutation and alternative mechanisms of nucleotide variation, and ii) three separate transmission events of V. cholerae into Lusaka, Zambia. We suggest that locally, case-area targeted invention strategies and regionally, well-coordinated plans be in place to effectively control future cholera outbreaks.

Cholera is a severe acute, highly transmissible diarrheal disease which affects many low- and middle-income countries. Outbreaks of cholera are confirmed using microbiological culture, and additional cases during the outbreak are generally identified based on clinical case definitions, rather than laboratory confirmation. Many low-resource areas where cholera occurs lack the capacity to perform culture in an expeditious manner. A simple, reliable, and low-cost rapid diagnostic test (RDT) would improve identification of cases allowing rapid response to outbreaks. Several commercial RDTs are available for cholera testing with two lines to detect either serotypes O1 and O139; however, issues with sensitivity and specificity have not been optimal with these bivalent tests. Here, we report an evaluation of a new commercially available cholera dipstick test which detects only serotype O1. In both laboratory and field studies in Kenya, we demonstrate high sensitivity (97.5%), specificity (100%), and positive predictive value (100%) of this new RDT targeting only serogroup O1. This is the first field evaluation for the new Crystal VC-O1 RDT; however, with these high-performance metrics, this RDT could significantly improve cholera outbreak detection and improve surveillance for better understanding of cholera disease burden.

Objectives: Few studies have evaluated determinants of multidrug-resistant (MDR) Vibrio cholerae O1 in older children and adults. This study aimed to characterize the prevalence of MDR V. cholerae O1 and associated risk factors among patients over five years of age in Bangladesh., Methods: Stool culture and antimicrobial susceptibility testing were performed as a part of a larger study at Dhaka Hospital in Bangladesh from March 2019-March 2020. Univariate statistics and multiple logistic regression were used to assess the association between a range of variables and MDR V. cholerae O1., Results: MDR was found in 175 of 623 (28.1%) V. cholerae O1 isolates. High levels of resistance were found to erythromycin (99.2%), trimethoprim-sulfamethoxazole (99.7%), and ampicillin (88.9%), while susceptibility was high to tetracyclines (99.7%), azithromycin (99.2%), ciprofloxacin (99.8%), and cephalosporins (98.6%). MDR was associated with prior antibiotic use, longer transport time to hospital, higher income, non-flush toilet use, greater stool frequency, lower blood pressure, lower mid-upper arm circumference, and lower percent dehydration., Conclusions: MDR V. cholerae O1 was common among patients over five in an urban hospital in Bangladesh. Significant factors associated with MDR may be actionable in identifying patients with a high likelihood of MDR., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cholera posed a significant threat causing outbreaks/epidemics with high morbidity and mortality in Odisha. This study envisages the characterisation of isolated pathogen from two cholera outbreaks reported in 2018 and 2019 from Bargarh and Rayagada districts of Odisha respectively. Vibrio cholerae O1 were isolated following standard techniques. The different virulent and drug resistant genes were detected by multiplex PCR assays; whereas the ctxB genotypes were characterised through double mismatch amplification mutation (DMAMA) PCR assay. The ctxB genes were further sequenced and pulse-field gel electrophoresis (PFGE) was done on some selected strains. The clinical and water isolates of Haitian variant (HCT) V. cholerae O1 Ogawa biotype El Tor with multi drug resistant strains were isolated from both the places. All the V. cholerae O1 strains were positive for virulence genes. The antibiotic resistant genes like dfrA1 (100%), strB (76.9%), intSXT (61.5%) were detected. The PFGE results on V. cholerae O1 strains exhibited two different pulsotypes. These cholera outbreaks were due to multidrug resistant HCT variant V. cholerae O1 strains which were circulating and caused the cholera outbreaks in Odisha. So continuous surveillance on diarrheal disorders is highly essential to prevent the future diarrheal outbreaks in this region.

Cholera is a severe diarrhoeal disease that spreads rapidly and affects millions of people each year, resulting in tens of thousands of deaths. The disease is caused by Vibrio cholerae O1 and is characterized by watery diarrhoea that can be lethal if not properly treated. Cholera had not been reported in South America from the late 1800s until 1991, when it was introduced in Peru, wreaking havoc in one of the biggest epidemics reported to date. Within a year, the disease had spread to most of the Latin American region, resulting in millions of cases and thousands of deaths in all affected countries. Despite its aggressive entry, cholera virtually disappeared from the continent after 1999. The progression of the entire epidemic was well documented, making it an ideal model to understand cholera dynamics. In this review, we highlight how the synergy of socioeconomic, political and ecological factors led to the emergence, rapid spread and eventual disappearance of cholera in Latin America. We discuss how measures implemented during the cholera epidemic drastically changed its course and continental dynamics. Finally, we synthesize our findings and highlight potential lessons that can be learned for efficient and standardized cholera management programmes during future outbreaks in non-endemic areas.

Vibrio cholerae serogroup O1 is responsible for epidemic and pandemic cholera and remains a global public health threat. This organism has been well established as a resident flora of the aquatic environment that alters its phenotypic and genotypic attributes for better adaptation to the environment. To reveal the diversity of clinical isolates of V. cholerae O1 in the Bay of Bengal, we performed whole-genome sequencing of isolates from Kolkata, India, and Dhaka, Bangladesh, collected between 2009 and 2016. Comparison with global isolates by phylogenetic analysis placed the current isolates in two Asian lineages, with lineages 1 and 2 predominant in Dhaka and Kolkata, respectively. Each lineage possessed different genetic traits in the cholera toxin B subunit gene, Vibrio seventh pandemic island II, integrative and conjugative element, and antibiotic-resistant genes. Thus, although recent global transmission of V. cholerae O1 from South Asia has been attributed only to isolates of lineage 2, another distinct lineage exists in Bengal. IMPORTANCE Cholera continues to be a global concern, as large epidemics have occurred recently in Haiti, Yemen, and countries of sub-Saharan Africa. A single lineage of Vibrio cholerae O1 has been considered to be introduced into these regions from South Asia and to cause the spread of cholera. Using genomic epidemiology, we showed that two distinct lineages exist in Bengal, one of which is linked to the global lineage. The other lineage was found only in Iran, Iraq, and countries in Asia and differed from the global lineage regarding cholera toxin variant and drug resistance profile. Therefore, the potential transmission of this lineage to other regions would likely cause worldwide cholera spread and may result in this lineage replacing the current global lineage., (Copyright © 2020 Morita et al.)

