Contrasts, contradictions and control of cholera.

Cholera has been extremely pervasive during the past four decades and continues to remain a significant public health concern. The disease has plagued humankind in the form of seven pandemics since the last two centuries. There is considerable scientific evidence based on research on cholera and its etiologic agent Vibrio cholerae, however we are still unable to accurately forecast and pre-empt the occurrence of cholera outbreaks. The commentary discusses the contrasts and contradictions of cholera, its control and its unpredictable nature. Through a multi-sectoral approach and broad stakeholder collaboration cholera control is possible with meticulous country-level planning for early detection and response to outbreaks. The commentary reiterates that every potential death on account of cholera is preventable because of the available knowledge and tools to effectively prevent and treat cholera.

Detection of Cholera Toxin by an Immunochromatographic Test Strip.

As cholera toxin (CT) is responsible for most of the symptoms induced by Vibrio cholerae O1 or O139 infection, detection of CT is an important biomarker for diagnosis of the disease. The procedure for pathogenicity analysis of V. cholerae isolates must be carefully developed for the reason that the amount of CT produced by V. cholerae varies according to the medium used and culture conditions (i.e. temperature and aeration status) applied. Here we describe a reproducible rapid method for analysis of CT production by toxigenic V. cholerae with an immunochromatographic test strip that can detect as low as 10 ng/mL of purified recombinant CT.

Cholera outbreaks in the El Tor biotype era and the impact of the new El Tor variants.

Vibrio cholerae O1, the causative agent of the disease cholera, has two biotypes namely the classical and El Tor. Biotype is a subspecific taxonomic classification of V. cholerae O1. Differentiation of V. cholerae strains into biotype does not alter the clinical management of cholera but is of immense public health and epidemiological importance in identifying the source and spread of infection, particularly when V. cholerae is first isolated in a country or geographic area. From recorded history, till date, the world has experienced seven pandemics of cholera. Among these, the first six pandemics are believed to have been caused by the classical biotype whereas the ongoing seventh pandemic is caused by the El Tor biotype. In recent years, new pathogenic variants of V. cholerae have emerged and spread throughout many Asian and African countries with corresponding cryptic changes in the epidemiology of cholera. In this chapter, we describe the outbreaks during the seventh pandemic El Tor biotype era spanning more than five decades along with the recent advances in our understanding of the development, evolution, spread, and impact of the new variants of El Tor strains.

Dynamics in genome evolution of Vibrio cholerae.

Vibrio cholerae, the etiological agent of the acute secretary diarrheal disease cholera, is still a major public health concern in developing countries. In former centuries cholera was a permanent threat even to the highly developed populations of Europe, North America, and the northern part of Asia. Extensive studies on the cholera bug over more than a century have made significant advances in our understanding of the disease and ways of treating patients. V. cholerae has more than 200 serogroups, but only few serogroups have caused disease on a worldwide scale. Until the present, the evolutionary relationship of these pandemic causing serogroups was not clear. In the last decades, we have witnessed a shift involving genetically and phenotypically varied pandemic clones of V. cholerae in Asia and Africa. The exponential knowledge on the genome of several representatives V. cholerae strains has been used to identify and analyze the key determinants for rapid evolution of cholera pathogen. Recent comparative genomic studies have identified the presence of various integrative mobile genetic elements (IMGEs) in V. cholerae genome, which can be used as a marker of differentiation of all seventh pandemic clones with very similar core genome. This review attempts to bring together some of the important researches in recent times that have contributed towards understanding the genetics, epidemiology and evolution of toxigenic V. cholerae strains.

The cholera outbreak in Haiti: where and how did it begin?

In October 2010, cholera appeared in Haiti for the first time in nearly a century. The Secretary-General of the United Nations formed an Independent Panel to "investigate and seek to determine the source of the 2010 cholera outbreak in Haiti". To fulfill this mandate, the Panel conducted concurrent epidemiological, water and sanitation, and molecular analysis investigations. Our May 2011 findings indicated that the 2010 Haiti cholera outbreak was caused by bacteria introduced into Haiti as a result of human activity; more specifically by the contamination of the Meye Tributary System of the Artibonite River with a pathogenic strain of the current South Asian type Vibrio cholerae. Recommendations were presented to assist in preventing the future introduction and spread of cholera in Haiti and worldwide. In this chapter, we discuss both the results of the Independent Panel's investigation and the context the report sat within; including background information, responses to the report's release, additional research subsequent to our report, and the public health implications of the Haiti cholera epidemic.

