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Cholera remains a major contributor of diarrheal diseases and leads to substantial morbidity and mortality, particularly in low socioeconomic settings. Nonavailability of a national cholera control plan in India, compounded by underreporting of cholera cases and deficient accurate cholera hotspot estimates, has made cholera control a challenge. Obstacles in the programmatic introduction of oral cholera vaccine (OCV) lie within the infrastructure—stockpile, costing, distribution system, cold-chain mechanism, vaccine logistics, and lack of strengthened surveillance systems for adverse events following immunization. Sustained political commitment along with collaboration of people working in the media will also determine the policy outcome of OCV introduction in India.

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.

We identified 281 Vibrio cholerae non-O1, non-O139 strains from patients with diarrhea in Kolkata, India. Cholera-like diarrhea was the major symptom (66.0%); some patients (20.3%) had severe dehydration. These strains lacked the ctxA gene but many had hlyA, rtxA, and rtxC genes. Pulsed-field gel electrophoresis showed no genetic link among strains.

Culture-independent identification of diarrhoeal aetiological agents was performed using DNA harvested from diarrhoeal stool specimens with SYBR-Green-based real-time PCR targeting Vibrio cholerae. Vibrio parahaemolyticus, Campylobacter spp., Shigella spp. and three different pathotypes of diarrhoeagenic Escherichia coli. Conventional culture-dependent methods detected bacterial enteropathogens in 68 of 122 diarrhoeal stool specimens. Of 68 specimens, 59 (86.8%) had a single pathogen and the remaining nine (13.2%) had polymicrobial infections with multiple pathogens. Re-analysis of the 68 specimens by culture-independent real-time PCR methods showed that 25 (36.8%) specimens contained single pathogen and 43 (63.2%) specimens contained mixed infections with multiple pathogens. The prevalence of such high levels of polymicrobial infections would not have been detected without using real-time PCR. Culture-dependent analysis assigned 54 of the 122 selected archived specimens as ‘no known aetiology’. However, re-analysis of these samples by real-time PCR showed the presence of single or multiple pathogens among 34 (63%) of these specimens. Estimation of relative pathogen load by real-time PCR in the stool specimens indicated that the inability of conventional culture-dependent methods to detect the pathogens was related to lower colony-forming units of the pathogen, as reflected by lower Ct values. Detection of high levels of polymicrobial infection by real-time PCR indicates that in the settings like Kolkata and its surroundings, where cholera and other enteric diseases are endemic, the concept of one pathogen one disease might need to be re-evaluated.

Vibrio cholerae causes cholera outbreaks in endemic regions where the water quality and sanitation facilities remain poor. Apart from biotype and serotype changes, V. cholerae undergoes phase variation, which results in the generation of two morphologically different variants termed smooth and rugose. In this study, 12 rugose (R-VC) and 6 smooth (S-VC) V. cholerae O1 Ogawa isolates were identified in a cholera outbreak that occurred in Hyderabad, India. Antimicrobial susceptibility results showed that all the isolates were resistant to ampicillin, furazolidone and nalidixic acid. In addition, R-VC isolates were resistant to ciprofloxacin (92 %), streptomycin (92 %), erythromycin (83 %), trimethoprim-sulfamethoxazole (75 %) and tetracycline (75 %). Based on the ctxB gene analysis, all the isolates were identified as El Tor variant with mutation in two positions of ctxB, similar to the classical biotype. The R-VC isolates specifically showed excessive biofilm formation and were comparatively less motile. In addition, the majority of these isolates (~83 %) displayed random mutations in the hapR gene, which encodes haemagglutinin protease regulatory protein. In the PFGE analysis, R-VC and S-VC were placed in distinct clusters but remained clonally related. In the ribotyping analysis, all the R-VC isolates exhibited R-III pattern, which is a prevailing type among the current El Tor isolates. A hapR deletion mutant generated using an S-VC isolate expressed rugose phenotype. To our knowledge, this is the first report on the association of rugose V. cholerae O1 in a large cholera outbreak with extended antimicrobial resistance and random mutations in the haemagglutinin protease regulatory protein encoding gene (hapR).

