Genomic profile of antibiotic resistant, classical ctxB positive Vibrio cholerae O1 biotype El Tor isolated in 2003 and 2005 from Puri, India: A retrospective study.

OBJECTIVES: To examine eight strains of Vibrio cholerae O1 isolated in 2003 and 2005 from Puri, India, for antibiotic susceptibility, presence of virulence and regulatory genes, cholera toxin (CT) production, CTX arrangement and genomic profiles. MATERIALS AND METHODS: Bacterial strains were tested for antibiotic susceptibility using disc diffusion assay. Polymerase chain reaction determined the presence of antibiotic resistance, virulence and regulatory genes. To determine the type of cholera toxin subunit B (ctxB), nucleotide sequencing was performed. Southern hybridisation determined the number and arrangement of CTXΦ. Ribotyping and pulsed-field gel electrophoresis (PFGE) were used to determine the genomic profile of isolates. RESULTS: All the eight strains, except one strain, showed resistant to nalidixic acid, sulphamethoxazole, streptomycin and trimethoprim and possessed the sullI, strB, dfrA1 and int SXT genes. All the strains carried the toxin-co-regulated pilus pathogenicity island, the CTX genetic element, the repeat in toxin and produced CT. Restriction fragment length polymorphism (RFLP) analysis showed that V. cholerae O1 possess a single copy of the CTX element flanked by tandemly arranged RS element. Nucleotide sequencing of the ctxB gene showed the presence of classical ctxB. RFLP analysis of conserved rRNA gene showed two ribotype patterns. PFGE analysis also showed at least three PFGE patterns, irrespective of year of isolations, indicating the genomic relatedness among them. CONCLUSION: Overall, these data suggest that classical ctxB-positive V. cholerae O1 El Tor strains that appeared in 2003 continue to cause infection in 2005 in Puri, India, and belong to identical ribotype(s) and/or pulsotype(s). There is need to continuous monitor the emergence of variant of El Tor because it will improve our understanding of the evolution of new clones of variant of V. cholerae.

Rapid spread of Vibrio cholerae O1 El Tor variant in Odisha, Eastern India, in 2008 and 2009.

The emergence and spread of Vibrio cholerae O1 El Tor variant strains causing severe diarrhea has been witnessed worldwide in recent years. In the state of Odisha, India, the spread of the V. cholerae O1 El Tor variant strains was studied during outbreaks in 2008 and 2009. Analysis of 194 V. cholerae O1 Ogawa strains revealed that V. cholerae O1 El Tor variant strains are spreading gradually throughout the state, causing outbreaks replacing typical V. cholerae O1 El Tor biotype strains.

Large outbreak of cholera caused by El Tor variant Vibrio cholerae O1 in the eastern coast of Odisha, India during 2009.

A large outbreak of cholera reported during April-July 2009 in the Kendrapada district of Odisha, India was investigated. Forty-one rectal swabs and 41 water samples, collected from diarrhoeal patients and from different villages were bacteriologically analysed for the isolation of bacterial enteriopathogens, antibiogram profile and detection of various toxic genes. The bacteriological analysis of rectal swabs and environmental water samples revealed the presence of V. cholerae O1 Ogawa biotype El Tor. The V. cholerae strains were resistant to ciprofloxacin, co-trimoxazole, chloramphenicol, streptomycin, ampicillin, furazolidone and nalidixic acid. The multiplex polymerase chain reaction (PCR) assay on V. cholerae strains revealed the presence of ctxA and tcpA genes. The mismatch amplification of mutation assay (MAMA) PCR on clinical and environmental isolates of V. cholerae revealed that the strains were El Tor biotype, which harboured the ctxB gene of the classical strain. The random amplified polymorphic DNA PCR analysis and pulsed-field gel electrophoresis results indicated that the V. cholerae isolates belonged to the same clone. This investigation gives a warning that the El Tor variant of V. cholerae has spread to the coastal district causing a large outbreak that requires close monitoring and surveillance on diarrhoeal outbreaks in Odisha.

Epidemics of severe cholera caused by El Tor Vibrio cholerae O1 Ogawa possessing the ctxB gene of the classical biotype in Orissa, India.

