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The Infectious Diseases and Beliaghata General Hospital, Kolkata, India witnessed a sudden increase in admissions of diarrhoea cases during the first 2 weeks of August 2015 following heavy rainfall. This prompted us to investigate the event. Cases were recruited through hospital-based surveillance along with the collection of socio-demographic characteristics and clinical profile using a structured questionnaire. Stool specimens were tested at bacteriological laboratory of the National Institute of Cholera and Enteric Diseases (NICED), Kolkata. Admission of 3003 diarrhoea cases, clearly indicated occurrence of outbreak in Kolkata municipal area as it was more than two standard deviation of the mean number (911; s.d. = 111) of diarrhoea admissions during the same period in previous 7 years. Out of 164 recruited cases, 25% were under-5 children. Organisms were isolated from 80 (49%) stool specimens. Vibrio cholerae O1 was isolated from 50 patients. Twenty-eight patients had this organism as the sole pathogen. Among 14 infants, five had cholera. All V. cholerae O1 isolates were resistant to nalidixic acid, followed by co-trimoxazole (96%), streptomycin (92%), but sensitive to fluroquinolones. We confirmed the occurrence of a cholera outbreak in Kolkata during August 2015 due to V. cholerae O1 infection, where infants were affected.

These strains might acquire the blaNDM-1 gene without exposure to antimicrobial drugs., Carbapenems have been used for many years to treat severe nosocomial Enterobacteriaceae infections. The spread of resistance to these drugs among other bacterial families is an emerging problem worldwide, mostly caused by New Delhi metallo-β-lactamase (NDM-1). We screened for the prevalence of NDM-1–expressing enteric pathogens from hospitalized patients with acute diarrhea in Kolkata, India, and identified 27 Vibrio fluvialis–harboring blaNDM-1 (NDM-VF) strains. These isolates were also resistant to all the tested antimicrobial drugs except doxycycline. The large plasmid of V. fluvialis harboring blaNDM-1 could be easily transferred to other enteric pathogens. Genes flanking the blaNDM-1 were found to be identical to the reported sequence from an Escherichia coli isolate. Analyses showed that the V. fluvialis possessing the NDM-VF region belonged to different clones. The pathogenicity of V. fluvialis to humans and its ubiquitous presence in the environment call for constant monitoring of this species for emerging antimicrobial drug resistance.

Background Rotavirus infections, prevalent in human populations worldwide are mostly caused by Group A viruses. Live attenuated rotavirus vaccines are highly effective in preventing severe rotavirus gastroenteritis. However, the cost of these vaccines and local availability can be a barrier for widespread adoption in public health programs in developing countries where infants suffer a heavy burden of rotavirus related morbidity and mortality. A phase I/II study was carried out with the long term aim to produce a locally licensed vaccine which is equally safe and immunogenic as compared to available licensed vaccines. Methods This study was conducted in two cohorts. In the first cohort, 20 healthy adults were administered a single dose of the rotavirus vaccine (highest antigen concentration planned for infants) or placebo and were followed up for 10 days for safety. Following demonstration of safety in adult volunteers, 100 healthy infants were recruited (cohort 2) and randomly divided into five equal study groups. They were administered three doses of either the investigational rotavirus vaccine (BRV-TV) at one of the three antigen concentrations or Rotateq or Placebo at 6–8, 10–12 and 14–16 weeks of age. All infants were followed up for safety till 28 days after the third dose. Immune response to the vaccine, in terms of seroresponse and geometric mean concentrations, was compared across the five study groups. Results Increase in anti-rotavirus serum IgA antibodies from baseline, demonstrated higher immune response for all the three antigen concentrations of BRV-TV vaccine and RotaTeq in comparison with the placebo. Sero-response rates for placebo, BRV-TV dose-levels 10 5.0 FFU, 10 5.8 FFU, 10 6.4 FFU, and Rotateq at 28 days post third dose were 11.1%, 27.8%, 41.2%, 83.3%, and 63.2% respectively using the four-fold or more criteria. The BRV-TV vaccine arm corresponding to the highest antigen concentration of 10 6.4 FFU had a higher sero-response rate compared to the active comparator arm (RotaTeq), 28 days post each vaccine dose. The safety profile was comparable across the treatment groups. Conclusions Overall, the results showed that all three doses of BRV-TV vaccine were safe, well tolerated and displayed good immunogenicity (dose–response) in healthy Indian infants.

