Phylogenetic and antimicrobial drug resistance analysis of Vibrio cholerae O1 isolates from Ghana.

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.

Laboratory evaluation of the rapid diagnostic tests for the detection of Vibrio cholerae O1 using diarrheal samples.

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.

Three transmission events of Vibrio cholerae O1 into Lusaka, Zambia.

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.

Laboratory and Field Evaluation of the Crystal VC-O1 Cholera Rapid Diagnostic Test.

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.

Clinical and socio-environmental determinants of multidrug-resistant vibrio cholerae 01 in older children and adults in Bangladesh.

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.

Cholera dynamics: lessons from an epidemic.

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.

Spread of Haitian Variant Vibrio cholerae O1 Causing Cholera Outbreaks in Odisha, India.

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.

Immunodiagnostic of Vibrio cholerae O1 using localized surface plasmon resonance (LSPR) biosensor.

V. cholerae O1 is a gram-negative bacilli that causes an acute gastrointestinal disease called cholera. V. cholerae can enter into the biofilm phase in a period of life; hence, it is challenging to recognize these bacteria. Accordingly, using localized surface plasmon resonance (LSPR) features of the nanoparticles, an accurate detection method based on the antigen-antibody reaction was used. Ordinarily, immobilization of plasmonic nanoparticles by monoclonal antibodies was performed and UV-visible spectroscopy, dynamic light scattering (DLS), and zeta potential (Zp) measurements verified the conjugation process. In the vicinity of several concentrations of V. cholerae O1, the consistency of the engineered nanobioprobe was then investigated using LSPR monitoring and colorimetric assay. Finally, the ELISA and PCR techniques contrasted the sensitivity of nanobiosensors. The results showed that by applying monoclonal antibodies as a sensor feature, the nanobioprobe showed high sensitivity to target bacterial analysis. Thus, the limit of detection in this immunoassay-based biosensor was calculated to be a sharp reduction in the absorption of 10 CFU/mL of V. cholerae O1 with approximately 5 nm of redshift, while the shift of light refraction in the LSPR band was extended to approximately 18 nm by raising the antigen concentration to 104 CFU/mL. This LSPR biosensor can therefore be used for V. cholerae O1 (Inaba strain) detection as a simple, sensitive, and reliable diagnostic tool. In conclusion, the built biosensor will facilitate and speed up V. cholerae O1 (Inaba strain) classification by controlling the specific antigen to prevent the unintended spread of cholera disease.

Whole-Genome Analysis of Clinical Vibrio cholerae O1 in Kolkata, India, and Dhaka, Bangladesh, Reveals Two Lineages of Circulating Strains, Indicating Variation in Genomic Attributes.

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.

Cholera outbreak investigation, Bhadola, Delhi, India, April-May 2018.

BACKGROUND: In the Gangetic plains of India, including Delhi, cholera is endemic. On 10 May 2018, staff at the north Delhi district surveillance unit identified a laboratory-confirmed cholera outbreak when five people tested positive for Vibrio cholerae O1 Ogawa serotype in Bhadola. We investigated to identify risk factors and recommend prevention measures. METHODS: We defined a case as ≥3 loose stools within 24 h in a Bhadola resident during 1 April-29 May 2018. We searched for cases house-to-house. In a 1 : 1 unmatched case control study, a control was defined as an absence of loose stools in a Bhadola resident during 1 April-29 May 2018. We selected cases and controls randomly. We tested stool samples for Vibrio cholerae by culture. We tested drinking water for fecal contamination. Using multivariable logistic regression we calculated adjusted ORs (aORs) with 95% CIs. RESULTS: We identified 129 cases; the median age was 14.5 y, 52% were females, 27% were hospitalized and there were no deaths. Symptoms were abdominal pain (54%), vomiting (44%) and fever (29%). Among 90 cases and controls, the odds of illness were higher for drinking untreated municipal water (aOR=2.3; 95% CI 1.0 to 6.2) and not knowing about diarrhea transmission (aOR=4.9; 95% CI 1.0 to 21.1). Of 12 stool samples, 6 (50%) tested positive for Vibrio cholerae O1 Ogawa serotype. Of 15 water samples, 8 (53%) showed growth of fecal coliforms. CONCLUSIONS: This laboratory-confirmed cholera outbreak associated with drinking untreated municipal water and lack of knowledge of diarrhea transmission triggered public health action in Bhadola, Delhi.

