Piperine, an active ingredient of white pepper, suppresses the growth of multidrug-resistant toxigenic Vibrio cholerae and other pathogenic bacteria.

Emergence and rapid spread of multidrug-resistant (MDR) bacteria including Vibrio cholerae are a global public health issue. Much attention has been paid to natural compounds, such as spices and herbs to find novel antimicrobial compounds as they are considered to be cheaper alternatives to develop as a drug. Here, we show that methanol extract of white pepper could inhibit the growth of V. cholerae O1 El Tor variant, responsible for the recent outbreaks/epidemics. Furthermore, we demonstrate for the first time that piperine, the major component of white pepper, showed a dose-dependent bactericidal effect on V. cholerae growth irrespective of their biotypes and serogroups in the presence of 200 and 300 µg ml-1 of piperine, respectively. Piperine also inhibited the growth of MDR strains of Pseudomonas aeruginosa, Escherichia coli isolated from poultry and enterohemorrhagic/enteroaggregative E. coli O104 in the presence of 200 µg ml-1 . Interestingly, we did not observe any significant inhibitory effect of piperine on E. coli strains isolated from healthy person even up to 200 µg ml-1 . Our data suggest that piperine could be a novel antimicrobial agent in therapeutic and preventive applications against infections caused by pathogenic bacteria including MDR strains.

Characteristics of Salmonella Recovered From Stools of Children Enrolled in the Global Enteric Multicenter Study.

BACKGROUND: The Global Enteric Multicenter Study (GEMS) determined the etiologic agents of moderate-to-severe diarrhea (MSD) in children under 5 years old in Africa and Asia. Here, we describe the prevalence and antimicrobial susceptibility of nontyphoidal Salmonella (NTS) serovars in GEMS and examine the phylogenetics of Salmonella Typhimurium ST313 isolates. METHODS: Salmonella isolated from children with MSD or diarrhea-free controls were identified by classical clinical microbiology and serotyped using antisera and/or whole-genome sequence data. We evaluated antimicrobial susceptibility using the Kirby-Bauer disk-diffusion method. Salmonella Typhimurium sequence types were determined using multi-locus sequence typing, and whole-genome sequencing was performed to assess the phylogeny of ST313. RESULTS: Of 370 Salmonella-positive individuals, 190 (51.4%) were MSD cases and 180 (48.6%) were diarrhea-free controls. The most frequent Salmonella serovars identified were Salmonella Typhimurium, serogroup O:8 (C2-C3), serogroup O:6,7 (C1), Salmonella Paratyphi B Java, and serogroup O:4 (B). The prevalence of NTS was low but similar across sites, regardless of age, and was similar among both cases and controls except in Kenya, where Salmonella Typhimurium was more commonly associated with cases than controls. Phylogenetic analysis showed that these Salmonella Typhimurium isolates, all ST313, were highly genetically related to isolates from controls. Generally, Salmonella isolates from Asia were resistant to ciprofloxacin and ceftriaxone, but African isolates were susceptible to these antibiotics. CONCLUSIONS: Our data confirm that NTS is prevalent, albeit at low levels, in Africa and South Asia. Our findings provide further evidence that multidrug-resistant Salmonella Typhimurium ST313 can be carried asymptomatically by humans in sub-Saharan Africa.

Changing facades of Vibrio cholerae: An enigma in the epidemiology of cholera.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, has ravaged humanity from time immemorial. Although the disease can be treated using antibiotics along with administration of oral rehydration salts and controlled by good sanitation, cholera is known to have produced mayhems in ancient times when little was known about the pathogen. By the 21st century, ample information about the pathogen, its epidemiology, genetics, treatment and control strategies was revealed. However, there is still fear of cholera outbreaks in developing countries, especially in the wake of natural calamities. Studies have proved that the bacterium is mutating and evolving, out-competing all our efforts to treat the disease with previously used antibiotics and control with existing vaccines. In this review, the major scientific insights of cholera research are discussed. Considering the important role of biofilm formation in the V. cholerae life cycle, the vast availability of next-generation sequencing data of the pathogen and multi-omic approach, the review thrusts on the identification of suitable biofilm-inhibiting targets and the discovery of anti-biofilm drugs from nature to control the disease.