Introduction India accounts for an estimated 457,000–884,000 hospitalizations and 2 million outpatient visits for diarrhea. In spite of the huge burden of rotavirus (RV) disease, RV vaccines have not been introduced in national immunization programme of India. Therefore, continuous surveillance for prevalence and monitoring of the circulating genotypes is needed to assess the disease burden prior to introduction of vaccines in this region. Methods During January 2011 through December 2013, 830 and 1000 stool samples were collected from hospitalized and out-patient department (OPD) patients, respectively, in two hospitals in Kolkata, Eastern India. After primary screening, the G-P typing was done by multiplex semi-nested PCR using type specific primers followed by sequencing. Phylogenetic analysis for the VP7 gene of 25 representative strains was done. Results Among hospitalized and OPD patients, 53.4% and 47.5% cases were positive for rotaviruses, respectively. Unlike previous studies where G1 was predominant, in hospitalized cases G9 rotavirus strains were most prevalent (40%), followed by G2 (39.6%) whereas G1 and G12 occurred at 16.4% and 5.6% frequency. In OPD cases, the most prevalent strain was G2 (40.3%), followed by G1, G9 and G12 at 25.5%, 22.8%, 9.3%, respectively. Phylogenetically the G1, G2 and G9 strains from Kolkata did not cluster with corresponding genotypes of Rotarix, RotaTeq and Rotavac (116E) vaccine strains. Conclusion The study highlights the high prevalence of RV in children with gastroenteritis in Kolkata. The circulating genotypes have changed over the time with predominance of G9 and G2 strains during 2011-2013. The current G2, G9 and G1 Kolkata strains shared low amino acid homologies with current vaccine strains. Although there is substantial evidence for cross protection of vaccines against a variety of strains, still the strain variation should be monitored post vaccine introduction to determine if it has any impact on vaccine effectiveness.

The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needed to be explored. Proper identification and genetic characterization of Entamoeba histolytica clinical isolates is an effective tool for exploring this relation. Along with conventional polymorphic marker (Chitinase), tRNA linked short tandem repeat (STR) loci has been employed as multilocus genotyping tool due to its better resolution and evolutionary significance. Some common as well as exclusive repeat patterns showing significant relation with disease outcome have been identified. Phylogenetic analysis revealed that repeat patterns exclusively found in asymptomatic and amoebic liver abscess derived isolates are placed in a common lineage and has similar association pattern with the disease outcome. Assumption can be made that isolates of E. histolytica remaining asymptomatic is genetically closer siblings of those causing liver abscess rather than the diarrheal isolates.

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.

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.

Mutation and recombination are recognized as important driving forces of evolution among RNA viruses. An intergenogroup recombinant norovirus strain [Hu/Kol/NLV/L8775/AB290150/2006/India] was detected in the faecal specimen of a 17 year old male, who had suffered from acute watery diarrhea and severe dehydration. Sequence analysis confirmed that this novel recombinant strain had a polymerase gene fragment that closely resembled a Norovirus (NoV) genogroup-I genotype-3 virus (HuCV/NLV/GI.3/VA98115/AY038598/1998/USA) and a capsid gene resembling NoV genogroup-II genotype-4 virus (NoV/Hu/GII.4/Terneuzen70/EF126964/2006/NL). The crossing over and recombination was observed at nucleotide (nt) 790 of NoV GI VA98115 strain and nt808 of NoV GII Terneuzen70 strain. In both parent strains conserved nucleotide sequence and hairpin structure (DNA secondary structure) were reported at the junction point of ORF1 and ORF2, exhibiting the mechanism of recombination in these viruses. Thus this novel recombinant NoV is another step in evolution among NoVs, indicating that constant surveillance is important to successfully monitor emergence of these strains.

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.

The protective efficacy of an orally administered heat-killed virulent strain of Shigella flexneri 2a (5 weekly oral doses) was evaluated in 25 rabbits (14 immunized and 11 non-immunized controls) against challenge with the same strain of Shigella using the rabbit model of shigellosis. All 11 non-immunized rabbits developed bloody diarrhoea following challenge and 6 (54%) died. None of the 14 immunized rabbits developed diarrhoea (all had pellet stools) but 3 (21%) died from causes not associated with diarrhoea. Protection from diarrhoea and dysentery following oral immunization with a killed Shigella species was 100% and highly significant. Death following challenge was 2.5-fold higher in the non-immunized group (p= 0.115) but was not significant. These promising results suggest that further studies should be undertaken to develop a killed oral vaccine against shigellosis.