BACKGROUND: We investigated the epidemic of cholera that occurred in Kashipur and Dasmantpur blocks of Orissa, reported during July-September 2007. METHODS: Sixty-two rectal swabs and 28 water samples collected from diarrhea patients at different hospitals and villages were bacteriologically analyzed for the identification, antibiogram, and detection of toxic genes of Vibrio cholerae. RESULTS: The cholera outbreaks were caused by V. cholerae O1 Ogawa biotype El Tor in both Kashipur and Dasmantpur blocks. All the V. cholerae isolates from the clinical and environmental samples were sensitive to tetracycline, gentamicin, azithromycin, and chloramphenicol, but were resistant to ampicillin, ciprofloxacin, norfloxacin, co-trimoxazole, nalidixic acid, neomycin, and furazolidone, except the water isolates, which were sensitive to ciprofloxacin and norfloxacin. The multiplex PCR assay revealed that all the clinical and environmental V. cholerae isolates were positive for the ctxA and tcpA genes, showing biotype El Tor. Interestingly, 88% of the clinical and environmental isolates of V. cholerae were El Tor biotype with mutation at the ctxB gene of the classical strain, as confirmed by mismatch amplification of mutation (MAMA)-PCR assay. CONCLUSIONS: This is the first report of the El Tor variant of V. cholerae O1 Ogawa having the ctxB gene of the classical strain with altered antibiogram causing epidemics of cholera in Orissa, India.

An outbreak of cholera associated with an unprotected well in Parbatia, Orissa, Eastern India.

In November 2003, an outbreak (41 cases; attack rate-4.3%; no deaths) of severe diarrhoea was reported from a village in Orissa, eastern India. Thirteen of these cases were hospitalized. A matched case-control study was conducted to identify the possible exposure variables. Since all wells were heavily chlorinated immediately after the outbreak, water samples were not tested. The cases were managed symptomatically. Descriptive epidemiology suggested clustering of cases around one public well. Vibrio cholerae El Tor O1, serotype Ogawa was isolated from four of six rectal swabs. The water from the public well was associated with the outbreak (matched odds ratio: 12; 95% confidence interval 1.2-44.1). On the basis of these conclusions, access to the well was barred immediately, and it was protected. This investigation highlighted the broader use of field epidemiology methods to implement public-health actions guided by epidemiologic data to control a cholera epidemic.

Two sequential outbreaks in two villages illustrate the various modes of transmission of cholera.

We investigated two sequential outbreaks of severe diarrhoea in two neighbouring villages of Orissa, in 2005. We conducted descriptive and matched case-control studies. The attack rates were 5.6% (n=62) and 5.2% (n=51), respectively, in the first and second villages. One death was reported in the second village (case fatality 2%). We identified that consumption of milk products prepared in the household of the index case [matched odds ratio (mOR) 5.7, 95% confidence interval (CI) 1.7-30] in the first village, and drinking well water in the second village were associated with the illness (mOR 4.7, 95% CI 1.6-19). We isolated Vibrio cholerae El Tor O1 Ogawa from stool samples from both the villages. Mishandling of milk products led to a cholera outbreak in the first village, which led to sewerage contamination of a well and another outbreak in the second village. Environmental contamination should be expected and prevented during cholera outbreaks.

Drug abusers - a new high risk population for HIV infection in Orissa.

The present study reports the prevalence of HIV infection among the drug addicts undergoing counselling and treatment in a drug deaddiction centre located in Bhubaneswar, during July 1996 to August 1997. All subjects were males. The coded serum samples were tested by ELISA and rapid spot test for the detection of HIV antibodies. The positive samples were finally confirmed by the line immunoassay for HIV infection. A high prevalence of 7% HIV infection was noticed among the drug addicts (n=100). Oral drug abusers and IDUs were positive for HIV-1 infection in 5.26% and 21.74% cases respectively. The present study reveals a high prevalence of HIV infection among the drug addicts for the first time from Orissa which needs a careful monitoring and surveillance.