Retrospective genomic analysis of Vibrio cholerae O1 El Tor strains from different places in India reveals the presence of ctxB-7 allele found in Haitian isolates.

A total of 45 strains of Vibrio cholerae O1 isolated from 10 different places in India where they were associated with cases of cholera between the years 2007 and 2008 were examined by molecular methods. With the help of phenotypic and genotypic tests the strains were confirmed to be O1 El Tor biotype strains with classical ctxB gene. Polymerase chain reaction (PCR) analysis by double - mismatch amplification mutation assay PCR showed 16 of these strains carried the ctxB-7 allele reported in Haitian strains. Sequencing of the ctxB gene in all the 45 strains revealed that in 16 strains the histidine at the 20th amino acid position had been replaced by asparagine and this single nucleotide polymorphism did not affect cholera toxin production as revealed by beads enzyme-linked immunosorbent assay. This study shows that the new ctxB gene sequence was circulating in different places in India. Seven representatives of these 45 strains analysed by pulsed - field gel electrophoresis showed four distinct Not I digested profiles showing that multiple clones were causing cholera in 2007 and 2008.

Evidence for several waves of global transmission in the seventh cholera pandemic.

Vibrio cholerae is a globally important pathogen that is endemic in many areas of the world and causes 3-5 million reported cases of cholera every year. Historically, there have been seven acknowledged cholera pandemics; recent outbreaks in Zimbabwe and Haiti are included in the seventh and ongoing pandemic. Only isolates in serogroup O1 (consisting of two biotypes known as 'classical' and 'El Tor') and the derivative O139 can cause epidemic cholera. It is believed that the first six cholera pandemics were caused by the classical biotype, but El Tor has subsequently spread globally and replaced the classical biotype in the current pandemic. Detailed molecular epidemiological mapping of cholera has been compromised by a reliance on sub-genomic regions such as mobile elements to infer relationships, making El Tor isolates associated with the seventh pandemic seem superficially diverse. To understand the underlying phylogeny of the lineage responsible for the current pandemic, we identified high-resolution markers (single nucleotide polymorphisms; SNPs) in 154 whole-genome sequences of globally and temporally representative V. cholerae isolates. Using this phylogeny, we show here that the seventh pandemic has spread from the Bay of Bengal in at least three independent but overlapping waves with a common ancestor in the 1950s, and identify several transcontinental transmission events. Additionally, we show how the acquisition of the SXT family of antibiotic resistance elements has shaped pandemic spread, and show that this family was first acquired at least ten years before its discovery in V. cholerae.

Characterization of oligomeric assembly of colonization factor CS6 from enterotoxigenic Escherichia coli.

The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB)n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.

Molecular characteristics of Clostridium difficile isolates from human and animals in the North Eastern region of India.

A total of 1034 samples were collected from different sources and C. difficile was isolated from 18 (9.04%) of 199 human, 9 (4.89%) of 184 cattle, 29 (12.44%) of 233 pig, and from 23 (13.94%) of 165 poultry samples. Variations were observed on the rate of isolation according to age and clinical conditions (diarrhoea). None of the samples from cow, sheep, goat, local chicken, and wild animals yielded any C. difficile. Out of those isolates, 8, 2, 19 and 6 isolates from human, cattle, pig and poultry, respectively were toxigenic. The toxigenic isolates carried both tcdA, and tcdB (A+B+) and most of the human and the pig isolates were also positive for binary toxin genes (cdtA and cdtB). The A+B+ isolates belonged to three different toxinotypes (0, VI and XXXIII). Human and pig A+B+ isolates belonged to three (045, 126 and ACD 019) and four (046, 087, 126 and ACD 011) different ribotypes, respectively and the ribotypes of two cattle isolates were 014 and ACD 010. Six A+B+ avian isolates belonged to six different ribotypes (014, 087, SLO 134, SLO 160, ACD 012, ACD 014). The non-toxigenic isolates from human, cattle, pig and poultry were grouped into 7, 4, 4 and 7 different ribotypes, respectively. PFGE analysis could not differentiate similar ribotypes/toxinotypes of toxigenic isolates. All the toxigenic isolates showed cytopathic effect on Vero and Hela cell monolayers at 1:100 dilutions of cell-free culture supernatants within 18-20 h of inoculation.