Zinc deficiency common in developing countries is associated with decreased immunocompetence. Zinc supplementation benefits children with acute and persistent diarrhea and prevents pneumonia. Most deaths from vaccine-preventable diseases are from measles and whooping cough; pneumonia is the most common complication of measles and often the proximate cause of related deaths. We evaluated the effect of zinc supplementation on episodes of illness in children with measles accompanied by pneumonia. In a double-blind randomized controlled trial children aged 9 mo-15 y who were admitted to the Infectious Diseases Hospital in Calcutta with clinically severe measles accompanied by pneumonia and who had been ill for = 7 d were randomly assigned to receive zinc (20 mg in elemental form as acetate twice daily for 6 d) or a placebo. All patients received standard treatment with antibiotics and an initial 100 000-IU dose of vitamin A (as palmitate) by mouth. Time-to-event analysis using the Cox proportional hazards model (42 in the zinc group and 43 in the placebo group) showed that the time needed for the resolution of fever and tachypnea the return of appetite and the achievement of a "much improved" or "cured" status was not different between the 2 groups. A high proportion of children had low serum retinol and zinc concentrations. Improvement in serum zinc and retinol concentrations after 6 d of treatment was not different between the 2 groups. Children with severe measles accompanied by pneumonia treated with antibiotics and vitamin A did not show any additional benefit from also receiving a zinc supplement. (authors)

The study was carried out to detect the prevalence of pulmonary tuberculosis among HIV-seropositive individuals (HIV/TB co-infection) who attended counseling center of National Institute of Cholera and Enteric Diseases, Kolkata. A total of 109 HIV-seropositive individuals were screened. Of them, 36 (33%) had HIV/TB co-infection diagnosed by chest X-ray and presence of acid fast bacillus (AFB) detected by repeated microscopic examination of sputum. Blood samples were examined for CD4 and CD8 counts and ratio. Findings of blood examination showed that low CD4 count (

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 a randomized controlled clinical trial the efficacy of a low-sodium low-glucose oral rehydration solution (ORS) and a low-sodium rice-based ORS was compared with standard WHO glucose ORS in the treatment of severe cholera in children aged 2-10 y. In total 120 children were evaluated for the study of whom 58 patients were positive for Vibrio cholerae and were included in the study. Of these 58 cases 19 received rice-based hypo-osmolar ORS 20 received WHOORS and 19 received glucose-based hypo-osmolar ORS. The clinical characteristics (age preadmission duration of diarrhoea frequency of stool before admission incidence of vomiting body weight and volume of initial fluid requirement) were comparable in the three treatment groups. All patients received tetracycline in a dose of 50 mg/kg/d of body weight in 4 divided doses for 3 d. Conclusions: Patients who received rice-based hypo-osmolar ORS had subsequently reduced (p < 0.05) stool output ORS consumption and diarrhoea duration than the patients who received either WHO-ORS or glucose-based hypo-osmolar ORS. (authors)

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)

Aim: To evaluate the efficacy of azithromycin in the treatment of cholera in children. Methods: A double-blind, randomized, controlled clinical trial on 80 children with acute watery diarrhoea and moderate to severe dehydration compared the efficacy of azithromycin and erythromycin in treating cholera. Data were analysed for 56 patients who were stool culture positive for Vibrio cholerae. In conjunction with rehydration therapy, 29 patients received azithromycin and 27 patients received erythromycin. Patients in the two treatment groups had comparable clinical and blood biochemical characteristics on admission. Results: Patients who received azithromycin had significantly less stool output, shorter duration of diarrhoea and lower fluid intake compared with patients who received erythromycin. Conclusion: Azithromycin appears to be superior to erythromycin for treating cholera in children.

We reviewed data during 1991-94 from a systematic 4 per cent subsample of all patients who presented with diarrhoea to our facility, in which there were 1949 cases of acute diarrhoea in children between the ages of birth to 59 months. Cryptosporidia oocysts were detected in the stools of 68 (3.5 per cent) of these children. A case-control study was designed using surveillance data which included the 68 children with stool positive for Cryptosporidium as cases. Two hundred and four children who did not have Cryptosporidium were randomly selected to serve as controls. The most common presentations were watery diarrhoea (91 per cent), dehydration (81 per cent), and vomiting (71 per cent), and Cryptosporidium was detected throughout the year, but was most frequently isolated during April to October. Lowest rates of detection were observed in the months of November, December, and January. Age below 2 years, non-breastfeeding, and stunting were significantly associated with Cryptosporidium infection. In multivariate analysis of our study we found that only stunted (P = 0.031) and non-breastfed children (P = 0.022) had a greater risk of having Cryptosporidium infection.