The efficacy of commonly used antibiotics for treating severe cholera has been compromised over time because of the reduced antibiotic susceptibility. This study aimed to describe the rate of detection of Vibrio cholerae O1 from fecal samples and antimicrobial susceptibility profiles of V. cholerae O1 serotypes to commonly used antibiotics. During January 2000-December 2018, V. cholerae O1 was detected in fecal samples of 7,472 patients. Vibrio cholerae O1 Inaba serotype was predominant, ranging from 60% to 86% during the period 2000-2006 except for 2003 and 2005 when the Ogawa serotype was predominant. Later on, the Ogawa serotype became predominant from 2007 to 2015, fluctuating between 52% and 100%. However, in 2016 and 2017, isolation rates declined to 2% and 1%, respectively, but surged again to 75% in 2018. Nearly 100% of V. cholerae O1 strains were sensitive to tetracycline during 2000-2004. Thereafter, a declining trend of sensitivity was observed to be continued and dropped down to < 6% during 2012-2017 and again increased to 76% in 2018. Susceptibility to azithromycin and ciprofloxacin was nearly 100%, and susceptibility to cotrimoxazole and furazolidone was 01% throughout the study period. We also found the emergence of resistance to erythromycin in 2005 and sensitivity to cotrimoxazole in 2018. Thus, the rapid decline of the sensitivity of V . cholerae O1 to tetracycline and a reversed peak after 6 years need continued monitoring and reporting.

Background: Cholera remains a significant public health problem in more than one-third of the countries of the world. Cholera outbreak has become more common in Addis Ababa particularly in the rainy seasons; however, there is a paucity of data on risk factors associated with cholera outbreaks rendering interventions difficult. We investigated the outbreak to identify its etiology, source, risk factors and in order to control the outbreak., Methods: We compared cases with health center-based unmatched controls (1:2). Cases were patients aged ≥5 years with acute watery diarrhea, with or without vomiting while controls were persons aged ≥5 years without history of acute watery diarrhea. We interviewed our study participants using structured questionnaire to collect demographic and cholera risk factors data. We described the outbreak over time, and then tested our hypotheses using unconditional logistic regression., Results: The outbreak began on 7 September, 2017 reaching its peak on 23 September, 2017 and ended on 01 October, 2017. We identified a total of 25 cases (Median age: 38 years; IQR: 20 years) and recruited 50 controls (Median age: 35 years; IQR: 29 years). All case-patients had acute watery diarrhea and dehydration requiring intravenous fluids. All cases were admitted to cholera treatment center but there were no deaths. Stool and water samples yielded isolates of Vibrio cholerae O1 of serological subtype Ogawa. Consumption of contaminated holy water (AOR: 20.5, 95%CI: 3.50, 119.61) and raw vegetables (AOR: 15.3, 95%CI: 3, 81.51) were independent risk factors whereas washing hands with soap after visiting latrine (AOR: 0.04, 95%CI: 0.01, 0.25) was independent protective factor., Conclusion: Our findings demonstrated cholera foodborne transmission via consumption of raw vegetables, and its waterborne transmission via consumption of contaminated holy water. Washing hands with soap after visiting latrine was protective. We recommended cooking of vegetables and promoting hand washing., Competing Interests: The authors have declared that no competing interests exist.

Objective: On 24th October 2016, the Central Regional Health Directorate received report of a suspected cholera outbreak in the Cape Coast Metropolis (CCM). We investigated to confirm the diagnosis, identify risk factors and implement control measures., Design: We used a descriptive study followed by 1:2 unmatched case-control study., Data Source: We reviewed medical records, conducted active case search and contact tracing, interviewed case-patients and their contacts and conducted environmental assessment. Case-patients' stool samples were tested with point of care test kits (SD Bioline Cholera Ag 01/0139) and sent to the Cape Coast Teaching Hospital Laboratory for confirmation., Main Outcomes: Cause of outbreak, risk factors associated with spread of outbreak., Results: Vibrio cholerae serotype Ogawa caused the outbreak. There was no mortality. Of 704 case-patients, 371(52.7%) were males and 55(7.8%) were aged under-five years. The median age was 23 years (interquartile range: 16-32 years). About a third 248(35.2%) of the case patients were aged 15-24 years. The University of Cape Coast subdistrict was the epicenter with 341(48.44%) cases. Compared to controls, cholera case-patients were more likely to have visited Cholera Treatment Centers (CTC) (aOR=12.1, 95%CI: 1.5-101.3), drank pipe-borne water (aOR=11.7, 95%CI: 3.3-41.8), or drank street-vended sachet water (aOR=11.0, 95%CI: 3.7-32.9). Open defecation and broken sewage pipes were observed in the epicenter., Conclusion: Vibrio cholerae serotype Ogawa caused the CCM cholera outbreak mostly affecting the youth. Visiting CTC was a major risk factor. Prompt case-management, contact tracing, health education, restricting access to CTC and implementing water sanitation and hygiene activities helped in the control., Funding: This work was supported by Ghana Field Epidemiology and Laboratory Training Program (GFELTP), University of Ghana., Competing Interests: Conflict of interest: None declared, (Copyright © The Author(s).)

Introduction. The emergence of novel strains of Vibrio cholerae O1 El Tor biotype has gained attention due to causing several epidemics around the world. Variant strains have evolved as a result of the acquisition of genes that confer extended virulence and pathogenicity. Aim. This study aimed to determine the presence of the most recently emerging Haitian-like genetic traits among the isolates from Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, Southern India. We also wanted to detect the prevalence of the sulfamethoxazole and trimethoprim (SXT) element, which is an integrating conjugative element (ICE) and the antimicrobial resistance genes present in our isolates. Methodology. Identification of Haitian-specific alleles was done by mismatched amplification mutation assay PCR (MAMA-PCR). The presence of SXT elements was carried out by PCR by detecting int, eex, att-prfC and setR genes. Detection of antibiotic resistance determinant, sul(1,2,3); dfr(A1,18,5 ) for trimethoprim resistance, tet(A,B,C,D,E,Y,G,M), tet34 for tetracycline resistance and erm(A,B,C), mph(A,B), ere(A,B), msr(A,D ) for azithromycin resistance were targeted by PCR. The MIC of tetracycline, ciprofloxacin and azithromycin was determined by the E-test method. Results. Of the 95 isolates, 60 % of the isolates were found to carry Haitian-specific alleles of ctxB , tcpA and rtxA gene , 100 % of the isolates were found to carry SXT elements. All the isolates harboured the four conserved genes of the SXT element, except one which had only eex , att-prfC , setR genes. About 99 % harboured sul2 and dfrA1 genes. No tet and macrolide genes were detected. We observed a progressive increase in the MIC of azithromycin ranging from 0.75 µg ml -1 to 2 µg ml -1 . Conclusion. None of the isolates were the prototype El Tor biotype. All the isolates were a Haitian variant. The presence of SXT elements across all our isolates and their creeping MIC of azithromycin is a matter of concern. Further testing for other genetic determinants of resistance will be carried out in our future studies.

