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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.

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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-ß-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.

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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.

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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 ß-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.

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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.

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Salmonella enterica serovar Weltevreden and Vibrio fluvialis were identified as etiological agents of a foodborne gastroenteritis outbreak after an Iftar feast in North Dumdum. Of the 278 cases admitted to the Infectious Diseases Hospital, Kolkata, 44 stool samples were tested for the enteric pathogens. Six were positive for Salmonella Weltevreden, 5 for Vibrio fluvialis, and 8 contained both of the pathogens. Consumption of mutton-ghogni might have been the likely vehicle of this outbreak. In the pulsed-field gel electrophoresis, Salmonella Weltevreden was identified as a single clone but the V. fluvialis strains were heterogeneous.

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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.

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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.

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We identified 131 strains of Vibrio fluvialis among 400 nonagglutinating Vibrio spp. isolated from patients with diarrhea in Kolkata, India. For 43 patients, V. fluvialis was the sole pathogen identified. Most strains harbored genes encoding hemolysin and metalloprotease; this finding may contribute to understanding of the pathogenicity of V. fluvialis.

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Two conserved genomic fragments viz. 289bp of ORF1a and 449bp of ORF2 amplified by RT-PCR showed emergence of interesting recombinant strains representing new and novel genetic variants (n=5) within eight different genotypes of astroviruses known to date. HAstV-positive cases with ORF1a [HAstV genotype G2 or G8] and ORF2 [HAstV genotype G1, G2, or G3] were detected as sole or mixed infection among infants, children and adults in Kolkata with severe illness owing to acute gastroenteritis that required hospitalization for treatment between 2007 and 2009. The twelve interesting recombinants were of type HAstV _ ORF1a _ ORF2 as HAstV _ G8_ G2 (n=1), HAstV _ G8_ G1 (n=10) and HAstV _ G2_ G3 (n=1).

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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.

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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 (<50/µl) had statistically significant association (P = 0.007) with HIV/TB co-infection as compared to HIV infection only. However, no significant correlation with CD4:CD8 ratio in HIV/TB co-infection was observed.

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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.

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We evaluated the Crystal VC, a commercially produced dipstick, for the rapid detection of Vibrio cholerae serotypes O1 and O139 directly from the stool samples of hospitalized diarrheal patients using the conventional bacteriological method as gold standard. The sensitivity and specificity of the dipsticks were about 92 and 73%, respectively. Introduction of the PCR-based method along with the classical bacteriological method as the gold standard for the evaluation of a kit may improve the sensitivity and specificity of the assay. The dipstick method requires minimal technical skill, and the test can be read in about 10 min. This dipstick test has the potential to act as an early warning system for cholera in many developing countries, especially during the start of an outbreak, which would ultimately lead to a decrease in the spread of the disease as well as the case fatality rate. Furthermore, the use of a rapid detection test will improve surveillance and thus reduce the burden of disease estimates, especially in remote settings.

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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.

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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.

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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.

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BACKGROUND: 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. OBJECTIVE: We evaluated the effect of zinc supplementation on episodes of illness in children with measles accompanied by pneumonia. DESIGN: 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

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