The Ganges delta has been well known for its high endemicity for cholera for a long time. Several pandemics of cholera originated from this region. A year-long hospital-based surveillance for cholera was undertaken in two major government hospitals in Howrah the twin city to Kolkata (capital of West Bengal India) situated on the banks of the river Ganges. Bi-weekly stool samples were collected from all diarrhoea cases admitted to these two hospitals and were screened for Vibrio cholerae using a standard microbiological technique. The antibiotic susceptibility of the isolates was tested by the Kirby-Bauer disc diffusion technique using commercially available discs. (excerpt)

In a case-control study to understand the risk factors for development of life-threatening dehydration, a total of 379 children comprising 243 cases (moderate or severe dehydration) and 136 controls (non or mild dehydration) up to 2 years of age suffering from acute watery diarrhoea were studied. By univariate analysis, the presence of vibrios in stool, withdrawal of breast feeding during diarrhoea, not giving fluids, including oral rehydration solution (ORS), during diarrhoea, frequent purging (8/day), vomiting (2/day) and undernutrition were identified as risk factors. However, by multivariate analysis after controlling for confounders, withdrawal of breast feeding during diarrhoea (odds ratio (OR) = 6.8, p0.00001) and not giving ORS during diarrhoea (OR = 2.1, p0.006) were identified as significant risk factors. The confounding variables which also contributed significantly to increasing the risk were age (or = 12 months; OR = 2.7, p = 0.001), frequent purging (8/day; OR = 4.1, p0.00001), vomiting (2/day; OR = 2.4, p = 0.001) and severe undernutrition (%medianor = 60 weight-for-age of Indian Academy of Paediatrics classification; OR = 3.1, p = 0.001). We feel that these findings will be useful for Global and National Diarrhoeal Diseases Control Programmes for formulating intervention strategies for preventing death due to diarrhoeal dehydration.

Human astroviruses (HAstVs) have now emerged as another common cause of non-bacterial acute gastroenteritis (AGE) in humans worldwide. This study investigated the epidemiology and genetic diversity of human astrovirus strains circulating among infants, younger children (up to 6 years), older children and adolescents (>6–17 years) and adults (18 years and above) hospitalized for diarrhea and their role in AGE in Kolkata, India. A total of 2535 fecal samples were screened for the presence of known enteric viral, bacterial and parasitic etiologies by conventional microbiological assays and molecular methods. The overall incidences of sole or mixed infection of HAstV with known enteric viral, bacterial and parasitic pathogens were detected in 60 cases (2.4%) among all age groups. The clinical symptoms of astrovirus-associated acute watery diarrhea cases were recorded for all sole and mixed infection cases. A high number of sole (n = 13/60 [21.7%]) and mixed infection cases (n = 22/60 [36.7%]) were observed in adults (18 years old or more). Considering all age groups, 18 sole infection cases (n = 18/60 [30%]) and 42 mixed infection cases (n = 42/60 [70%]) with Rotavirus (n = 11/25 [44%]), Vibrio cholerae O1 (n = 6/24 [25%]) Cryptosporidium spp and Giardia lamblia (n = 5/13 [38.4%]) were observed. Further, eleven HAstV samples from infants and children (up to 6 years), children and adolescents (>6–17 years) and adults (18 years and above) were analyzed for their sequences of overlap region between ORF1b (RdRp) and ORF2 (capsid). Among these, ten strains were found to have close genetic relatedness to the Japanese strain HAstV_G1 [AB009985]. Additionally, the IDH2211 Kolkata strain showed a close genetic match with the Thai HAstV_G3 strain [EU363889]. Our study reports show that HAstVs as the sole agent and as mixed infection with other known enteric viral, bacterial, parasitic pathogens are also responsible for AGE among infants, children, adolescents and adults in Kolkata, India.

Vibrio cholerae serogroup O139 Bengal, a novel strain with epidemic potential, completely displaced V. cholerae serogroup 01 in Calcutta in January 1993, which was followed by an epidemic caused by V. cholerae O139 in March-May 1993. From November 1992 to July 1993, 95.6% of 916 V. cholerae isolates submitted to the National Institute of Cholera and Enteric Diseases from 28 locations in India were confirmed as serogroup O139. As of July 1993, V. cholerae O139 had been isolated from 13 Indian states and a union territory, and large outbreaks of cholera caused by the O139 serogroup had occurred in several cities. The extent of spread of V. cholerae O139 Bengal in India and its ability to swiftly disseminate leaves little doubt that this is the beginning of the eighth pandemic of cholera.