Introduction: In 2009 and 2010, more than 6,000 cholera cases were recorded during these outbreaks with more than 80% of cases recorded in Lusaka province. After a five-year break, in 2016 an outbreak occurred in Lusaka, causing more than 1,000 cases of cholera. This study will strengthen the epidemiological information on the changing characteristics of the cholera outbreaks, for treatment, prevention and control of the disease., Methods: This was a laboratory-based descriptive cross-sectional study conducted at the University Teaching Hospital in Lusaka, Zambia. A total of 83 V. cholerae O1 isolates were characterised by biochemical testing, serotyping, antimicrobial susceptibility testing, and macrorestriction analysis using Pulsed-Field Gel Electrophoresis., Results: Macrorestriction analysis of the isolates demonstrated high genetic diversity among the isolates with 16 different patterns. The largest pattern comprised 9 isolates while the smallest one had 1 isolate. 2009 and 2010 isolates were highly resistant to nalidixic acid and cotrimoxazole, but highly sensitive to azithromycin and ampicillin. Of the fifty-two isolates from the 2016 cholera outbreak, 90% (47) were sensitive to cotrimoxazole, 94% (49) to tetracycline, and 98% (51) to azithromycin, while 98% (51) were resistant to nalidixic acid and 31(60%) to ampicillin., Conclusion: macrorestriction analysis demonstrated high genetic diversity among the V. cholerae O1 strains, suggesting that these isolates were probably not from a similar source. This study also revealed the emergence of multidrug resistance among the 2016 V. cholerae outbreak isolates but were susceptible to cotrimoxazole, tetracycline, and azithromycin, which can be used for treatment of the cholera cases., Competing Interests: The authors declare no competing interests., (© Kapambwe Mwape et al.)

Background: Tanzania is one of seven countries with the highest disease burden caused by cholera in Africa. We studied the evolution of Vibrio cholerae O1 isolated in Tanzania during the past three decades., Methodology/principal Findings: Genome-wide analysis was performed to characterize V. cholerae O1 responsible for the Tanzanian 2015-2017 outbreak along with strains causing outbreaks in the country for the past three decades. The genomes were further analyzed in a global context of 590 strains of the seventh cholera pandemic (7PET), as well as environmental isolates from Lake Victoria. All Tanzanian cholera outbreaks were caused by the 7PET lineage. The T5 sub-lineage (ctxB3) dominated outbreaks until 1997, followed by the T10 atypical El Tor (ctxB1) up to 2015, which were replaced by the T13 atypical El Tor of the current third wave (ctxB7) causing most cholera outbreaks until 2017 with T13 being phylogenetically related to strains from East African countries, Yemen and Lake Victoria. The strains were less drug resistant with approximate 10-kb deletions found in the SXT element, which encodes resistance to sulfamethoxazole and trimethoprim. Nucleotide deletions were observed in the CTX prophage of some strains, which warrants further virulence studies. Outbreak strains share 90% of core genes with V. cholerae O1 from Lake Victoria with as low as three SNPs difference and a significantly similar accessory genome, composed of genomic islands namely the CTX prophage, Vibrio Pathogenicity Islands; toxin co-regulated pilus biosynthesis proteins and the SXT-ICE element., Conclusion/significance: Characterization of V. cholerae O1 from Tanzania reveals genetic diversity of the 7PET lineage composed of T5, T10 and T13 sub-lineages with introductions of new sequence types from neighboring countries. The presence of these sub-lineages in environmental isolates suggests that the African Great Lakes may serve as aquatic reservoirs for survival of V. cholerae O1 favoring continuous human exposure., Competing Interests: The authors have declared that no competing interests exist.

Background: Antibodies targeting O-specific polysaccharide (OSP) of Vibrio cholerae may protect against cholera; however, little is known about this immune response in infected immunologically naïve humans., Methodology: We measured serum anti-OSP antibodies in adult North American volunteers experimentally infected with V. cholerae O1 Inaba El Tor N16961. We also measured vibriocidal and anti-cholera toxin B subunit (CtxB) antibodies and compared responses to those in matched cholera patients in Dhaka, Bangladesh, an area endemic for cholera., Principal Findings: We found prominent anti-OSP antibody responses following initial cholera infection: these responses were largely IgM and IgA, and highest to infecting serotype with significant cross-serotype reactivity. The anti-OSP responses peaked 10 days after infection and remained elevated over baseline for ≥ 6 months, correlated with vibriocidal responses, and may have been blunted in blood group O individuals (IgA anti-OSP). We found significant differences in immune responses between naïve and endemic zone cohorts, presumably reflecting previous exposure in the latter., Conclusions: Our results define immune responses to O-specific polysaccharide in immunologically naive humans with cholera, find that they are largely IgM and IgA, may be blunted in blood group O individuals, and differ in a number of significant ways from responses in previously humans. These differences may explain in part varying degrees of protective efficacy afforded by cholera vaccination between these two populations., Trial Registration Number: ClinicalTrials.gov NCT01895855., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Jakub K. Simon, Michael Lock, and Marc Gurwith were employed by PaxVax, Inc. Wilbur H. Chen, Caroline E. Lyon, Beth D. Kirkpatrick, Mitchell Cohen, and Myron M. Levine received research funding from PaxVax, Inc. Jakub K. Simon is currently employed by Merck & Co. All other authors report no potential conflicts.