Molecular characterization of Shiga-toxigenic Escherichia coli isolated from diverse sources from India by multi-locus variable number tandem repeat analysis (MLVA).

In a first study from India, a diverse collection of 140 environmental and clinical non-O157 Shiga-toxigenic Escherichia coli strains from a large geographical area in north India was typed by multi-locus variable number tandem repeat analysis (MLVA). The distribution of major virulence genes stx1, stx2 and eae was found to be 78%, 70% and 10%, respectively; 15 isolates were enterohaemorrhagic E. coli (stx1 +/stx2 + and eae +). By MLVA analysis, 44 different alleles were obtained. Dendrogram analysis revealed 104 different genotypes and 19 MLVA-type complexes divided into two main lineages, i.e. mutton and animal stool. Human isolates presented a statistically significant greater odds ratio for clustering with mutton samples compared to animal stool isolates. Five human isolates clustered with animal stool strains suggesting that some of the human infections may be from cattle, perhaps through milk, contact or the environment. Further epidemiological studies are required to explore these sources in context with occurrence of human cases.

Factors associated with diarrhoea in young children and incidence of symptomatic rotavirus infection in rural West Bengal, India.

Socio-behavioural factors and pathogens associated with childhood diarrhoea are of global public health concern. Our survey in 696 children aged ⩽2 years in rural West Bengal detected rotavirus as sole pathogen in 8% (17/199) of diarrhoeic stool specimens. Other organisms were detected along with rotavirus in 11% of faecal specimens. A third of the children with rotavirus diarrhoea, according to Vesikari score, had severe illness. The top four rotavirus genotypes were G9P[4] (28%), G1P[8] (19%), G2P[4] (14%) and G8P[4] (8%). In the multivariate model, the practice of 'drawing drinking water by dipping a pot in the storage vessel' [adjusted odds ratio (aOR) 2·21, 95% confidence interval (CI) 1·03-4·74, P = 0·041], and 'children aged ⩽6 months with non-exclusive breastfeeding' (aOR 2·07, 95% CI 1·1-3·82, P = 0·024) had twice the odds of having diarrhoea. Incidence of rotavirus diarrhoea was 24/100 child-years in children aged >6-18 months, 19/100 child-years in children aged >18-24 months and 5/100 child-years in those aged ⩽6 months. Results have translational implications for future interventions including vaccine development.

Outbreak of waterborne acute diarrheal disease in a South District village of Tripura: A public health emergency in the Northeast region of India.

Food and waterborne outbreaks are a neglected public health problem in India. However, it is important to identify the source of infection and the causative pathogen to curb the outbreak quickly and minimize mortality and morbidity. A retrospective descriptive study was conducted with a line list of 130 diarrheal cases. Epidemiological investigation and laboratory investigation were done. Data were collected from hospital case report forms as well as interviewed affected cases. A case of acute diarrheal disease was reported among the people in the village with abdominal pain, vomiting, and diarrhea from December 31, 2022 to January 3, 2023. Out of a total of 130 recorded cases, 33 stool samples were collected and were positive for Enteroaggregative Escherichia coli, Shigella flexneri 3a, and Shigella sonnei by cultural and molecular tests. The presumptive fecal pollution indicator assay indicated high coliform counts in the water samples (most probable number [MPN]-05) and the presence of Escherichia coli. The identified pathogens showed susceptibility to gentamicin and meropenem. People who used public drinking water were found to be infected with acute diarrheal disease (ADD). Quick identification of the causative pathogens and their antimicrobial resistance pattern helped correct antibiotic prescriptions and quick recovery of the patients without any deaths. Thus, a timely implementation of food and waterborne outbreak investigation is crucial to saving lives and preventing the spread of infection.