Background Entamoeba histolytica infection is found almost all over the world and is highly endemic and a major cause of parasitic diarrhoea particularly in the developing countries. Methods A systemic surveillance was set up at the Infectious Disease hospital, Kolkata, India between November 2007 and October 2009 for understanding the trend of E. histolytica infection in Kolkata. Fecal samples were collected from diarrhoeal patients attending the hospital, under the surveillance system and processed for detection of E. histolytica. Results During the last two years about 2500 diarrhoeal samples were collected and screened for E. histolytica. About 3.6% were positive for E. histolytica. As compared to the earlier years, E. histolytica infection was observed to be less amongst patients screened during the last two years. No seasonality was observed in Kolkata although in the neighboring tropical country Bangladesh, a typical seasonality of E. histolytica infection was noticed. Conclusion The study indicates that the detection rate of E. histolytica infection amongst diarrhoeal patients in Kolkata is decreasing during the last two years than that of Bangladesh.

Immune responses after one and two doses of the reformulated killed oral cholera vaccine were measured in a double-blind, randomized, placebo-controlled trial of 77 adults aged 18-40 years and 77 children aged 1-17 years residing in Kolkata, India. 65% of adults and 87% of children and 46% of adults and 82% of children exhibited aor =4-fold rise in serum Vibrio cholerae O1 vibriocidal antibody titers from baseline following dose 1 and 2, respectively. Responses to V. cholerae O139 were less pronounced but followed a similar pattern. We demonstrate that in a cholera-endemic area, the vaccine elicited vibriocidal responses after a single-dose of the vaccine.

The causative agent of cholera, Vibrio cholerae , has been shown to be autochthonous to riverine, estuarine, and coastal waters along with its host, the copepod, a significant member of the zooplankton community. Temperature, salinity, rainfall and plankton have proven to be important factors in the ecology of V. cholerae , influencing the transmission of the disease in those regions of the world where the human population relies on untreated water as a source of drinking water. In this study, the pattern of cholera outbreaks during 1998–2006 in Kolkata, India, and Matlab, Bangladesh, and the earth observation data were analyzed with the objective of developing a prediction model for cholera. Satellite sensors were used to measure chlorophyll a concentration (CHL) and sea surface temperature (SST). In addition, rainfall data were obtained from both satellite and in situ gauge measurements. From the analyses, a statistically significant relationship between the time series for cholera in Kolkata, India, and CHL and rainfall anomalies was determined. A statistically significant one month lag was observed between CHL anomaly and number of cholera cases in Matlab, Bangladesh. From the results of the study, it is concluded that ocean and climate patterns are useful predictors of cholera epidemics, with the dynamics of endemic cholera being related to climate and/or changes in the aquatic ecosystem. When the ecology of V. cholerae is considered in predictive models, a robust early warning system for cholera in endemic regions of the world can be developed for public health planning and decision making.

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 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.

The intracellular parasite Cryptosporidium is responsible for severe diarrhea in immunocompromised persons in developing countries. Few studies on the characterization of the parasite in India are available. In this study, molecular characterization of the parasite from diarrheic children was carried out by PCR-restriction fragment length polymorphism analysis. At least three genotypes were identified. Out of 40 positive samples, 35 were positive for C. hominis , 4 were positive for C. parvum , and 1 was positive for C. felis . This study clearly suggests that cryptosporidiosis in this region is caused largely by anthroponotic transmission.