Background: In Nepal, cases of Cholera occur annually either as sporadic or as outbreaks claiming the lives of many in rural areas. The present study is a laboratory based surveillance which aims to analyze the changing epidemiology and antimicrobial susceptibility trend of V. cholerae strains isolated or referred to National Public Health Laboratory (NPHL) over a period of 11 years (2006-2016)., Methods: Specimens of fresh stool /rectal swab either received at sentinel sites or NPHL were processed following standard microbiological techniques. Suspected colonies on selective medium were identified using routine biochemical tests and confirmed by serotyping. Antimicrobial susceptibility testing was performed following Kirby Baeur disc diffusion method., Results: Of the 836 confirmed isolates, 87% (728/836) were V.cholerae O1 Ogawa,12% (103/836) were V.cholerae O1 Inaba and only 6 isolates were V.cholerae O1 Hikojima. In 2006 all the Vibrio isolates were of Inaba serotype, followed by all 3 serotypes during 2007.During 2008-2014 only Ogawa serotype was isolated while few cases of Inaba again surfaced in 2015. Resistance to ampicillin decreased from 93% in 2006 to 18% by 2010 and again raised to 100% by 2016.Cotrimoxazole resistance remained at constant range (77-100%).Nalidixic acid resistance was 100% since 2006.Ciprofloxacin and tetracycline resistance emerged in 2007, reached a peak during 2010-2012 and declined to 0 by 2016.Susceptibility to Furazolidone has re-emerged.63.6% of the isolates were Multi drug resistant., Conclusion: With changing epidemiology and antibiogram of V.cholerae in Nepal, the present study reflects the importance of continuous monitoring, which could be used by policy makers and health professionals for better management of outbreaks. Decline in tetracycline and ciprofloxacin resistance along with emerging sensitivity to furazolidone shows that these drugs could make an effective comeback in future.

Background: Cholera is an important health problem in Sabah, a Malaysian state in northern Borneo; however, Vibrio cholerae in Sabah have never been characterized. Since 2002, serogroup O1 strains having the traits of both classical and El Tor biotype, designated as atypical El Tor biotype, have been increasingly reported as the cause of cholera worldwide. These variants are believed to produce clinically more severe disease like classical strains., Purpose: The purpose of this study is to investigate the genetic diversity of V.cholerae in Sabah and whether V.cholerae in Sabah belong to atypical El Tor biotype., Methods: ERIC-PCR, a DNA fingerprinting method for bacterial pathogens based on the enterobacterial repetitive intergenic consensus sequence, was used to study the genetic diversity of 65 clinical V.cholerae O1 isolates from 3 districts (Kudat, Beluran, Sandakan) in Sabah and one environmental isolate from coastal sea water in Kudat district. In addition, we studied the biotype-specific genetic traits in these isolates to establish their biotype., Results: Different fingerprint patterns were seen in isolates from these three districts but one of the patterns was seen in more than one district. Clinical isolates and environmental isolate have different patterns. In addition, Sabah isolates harbor genetic traits specific to both classical biotype (ctxB-1, rstR Cla ) and El Tor biotype (rstR ET , rstC, tcpA ET , rtxC, VC2346)., Conclusion: This study revealed that V.cholerae in Sabah were genetically diverse and were atypical El Tor strains. Fingerprint patterns of these isolates will be useful in tracing the origin of this pathogen in the future., (Copyright © 2018. Published by Elsevier B.V.)

Background: Toxigenic Vibrio cholerae serogroup O1 is the causative pathogen in the sixth and seventh cholera pandemics. Cholera toxin is the major virulent factor but other virulence and virulence-related factors play certain roles in the pathogenesis and survival in the host. Along with the evolution of the epidemic strains, the virulence-related genes also experience variation, gain and loss, and lead to genetic divergence in different strains., Results: In this study, we analyzed the virulence-related gene profiles in the toxigenic serogroup O1 strains isolated from 1923 to 2015, the genomes of which were publicly available. The virulence-related genes of the V. cholerae O1 strains were annotated based on the Virulence Factors Database (VFDB). An average of 230.1 virulence-related genes per strain were identified; significant differences in the average numbers were found between the classical and El Tor biotypes, and increasing trends in the number of virulence-related genes along with the isolation years were observed in the El Tor biotype strains. A total of 176 homologs of virulence-related genes were found from these strains, of which 25 belonged to the core genes, suggesting their conservative and necessary roles in V. cholerae pathogenesis. We described the diversities of the homologs by defining gene sequence type, and illustrated its association with gene duplication; we found that gene duplication clearly increased the complexity of the gene sequence types in the core virulence-related genes. In addition, we provided virulence-related gene profiles whose genetic characteristic depend on the isolation years from the view of gene gain and loss, variation, gene duplication and gene sequence type number., Conclusions: Our study reveals the comprehensive variation dynamics of the virulence-related genes in toxigenic V. cholerae serogroup O1 during epidemics. The increasing trend for the virulence-related genes may suggest the evolutional advantage of strains by gaining virulence-related genes with diverse functional categories.

Background: Cholera, an acute diarrheal disease caused by Vibrio cholerae (V. cholerae), is an endemic disease and a major public health problem in Iran. Antibiotic therapy can decrease duration of the disease, transmission of infection and contamination of the environment. Considering different pattern of V. cholerae antibiotic resistance around the world, the aim of the current systematic review and meta-analysis was to evaluate the prevalence of antibiotic resistance of V. cholerae in Iran., Methods: A systematic review of the literature was performed using related keywords in the electronic national and international databases including SID, Irandoc, Iran Medex and Magiran as well as PubMed, Scopus, Google Scholar and ISI web of knowledge. Up to July 31, 2018, 27 eligible papers were included in our meta-analysis based on the defined inclusion criteria., Results: V. cholerae O1 was the most prevalent strain isolated in Iran and exhibited a high resistance rate against numerous antibiotics including chloramphenicol (33.6%), oxytetracycline (40.2%), trimethoprim/sulphamethoxazole (86%), tetracycline (34.5%), furazolidone (69.8%), streptomycin (93.8%), polymyxin (80.7%), ampicillin (32.1%), nalidixic acid (88.9%), kanamycin (29%) and amoxicillin (30.5%)., Conclusions: According to the meta-analysis results, antibiotic therapy with ciprofloxacin, doxycycline, erythromycin, gentamicin, azithromycin, cefixime and cefepime could be effective for the treatment of severe cases of cholera in Iran.

The Bay of Bengal is known as the epicenter for seeding several devastating cholera outbreaks across the globe. Vibrio cholerae , the etiological agent of cholera, has extraordinary competency to acquire exogenous DNA by horizontal gene transfer (HGT) and adapt them into its genome for structuring metabolic processes, developing drug resistance, and colonizing the human intestine. Antimicrobial resistance (AMR) in V. cholerae has become a global concern. However, little is known about the identity of the resistance traits, source of AMR genes, acquisition process, and stability of the genetic elements linked with resistance genes in V. cholerae Here we present details of AMR profiles of 443 V. cholerae strains isolated from the stool samples of diarrheal patients from two regions of India. We sequenced the whole genome of multidrug-resistant (MDR) and extensively drug-resistant (XDR) V. cholerae to identify AMR genes and genomic elements that harbor the resistance traits. Our genomic findings were further confirmed by proteome analysis. We also engineered the genome of V. cholerae to monitor the importance of the autonomously replicating plasmid and core genome in the resistance profile. Our findings provided insights into the genomes of recent cholera isolates and identified several acquired traits including plasmids, transposons, integrative conjugative elements (ICEs), pathogenicity islands (PIs), prophages, and gene cassettes that confer fitness to the pathogen. The knowledge generated from this study would help in better understanding of V. cholerae evolution and management of cholera disease by providing clinical guidance on preferred treatment regimens., Competing Interests: The authors declare no conflict of interest.