Developing countries are burdened with Shigella diarrhea. Understanding mucosal immune responses associated with natural Shigella infection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens, p an- S higella s urface p rotein antigen 1 (PSSP1) and PSSP2, common to all virulent Shigella strains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α 4 β 7 + ) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive for Shigella infection by microbiological and real-time PCR assays, respectively. The frequency of α 4 β 7 + IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of the Shigella serotype isolated from these patients. Thus, α 4 β 7 + ASC responses to Ipas may be considered an indirect marker of Shigella infection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by natural Shigella infection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude.

Analysis of 75 Vibrio cholerae O1 strains isolated from hospitalized patients in Kolkata from 1989 to 1994 revealed the existence of true El Tor along with El Tor variants that possessed the classical allele of ctxB (ctxB(cl)) in strains having an El Tor backbone. Based on the existence of different combinations of ctxB and rstR alleles and their localization sites in the genome, these strains were classified into multiple genetic groups. Of 75 clinical strains, 11 were identified as non-toxigenic. These 8 strains were also devoid of pTLC, which is uncommon among the O1 strains. However, Mozambique variants isolated in 2004 were typically negative for pTLC, but these strains possessed tandemly arranged CTX prophages with ctxB(cl) in the small chromosome. Genetic manipulation studies with laboratory-generated kanamycin-tagged pCTX-Kan (derived from tandemly arranged small chromosome-localized ctxB(cl) bearing CTX prophages of 1992 VC53, a progenitor strain of the Mozambique variant) demonstrated that integration specificity of the pCTX-Kan was somewhat towards small chromosome. Such integration could be the prime step towards generation of the Mozambique variant. Based on the existence of multiple alleles of CTXϕ and their infections with non-toxigenic strains, we propose that the El Tor variant strains could have emerged following these genetic events. This study demonstrated existence of different 'intermediate strains' in a time frame that overlapped with a period of V. cholerae O139 emergence. Identification of these intermediate strains gave impetus to believe stepwise generation of the El Tor variants, and all these events profoundly influenced V. cholerae epidemiology in the following years.

A live oral cholera vaccine developed from a non-toxigenic Vibrio cholerae O1 El Tor strain VA1.3 was tested in a double-blind randomized placebo controlled study for safety and immunogenicity in 304 men aged between 16 and 50 years from Kolkata, India. A dose of 5 x 10(9)CFU (n=186) or a placebo (n=116) containing the diluent buffer was administered. The vaccine did not elicit adverse events except in two vaccine recipients with mild diarrhoea and vomiting. None excreted the vaccine strain. Vibriocidal antibody response developed in 105/186 (57%) and 5/116 (4%) in vaccine and placebo recipients, respectively. In a subgroup, anti-CT antibody rose (or =2-folds) in 23/30 (77%) and 6/19 (32%) in vaccine and placebo recipients, respectively. These studies demonstrate that VA1.3 at a dose of 5 x 10(9) is safe and immunogenic in adults from a cholera endemic region.

The incidence of Vibrio cholerae non-O1, non-O139 strains from hospitalized patients with acute diarrhea constituted 27.4% ( n = 54) of the total 197 V. cholerae strains isolated from patients in Kolkata, India, in 2003. Of 197 strains, 135 were identified as O1 serotype Ogawa and 2 were identified as O139. In the same time period, six O1 background rough strains that possessed all known virulence factors were identified. Serotype analysis of the non-O1, non-O139 strains placed 42 strains into 19 serogroups, while 12 remained O nontypeable (ONT); the existing serotyping scheme involved antisera to 206 serogroups. Detection of a good number of ONT strains suggested that additional serogroups have arisen that need to be added to the current serotyping scheme. The non-O1, non-O139 strains were nontoxigenic except for an O36 strain (SC124), which regulated expression of cholera toxin as O1 classical strains did. Additionally, strain SC124 carried alleles of tcpA and toxT that were different from those of the O1 counterpart, and these were also found in five clonally related strains belonging to different serogroups. Strains carrying tcpA exhibited higher colonization in an animal model compared to those lacking tcpA . PCR-based analyses revealed remarkable variations in the distribution of other virulence factors, including hlyA , rtxA , Vibrio seventh pandemic island I (VSP-I), VSP-II, and type III secretion system (TTSS). Most strains contained hlyA (87%) and rtxA (81.5%) and secreted cytotoxic factors when grown in vitro. Approximately one-third of the strains (31.5%) contained the TTSS gene cluster, and most of these strains were more motile and hemolytic against rabbit erythrocytes. Partial nucleotide sequence analysis of the TTSS-containing strains revealed silent nucleotide mutations within vcsN2 (type III secretion cytoplasmic ATPase), indicating functional conservation of the TTSS apparatus.