Gravedad de la gastroenteritis causada por Vibrio parahaemolyticus del grupo pandémico en el Perú / Severity of the gastroenteritis caused by Vibrio parahaemolyticus of the pandemic group in Peru

Objetivos. Determinar las características epidemiológicas y clínicas de la gastroenteritis causada por Vibrio parahaemolyticus del grupo pandémico en el Perú. Materiales y métodos. Se examinó las historias clínicas y registros de laboratorio de cien casos de gastroenteritis en los cuales se aisló V. parahaemolyticus del grupo pandémico y no pandémico. Se recolectó información epidemiológica y clínica y se realizó el análisis estadístico de los datos para evaluar si la gravedad de la enfermedad se asoció con la presencia de las cepas del grupo pandémico. Resultados. Se logró colectar información epidemiológica en 85 por ciento de los casos e información clínica sólo en 37 por ciento de los casos, principalmente de los hospitalizados. Los casos del grupo pandémico tuvieron una mayor probabilidad de tener deposiciones líquidas (96,3 por ciento frente a 62,5 por ciento, p<0,05), presentar deshidratación moderada o grave (100 por ciento frente a 60 por ciento, p<0,05) y requerir atención hospitalaria (98 por ciento frente a 42,9 por ciento, p<0,0001). Fue más probable aislar una cepa pandémica en personas de 30 o más años de edad (63 por ciento frente a 39,5 por ciento, p<0,05). Conclusiones. El Vibrio parahaemolyticus del grupo pandémico causa enfermedad gastrointestinal de mayor gravedad que las cepas no pandémicas, con mayor probabilidad de requerir atención hospitalaria. Basados en este reporte, se recomienda incluir la identificación de V. parahaemolyticus en el diagnóstico etiológico de agentes causantes de gastroenteritis grave en el sistema de salud del Perú.

Objective. To determine the epidemiological and clinic characteristics of gastroenteritis caused by Vibrio parahaemolyticus strains of the pandemic group in Peru. Material and methods. Clinical and laboratory records were searched in 100cases of gastroenteritis caused by V parahaemolyticus, either of the pandemic or non pandemic group. Clinical and epidemiological data were collected and statistical analysis was done to evaluate if the severity of illness was associated with the pandemic group. Results. Epidemiological data were collected in 85 per cent of cases, and clinical data were only available in 37 per cent of cases, mainly on those hospitalized. Cases associated with the pandemic strains had a higher probability of liquid stools (96.3 per cent vs. 62.5 per cent, p<0.05), moderate or severe dehydration (100 per cent vs. 60 per cent, p<0.05), and hospital care (98 per cent vs. 42.9 per cent, p<0.0001). Cases aged thirty or older were associated with the pandemic strains (63 per cent vs. 39.5 per cent, p<0.05). Conclusions. Vibrio parahaemolyticus of the pandemic group causes more severe gastrointestinal disease than none pandemic strains, with higher probability of requiring hospital care. Based on this report, it is advisable to include the identification of V. parahaemolyticus in the etiological diagnosis of agents causing severe gastroenteritis in the Peruvian health system.

Genetic characterization of Vibrio cholerae strains by inter simple sequence repeat-PCR.

The utility of inter simple sequence repeat-PCR (ISSR-PCR) assay in the characterization and elucidation of the phylogenetic relationship between the pathogenic and nonpathogenic isolates of Vibrio cholerae is demonstrated. A total of 45 V. cholerae strains including 15 O1 El Tor, nine O139 and 21 non-O1/non-O139 strains were analyzed using eight ISSR primers. These primers, which are essentially simple sequence repeats (SSR) with additional nonrepeat bases at the 5' or 3' end, amplify genomic regions interspersed between closely spaced SSRs. Neighbor-joining analysis showed that the strains belonging to the same serogroup clustered together with the exception of one O1 and two O139 strains. The absence of pathogenicity islands in these strains, as confirmed by PCR, suggested their non-O1/non-O139 origin. Thus the ISSR-PCR-based phylogeny was consistent with the classification of V. cholerae based on serological methods. A finer resolution of the clustering of the toxinogenic O1 El Tor and toxinogenic O139 subtypes was obtained by ISSR-PCR analysis as compared with the Enterobacterial Repetitive Intergenic Consensus sequences-based PCR analysis for the same set of strains. Thus, it is proposed that ISSR-PCR is an efficient tool in phylogenetic classification of prokaryotic genomes in general and diagnostic genotyping of microbial pathogens in particular.