Analyses of stool from patients with acute watery diarrhea (AWD) using sensitive molecular diagnostics have challenged whether fecal microbiological cultures have acceptably high sensitivity for cholera diagnosis. If true, these findings imply that current estimates of the global burden of cholera, which rely largely on culture-confirmation, may be underestimates. We conducted a vaccine probe study to evaluate this possibility, assessing whether an effective killed oral cholera vaccine (OCV) tested in a field trial in a cholera-endemic population conferred protection against cholera culture-negative AWD, with the assumption that if cultures are indeed insensitive, OCV protection in such cases should be detectable. We re-analysed the data of a Phase III individually-randomized placebo-controlled efficacy trial of killed OCVs conducted in Matlab, Bangladesh in 1985. We calculated the protective efficacy (PE) of a killed whole cell-only (WC-only) OCV against first-episodes of cholera culture-negative AWD during two years of post-dosing follow-up. In secondary analyses, we evaluated PE against cholera culture-negative AWD by age at vaccination, season of onset, and disease severity. In this trial 50,770 people received at least 2 complete doses of either WC-only OCV or placebo, and 791 first episodes of AWD were reported during the follow-up period, of which 365 were culture-positive for Vibrio cholerae O1. Of the 426 culture-negative AWD episodes, 215 occurred in the WC group and 211 occurred in the placebo group (adjusted PE = -1.7%; 95%CI -23.0 to 13.9%, p = 0.859). No measurable PE of OCV was observed against all or severe cholera culture-negative AWD when measured overall or by age and season subgroups. In this OCV probe study we detected no vaccine protection against AWD episodes for which fecal cultures were negative for Vibrio cholera O1. Results from this setting suggest that fecal cultures from patients with AWD were highly sensitive for cholera episodes that were etiologically attributable to this pathogen. Similar analyses of other OCV randomized controlled trials are recommended to corroborate these findings., Competing Interests: The authors have declared that no competing interests exist.

Objective: Diarrheal diseases, including cholera, remain a major public health concern in developing countries like Nepal. This study investigated a diarrheal outbreak that affected over 1500 people in Gaidatar village of Rautahat district in central Nepal and sought to identify the source and causation of the disease. Stool samples were collected from individuals with acute diarrheal illness (n = 16) and healthy non-diarrheal children (n = 39), along with samples from local drinking water sources (n = 8) and their sewage system (n = 10). None of the individuals were sampled multiple times. Diarrheic stool and sewage samples were analysed for the presence of Vibrio cholerae, while coliforms were tested in drinking water samples following standard microbiological protocols. Enteric parasites were tested in both diarrheic and non-diarrheic stool samples., Results: Vibrio cholerae O1 Ogawa serotype was isolated in 18.7% of the diarrheic stool and 20.0% of the sewage. Coliforms were found in all drinking water samples, with 87.5% testing positive for fecal coliform. Additionally, 43.6% of the stool samples (n = 55) had at least one of the intestinal parasites tested, primarily Giardia lamblia (21.8%). However, almost all parasites were found in non-diarrheal stool. Taken together, our results provide evidence that the diarrheal outbreak was associated with V. cholerae O1 Ogawa serotype, possibly transmitted through the drinking water sources contaminated with fecal matters from their sewage (drainage) system. These findings warrant regular surveillance of drinking water sources to help prevent future outbreaks.

Cholera has affected Haiti with damping waves of outbreaks since October 2010. However, mechanisms behind disease persistence during lull periods remain poorly understood. By mid 2014, cholera transmission seemed to only persist in the northern part of Haiti. Meanwhile, cholera appeared nearly extinct in the capital, Port-au-Prince, where it eventually exploded in September 2014. This study aimed to determine whether this outbreak was caused by local undetected cases or by re-importation of the disease from the north. Applying an integrated approach between November 2013 and November 2014, we assessed the temporal and spatial dynamics of cholera using routine surveillance data and performed population genetics analyses of 178 Vibrio cholerae O1 clinical isolates. The results suggest that the northern part of the country exhibited a persisting metapopulation pattern with roaming oligoclonal outbreaks that could not be effectively controlled. Conversely, undetected and unaddressed autochthonous low-grade transmission persisted in the Port-au-Prince area, which may have been the source of the acute outbreak in late-2014. Cholera genotyping is a simple but powerful tool to adapt control strategies based on epidemic specificities. In Haiti, these data have already yielded significant progress in cholera surveillance, which is a key component of the strategy to eventually eliminate cholera.

Purpose: Two natural epidemic biotypes of Vibrio cholerae O1, classical and El Tor, exhibit different patterns of sensitivity against the antimicrobial peptide polymyxin B. This difference in sensitivity has been one of the major markers in biotype classification system for several decades. A recent report regarding the emergence of polymyxin B-sensitive El Tor V. cholerae O1 in Kolkata has motivated us to track the spread of the strains containing this important trait, along with Haitian-like genetic content, in different parts of India., Methodology: We have collected 260 clinical V. cholerae O1 strains from 12 states in India and screened them for polymyxin B susceptibility. Genetic characterization was also performed to study the tcpA, ctxB and rtxA genotypes by allele-specific polymerase chain reaction (PCR) and nucleotide sequencing., Results: Interestingly, 88.85 % of the isolates were found to be sensitive to polymyxin B. All of the states, with the exception of Assam, had polymyxin B-sensitive V. cholerae strains and complete replacement with this strain was found in eight of the states. However, from 2016 onwards, all the strains tested showed sensitivity to polymyxin B. Allele-specific PCR and sequencing confirmed that all strains possessed Haitian-like genetic traits., Conclusion: Polymyxin B-sensitive strains have begun to spread throughout India and may lead to the revision of the biotype classification. The dissemination of these new variant strains needs to be carefully monitored in different endemic populations through active holistic surveillance to understand their clinical and epidemiological consequences.