Comparison of chitin-induced natural transformation in pandemic Vibrio cholerae O1 El Tor strains.

The human pathogen Vibrio cholerae serves as a model organism for many important processes ranging from pathogenesis to natural transformation, which has been extensively studied in this bacterium. Previous work has deciphered important regulatory circuits involved in natural competence induction as well as mechanistic details related to its DNA acquisition and uptake potential. However, since competence was first reported for V. cholerae in 2005, many researchers have struggled with reproducibility in certain strains. In this study, we therefore compare prominent seventh pandemic V. cholerae isolates, namely strains A1552, N16961, C6706, C6709, E7946, P27459, and the close relative MO10, for their natural transformability and decipher underlying defects that mask the high degree of competence conservation. Through a combination of experimental approaches and comparative genomics based on new whole-genome sequences and de novo assemblies, we identify several strain-specific defects, mostly in genes that encode key players in quorum sensing. Moreover, we provide evidence that most of these deficiencies might have recently occurred through laboratory domestication events or through the acquisition of mobile genetic elements. Lastly, we highlight that differing experimental approaches between research groups might explain more of the variations than strain-specific alterations.

Cholera outbreak in Addis Ababa, Ethiopia: A case-control study.

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.

Characterisation of Vibrio cholerae isolates from the 2009, 2010 and 2016 cholera outbreaks in Lusaka province, Zambia.

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.

Investigation of an imported cholera case in China with whole genome sequencing.

Determining the source and genetic characteristics of the imported pathogen is critical in the control of infectious diseases. Here, we reported the investigation of an imported cholera case in China in 2018 with a recent travel history in Nepal and India. Stool culture from the patient was identified as Vibrio cholerae serogroup O1, biotype El Tor, serotype Ogawa. The strain 2018HL24 possessed intact Vibrio seventh pandemic island I (VSP-I), Vibrio pathogenicity Island 1 and 2 (VPI-1, VPI-2). A VSP-II variant with a 13 kb deletion was also detected, which was identical to those observed in V. cholerae in cluster "Nepal-4". Phylogenetic analysis based on the core genome SNPs showed that the isolate was most closely related to the V. cholerae isolated in northern India not far from the border of Nepal in 2012 (16 SNPs). Combining the epidemiological data with phylogenetic analysis results, we speculate that the patient may got infected in Nepal-India region.

Changing Susceptibility Pattern of Vibrio cholerae O1 Isolates to Commonly Used Antibiotics in the Largest Diarrheal Disease Hospital in Bangladesh during 2000-2018.

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.

Quorum sensing regulation confronts the development of a viable but non-culturable state in Vibrio cholerae.

Vibrio cholerae can enter a viable but non-culturable (VBNC) state when it encounters unfavourable environments; VBNC cells serve as important reservoirs and still pose threats to public health. The genetic regulation of V. cholerae entering its VBNC state is not well understood. Here, we show a confrontation strategy adapted by V. cholerae O1 in which it utilizes a quorum sensing (QS) system to prevent transition into a VBNC state under low nutrition and temperature conditions. The upregulation of hapR resulted in a prolonged culturable state of V. cholerae in artificial sea water at 4°C, whereas the mutation of hapR led to fast entry into the VBNC state. We also observed that different V. cholerae O1 natural isolates with distinct QS functions present a variety of abilities to maintain culturability during the transition to a VBNC state. The strain groups with higher or constitutive expression of QS genes exhibit a greater tendency to maintain the culturable state during VBNC induction than those lacking QS functional groups. In summary, HapR-mediated QS regulation is associated with the transition to the VBNC state in V. cholerae. HapR expression causes V. cholerae to resist VBNC induction and become dominant over competitors in changing environments.

[Value of the syndromic approach for the diagnosis of an imported case of cholera]. / Intérêt de l'approche syndromique dans le diagnostic d'un cas importé de choléra.