Summary Objectives To study the phenotypic and genotypic characterization of newly emerged V. cholerae O1, Inaba strains isolated from patients with diarrhoea. Methods Bacterial characterization was made using polymerase chain reaction, ribotyping, PFGE and RFLP. Results After its first appearance in July 2004, O1 Inaba became the dominant serotype by March 2005 and totally replaced the former dominant serotype, Ogawa from May 2005. Most of the Inaba isolates belong to a new ribotype RIV. Ogawa and also some Inaba strains isolated during the same period were identified as RIII. Similarly, the majority of the Inaba isolates belong to ‘H1' pulsotype and one isolate is type ‘H', while the Ogawa isolates were mostly ‘H' pulsotype. Presence of CTX prophage was detected in a single site of the chromosome with at least two RS elements. Conclusions There has been a switch of dominant serotype from Ogawa to Inaba in the Kolkata region. This is not necessarily due to emergence of a new clone but does serve as an epidemiological marker. Further analysis at the molecular level will be required to define this trend and to monitor future spread to other regions.

SUMMARYIn this study, we have analysed the epidemiological significance of the concurrent infections caused by Vibrio cholerae and intestinal parasites among different age groups of hospitalized diarrhoeal patients in Kolkata. A total of 3556 stool samples collected during 1996–2004 were screened for vibrios and parasites. The seasonality of V. cholerae and parasitic infections were studied in detail. The detection rates for Ascaris lumbricoides and Giardia lamblia infection were more than for the hookworm, Trichuris trichiura and Entamoeba histolytica. V. cholerae O1 was identified as the predominant serogroup among diarrhoeal patients. The highest rates for V. cholerae infection were in the 2–5 years age group and combined infection of V. cholerae and parasites was recorded among children aged between 2 and 10 years.

Sixteen of the 18 Vibrio cholerae rough strains isolated from hospitalised diarrhoea patients were found to contain O1 serotype-specific (wbe) genes and all currently known virulence genes. Expression of the regulatory element ToxR was evident in these strains. Cholera toxin production ability of the rough strains was found to be higher (c. three- to five-fold) as compared to the smooth counterparts and this was transcriptionally regulated. Strains exhibiting the rough phenotype were more amenable to the uptake of CTXphi, which led us to consider that the rough phenotype could play a role in the generation of genetic diversity among V. cholerae strains.

We investigated the presence of cagA- like gene of Helicobacter pylori in environmental isolates of Aeromonas spp. from different water samples of Calcutta, India, by colony hybridization using a cagA -specific DNA probe and by PCR with cagA -specific primers. Nucleotide sequencing of five PCR products revealed 97 to 98% homology to canonical cagA of H. pylori 26695 as well as to four clinical H. pylori strains from Calcutta. The cagA -like gene of the environmental isolates was unstable in laboratory conditions and tended to be lost upon subculturing.

Diarrhoeal infection caused by Escherichia coli is common in India with occasional outbreaks. However, association of different pathotypes of diarrhoeagenic E. coli (DEC) with the disease and its phenotypic and genotypic characteristics are not fully demonstrated. In this study, E. coli strains from sporadic cases and outbreaks of diarrhoea during 2000-2001 were confirmed as DEC by polymerase chain reaction (PCR) targeting the specific virulence genes. DEC represented by enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and enteroaggregative E. coli (EAggEC) were mostly belonged to O serogroups 25, 86a, 114 and 146. The gene astA was frequently detected among ETEC and EAggEC than EPEC. After initial screening of 200 DEC strains with serology and antibiotic susceptibility test, 32 strains representing ETEC, EPEC, and EAggEC isolated from different areas of India were included in the pulsed-field gel electrophoresis (PFGE) analysis. Using the PFGE results, the hierarchical representation of different linkage levels between the DEC strains were determined by unweighed pair-group arithmetic mean (UPGAMA) method. Except for few strains, clonotyping by PFGE revealed no correlation between pathotypes and serogroups as well as the place of isolation of the DEC strains. The prevailing clonal diversity among the different categories of DEC strains suggests that the pathotypes of DEC belonged to diverse clones.