Dadaab Refugee camp in Garissa County, Kenya, hosts nearly 340,000 refugees in five subcamps (Dagahaley, Hagadera, Ifo, Ifo2, and Kambioos) (1). On November 18 and 19, 2015, during an ongoing national cholera outbreak (2), two camp residents were evaluated for acute watery diarrhea (three or more stools in ≤24 hours); Vibrio cholerae serogroup O1 serotype Ogawa was isolated from stool specimens collected from both patients. Within 1 week of the report of index cases, an additional 45 cases of acute watery diarrhea were reported. The United Nations High Commissioner for Refugees and their health-sector partners coordinated the cholera response, community outreach and water, sanitation, and hygiene (WASH) activities; Médecins Sans Frontiéres and the International Rescue Committee were involved in management of cholera treatment centers; CDC performed laboratory confirmation of cases and undertook GIS mapping and postoutbreak response assessment; and the Garissa County Government and the Kenya Ministry of Health conducted a case-control study. To prevent future cholera outbreaks, improvements to WASH and enhanced disease surveillance systems in Dadaab camp and the surrounding area are needed., Competing Interests: All authors have completed and submitted the ICMJE form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Background: For almost 50 years sub-Saharan Africa, including Uganda, has experienced several outbreaks due to Vibrio cholerae. Our aim was to determine the genetic relatedness and spread of strains responsible for cholera outbreaks in Uganda., Methodology/principal Findings: Sixty-three V. cholerae isolates collected from outbreaks in Uganda between 2014 and 2016 were tested using multiplex polymerase chain reaction (PCR), multi-locus variable number of tandem repeat analysis (MLVA) and whole genome sequencing (WGS). Three closely related MLVA clonal complexes (CC) were identified: CC1, 32% (20/63); CC2, 40% (25/63) and CC3, 28% (18/63). Each CC contained isolates from a different WGS clade. These clades were contained in the third wave of the 7th cholera pandemic strain, two clades were contained in the transmission event (T)10 lineage and other in T13. Analysing the dates and genetic relatedness revealed that V. cholerae genetic lineages spread between districts within Uganda and across national borders., Conclusion: The V. cholerae strains showed local and regional transmission within Uganda and the East African region. To prevent, control and eliminate cholera, these countries should implement strong cross-border collaboration and regional coordination of preventive activities., Competing Interests: The authors have declared that no competing interests exist.

Competing Interests: No conflicts of interest were reported.

Introduction: Cholera outbreaks in rural communities are associated with high morbidity and mortality. Effective interventions to control these outbreaks require identification of source and risk factors for infection. In September, 2010 we investigated a cholera outbreak in Bashuri, a cholera naïve rural community in northern Nigeria to identify the risk factors and institute control measures., Methods: We conducted an unmatched case-control study. We defined a case as any resident of Bashuri community two years and above with acute watery diarrhea with or without vomiting and a control as any resident two years and above without acute watery diarrhea and vomiting. We recruited 80 hospital-based cases and 80 neighborhood controls. We collected and analyzed data on demographic characteristics, clinical information and risk factors. Laboratory analysis was performed on 10 stool samples and 14 open-well samples., Results: Mean age was 29 years (± 20 years) for cases and 32 years (± 16 years) for controls; 38 (47.5%) of cases and 60 (75%) of controls were males. Compared to controls, cases were less likely to have washed hands with soap before eating (age-adjusted odds ratio (AAOR) = 0.27, 95% confidence interval (CI): 0.10-0.72) and less likely to have washed hands with soap after using the toilet (AAOR = 0.34, 95% CI: 0.15-0.75). Vibrio cholerae O1 was isolated from six stool samples but not from any open-well samples., Conclusion: Unhygienic hand washing practices was the key risk factor in this outbreak. We educated the community on personal hygiene focusing on the importance of hand washing with soap.

Recognizing cholera cases early, especially in the initial phase of an outbreak and in areas where cholera has not previously circulated, is a high public health priority. Laboratory capacity in such settings is often limited. To address this, we have developed a rapid diagnostic test (RDT) termed Cholkit that is based on an immunochromatographic lateral flow assay for the diagnosis of cholera cases using stool. Cholkit contains a monoclonal antibody (ICL-33) to the O-specific polysaccharide (OSP) component of V. cholerae O1 lipopolysaccharide, and recognizes both Inaba and Ogawa serotypes. We tested the Cholkit dipstick using fresh stool specimens of 76 adults and children presenting with acute watery diarrhea at the icddr,b hospital in Dhaka, Bangladesh. We compared Cholkit's performance with those of microbial culture, PCR (targeting the rfb and ctxA genes of V. cholerae) and the commercially available RDT, Crystal VC (Span Diagnostics; Surat, India). We found that all stool specimens with a positive culture for V. cholerae O1 (n = 19) were positive by Cholkit as well as Crystal VC. We then used Bayesian latent class modeling to estimate the sensitivity and specificity of each diagnostic assay. The sensitivity of Cholkit, microbiological culture, PCR and Crystal VC was 98% (95% CI: 88-100), 71% (95% CI: 59-81), 74% (95% CI: 59-86) and 98% (95% CI: 88-100), respectively. The specificity for V. cholerae O1 was 97% (95% CI: 89-100), 100%, 97% (95% CI: 93-99) and 98% (95% CI: 92-100), respectively. Of note, two Crystal VC dipsticks were positive for V. cholerae O139 but negative by culture and PCR in this area without known circulating epidemic V. cholerae O139. In conclusion, the Cholkit dipstick is simple to use, requires no dedicated laboratory capacity, and has a sensitivity and specificity for V. cholerae O1 of 98% and 97%, respectively. Cholkit warrants further evaluation in other settings.

Objective: The purpose of this study was to investigate the virulent factors of Vibrio cholerae which caused an unprecedented large cholera outbreak in Ghana in 2014 and progressed into 2015, affected 28,975 people with 243 deaths., Results: The V. cholerae isolates were identified to be the classical V. cholerae 01 biotype El Tor, serotype Ogawa, responsible for the large cholera outbreak in Ghana. These El Tor strains bear CtxAB and Tcp virulent genes, making the strains highly virulent. The strains also bear SXT transmissible element coding their resistance to antibiotics, causing high proportions of the strains to be multidrug resistant, with resistant proportions of 95, 90 and 75% to trimethoprim/sulfamethoxazole, ampicillin and ceftriaxone respectively. PFGE patterns indicated that the isolates clustered together with the same pattern and showed clusters similar to strains circulating in DR Congo, Cameroun, Ivory Coast and Togo. The strains carried virulence genes which facilitated the disease causation and spread. This is the first time these virulent genes were determined on the Ghanaian Vibrio strains.

