Post-monsoon waterlogging-associated upsurge of cholera cases in and around Kolkata metropolis, 2015.

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.

Carbapenem Resistance in Clonally Distinct Clinical Strains of Vibrio fluvialis Isolated from Diarrheal Samples.

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.

Flexibility of oral cholera vaccine dosing-a randomized controlled trial measuring immune responses following alternative vaccination schedules in a cholera hyper-endemic zone.

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.

Hospital based surveillance and genetic characterization of rotavirus strains in children (<5 years) with acute gastroenteritis in Kolkata, India, revealed resurgence of G9 and G2 genotypes during 2011-2013.

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.

Safety and immunogenicity of a live oral recombinant cholera vaccine VA1.4: a randomized, placebo controlled trial in healthy adults in a cholera endemic area in Kolkata, India.

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.

Trends in the epidemiology of pandemic and non-pandemic strains of Vibrio parahaemolyticus isolated from diarrheal patients in Kolkata, India.

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.

Multilocus sequence typing system (MLST) reveals a significant association of Entamoeba histolytica genetic patterns with disease outcome.

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.

An outbreak of foodborne gastroenteritis caused by dual pathogens, Salmonella enterica serovar Weltevreden and Vibrio fluvialis in Kolkata, India.

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.

Vibrio cholerae non-O1, non-O139 serogroups and cholera-like diarrhea, Kolkata, India.

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.

Trends in the prevalence of diarrheagenic Escherichia coli among hospitalized diarrheal patients in Kolkata, India.

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.

Vibrio fluvialis in patients with diarrhea, Kolkata, India.

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.

Emerging trends in the epidemiology of human astrovirus infection among infants, children and adults hospitalized with acute watery diarrhea in Kolkata, India.

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.

Emergence of unique variants and inter-genotype recombinants of human astroviruses infecting infants, children and adults in Kolkata, India.

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).

Emergence of novel Norovirus recombinants with NVGII.1/NVGII.5 RdRp gene and NVGII.13 capsid gene among children and adults in Kolkata, India.

Norovirus (NoV) is a leading cause of non bacterial acute gastroenteritis in human beings. Molecular characterization of NoVs following continuous, stringent surveillance had earlier shown that novel strains representing an intergenogroup as well as GII NoV intergenotype recombinants were in circulation among acute watery diarrhoea cases in Kolkata, India. The present study documents characterization of two recombinant NoV strains (Hu/NoV/ IDH1501/2009/IND and Hu/NoV/IDH1873/2009/IND) along with other interesting GII NoV strains. Similarity plot and phylogenetic analysis confirmed the strain Hu/NoV/IDH1501/2009/IND as a NoV recombinant strain with genes for RNA dependent RNA polymerase (RdRp) GII.1-like and capsid GII.13-like; the strain Hu/NoV/IDH1873/2009/IND was a NoV recombinant strain with its RdRp gene GII.5-like and capsid gene being GII.13-like. Clinical symptoms chiefly associated with the cases that had NoV infection was varying duration of diarrohea and vomiting with some dehydration.

Rapid situation & response assessment of diarrhoea outbreak in a coastal district following tropical cyclone AILA in India.

BACKGROUND & OBJECTIVES: Cyclone AILA hit Indian States on eastern coast on May 25, 2009. An investigation was conducted to examine if AILA was responsible for increased reporting of diarrhoea cases from the district of East-Medinipur in West Bengal. Identifying causative organisms for diarrhoea and assessing their antibiotic susceptibility profile were other objectives. METHODS: Rapid situation and response assessment technique was employed to triangulate primary and secondary data collected through field visits. Prescription audit was also conducted. RESULTS: Significantly increased occurrence of diarrhoea was observed in June 2009 in two subdivisions namely Haldia and Egra (OR 1.6 and 1.3 respectively; 95% CI 1.52-1.65 and 1.21-1.32 P<0.001) considering 2007 as baseline. Vibrio cholerae grew from 54 per cent of the stool samples (21/39; 17 V. cholerae O1-Ogawa and 4 non-O1-non-O139), confirming a community outbreak of cholera. Shigella flexneri 3a was isolated from 5 per cent stool specimens. Increased rate of admission in treatment centres due to diarrhoea in the whole district coincided with the formation of cyclone and showed over two-fold rise compared to the admission recorded 6 days ago. Haldia subdivision had the highest attack rate of 9 per 1000 in the month of June, 2009 whereas for the whole district it was 5 per 1000 in the same month. All the isolates of V. cholerae were resistant to ampicillin and furazolidone and sensitive to norfloxacin and azithromycin. INTERPRETATION & CONCLUSIONS: Pre-AILA changes in the environment, AILA and seasonality of diarrhoea in the study district interplayed towards increased occurrence of diarrhoea. Continuous tracking of 'seasonality of diarrhoea in the community with vulnerability assessment of potential hosts', 'antibiotic sensitivity profile of the causative microorganisms', and 'prescription practice of physicians' would help appropriate disaster management.

Recent pattern of Co-infection amongst HIV seropositive individuals in tertiary care hospital, Kolkata.

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.

Pulmonary tuberculosis among HIV seropositives attending a counseling center in Kolkata.

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.

Trend of Entamoeba histolytica infestation in Kolkata.

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.

Evaluation of a rapid immunochromatographic dipstick kit for diagnosis of cholera emphasizes its outbreak utility.

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.