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region., Author Summary Vibrio parahaemolyticus has been associated with several epidemics of foodborne diarrheal infection. Recent observations in several counties have shown the emergence of pandemic strains of V. parahaemolyticus with unique genetic features and their role in diarrheal outbreaks. Unlike other enteric pathogens, the appearance of pandemic strains of V. parahaemolyticus has not been associated with the economic/hygiene status of the population. The pandemic strains of V. parahaemolyticus continue to prevail in Kolkata, India since its appearance during 1996. The present communication describes not only the prevalence of pandemic serovars of V. parahaemolyticus, but also the appearance of novel serovars under the pandemic strain category. In addition, the trh gene was detected in some of the pandemic strains for the first time. In the newly emerged serovars genetic changes have occurred, as evidenced from the PFGE analysis. Overall, the antimicrobial susceptibility of pandemic strains remains unchanged for the past 20 years. The observations made in this study re-emphasize the importance of this pathogen and shows the recent genetic and serovar changes in the epidemiology of V. parahaemolyticus-mediated diarrhea.

In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V. cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000. All of the representative V. cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of V. cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V. cholerae O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the V. cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the E. coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which V. cholerae and ETEC were concomitantly involved.

This study examined established enteric pathogens, Arcobacter species and enterotoxigenic Bacteroides fragilis (ETBF), in 201 U.S. and European travelers with acute diarrhea acquired in Mexico, Guatemala, and India. Arcobacter butzleri and ETBF were detected in 8% and 7% of diarrhea cases, respectively.

Since 1996 infections caused by Vibrio parahaemolyticus have increased globally. Outbreak and/or sporadic cases of diarrhea caused by V. parahaemolyticus have been reported from India Indonesia Japan Taiwan the USA Laos Korea Chile Bangladesh Russia Thailand Spain France and Vietnam. This increase in incidence appears to be related to the emergence of a new clone belonging to the O3:K6 serovar which has pandemic potential. Due to its spread in many countries with identical phenotypic and genotypic features the recently emerged V. parahaemolyticus has now been termed a pandemic strain which can be identified by group-specific GSPCR based on the sequence variation in the toxRS gene. In addition the pandemic strains have a novel open reading frame orf8 which corresponds to a filamentous phage f237. On September 14 2003 an outbreak of diarrhea occurred near Dumdum in Kolkata India. About 200 people had diarrhea after consumption of rice with meat served at the blood donation camp. Twenty-one patients were admitted to the Infectious Diseases Hospital (IDH) Kolkata from this outbreak-affected area. Stool specimens were collected from all the patients and screened for common enteric pathogens within two hours of collection using standard methods. In addition 3--5 Escherichia coli colonies grown in MacConkey agar from each stool specimen were screened for diarrheagenic E. coli (DEC) by PCR.4 In this assay all the tested E. coli isolates were negative for common DEC such as enterotoxigenic E. coli enteropathogenic E. coli and enteroaggregative E. coli. (excerpt)

Summary. Vibrio cholerae 0139 organisms isolated from different parts of India and from Bangladesh were characterised with respect to their haemagglutination (HA) activity, plasmid content, cholera toxin (CT) production, cell surface protein and lipopolysaccharide (LPS) profiles, and antigenic properties. Of 28 V. cholerae 0139 isolates tested, 14 (50 YO) were shown to agglutinate chicken erythrocytes; the HA activity was sensitive to D-mannose 0.1 %. In parallel experiments, 12 (92.3%) of 13 V. cholerae 01 (El Tor) and 12 (75%) of 16 non-0 1, non-0 139 strains agglutinated chicken erythrocytes. Plasmid analysis of 32 0 139 isolates showed that 12 (37.5%) carried one or more plasmids of 35-8-2.6 MDa. Plasmids were not detected in any of the V. cholerae 01 strains, although plasmids were demonstrable in 35% of the non-01, non-0139 strains tested. V. cholerae 0139 isolates showed an ability to produce CT that depended on media composition and other cultural conditions. A comparison of envelope and outer-membrane protein profiles between 0 1 and 0 139 isolates failed to show any significant differences. LPS analysis of 0139 isolates revealed that these organisms were devoid of long " 0 " side-chain polysaccharides. Some of the non-0 1, non-0139 strains also showed similar LPS profiles whereas others showed the presence of long repetitive " 0 " side-chain polysaccharides similar to those seen in 0 1 organisms. An antiserum raised against V. cholerae 01 strain 0395 did not show any significant reactivity towards 0139 and non-01, non-0139 strains although it reacted with other 01 strains. Furthermore, the anti-01 serum induced marked protection against challenge with an 01 strain but not with an 0 139 strain in passive protection experiments. All these results indicate that, despite sharing some common properties with V. cholerae 01, the 0139 isolates posses certain characteristics that make them distinct from their 0 1 counterparts.

