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.

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.

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 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.

Emerging trends in the etiology of enteric pathogens as evidenced from an active surveillance of hospitalized diarrhoeal patients in Kolkata, India.

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 <5 year age group. Atypical EPEC was comparatively higher than the typical EPEC. Multidrug resistance was common among 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 <2 years age group, resistance of 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.

Hospital-based surveillance of enteric parasites in Kolkata.

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 >or= 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.

Environmental signatures associated with cholera epidemics.

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.

Emergence of tetracycline-resistant Vibrio cholerae O1 serotype Inaba, in Kolkata, India.

Out of 2,235 diarrheal stool samples collected from patients admitted to the Infectious Diseases Hospital, Kolkata, 343 cases were positive for Vibrio cholerae (341, V. cholerae O1 and 2, O139). During the year 2004, infections caused by V. cholerae serotype Ogawa and Inaba were 93 and 7%, respectively, while in 2005, the Inaba isolation rate rose to 88% as compared to 12% for Ogawa. Susceptibility to antimicrobial agents revealed that the O1 strains were resistant to multiple antibiotics (ampicillin, co-trimoxazole, furazolidone, nalidixic acid and streptomycin) with reduced susceptibility to ciprofloxacin. Increased isolation of tetracycline-resistant strains (27.3% for Ogawa and 15% for Inaba) was noted in 2005. It appears that the population might be at risk of infection by the Inaba serotype and that tetracycline may not be useful for the treatment.

Phenotypic and genotypic traits and epidemiological implication of Vibrio cholerae O1 and O139 strains in India during 2003.

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 CTX(El), CTX(Cal) and truncated CTX(Cal) (devoid of ctxAB) prophages was unique among the majority of these O139 strains.

Increasing prevalence of Salmonella enterica serotype Paratyphi-A in patients with enteric fever in a periurban slum setting of Kolkata, India.

A sudden rise in the occurrence of Salmonella enterica serotype Paratyphi-A (SPTA) was noted in a longitudinal community-based study in Kolkata, India, during 2004 - 2005. We compared the incidence rate of Salmonella enterica serotype Typhi (ST) and SPTA and their antimicrobial susceptibility pattern. Rate of isolation of SPTA was 1.5 times higher than that of ST, a trend detected for the first time in that particular focus. Almost all the isolates were sensitive to Gentamycin and Norfloxacin. Most of the strains (90%) were sensitive to Ciprofloxacin. Two thirds of the strains were also sensitive to Chloramphenicol. The high occurrence of SPTA in the present study could be the signal of the emergence of SPTA as a pathogen in India. Quinolone derivatives (namely, Ciprofloxacin, Norfloxacin and Chloramphenicol) can be suggested as drugs of choice for treatment of enteric fever caused by SPTA. Future vaccination strategies should include bivalent vaccines with protection capacities against both ST as well as SPTA.