Management of Cheilitis Granulomatosa with Pica Disorder: A Rare Case Report.

Aim: To report a rare case of cheilitis granulomatosa (CG) with pica disorder presented as chronic persistent lip swelling with an unusual eating disorder. Background: Cheilitis granulomatosa or Miescher's cheilitis is a rare, persistent, or recurring nontender chronic painless swelling of one or both the lips due to granulomatous inflammation, which was first described by Miescher in 1945. It can present as a monosymptomatic form or as a part of other entities of Melkersson-Rosenthal syndrome (MRS). Case description: A 14-year-old female patient presented with persistent diffuse swelling of the upper lip for 1 year. She also had an unusual habit of eating clay, mud, and potentially harmful items like flakes of paint for 1.5 years. Clinically, chronic persistent lip swelling involving upper lip and gingival enlargements were present along with systemic manifestations. Patient counseling with combination therapy showed excellent response with no signs of relapse at 6 months of follow-up. Conclusion: Cheilitis granulomatosa is a unique rare inflammatory disorder with many possible contributory factors and exact etiology is poorly understood. Clinical significance: This case report draws attention to constant exposure to an allergen that could be one of the possible etiological factors to CG secondary to pica eating disorder. Though it is a rare disease, research on the pathogenesis of CG and clinical trials evaluating the efficacy of the various treatments are needed to enhance our understanding of this disease, to improve the quality of life, as patients are very disturbed by the unsightly and distressing nature of macrocheilitis. How to cite this article: Dayanarayana U, Shastry SP, Kempegowda RT, et al. Management of Cheilitis Granulomatosa with Pica Disorder: A Rare Case Report. Int J Clin Pediatr Dent 2024;17(4):483-489.

Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

Molecular characterization of high-level-cholera-toxin-producing El Tor variant Vibrio cholerae strains in the Zanzibar Archipelago of Tanzania.

Analysis of 1,180 diarrheal stool samples in Zanzibar detected 247 Vibrio cholerae O1, Ogawa strains in 2009. Phenotypic traits and PCR-based detection of rstR, rtxC, and tcpA alleles showed that they belonged to the El Tor biotype. Genetic analysis of ctxB of these strains revealed that they were classical type, and production of classical cholera toxin B (CTB) was confirmed by Western blotting. These strains produced more CT than the prototype El Tor and formed a separate cluster by pulsed-field gel electrophoresis (PFGE) analysis.

Effect of temperature, relative humidity and rainfall on rotavirus infections in Kolkata, India.

Rotavirus is a common viral cause of severe diarrhoea. For the underlying cause of rotavirus seasonality, the meteorological factor has been suspected, whereas quantitative correlation between seasonality and meteorological factor has not been fully investigated. In this study, we investigated the correlation of temporal patterns of the isolation rate of rotavirus with meteorological condition (temperature, relative humidity, rainfall) in Kolkata, India. We used time-series analysis combined with spectral analysis and least squares method. A 1-year cycle explained underlying variations of rotavirus and meteorological data. The 1-year cycle for rotavirus data was correlated with an opposite phase to that for meteorological data. Relatively high temperature could be associated with a low value of isolation rate of rotavirus in the monsoon season. Quantifying a correlation of rotavirus infections with meteorological conditions might prove useful in predicting rotavirus epidemics and health services could plan accordingly.

Comparative analysis of the genomes of Shigella dysenteriae type 2 & type 7 isolates.