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.

The toxin-coregulated pilus (TCP) is a crucial determinant of the pathogenicity of Vibrio cholerae ([1][1][–][2][3][3]). TCP is essential for intestinal colonization and serves as a receptor for CTX prophage ([4][4]). Whole-genome sequence analysis of a V. cholerae strain isolated from the Haitian

The incidence rates of travelers' diarrhea (TD) have remained high for the last 50 years. More recently, there have been increasing recommendations for self-initiated therapy and use of prophylactic drugs for TD. We last examined the in vitro susceptibilities of commonly used antibiotics against TD pathogens in 1997. We now examine 456 enteropathogens isolated from adult travelers to Mexico, India, and Guatemala with diarrhea acquired between 2006 and 2008 to determine changes in susceptibility against 10 different antimicrobials by the agar dilution method. Traditional antibiotics, such as ampicillin, trimethoprim-sulfamethoxazole, and doxycycline, continue to show high levels of resistance. Current first-line antibiotic agents, including fluoroquinolones and azithromycin, showed significantly higher MICs than in our earlier study, and MIC 90 levels were above the Clinical and Laboratory Standards Institute cutoffs for resistance. There were significant geographical differences in resistance patterns when Central America was compared with India. Entertoxigenic Escherichia coli (ETEC) isolates showed increased resistance to ciprofloxacin ( P = 0.023) and levofloxacin ( P = 0.0078) in India compared with Central America. Enteroaggregative E. coli (EAEC) isolates from Central America showed increased resistance to nearly all of the antibiotics tested. Compared to MICs of isolates 10 years prior, there were 4- to 10-fold increases in MIC 90 values for ceftriaxone, ciprofloxacin, levofloxacin, and azithromycin for both ETEC and EAEC. There were no significant changes in rifaximin MICs. Rising MICs over time imply the need for continuous surveillance of susceptibility patterns worldwide and geographically specific recommendations in TD therapy.

Chronological analysis of 125 Vibrio cholerae O139 strains isolated during 1993-2005 in Kolkata revealed the prevalence of two new genotypes of cholera toxin (CT) and novel combinations of ctxB and rstR alleles resulting in variant CTX prophages. One of the new genotypes of ctxB, which first appeared in 1996 with the re-emerged V. cholerae O139 strains that had CTX Calcutta phage, was designated as genotype 4. In 1998, another new genotype, designated as genotype 5, was detected that prevailed mostly in CTX phages with El Tor rstR. The prototype El Tor CTX phage with genotype 3 gradually disappeared in O139, and since 2002 the predominant CTX prophages in O139 are Calcutta phages with genotype 4 and El Tor phages with genotype 5. Results showed that V. cholerae O139 strains of Kolkata, isolated over a decade, harboured CTX prophages in the large chromosome having no RS1 downstream of CTX prophage. During the course of its intermittent incidence over a decade, five types of O139 strains were detected based on CT genotypes. Such abrupt genetic changes in O139 strains might not favour its continued prevalence in human cases in Kolkata, India.

Cholera is a public health threat in all developing countries. Kolkata, a city in eastern India, is an endemic zone for cholera. During the course of a comprehensive investigation on the distribution of phages of Vibrio cholerae O1 and O139 in freshwater bodies in Kolkata, we were able to isolate the phages of V. cholerae O1 and O139. Vibrio cholerae O1 phages were found at all the sites and exhibited a distinct seasonal cycle, with a primary peak (13.6-17.2 PFU mL(-1)) during monsoon (June to August) in both 2006 and 2007. Vibrio cholerae O139 phages were present in the environment and were predominant during monsoon in the year 2006, except for late winter and early summer from February to April. In contrast, in the year 2007, the O139 phages could be isolated only during July to December, with the highest counts of 12.0 PFU mL(-1) determined in August. The multiplex PCR results showed that 90 samples were positive for wbe of V. cholerae O1, 32 samples for O139 (wbf) and 18 samples for both. This study shows that surveillance of vibriophages indicates the presence of V. cholerae O1 and O139 in water bodies in and around Kolkata and could therefore serve as a powerful biomonitoring agent.