Cholera toxin is a major virulent agent of Vibrio cholerae, and it can rapidly lead to severe dehydration, shock, causing death within hours without appropriate clinical treatments. In this study, we present a method wherein unique and short peptides that bind to cholera toxin subunit B (CTX-B) were selected through M13 phage display. Biopanning over recombinant CTX-B led to rapid screening of a unique peptide with an amino acid sequence of VQCRLGPPWCAK, and the phage-displayed peptides analyzed using ELISA, were found to show specific affinities towards CTX-B. To address the use of affinity peptides in development of the biosensor, sequences of newly selected peptides were modified and chemically synthesized to create a series of affinity peptides. Performance of the biosensor was studied using plasmonic-based optical techniques: localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering (SERS). The limit of detection (LOD) obtained by LSPR with 3σ-rule was 1.89ng/mL, while SERS had a LOD of 3.51pg/mL. In both cases, the sensitivity was much higher than the previously reported values, and our sensor system was specific towards actual CTX-B secreted from V. cholera, but not for CTX-AB 5 ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: in February 2015, an outbreak of acute watery diarrhea was reported in two sub counties in western Kenya. Vibrio cholera e 01 serotype Ogawa was isolated from 26 cases and from water samples collected from a river mainly used by residents of the two sub-counties for domestic purposes. We carried out an investigation to determine factors associated with the outbreak., Methods: we conducted a frequency matched case control study in the community. We defined cases as episodes of watery diarrhea (at least three motions in 24 hours) in persons ≥ 2 years who were residents of Rongo or Ndhiwa sub-counties from January 23-February 25, 2015. Cases were systematically recruited from a cholera line list and matched to two controls (persons without diarrhea since January 23, 2015) by age category and residence. A structured questionnaire was administered to evaluate exposures in cases and controls and multivariable logistic regression done to determine independent factors associated with the outbreak., Results: we recruited 52 cases and 104 controls. Females constituted 61% (95/156) of all participants. Overall latrine coverage was 58% (90/156). Latrine coverage was 44% (23/52) for cases and 64% (67/104) for controls. Having no latrine at home (aOR = 10.9; 95% CI: 3.02-39.21), practicing communal hand washing in a basin (aOR = 6.5; 95% CI: 2.30-18.11) and vending of food as an occupation (aOR = 3.4; 95% CI: 1.06-10.74) were independently associated with the outbreak., Conclusion: poor latrine coverage and personal hygiene practices were identified as the main drivers of the outbreak. We recommended improved public health education on latrine usage and promotion of hand washing with soap and water in the community.

The large outbreak of cholera reported during July to September 2014 in the Narla block of Kalahandi district, India, was investigated to determine the causative organism. Rectal swabs collected from patients with diarrhea and environmental water samples were cultured following standard techniques. The causative organism was identified as Vibrio cholerae O1 Ogawa biotype El Tor, and analysis by double mismatch mutation assay PCR confirmed that all strains were the ctxB7 variant of Haitian V. cholerae O1. The environmental water samples were negative for V. cholerae. The V. cholerae O1 strains were sensitive to tetracycline, ciprofloxacin, norfloxacin, ofloxacin, doxycycline, and azithromycin, but were resistant to erythromycin, gentamicin, chloramphenicol, furazolidone, neomycin, cotrimoxazole, nalidixic acid, and ampicillin. In the 2014 cholera outbreak, the early reporting of the pathogen enabled the government authorities to implement adequate control measures in time to curtail the spread of the disease. That was the second large cholera outbreak due to Haitian variants of V. cholerae O1 after the 2010 Haiti cholera outbreak reported from Odisha, India, and other locations globally. Active surveillance is required to track the spread of this strain in the Odisha region.

Background: A nationwide outbreak of Vibrio cholerae occurred in Ghana in 2014 with Accra, the nation's capital as the epi-center. The outbreak spread to the Brong Ahafo Region (BAR) which is geographically located in the middle of the country. In this region a review of data collected during the outbreak was carried out and analyzed descriptively to determine the hot spots and make recommendations for effective response to future outbreaks., Methods: A review of patient records and line lists of cases of cholera reported in all hospitals during the period of the outbreak (July-December 2014) was conducted. Hospitals used IDSR (Integrated Disease Surveillance and Response system) standard case definitions to detect and report cases for management. The GPS coordinates of all districts and health facilities were collected and utilized in the construction of spot maps. We also obtained populations (denominators) from the BAR Health surveillance unit of the Ghana Health Service. All the data thus collected was analyzed descriptively and expressed as frequencies and rates., Results: A total of 1035 cases were reported, 550 (53.4%) were males and the rest females. Their ages ranged from 1 to 95 years; (mean age of 28.2 ± 19.6 years). The most affected (23.5%) was the 20-29 year old age group. On the 30th July, 2014, a 26 year old male (recorded as the index case of the cholera outbreak in the Brong Ahafo region) with a history of travel from Accra reported to the Nkoranza district hospital with a history of symptoms suggestive of cholera. The reporting of cholera cases reached their peak (17.3%) in week 15 of the outbreak (this lasted 25 weeks). An overall attack rate of 71/100,000 population, and a case fatality rate of 2.4% was recorded in the region. Asutifi South district however recorded a case fatality of 9.1%, the highest amongst all the districts which recorded outbreaks. The majority of the cases reported in the region were from Atebubu-Amanten, Sene West, Pru, and Asunafo North districts with 31.1, 26.0, 18.2 and 9.9% respectively. Vibrio cholerae serotype O1 was isolated from rectal swabs/stool samples tested., Conclusion: Vibrio cholerae serotype O1 caused the cholera-outbreak in the Brong Ahafo Region and mainly affected young adult-males. The most affected districts were Atebubu-Amanten, Sene west, Pru (located in the eastern part of the region), and Asunafo North districts (located in the south west of the region). Case Fatality Rate was higher (2.4%) than the WHO recommended rate (<1%). Active district level public health education is recommended on prevention and effective response for future outbreaks of cholera.