To the Editor: Campylobacter spp. infection is the leading cause of bacterial enteritis worldwide. The epidemiology of Campylobacter infection in developing countries differs substantially from that in industrialized countries. In many studies from the United States and other industrialized countries, Campylobacter spp. are among the most common bacterial causes of diarrhea (1), with an incidence of ≈10% in persons with diarrhea. Reports from developing countries also suggest that C. jejuni and C. coli have been isolated mostly from patients with diarrheal illness (2). We investigated for the prevalence of Campylobacter infection in patients hospitalized with diarrhea at the Infectious Disease Hospital in Kolkata, India, and their resistance patterns to different antimicrobial drugs. During January 2008–December 2010, we screened 3,186 fecal samples on brain–heart infusion agar with 5% defibrinated sheep blood and antimicrobial drugs (bacitracin, cycloheximide, colistin sulfate, cephazoline sodium, novobiocin) and incubated under microaerophillic environment (5% O2, 10% CO2, and 85% N2) at 37°C for 48 h. Each isolate was tested by Gram staining, cytochrome oxidase, and hippurate hydrolysis for presumptive identification and species-specific PCR (3) to identify 5 species from Campylobacter genus. The overall isolation rate of Campylobacter spp. was ≈7% (222/3,186). Sole infection with Campylobacter spp. accounted for only 40% of cases; others were mixed infection. C. jejuni was the predominant species (78%), with C. coli, C. fetus, C. lari, and C. upsaliensis isolated less frequently. Campylobacter infection prevailed throughout the year, with no seasonality. The C. jejuni isolation rate was significantly higher (10.0%; p97%) in most isolates. Multidrug resistance was frequent among many of the isolates: ampicillin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, and trimethoprim–sulfamethoxazole (19%); tetracycline, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, and trimethoprim–sulfamethoxazole (10.2%); and tetracycline, ampicillin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, and trimethoprim–sulfamethoxazole (6.8%). These results indicate that macrolides may be useful for treating campylobacteriosis in this region. Figure Antimicrobial drug susceptibility profile of 142 Campylobacter jejuni isolates, Kolkata, India, 2008–2010. Black bars, resistant; gray bars, intermediate resistance; white bars, susceptible. AMP, ampicillin; NAL, nalidixic acid; NOR, norfloxacin; ... The resistance patterns are influenced by various factors, possibly including pressure exerted by use of antimicrobial drugs. Various reports have stated that introduction of fluoroquinolones for use in veterinary practice has been associated with a dramatic rise in Campylobacter strains showing resistance to these drugs (9). Increasing antimicrobial drug resistance limits the number of therapeutic options, which makes empirical treatment more difficult. Therefore, constant monitoring of Campylobacter susceptibility to antimicrobial agents is essential. We could not detect any allele of plasmid-mediated quinolone resistance genes (qnr) among C. jejuni isolates and the different class of mobile genetic elements that generally carry the antimicrobial resistance gene cassettes. However, we found that most of the C. jejuni isolates had a mutation in the quinolone-resistance determining region of gyrA (Thr-86 to Ile), which led the isolates to become resistant for quinolone and fluoroquinolones. Recent microbiome analysis of the gut of a malnourished child residing in an urban slum in Kolkata showed 35 times more Campylobacter bacteria than in healthy child in the same setting (10). This finding suggests that intestinal inflammation may directly influence malabsorption of nutrients. Hence, it is essential to examine the effect of Campylobacter infection in the developing world in the context of many recent developments in the human gut microbiome.

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

Animal models using rabbits were developed to accumulate a variable body fat mass (FM) in two groups of animals while the fat-free mass (FFM), eg, total body protein, was maintained essentially similar between the groups. Thiobarbituric acid-reactive substances (TBARS) were measured as an index of lipid peroxidation and were found to be higher in the whole-body tissues of animals with a higher FM. Bacterial toxin (lipopolysaccharide [LPS]) also induced higher lipid peroxidation in animals with a higher FM, with a concomitant incidence of bloody mucous diarrhea. To our knowledge, this is the first evidence to show the effect of body FM to aggravate toxin-induced infections leading to diarrhea. The overall results suggest further investigations to explore the possible role of body fat in infectious diseases in humans.

One hundred and six patients suffering from severe dehydrating diarrhoea were studied of whom 36 patients were positive for Vibrio cholerae. Out of 36, 15 were positive for V. cholerae O1, 10 for V. cholerae O139 and 11 for V. cholerae non-O1 non-O139. O1 and O139 were positive for the 301-bp ctxA amplicon and 471-bp tcpA amplicon indicating that the strains possessed toxigenic capability whereas no non-O1 non-O139 strain possessed ctxA or tcpA genes. Post-admission severity of purging and amount of ORS required were less in the V. cholerae non-O1 non-O139 group (PV. cholerae O1 and O139 groups. It appears from this study that a cholera-like clinical condition can be caused in the absence of CT as exemplified by strains of non-O1 non-O139.

BACKGROUND: To analyse the trends in the prevalence of different pathogroups of diarrheagenic Escherichia coli (DEC) among hospitalized acute diarrheal patients. METHODOLOGY/PRINCIPAL FINDINGS: From the active surveillance of diarrheal disease at the Infectious Diseases Hospital, Kolkata, 3826 stool specimens collected during 2008-2011 were screened for DEC and other enteric pathogens. PCR was used in the detection of enterotoxigenic, enteropathogenic and enteroaggregative E. coli and 10 major colonization factor antigens (CFs) of enterotoxigenic E. coli. The relationship between DEC infected patient's age group and clinical symptoms were also investigated. Multiplex PCR assay showed that the prevalence of EAEC was most common (5.7%) followed by ETEC (4.2%) and EPEC (1.8%). In diarrheal children >2 year of age, EAEC and EPEC were detected significantly (p = 0.000 and 0.007, respectively). In children >2 to 5 and >5 to 14 years, ETEC was significantly associated with diarrhea (p = 0.000 each). EAEC was significantly associated with diarrheal patients with age groups >14 to 30 and >30 to 50 years (p = 0.001, and p = 0.009, respectively). Clinical symptoms such as vomiting, abdominal pain, watery diarrhea, were recorded in patients infected with ETEC. Dehydration status was severe among patients infected by ST-ETEC (19%) and EPEC (15%). CS6 was frequently detected (37%) among ETEC. CONCLUSIONS/SIGNIFICANCE: Hospital based surveillance reviled that specific pathogroups of DEC are important to certain age groups and among ETEC, CS6 was predominant.