Retrospective analysis led to the detection of twoVibrio choleraevariant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml−1) resistance, positivity in Voges–Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to KolkataV. choleraeO1 strains isolated on or before 1992.NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.

During 2003, Vibrio cholerae O1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identical wbeT region among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTXEl, CTXCal and truncated CTXCal (devoid of ctxAB) prophages was unique among the majority of these O139 strains.

Background The bivalent killed oral cholera vaccine (OCV) provides 65% cumulative protection over five years. It remains unknown whether a boosting regimen can maintain protection in previously immunized populations. This study examines the immunogenicity and safety of an OCV regimen given five years following initial dosing. Methodology/Principal Findings An open label controlled trial was conducted in 426 healthy Indian participants previously enrolled in a large efficacy trial. To assess whether an OCV regimen given after five years can elicit an antibody response equal to that of a primary series, we compared vibriocidal antibody titers in previously immunized participants receiving a two dose booster regimen to participants receiving a primary two dose immunization series. Among participants receiving a two dose primary series of OCV (n = 186), 69% (95% CI 62%-76%) seroconverted. In the intervention arm (n = 184), 66% (95% CI 59%-73%) seroconverted following a two dose boosting schedule given five years following the initial series. Following a single boosting dose, 71% (95% CI 64%-77%) seroconverted. Children demonstrated 79% (95% CI 69%-86%) and 82% (95% CI 73%-88%) seroconversion after primary and boosting regimens, respectively. Conclusions/Significance Administration of an OCV boosting regimen elicits an immune response similar to those receiving a primary series in endemic areas. Though a single boosting dose induces a strong immune response, further investigations are needed to measure if these findings translate to clinical protection., Author Summary The five year efficacy results of the bivalent, killed whole cell oral cholera vaccine (WC OCV) was shown to offer 65% protection in cholera endemic Kolkata. Further search strategies focused on natural boosting of immunity, since this trial assessed protection in a population that has endemic cholera at high rates every year. The efficacy demonstrated in this project reflected both vaccine and naturally induced immunity. Though efficacy is maintained for five years, no formal recommendations on a boosting regimen exist. This study suggests that a boosting regimen of killed OCV can elicit vibriocidal titers similar to those levels produced by a primary series in adults and children residing in endemic areas. A boosting recommendation could help to ease logistical challenges faced in maintaining protection in cholera endemic areas. These immunogenicity findings provide initial evidence to support the use of an OCV boosting regimen five years following the primary series, with consideration of a shorter interval for children under the age of 5 years due to a lower observed efficacy in field trials.

Background A bivalent killed whole cell oral cholera vaccine has been found to be safe and efficacious for five years in the cholera endemic setting of Kolkata, India, when given in a two dose schedule, two weeks apart. A randomized controlled trial revealed that the immune response was not significantly increased following the second dose compared to that after the first dose. We aimed to evaluate the impact of an extended four week dosing schedule on vibriocidal response. Methodology/Principal Findings In this double blind randomized controlled non-inferiority trial, 356 Indian, non-pregnant residents aged 1 year or older were randomized to receive two doses of oral cholera vaccine at 14 and 28 day intervals. We compared vibriocidal immune responses between these schedules. Among adults, no significant differences were noted when comparing the rates of seroconversion for V. cholerae O1 Inaba following two dose regimens administered at a 14 day interval (55%) vs the 28 day interval (58%). Similarly, no differences in seroconversion were demonstrated in children comparing the 14 (80%) and 28 day intervals (77%). Following 14 and 28 day dosing intervals, vibriocidal response rates against V. cholerae O1 Ogawa were 45% and 49% in adults and 73% and 72% in children respectively. Responses were lower for V. cholerae O139, but similar between dosing schedules for adults (20%, 20%) and children (28%, 20%). Conclusions/Significance Comparable immune responses and safety profiles between the two dosing schedules support the option for increased flexibility of current OCV dosing. Further operational research using a longer dosing regimen will provide answers to improve implementation and delivery of cholera vaccination in endemic and epidemic outbreak scenarios., Author Summary The five year efficacy results of the bivalent, killed whole cell oral cholera vaccine was shown to offer 65% protection in cholera endemic Kolkata. Currently, two oral cholera vaccines (OCV) are prequalified by the World Health Organization: the whole cell recombinant cholera toxin B subunit vaccine (Dukoral), and the bivalent killed whole cell only OCV (Shanchol). Shanchol, which is less expensive and possibly associated with longer protection, is recommended in a two dose schedule to be given at two weeks apart. Large scale cholera outbreaks often affect vulnerable populations with limited access to care. Strict dosing schedules can create further logistical barriers, hindering proper vaccine delivery to affected residents returning for their second OCV dose. In this study, 356 participants aged 1 year or older were randomized to receive two doses of OCV at 14 or 28 day intervals, for which vibriocidal immune responses were compared. Similar immune responses were demonstrated between a two and four week OCV dosing schedule, which can increase flexibility when offered as part of a targeted vaccination program. This can further serve to increase adherence and completion of the recommended dosing regimen, as well as providing a platform to increase coverage of other beneficial non-vaccine interventions.