Incidence and molecular analysis of Vibrio cholerae associated with cholera outbreak subsequent to the super cyclone in Orissa, India.

An epidemiological study was carried out to find out the aetiological agent for diarrhoeal disorders in the cyclone and flood affected areas of Orissa, India. Rectal swabs collected from 107 hospitalized diarrhoea patients were bacteriologically analysed to isolate and identify the various enteropathogens. Detection of toxic genes among E. coli and V. cholerae was carried out by polymerase chain reaction (PCR) assay. Of the 107 rectal swabs analysed, 72.3% were positive for V. cholerae O1 Ogawa, 7.2% for V. cholerae O139, 1.2% for E. coli (EAggEC) and 1.2% for Shigella flexneri type 6. Using multiplex PCR assay it was found that all V. cholerae isolates were ctxA positive and El Tor biotype. Strains of V. cholerae O1 were observed to be resistant to nalidixic acid, furazolidone, streptomycin, co-trimoxazole and ampicillin. Except for nalidixic acid, the resistance pattern for O139 was identical to that of O1 strains. Representative strains of V. cholerae were further characterized by randomly amplified polymorphic DNA (RAPD) analysis and ribotyping. Both O1 and O139 V. cholerae strains exhibited the R3 pattern of ribotype and belonged to a similar pattern of RAPD compared with that of Calcutta strains. Early bacteriological and epidemiological investigations have revealed the dominance of V. cholerae O1 among the hospitalized patients in cyclone affected areas of Orissa. Drinking water scarcity and poor sanitation were thought to be responsible for these diarrhoeal outbreaks. Timely reporting and implementation of appropriate control measures could contain a vital epidemic in this area.

Is immunoproliferative small intestinal disease uncommon in India?

Till date only three series of immunoproliferative small intestinal disease (IPSID) describing 22 patients have been reported from India. Seven patients with IPSID in two tertiary referral centers in India are included in the study. Diagnosis was based on typical clinical features [diarrhoea (7/7), weight loss (7/7), clubbing (6/7), fever (3/7), abdominal pain and lump (3/7)], biochemical evidence of malabsorption and duodenal biopsy findings. All patients were young males (mean age 29.8 +/- 11.8 years, range 17-53). Atypical features included gastric involvement (1/7), colonic involvement (1/7) and appearance of pigmented nails following anti-cancer chemotherapy (1/7) which disappeared six months after omitting doxorubin from chemotherapy regimen. Parasitic infestation was common. Ascaris lumbricoides (1/7), Giardia lamblia and hookworm (1/7), Strongyloides stercoralis and Trichuris trichura (1/7). In the latter patient S. stercoralis became disseminated after anti-malignant chemotherapy. One patient had gastric H. pylori infection. Four of the seven patients who were misdiagnosed as tropical sprue were treated with tetracycline. This raises doubt on efficacy of tetracycline alone in treatment of IPSID. One other patient was misdiagnosed and treated as intestinal tuberculosis. Early diagnosis and administration of chemotherapy may improve survival in this disease.

Colonic mucosal changes in portal hypertension.

Forty one consecutive patients with portal hypertension (PHT) were evaluated by colonoscopy to study the prevalence, type, extent and predictors of haemorrhoids, colorectal varices, and portal hypertensive colopathy. Specific inquiry and regular follow-up assessed frequency of hematochezia. Twenty five patients with obscure gastrointestinal bleeding without PHT who underwent colonoscopy served as controls. Haemorrhoids were seen in nine of 41 (21.9%) patients with PHT and four of 25 (16%) controls (p = ns). Colorectal varices were seen in 13/41 (31.7%) patients with PHT and none of the controls (p = 0.005). Portal colopathy was present in 15/41 (36.6%) patients with PHT and none of the controls (p = 0.0005). None of the parameters (e.g. aetiology of PHT, Child's class, oesophageal variceal eradication by EST with or without EVL, history of variceal bleeding, grade of oesophageal varices, presence of portal hypertensive gastropathy or gastric varices) predicted the occurrence of colorectal varices and portal hypertensive colopathy. Detection of colorectal varices but not portal hypertensive colopathy was associated with occurrence of hematochezia.