Eighteen children with bacteriologically confirmed severe typhoid fever were initially treated intravenously with ciprofloxacin (10 mg/kg of body weight per day). Clinical cure with eradication of multiresistant Salmonella typhi infection was observed in 17 patients (94.4%; 95% confidence interval [CI], 84 to 100%). Children regained normal consciousness within an average of 2 days (95% CI, 1.8 to 2.2 days). The temperatures of the children returned to normal within 3.3 days (95% CI, 3.1 to 3.5 days). Complications were not observed during the hospital stay or a 3-month follow-up period. Relapse and carrier state were also not encountered during the follow-up period.

Background This study was conducted to determine the etiology of diarrhoea in a hospital setting in Kolkata. Active surveillance was conducted for 2 years on two random days per week by enrolling every fifth diarrhoeal patient admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata. Results Most of the patients (76.1%) had acute watery diarrhoea in association with vomiting (77.7%) and some dehydration (92%). Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of diarrhoea. Among Shigella spp, S. flexneri 2a and 3a serotypes were most predominantly isolated. Enteric viruses, EPEC and EAEC were common in children V. cholerae O1 and Shigella spp including tetracycline and ciprofloxacin. Polymicrobial infections were common in all age groups and 27.9% of the diarrhoea patients had no potential pathogen. Conclusions Increase in V. cholerae O1 infection among V. cholerae O1 to tetracycline, rise of untypable S. flexnerii, higher proportion of atypical EPEC and G. lamblia and polymicrobial etiology are some of the emerging trends observed in this diarrhoeal disease surveillance.

In a randomized clinical trial, norfloxacin was compared with nalidixic acid in the treatment of acute invasive diarrhea, with particular reference to shigellosis in adults. Of 104 patients studied, 40 were positive for Shigella in stool cultures, of which 22 received norfloxacin and 18 received nalidixic acid. The patients in these two groups were comparable on admission. In the treatment of culture-positive shigellosis cases, the responses to therapy with both drugs were similar, except that the duration of fever, anorexia, and abdominal pain were less in those who received norfloxacin. Norfloxacin appeared to be superior to nalidixic acid in the treatment of shigellosis cases caused by Shigella strains resistant to nalidixic acid.

In a randomized, double-blind clinical trial, the efficacy and safety of norfloxacin were compared with nalidixic acid in the treatment of shigellosis in children. Out of 59 cases, Shigella spp. were isolated from 8 cases in the nalidixic acid group and 14 cases in the norfloxacin group. The norfloxacin group had significantly less duration of diarrhoea and presence of blood in stool as compared to the nalidixic acid group. No joint problem was encountered in this study at up to 4 months follow-up. Norfloxacin is safe and effective and showed no cartilage toxicity on short-term follow-up.

Cholera rarely occurs in children under 2 years of age. We describe diarrhea due to Vibrio cholerae 0139 Bengal, the newly described etiologic agent of cholera in a 4-day-old breast-fed baby. However, the diarrhea was mild and was successfully treated with rehydration therapy and erythromycin.Cholera due to Vibrio cholerae 01 has been endemic in Bangladesh. In endemic cholera, the most susceptible age group is children aged 3-5 years, while those under age 2 years are rarely affected. Breast-fed children are protected from cholera due to the lack of extraneous contamination of breast milk and the protective effect of breast milk antibodies. The authors describe the case of diarrhea due to Vibrio cholerae 0139 Bengal in a 4-day old infant admitted to the Clinical Research and Service Center of the International Center for Diarrheal Disease Research, Bangladesh, for treatment. Vibrio cholerae 0139 Bengal caused large epidemics of clinical cholera in India during 1992-93, and is now established as the second etiologic agent of cholera. The male baby from a poor, rural family of Bangladesh was born at term, weighed 2.1 kg, and was breast fed. He had a history of watery diarrhea for the preceding 2 days and evidence of jaundice, but no indication of abnormality based upon systemic examination. After isolating V. cholerae 0139 in a stool specimen, the baby was treated with a total of 300 ml IV cholera saline and oral erythromycin, after which he recovered from diarrhea and was discharged on the third day after admission.