Background A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India. Method A lyophilized dose of 1.9×109 CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14. Result The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1st dose) and 21 days (i.e. after 2nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine. Conclusion This study demonstrates that VA 1.4 at a single dose of 1.9×109 is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen. Trial Registration Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582

To the Editor: Among the 206 serogroups of Vibrio cholerae, O1 and O139 are associated with epidemic cholera. Serogroup O1 is classified into 2 biotypes, classical and El Tor. Conventionally, the 2 biotypes can be differentiated on the basis of a set of phenotypic traits. Comparative genomic analysis has shown variations in different genes between these biotypes (1). Cholera toxin (CT), the major toxin responsible for the disease cholera, has 2 epitypes or immunologic forms, CT1 and CT2 (2). Another classification recognizes 3 genotypes on the basis of the ctxB gene sequence variation (3). In the past few years, a new emerging form of V. cholerae O1, which possesses traits of both classical and El Tor biotypes, has been isolated in Bangladesh (4,5), Mozambique (6), Vietnam, Hong Kong, Japan, and Zambia (7). These strains were variously labeled as Matlab variants, hybrids, or altered El Tor strains. Our study analyzed, in chronological order, strains of V. cholerae O1 that were isolated over 17 years (1989–2005). We used strains isolated during diarrhea surveillance conducted at the Infectious Diseases Hospital, Kolkata (Calcutta), to determine precisely when the hybrid strains appeared in this region. A total of 171 strains of V. cholerae O1, which were selected to cover different months of each year, were included in this study, along with 2 reference strains for classical and El Tor biotypes. The V. cholerae strains were confirmed serologically by slide agglutination using a specific polyvalent antiserum to V. cholerae O1. We focused on the ctxB gene. The strains were examined by mismatch amplification mutation assay (MAMA)–based PCR for detecting the ctxB allele; a common forward primer was used for 2 alleles, FW-Com (5′-ACTATCTTCAGCATATGCACATGG-3′); and 2 allele-specific primers, Re-cla (5′-cctggtacttctacttgaaacg-3′) and Re-elt (5′-CCTGGTACTTCTACTTGAAACA-3′), were used for classical and El Tor biotypes, respectively (8). Results of the MAMA-PCR are summarized in the Table. All of the 123 V. cholerae O1 strains from 1995 through 2005 yielded only the classical type of ctxB, which indicates that since 1995 the classical type has completely replaced the El Tor type ctxB (Table). To reconfirm our PCR-based results, we selected 25 representative strains for DNA sequencing of the ctxB gene. The deduced amino acid sequences were aligned with the CtxB sequences of reference strains N16961 (El Tor) and O395 (classical). The deduced amino acid sequences of all 25 strains were identical to those of the classical reference strain; histidine was at position 39 and threonine was at position 68. Thus, the results from DNA sequencing of the ctxB gene confirmed the MAMA-PCR results. Table Prevalence of different types of ctxB alleles among Vibrio cholerae O1 strains, Kolkata, India, 1989–2005 Our results highlight a noteworthy event in the evolution of recent V. cholerae strains. Analysis of type ctxB that had been circulating in Kolkata for 17 years (1989–2005) showed that in 1989 only the El Tor allele of ctxB was present. Our results further indicate that classical type ctxB emerged in 1990, although El Tor type ctxB was still present in almost equal numbers during that year. During 1991, a unique event took place when the classical type became predominant, along with strains having both classical and El Tor type ctxB. In 1994, isolation of strains with El Tor ctxB became rare, and the major ctxB allele was of the classical type. V. cholerae O1 strains from 1995 onward were found to carry classical type ctxB, which totally replaced the El Tor type ctxB allele. Replacement of El Tor type ctxB by the classical allele has been reported in Bangladesh since 2001 (5), but this event seems to have occurred earlier in Kolkata. Perhaps the new type of El Tor strains arose when V. cholerae strains with typical seventh pandemic El Tor genetic background were replaced with strains having the ctxB gene, possibly driven by selective pressure to survive and adapt better in host intestines. Considering the increase in the global prevalence of cholera (9), the origin and spread of these new variants of V. cholerae strains should be tracked in the population by genome analysis. Finally, this study has described a brief period from February 1991 through December 1992 when El Tor strains had CTX prophages of both classical and El Tor types (data not shown), along with the ctxB of both biotypes. Notably, this period coincided with an unprecedented event in the history of cholera—the genesis of the O139 serogroup. After this serogroup reemerged in 1996, it harbored 2 types of CTX prophages, namely, El Tor and Calcutta (10). Furthermore, these strains with ctxB of both biotypes might also have had a pivotal role behind the emergence of El Tor strains with classical ctxB. Further studies are warranted to determine whether any distinct relationship exists between these overlapping events.