A 70-year-old woman with no relevant medical history presented banal clinical signs of infectious gastroenteritis on her return from a trip to the Republic of the Union of Myanmar. The appearance of her stools and clinical findings were not suggestive of a typical case of cholera, but Vibrio cholerae was nevertheless isolated from her stools in the laboratory. The National reference center (NRC) for vibrios and cholera identified a Vibrio cholerae serogroup O1 (serotype Inaba) strain. The health authorities were notified of an imported case of cholera, identified on the basis of clinical, biological and epidemiological data. The diagnostic strategy used in the laboratory was based on a two-step algorithm involving molecular biological screening followed by culture on selective media for species identification. It was this approach, benefiting from the complementarity of the different techniques, that made it possible to reach a reliable rapid biological diagnosis of this atypical, but frequent form of the disease. The diagnosis of imported cases is of the utmost importance, because the mandatory signaling and notification of cases trigger investigations to check for additional cases among other exposed individuals or contacts of the patient, even though the risk of secondary transmission appears to be low in France. It also supplies data to international surveillance networks for cholera, which remains a serious disease and a major problem globally. This case highlights the importance of interactions between the various biological personnel and clinicians.

Haitian-like genetic traits with creeping MIC of Azithromycin in Vibrio cholerae O1 isolates from Puducherry, India.

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.

Inland cholera in freshwater environs of north India.

In the freshwater environment of north India, cholera appears seasonally in form of clusters as well as sporadically, accounting for a significant piece of the puzzle of cholera epidemiology. We describe a number of cholera outbreaks with an average attack rate of 96.5/1000 but an overall low case fatality (0.17). Clinical cholera cases coincided with high rainfall and elevated temperatures, whereas isolation of V. cholerae non-O1 non-O139 from water was dependent on temperature (p < 0.05) but was independent of rainfall and pH (p > 0.05). However, isolation from plankton samples correlated with increased temperature and pH (p < 0.05). A lag period of almost a month was observed between rising temperature and increased isolation of V. cholerae from the environment, which in succession was followed by an appearance of cholera cases in the community a month later. Our results suggested that the aquatic environment can harbor highly divergent V. cholerae strains and serve as a reservoir for multiple V. cholera virulence-associated genes that may be exchanged via mobile genetic elements. In agreement with PFGE, AFLP data also proved that the V. cholerae O1 population was not clonal but was closely related. Our investigation did not support the concept that seasonal cholera outbreaks occur by movement of a single clonal strain across the region, as the clinical isolates from the same years were clearly different, implying that continuous evolution of V. cholerae O1 strains occurs in the cholera endemic area. Interestingly, the viable but non-culturable (VBNC) V. cholerae O1 cells were demonstrated in 2.21% samples from natural water bodies in addition to 40.69% samples from cholera-affected areas respectively. This suggests that aquatic environs do harbor the pathogenic O1 strain, though the isolation of culturable V. cholerae O1 is a rare event in the presence of relatively abundant non-O1 non-O139 isolates.

Diagnostic techniques for rapid detection of Vibrio cholerae O1/O139.

Cholera caused by the toxigenic Vibrio cholerae is still a major public health problem in many countries. This disease is mainly due to poor sanitation, hygiene and consumption of unsafe water. Several recent epidemics of cholera showed its increasing intensity, duration and severity of the illness. This indicates an urgent need for effective management and preventive measures in controlling the outbreaks and epidemics. In preventing and spread of epidemic cholera, rapid diagnostic tests (RDTs) are useful in screening suspected stool specimens, water/food samples. Several RDTs developed recently are considered as investigative tools in confirming cholera cases, as the culture techniques are difficult to establish and/or maintain. The usefulness of RDTs will be more at the point-of-care facilities as it helps to make appropriate decisions in the management of outbreaks or epidemiological surveillance by the public health authorities. Apart from RDTs, several other tests are available for the direct detection of either V. cholerae or its cholera toxin. Viable but non-culturable (VBNC) state of V. cholerae poses a great challenge in developing RDTs. The aim of this article is to provide an overview of current knowledge about RDT and other techniques with reference to their status and future potentials in detecting cholera/V. cholerae.