In a double-blind, randomized clinical trial with 78 adults with acute watery diarrhea and severe dehydration, 37 subjects were positive for Vibrio cholerae. In conjunction with rehydration therapy, 13 patients received norfloxacin, 12 received trimethoprim-sulfamethoxazole (TMP-SMX), and 12 received a placebo. Norfloxacin was superior to TMP-SMX and to the placebo in reducing stool output, duration of diarrhea, fluid requirements, and vibrio excretion. TMP-SMX was no better than the placebo.

The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.

Background Opportunistic Infections (OIs) and co-infections are the major cause of deaths amongst HIV infected individuals and this mostly depends upon the risk factors, type of exposure and geographic region. The commonest types of infections reported are tuberculosis, chronic diarrhoea, oral candidiasis, herpes simplex virus-2, cytomegalovirus, hepatitis B virus and hepatitis C virus. Due to the scarcity of OIs data available from this region, we had designed a study to determine the frequency of different OIs amongst HIV seropositive patients. Methods Analysis of the different spectrum of OIs/Co-infections were carried out with 204 HIV sero-positive patients (142 males and 62 females) who visited the HIV/AIDS Apex Clinic in a tertiary care hospital from March 2006 to March 2009. The CD4+ count was estimated using FACS Calibur, the routine smear test, serology, nested RT-PCR and DNA sequencing were carried out to determine the different OIs. Results In this study, HIV seropositive patients were mostly from middle age group (31-40 yrs) with CD4+ counts in majority of symptomatic AIDS patients below 200 cells/mm3. The common co-infections/opportunistic infections were OC (53.43%), CD (47.05%), HSV-2 (36.76%), TB (35.29%), CMV (26.96%), HBV (15.19%) and HCV (7.35%). Dual infections, like HSV-2 & CMV (15.38%), HSV-2 & TB (14.61%), HSV-2 & oral candidiasis (24.61%) and CMV & oral candidiasis (14.61%) were significant in follow-up patients. Triple infections were also common e.g., TB, CD, OC infection occurring frequently in about 14.21% of the study population. Multiple infections like OC, TB, CD amongst the viral co-infected patients with HSV-2, HCV, CMV and HBV are also reported in this study. The genotyping analysis of the HCV co-infected HIV individuals shows that two belonged to HCV genotype 1 and 8 belonged to genotype 3. Conclusions A wide spectrum of OIs were observed amongst HIV-infected patients in the HIV/AIDS Apex Clinic. Oral candidiasis, CD, CMV and HSV-2, were the common OIs in those patients. This study aims to provide a clearer picture regarding infections occurring amongst HIV seropositive individuals so that the scientific findings could be translated into sustainable prevention programmes and improved public health policies. Trial registration None

Background Diarrhoea is the second leading cause of illness and death in developing countries and the second commonest cause of death due to infectious diseases among children under five in such countries. Parasites, as well as bacterial and viral pathogens, are important causes of diarrhoea. However, parasitic infections are sometimes overlooked, leading after a period of time to an uncertain aetiology. In this paper we report the prevalence of Giardia lamblia, Entamoeba histolytica and Cryptosporidium sp. in and around Kolkata. Findings A hospital-based laboratory surveillance study was conducted among the patients admitted between November 2007 and October 2008 to the Infectious Diseases (ID) Hospital (Population = 1103) with diarrhoeal complaints. Of the 1103 samples collected, 147 were positive for Giardia lamblia, 84 for Cryptosporidium sp. and 51 for Entamoeba histolytica. For all these parasites there was a high rate of mixed infection with common enteric viruses and bacteria such as Rotavirus, Vibrio cholerae and Shigella sp. There were also cases of co-infection with all other diarrheogenic pathogens. The age group ≥ 5 years had the highest prevalence of parasites whereas the age group >5 – 10 years was predominantly infected with Giardia lamblia (p =< 0.001; Odds ratio (OR) = 3.937; 95% Confidence interval (CI) = 1.862 – 8.326) and with all parasites (p = 0.040; OR = 2.043; 95% CI = 1.033 – 4.039). The age group >10 – 20 years could also be considered at risk for G. lamblia (p = 0.009; OR = 2.231; 95% CI = 1.223 – 4.067). Month-wise occurrence data showed an endemic presence of G. lamblia whereas Cryptosporidium sp. and E. histolytica occurred sporadically. The GIS study revealed that parasites were more prevalent in areas such as Tangra, Tiljala and Rajarhat, which are mainly slum areas. Because most of the population surveyed was in the lower income group, consumption of contaminated water and food could be the major underlying cause of parasitic infestations. Conclusion This study provides important information on the occurrence and distribution of three important intestinal parasites and indicates their diarrheogenic capacity in Kolkata and surrounding areas.