Etiology and management of obscure gastrointestinal bleed--an appraisal from eastern India.

OBJECTIVES AND METHOD: Forty patients (mean age 45 years; 24 men) attending a tertiary care hospital in eastern India during the period 1996-2000 were investigated to evaluate the etiology and clinical spectrum of obscure gastrointestinal bleed. RESULTS: The patients presented to hospital after mean symptom duration of 2.5 years. They had received an average of 15 units of blood transfusion. Most patients presented with recurrent melena (85%); all had iron-deficiency anemia. A total of 230 investigations (89 gastroscopies, 54 colonoscopies, 25 double-contrast meal and follow-through studies, 14 small bowel enemas, 24 radionuclide scans, 16 mesenteric angiographies and 8 intraoperative endoscopies) yielded positive diagnosis in 87.5% of cases. The diseases encountered were small bowel and colonic angiodysplasias (32.5%), ileal Crohn's disease (20%), intestinal tuberculosis (10%), intestinal tumors (10%), nonspecific small bowel ulcers and strictures (7.5%), Meckel's diverticulum (5%) and hemobilia (2.5%). The etiology remained obscure in 5 (12.5%) cases. Overall success of surgery was 63%; in-hospital mortality was 7.5%. CONCLUSION: Though obscure gastrointestinal bleed is commonly caused by angiodysplasias, it can be an atypical presentation of Crohn's disease.

Randomized controlled trial of intrasphincteric botulinum toxin A injection versus balloon dilatation in treatment of achalasia cardia.

As the few randomized controlled trials available in the literature comparing botulinum toxin (BT) injection with established endoscopic treatment of achalasia cardia, i.e. pneumatic dilatation, showed conflicting results, we conducted a prospective randomized trial. Seventeen consecutive patients with achalasia cardia diagnosed during a period between December 1997 and February 2000 were randomized into two treatment groups [pneumatic dilatation by Rigiflex dilator (n=10), BT injection by sclerotherapy needle into four quadrants of lower esophageal sphincter (LES) (n=7) 80 units in five cases, 60 units in two cases] after dysphagia grading, endoscopy, barium esophagogram, and manometry, all of which were repeated 1 week after treatment. Patients were followed up clinically for 35.2+/-14 weeks. Chi-squares, Wilcoxon rank-sum test, Kaplan-Meier method and log-rank tests were used for statistical analysis. After 1 week, 6/7 (86%) BT-treated vs. 8/10 (80%) dilatation-treated patients improved (P=NS). There was no difference in LES pressure and maximum esophageal diameter in the barium esophagogram in the two groups before therapy. Both therapies resulted in significant reduction in LES pressure. The cumulative dysphagia-free state using the Kaplan-Meier method decreased progressively in BT-treated compared with dilatation-treated patients (P=0.027). Two patients with tortuous megaesophagus, one of whom had failed dilatation complicated by perforation previously, improved after BT. One other patient in whom pneumatic dilatation had previously failed improved in a similar manner. BT is as good as pneumatic dilatation in achieving an initial improvement in dysphagia of achalasia cardia. It is also effective in patients with tortuous megaesophagus and previous failed pneumatic dilatation. However, dysphagia often recurs during 1-year follow up.

Aetiological spectrum of chronic liver disease in eastern India.

AIM: To evaluate the aetiologic spectrum of chronic liver disease (CLD) in a tertiary referral center in Eastern India. METHODS: A total of 175 patients (cirrhosis 166, chronic hepatitis 9) diagnosed by clinical, biochemical, radiological and histopathological (42 cases) parameters were evaluated for aetiology. Investigations included: HBsAg and anti HCV (third generation) by ELISA. HBeAg and HBV DNA were tested in HBsAg positive patients. HCV RNA was tested in anti-HCV positive patients. Markers for autoimmune and Wilson's disease (anti-nuclear antibody, anti smooth muscle antibody, serum ceruloplasmin, urinary copper and slit lamp examination for KF ring) were done where clinically indicated. RESULTS: A total of 62 (35.4%) patients had HBV related CLD and 6 (9.7%) of them had pre-core mutant. HCV was present in 17/114 (14.9%) cases and none had infection with both viruses. Autoimmunity, Wilson's disease and alcohol were the aetiological factors in 5 (2.8%), 5 (2.8%) and 3 (1.7%) patients respectively. No aetilogy could be found in 18/114 (15.8%) patients. CONCLUSIONS: It is concluded that HBV is the commonest cause of CLD in Eastern India. Alcohol and HCV are uncommon in this part of the country.