A total of 874 fecal specimens (446 diarrheal cases and 428 controls) from diarrheal children admitted in the Infectious Diseases Hospital, Kolkata and age and sex matched asymptomatic subjects from an urban community were assessed for the prevalence of enterotoxigenic Bacteroides fragilis (ETBF). Isolates of B. fragilis were tested for the presence of enterotoxin gene (bft) by PCR. The detection rate of ETBF was 7.2% (63 of 874 specimens) that prevailed equally in diarrheal cases and controls (7.2% each; 32 of 446 cases and 31 of 428 controls). Male children up to one year age group was significantly (p 2 years of age in cases and controls. In 25 ETBF isolates, the bft gene was genotyped using PCR-RFLP and only two alleles were identified with prevalence rate of 40% and 60% for bft-1 and bft-3, respectively. All the ETBF isolates were susceptible for chloramphenicol and imipenem but resistant to clindamycin (48%), moxifloxacin (44%) and metronidazole (32%). Resistance of ETBF to moxifloxacin (44%) and metronidazole is an emerging trend. Pulsed-field gel electrophoresis (PFGE) revealed that majority of the ETBF isolates are genetically diverse. In the dendrogram analysis, two clusters were identified, one with ETBF resistant to 5–8 antimicrobials and the other cluster with metronidazole and moxifloxacin susceptible isolates from diarrheal cases. To our knowledge, this is the first detailed report on ETBF from India indicating its clinical importance and molecular characteristics.

Background Designing an appropriate data system is important to the success of a clinical study. However, little information is available on this topic. We share our experiences on designing, developing, and implementation of a data system for management of data and field activities of a complex clinical study. Methods The data system was implemented aiming at determining the biological basis for the underperformance of oral vaccines, such as polio and rotavirus vaccines in children at a site in Kolkata, India. The system included several functionalities to control data and field activities. It was restricted to authorized users based on their access privileges. A relational database platform was chosen, and Microsoft Visual FoxPro 7.0 (Microsoft Corporation, Seattle, WA, USA) was used to develop the system. The system was installed at the clinic and data office to facilitate both the field and data management activities. Results Data were doubly entered by two different data operators to identify keypunching errors in the data. Outliers, duplication, inconsistencies, missing entries, and linkage were also checked. Every modification and users log-in/log-out information was auto-recorded in an audit trail. The system offered tools for preparation of visit schedule of the participants. A visit considered as protocol deviation was documented by the system. The system alerted field staff to every upcoming visit date to organize the field activities and to inform participants which day to come. The system also produced a growth chart for evaluating nutritional status and referring the child to a specialized clinic if found to be severely malnourished. Conclusion The data system offered unique features for controlling for both data and field activities, which led to minimize drop-out rates as well as protocol deviations. Such system is warranted for a successful clinical study.