BACKGROUND & OBJECTIVES: The four species of the genus Shigella, namely, S. dysenteriae , S. flexneri, S. boydii and S. sonnei cause a wide spectrum of illness from watery diarrhoea to severe dysentery. Genomes of these four species show great diversity. In this study, NotI, XbaI or I-CeuI restriction enzyme digested genomes of two Shigella dysenteriae isolates belonging to the serotypes 2 and 7 were extensively analyzed to find their relatedness, if any, with the whole genome sequenced strains of S. dysenteriae type 1 and S. flexneri type 2a. METHODS: Pulsed-field gel electrophoresis (PFGE) technique was used to determine the diversity of Shigella genomes by rapid construction of physical maps. DNA end labelling, Southern hybridization and PCR techniques were also applied for mapping purposes. RESULTS: The intron-coded enzyme I-CeuI cuts the bacterial genome specifically at its rrn operon. PFGE of I-CeuI digested S. dysenteriae genomes were found to carry seven rrn operons. However, I-CeuI profiles showed distinct restriction fragment polymorphism (RFLP) between the isolates as well as with the whole genome sequenced isolates. Further studies revealed that the genome sizes and I-CeuI linkage maps of the S. dysenteriae type 7 and type 2 isolates were similar to that of S. dysenteriae type 1 and S. flexneri type 2a genomes, respectively. INTERPRETATION & CONCLUSIONS: Our findings indicate that the type 7 and type 1 isolates of S. dysenteriae were probably evolved from a same precursor, while the type 2 and S. flexneri type 2a were probably evolved and diversified from a common progenitor.

Diagnostic microbiologic methods in the GEMS-1 case/control study.

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged <5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzyme immunoassays on stool samples. Samples positive for adenovirus were further evaluated for adenovirus serotypes 40 and 41. We developed a novel multiplex assay to detect norovirus (types 1 and 2), astrovirus, and sapovirus. The portfolio of diagnostic assays used in the GEMS study can be broadly applied in developing countries seeking robust cost-effective methods for enteric pathogen detection.

Development and evaluation of a PCR assay for tracking the emergence and dissemination of Haitian variant ctxB in Vibrio cholerae O1 strains isolated from Kolkata, India.

A PCR-based assay was developed to discriminate the classical, El Tor, and Haitian types of ctxB alleles. Our retrospective study using this newly developed PCR showed that Haitian ctxB first appeared in Kolkata during April 2006, and 93.3% of strains isolated during 2011 carried the new allele. Dendrogram analysis showed a pulsed-field gel electrophoresis (PFGE) pattern of the new variant strains isolated recently that was distinct from the PFGE pattern of the strains carrying classical ctxB that closely matched the 2006 to 2007 variant strains.

Real-time PCR-based mismatch amplification mutation assay for specific detection of CS6-expressing allelic variants of enterotoxigenic Escherichia coli and its application in assessing diarrheal cases and asymptomatic controls.

Enterotoxigenic Escherichia coli (ETEC) expressing the colonization factor CS6 is widespread in many developing countries, including India. The different allelic variants of CS6, caused by point mutations in its structural genes, cssA and cssB, are designated AIBI, AIIBII, AIIIBI, AIBII, and AIIIBII. A simple, reliable, and specific mismatch amplification mutation assay based on real-time quantitative PCR (MAMA-qPCR) was developed for the first time for the detection of CS6-expressing ETEC, along with the identification of allelic variations. The assay was based on mismatched nucleotide incorporation at the penultimate base at the 3' ends of the reverse primers specific for cssA and cssB and was validated using 38 CS6-expressing ETEC isolates. This strategy was effective in detecting all the alleles containing single-nucleotide polymorphisms. Using MAMA-qPCR, we also tested CS6 allelic variants in 145 ETEC isolates from children with acute diarrhea and asymptomatic infections, with the latter serving as controls. We observed that the AIBI and AIIIBI allelic variants were mostly associated with cases rather than controls, whereas the AIIBII variants were detected mostly in controls. In addition, the AIBI and AIIIBI alleles were frequently associated with ETEC harboring the heat-stable toxin gene (est) alone or with the heat-labile toxin gene (elt), whereas the AIIBII allele was predominant in ETEC isolates harboring the elt gene. This study may help in understanding the association of allelic variants in CS6-expressing ETEC with the clinical features of diarrhea, as well as in ETEC vaccine studies.