Esophageal motility changes after endoscopic intravariceal sclerotherapy with absolute alcohol.

Endoscopic sclerotherapy (EST) leads to structural and motility changes in the esophagus; the former are thought to be commoner after EST with absolute alcohol (AA), which is a commonly used sclerosant in India as it is cheap and effective. There are no previous studies on changes in esophageal motility after EST with AA. Accordingly, we studied patients with portal hypertension before (n = 24) and after (n = 22) variceal obliteration by EST with AA using a water perfusion esophageal manometry system. Contraction amplitude in the distal esophagus was reduced in the post-EST group compared with the pre-EST group (63.4 +/- 24.9 vs. 18.2 +/- 14.3 mmHg, p < 0.01). Duration of esophageal contraction in both the proximal and distal esophagus became prolonged in the post-EST compared with the pre-EST group (3.3 +/- 0.8 vs. 5.4 +/- 2.6 and 4.3 +/- 1.1 vs. 6.6 +/- 2.3 s, p < 0.001 for both). Lower esophageal sphincter (LES) pressure was reduced in the post-EST compared with the pre-EST group, although the difference was not significant statistically. Abnormal contraction waveforms were more frequent in the post-EST group. One patient in the post-EST group had persistent dysphagia in the absence of endoscopically documented stricture at the time of manometric study. This study shows frequent occurrence of esophageal dysmotility after EST with AA; however, esophageal dysmotility after EST was infrequently associated with motor dysphagia.

Seroprevalence of HIV infection among jail inmates in Orissa.

Seroprevalence of human immunodeficiency virus (HIV) infection was carried out in three prisons in Orissa from March 1994 to December 1995. All the prisoners of Indian origin (300), housed in these jails tested negative for HIV infection. On the other hand, 33.8 per cent (26/77) of jail inmates from foreign countries (Thailand and Myanmar), serving short terms in Orissa jails were found positive for HIV infection. While all the 26 HIV positive prisoners had HIV-1 infection, five of them had antibodies for HIV-1 and HIV-2 viruses. Such a high prevalence of HIV infection and detection of HIV-2 virus is being reported for the first time from Orissa.

Mutagenicity testing of a rodenticide, zinctox (zinc phosphide) in a mouse in vivo system.

The genotoxic potential of zinctox (zinc phosphide) was evaluated in mice in vivo employing different bioassays: bone marrow chromosome aberration, micronucleus and sperm shape abnormality. A significant increase of chromosome aberrations was induced by three doses. Interestingly, the highest percentage was induced by the middle dose. In a route-responsive study the highest percentage of chromosome aberration was induced by the oral route. A dose-dependent increase in the incidence of micronuclei was noticed in the bone marrow cells. In the sperm shape abnormality test a high percentage of abnormal sperm was induced by all three dose levels. The results revealed the genotoxic potential of zinctox in the tested mouse in vivo system.

Cryptosporidiosis in children of eastern India.

Cryptosporidium infection was found in association with acute diarrhoeal disease in ten patients (13%) of a total of 77 children below the age of 8 years admitted over a period of 1 year to Bhubaneswar Capital Hospital, Orissa state, India. Contrary to another report from South India, there were no asymptomatic cryptosporidium oocyst excretors detected either in the matched control group of 42 children without diarrhoea or in a group of 113 healthy school children investigated. A clustering of seven cases occurred in the months of the rainy season and of three cases in the winter months. Nine out of 63 children (14.3%) below the age of 2 years were infected. Seven of the ten infected children were significantly below their expected weight-for-age, but the mean weight-for-age did not differ from those of the uninfected children. The possible epidemiological factors in the transmission of cryptosporidium are discussed.