Multiply antibiotic-resistant Vibrio cholerae O1 biotype El Tor strains emerge during cholera outbreaks in Zambia.

Antibiotic resistance data, made available from laboratory records during eight cholera outbreaks between 1990 and 2004 showed Vibrio cholerae serogroup O1 to have a low level of resistance (2-3%) to tetracycline during 1990-1991. Resistance increased for tetracycline (95%), chloramphenicol (78%), doxycycline (70%) and trimethoprim-sulphamethoxazole (97%) in subsequent outbreaks. A significant drop in resistance to tetracycline and chloramphenicol followed the adoption of a national policy to replace tetracycline with erythromycin for treating cholera. Sixty-nine strains from cholera outbreaks in Zambia between 1996 and 2004, were examined for antibiotic resistance and basic molecular traits. A 140 MDa conjugative, multidrug-resistant plasmid was found to encode tetracycline resistance in strains from 1996/1997 whereas strains from 2003/2004 were resistant to furazolidone, but susceptible to tetracycline, and lacked this plasmid. PCR revealed 25 of 27 strains from 1996/1997 harboured the intl1 class 1 integron but lacked SXT, a conjugative transposon element. Similar screening of 42 strains from 2003/2004 revealed all carried SXT but not the intl1 class 1 integron. All 69 strains, except two, one lacking ctxA and the other rstR and thus presumably truncated in the CTX prophage region, were positive for important epidemic markers namely rfbO1, ctxA, rstR2, and tcpA of El Tor biotype. Effective cholera management is dependent on updated reports on culture and sensitivity to inform the choice of antibiotic. Since the emergence of antibiotic resistance may significantly influence strategies for controlling cholera, continuous monitoring of epidemic strains is crucial.

Isolation and molecular characterization of toxigenic Vibrio parahaemolyticus from the Kii Channel, Japan.

Studies were conducted on the ecology of potentially pathogenic Vibrio parahaemolyticus in three coastal areas of Kii Channel, Tokushima, Japan. Seawater and seaweed samples were collected seasonally between June 2003 and May 2004. Total and toxigenic strains of V. parahaemolyticus were isolated using most probable number culture and colony blot hybridization. Toxigenic strains were serotyped and further characterized by random amplified polymorphic DNA (RAPD) and ribotyping. Six thousand strains of V. parahaemolyticus were isolated and 18 were found positive for tdh. V. parahaemolyticus were detected in all samples during summer and autumn, and from some samples during winter and spring. Among the toxigenic strains seven serotypes, five ribotypes and RAPD patterns were observed. Seven strains belonged to O3:K6 clone with identical ribotypes and RAPD patterns to that of a pandemic reference strain. The presence of toxigenic V. parahaemolyticus with pandemic potential might indicate a human health risk due to consumption of marine food sources.

Assessing clonality of Vibrio cholerae Inaba isolates by characterization of nonsense mutations in wbeT.

The transferase gene wbeT of six clinical isolates of Vibrio cholerae O1 biotype El Tor was analysed. Two unique mutations were identified in the wbeT gene of three Inaba isolates. Due to their random nature, mutations in wbeT can be used to determine the clonal origin of clinical Inaba isolates.

Virulence genes and neutral DNA markers of Helicobacter pylori isolates from different ethnic communities of West Bengal, India.

Virulence-associated genes and neutral DNA markers of Helicobacter pylori strains from the Santhal and Oroan ethnic minorities of West Bengal, India, were studied. These people have traditionally been quite separate from other Indians and differ culturally, genetically, and linguistically from mainstream Bengalis, whose H. pylori strains have been characterized previously. H. pylori was found in each of 49 study participants, although none had peptic ulcer disease, and was cultured from 31 of them. All strains carried the cag pathogenicity island and potentially toxigenic s1 alleles of vacuolating cytotoxin gene (vacA) and were resistant to at least 8 micro g of metronidazole per ml. DNA sequence motifs in vacA mid-region m1 alleles, cagA, and an informative insertion or deletion motif next to cagA from these strains were similar to those of strains from ethnic Bengalis. Three mobile elements, IS605, IS607, and ISHp608, were present in 29, 19, and 10%, respectively, of Santhal and Oroan strains, which is similar to their prevalence in Bengali H. pylori. Thus, there is no evidence that the gene pools of H. pylori of these ethnic minorities differ from those of Bengalis from the same region. This relatedness of strains from persons of different ethnicities bears on our understanding of H. pylori transmission between communities and genome evolution.

Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs.

We evaluated the recently developed dipsticks for the rapid detection of Vibrio cholerae serotypes O1 and O139 from rectal swabs of hospitalized diarrheal patients after enrichment for 4 h in alkaline peptone water. The sensitivity and specificity of the dipsticks were above 92 and 91%, respectively. The dipsticks represent the first rapid test which has been successfully used to diagnose cholera from rectal swabs, and this would immensely improve surveillance for cholera, especially in remote settings.

Environmental isolates of Citrobacter braakii that agglutinate with Escherichia coli O157 antiserum but do not possess the genes responsible for the biosynthesis of O157 somatic antigen.

While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum. The five C. braakii isolates gave positive results when a sensitive dot-ELISA was performed with E. coli O157 monoclonal antibody. Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C. braakii isolates. Apart from weak reactions with two or three of the DNA probes, all the C. braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis. These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E. coli (STEC) specially the serotype O157: H7. Therefore, it appears that the serological cross-reaction between C. braakii and E. coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C. braakii and E. coli O157.

Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup.

Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment. The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease. Of the 559 environmental strains of V. cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn). Six of the 9 strains belonged to the O14 serogroup. Thus, V. cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup. Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V. cholerae strains. A strain that possessed the E1 Tor type tcpA also had the CTX prophage. Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V. cholerae were not clonal.

Differences in genotypes of Helicobacter pylori from different human populations.

DNA motifs at several informative loci in more than 500 strains of Helicobacter pylori from five continents were studied by PCR and sequencing to gain insights into the evolution of this gastric pathogen. Five types of deletion, insertion, and substitution motifs were found at the right end of the H. pylori cag pathogenicity island. Of the three most common motifs, type I predominated in Spaniards, native Peruvians, and Guatemalan Ladinos (mixed Amerindian-European ancestry) and also in native Africans and U.S. residents; type II predominated among Japanese and Chinese; and type III predominated in Indians from Calcutta. Sequences in the cagA gene and in vacAm1 type alleles of the vacuolating cytotoxin gene (vacA) of strains from native Peruvians were also more like those from Spaniards than those from Asians. These indications of relatedness of Latin American and Spanish strains, despite the closer genetic relatedness of Amerindian and Asian people themselves, lead us to suggest that H. pylori may have been brought to the New World by European conquerors and colonists about 500 years ago. This thinking, in turn, suggests that H. pylori infection might have become widespread in people quite recently in human evolution.

Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India.

Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country. The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V. cholerae O1 which emerged in Calcutta in 1994. Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1. The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V. cholerae 0139 in Calcutta. However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of 0139 in Calcutta. The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V. cholerae O1. None of the strains in this study carried plasmids. Molecular studies clearly show that the new expanded drug resistant clone of V. cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

Heterogeneity in the organization of the CTX genetic element in strains of Vibrio cholerae O139 Bengal isolated from Calcutta, India and Dhaka, Bangladesh and its possible link to the dissimilar incidence of O139 cholera in the two locales.

After a lapse of 33 months, Vibrio cholerae O139, the new serogroup associated with cholera, has re-emerged in Calcutta, India and has become the dominant serogroup causing cholera from September 1996. In neighbouring Bangladesh, V. cholerae O1 biotype El Tor continues to be the dominant cause of cholera with the O139 serogroup accounting for only a small proportion of cases. Comparison of the phenotypic traits of representative O139 strains from Calcutta and Dhaka isolated between December 1996 and April 1997 showed similar phenotypic traits with the exception that Dhaka O139 strains were susceptible to streptomycin whilst Calcutta O139 strains were resistant. The Dhaka and Calcutta O139 strains displayed identical ribotypes but showed remarkable differences in the structure and organization of the CTX genetic element. In the Dhaka O139 strains, two copies of the CTX element were arranged in tandem and this resembled the pattern displayed by the 1992 epidemic strains of O139. The Calcutta O139 strains, in contrast, carried three copies of the CTX genetic element arranged in tandem with the loss of a conserved BglII restriction site in the RS1 element and the appearance of a new HindIII site in the same region. While there may be other factors, it appears that the reorganization of the CTX genetic element in the Calcutta O139 strains may have contributed to the resurgence of this serogroup in Calcutta.