Rationale: Mozambique has recorded cyclically epidemic outbreaks of cholera. Antibiotic therapy is recommended in specific situations for management and control of cholera outbreaks. However, an increase in resistance rates to antibiotics by Vibrio cholerae has been reported in several epidemic outbreaks worldwide. On the other hand, there are few recent records of continuous surveillance of antibiotics susceptibility pattern of V. cholerae in Mozambique., Goals: The purpose of this study was to evaluate antibiotics resistance pattern of Vibrio cholerae O1 Ogawa isolated during Cholera outbreaks in Mozambique to commonly used antibiotics., Methodology: We analyzed data from samples received in the context of surveillance and response to Cholera outbreaks in the National Reference Laboratory of Microbiology from the National Institute of Health of Mozambique, 159 samples suspected of cholera from cholera treatment centers of, Metangula (09), Memba (01), Tete City (08), Moatize (01), Morrumbala (01) districts, City of Quelimane (01), Lichinga (06) and Nampula (86) districts, from 2012 to 2015. Laboratory culture and standard biochemical tests were employed to isolate and identify Vibrio cholerae; serotypes were determined by antisera agglutination reaction in blade. Biotype and presence of important virulence factors analysis was done by PCR. Antibiotics susceptibility pattern was detected by disk diffusion method Kirby Bauer. Antibiotic susceptibility and results were interpreted by following as per recommendations of CLSI (Clinical and Laboratory Standards Institute) 2014. All samples were collected and tested in the context of Africhol Project, approved by the National Bioethics Committee for Health., Results: Among isolates from of Vibrio cholerae O1 El Tor Ogawa resistance to Sulphamethoxazole-trimethropim was 100% (53/53) to Trimethoprim-, being 100% (54/54) for Ampicillin, 99% (72/74) for Nalidixic Acid, 97% (64/66) to Chloramphenicol, 95% (42/44) for Nitrofurantoin and (19/20) Cotrimoxazole, 83% (80/97) Tetracycline, 56% (5/13) Doxycycline, 56% (39/70) Azithromycin and 0% (0/101) for Ciprofloxacin. PCR analysis suggested strains of V. cholerae O1 being descendants of the current seventh pandemic V. cholerae O1 CIRS 101 hybrid variant. The V. cholerae O1 currently causing cholera epidemics in north and central Mozambique confirmed a CTXΦ genotype and a molecular arrangement similar to the V. cholerae O1 CIRS 101., Conclusion: Although V. cholerae infections in Mozambique are generally not treated with antibiotics circulating strains of the bacteria showed high frequency of in vitro resistance to available antibiotics. Continuous monitoring of antibiotic resistance pattern of epidemic strains is therefore crucial since the appearance of antibiotic resistance can influence cholera control strategies.

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL-1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL-1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

Background: After more than a decade of steadily declining notifications, the number of reported cholera cases has recently increased in Vietnam. We conducted a matched case-control study to investigate transmission of cholera during an outbreak in Ben Tre, southern Vietnam, and to explore the associated risk factors., Methodology/principal Findings: Sixty of 71 diarrheal patients confirmed to be infected with cholera by culture and diagnosed between May 9 and August 3, 2010 in Ben Tre were consecutively recruited as case-patients. Case-patients were matched 1:4 to controls by commune, sex, and 5-year age group. Risk factors for cholera were examined by multivariable conditional logistic regression. In addition, environmental samples from villages containing case-patients were taken to identify contamination of food and water sources. The regression indicated that drinking iced tea (adjusted odds ratio (aOR) = 8.40, 95% confidence interval (CI): 1.84-39.25), not always boiling drinking water (aOR = 2.62, 95% CI: 1.03-6.67), having the main source of water for use being close to a toilet (aOR = 4.36, 95% CI: 1.37-13.88), living with people who had acute diarrhea (aOR = 13.72, 95% CI: 2.77-67.97), and little or no education (aOR = 4.89, 95% CI: 1.18-20.19) were significantly associated with increased risk of cholera. In contrast, drinking stored rainwater (aOR = 0.17, 95% CI: 0.04-0.63), eating cooked seafood (aOR = 0.27, 95% CI: 0.10-0.73), and eating steamed vegetables (aOR = 0.22, 95% CI: 0.07-0.70) were protective against cholera. Vibrio cholerae O1 Ogawa carrying ctxA was found in two of twenty-five river water samples and one of six wastewater samples., Conclusions/significance: The magnitude of the cholera outbreak in Ben Tre was lower than in other similar settings. This investigation identified several risk factors and underscored the importance of continued responses targeting cholera prevention in southern Vietnam. The association between drinking iced tea and cholera and the spread of V. cholerae O1, altered El Tor strains warrant further research. These findings might be affected by a number of limitations due to the inability to capture asymptomatic or mildly symptomatic infections, the possible underreporting of personal unhygienic behaviors, and the purposive selection of environmental samples.

The rapid and accurate quantification of the pathogenic bacteria is extremely critical to decrease the bacterial infections in all areas related to health and safety. We have developed an electrochemical strategy for simultaneous ultrasensitive detection of E. coli O157:H7 and Vibrio cholerae O1. This approach was based on the specific immune recognition of different pathogenic bacteria by multifunctional nanoconjugates and subsequent signal amplification. By employing the proposed biosensor, the concentrations of these pathogenic bacteria could be established on a single interface in a single run with improved sensitivity and accuracy. The successful approach of the simultaneous detection and quantification of two bacteria by an electrochemical biosensor demonstrated here could be readily expanded for the estimation of a variety of other pathogenic bacteria, proteins, and nucleotides. Because of their high sensitivity, electrochemical biosensors may represent a new avenue for early diagnosis of diseases., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Background: Cholera outbreaks have occurred in Tanzania since 1974. To date, the genetic epidemiology of these outbreaks has not been assessed., Methods: 96 Vibrio cholerae O1 isolates from five regions were characterized, and their genetic relatedness assessed using multi-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS)., Results: Of the 48 MLVA genotypes observed, 3 were genetically unrelated to any others, while the remaining 45 genotypes separated into three MLVA clonal complexes (CCs) - each comprised of genotypes differing by a single allelic change. In Kigoma, two separate outbreaks, 4 months apart (January and May, 2015), were each caused by genetically distinct strains by MLVA and WGS. Remarkably, one MLVA CC contained isolates from both the May outbreak and ones from the 2011/2012 outbreak in Dar-es-Salaam. However, WGS revealed the isolates from the two outbreaks to be distinct clades. The outbreak that started in August 2015 in Dar-es-Salaam and spread to Morogoro, Singida and Mara was comprised of a single MLVA CC and WGS clade. Isolates from within an outbreak were closely related differing at fewer than 5 nucleotides. All isolates were part of the 3 rd wave of the 7 th pandemic and were found in four clades related to isolates from Kenya and Asia., Conclusions: We conclude that genetically related V. cholerae cluster in outbreaks, and distinct strains circulate simultaneously.