Molecular epidemiology of Vibrio cholerae causing outbreaks & sporadic cholera in northern India.

BACKGROUND & OBJECTIVES: Several outbreaks of cholera have been reported in Chandigarh region during a span of seven years from 2002-2008. The genetic characteristics of Vibrio cholerae isolates obtained during these outbreaks have not been adequately studied. The aim of this study was to do molecular typing of V. cholerae isolated from the sporadic and outbreak cases by pulsed-field gel electrophoresis (PFGE), Rep-PCR and ribotyping. METHODS: Fifty representative isolates of V. cholerae from outbreak as well as sporadic cases were subjected to molecular typing by PFGE, 173 isolates (163 clinical and 10 environmental) were typed by rep-PCR and ribotyping. Ribotyping was done by determination of rRNA restriction pattern of BglI restriction digestion and hybridization with 7.2 kb rRNA probe of pKK3535 plasmid using DIG DNA labelling and detection kit. Universal VC1 primer was used for rep-PCR. RESULTS: PFGE generated 15 pulsotypes, of which four matched the published pulsotypes and there were 11 new pulsotypes. PFGE was the most discriminatory method that could differentiate between isolates belonging to single ribotype. Pulsotype P1 corresponding to known pulsotype H1 was the major pulsotype till 2003. Pulsotype P3 corresponding to known pulsotype L emerged in 2004. The 2007 outbreaks in Punjab and Haryana were caused by P5 though P1 and P3 were isolated from the sporadic cases from the same region. The 2008 outbreak was caused by pulsotypes P6 and P7. Ribotype IV was the most predominant followed by RIII. This ribotype was not isolated after 2003 and ribotype IV became the most predominant 2004 onwards. Of the two unknown ribotypes (UNI and UN2), UNI was more common (27 isolates). Rep-PCR was the least discriminatory and divided all clinical isolates into four major profiles. The dendrogram analysis of PFGE revealed similarity of some clinical isolates with environmental isolates indicating the genetic relatedness. INTERPRETATION & CONCLUSION: Our findings showed that Rep-PCR was least discriminatory method. Ribotyping was a reliable and reproducible method. Ribotype IV was predominant ribotype followed by RIII. A total of 15 pulsotypes were generated and 11 of these were not reported earlier. Genetic relatedness was shown by clinical and environmental isolates which needs to be confirmed in future studies.

Evaluation of a rapid dipstick test for identifying cholera cases during the outbreak.

BACKGROUND & OBJECTIVES: Intermittent cholera outbreaks are major problem in many of the states of India. It is essential to identify cholera at the earliest for timely mobilization of public health responses and to abort the outbreaks. The present study was a part of a diarrhoeal outbreak investigation in Secunderabad, India, during May 2009 where the usefulness of Crystal VC rapid dipstick kit was assessed for detecting the aetiologic agent of the outbreak. METHODS: Stool specimens were collected from 15 hospitalized patients with acute watery diarrhoea and analyzed for detection of cholera vibrios using Crystal VC rapid dipstick kit and the usefulness of the kit was determined by comparative analysis of the same set of specimens using both microbiological and real-time PCR (RT-PCR) based assays. RESULTS: Detection of Vibrio cholerae O1 from 10 of 15 specimens was recorded using dipstick assay. Microbiological methods detected V. cholerae O1 positivity among 11 specimens. However, RT-PCR based assay showed all 15 specimens positive for the presence of V. cholerae O1. In addition, the same assay showed that the pathogen load in the dipstick as well as RT-PCR positive specimens ranged from 10 6 colony forming units (cfu)/ml or more. INTERPRETATION & CONCLUSIONS: Crystal VC kit had the potential to identify cholera cases in 10 min in field conditions without having good laboratory support. Therefore, dipstick kit may be considered as cholera detecting tool in diarrhoeal outbreak investigations. Specimens from clinically typical cholera cases, if negative by dipstick, should be reanalyzed by culture based methods.