of multiplex polymerase chain reaction using IS6110 and Protein b for the diagnosis of extra-pulmonary tuberculosis: North India.

Background: Prompt and accurate diagnosis of extra-pulmonary tuberculosis (TB) is highly challenging. Current conventional techniques lack sensitivity and are time-consuming. Here, we report our experience with multiplex polymerase chain reaction (MPCR) using two targets namely IS6110 and protein antigen b in the diagnosis of extra-pulmonary TB. Materials and Methods: A total of 150 patients of extra-pulmonary TB visiting tertiary care center in north India between September 2008 and December 2009 were included in the study. Sixty-six biopsy samples and 84 were body fluids from these patients were subjected for microscopy (Ziehl-Neelsen), culture on LJ medium and for Multiplex PCR using IS6110 and Protein b antigen. Results: Smear positivity was noted in 11 samples (7.33%), and LJ culture yielded Mycobacterium tuberculosis in 8 biopsies and 9 body fluids with overall positivity of 11.3%. The multi-targeted PCR could detect M. tuberculosis in a total of 112 samples. Of 112 positive samples, only Protein b band was detected in 7 samples and only IS6110 was detected in 5 samples. Overall Protein b, PCR could detect 71.33% of the cases, whereas IS6110 was positive in 66.6% of the cases. Overall the sensitivities of microscopy, culture, IS6110 PCR, Protein b PCR and MPCR were 7.33%, 11.3%, 66.67%, 71.3% and 74.6%, respectively. Thus by using more than two targets the sensitivity increased from 66.67% of IS6110 to 74.6% in MPCR. Conclusion: Multiplex polymerase chain reaction using IS6110 and Protein b antigen is a highly sensitive and specific tool in the diagnosis of pauci-bacillary conditions like extra-pulmonary TB.

Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis.

Background: Reactive arthritis (ReA)/Reiter’s syndrome (RS) may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. Aim: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. Materials and Methods: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects) were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. Results and Conclusions: A common 30 kDa antigen was found to be specifi cally present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients’ sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with postdysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the fi rst time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

Fecal Contamination of Drinking Water Supplies in and around Chandigarh and Correlation with Acute Gastroenteritis.

The study evaluated drinking water from localities in and around Chandigarh for fecal coliforms, V.cholerae and Enterotoxigenic E.coli and correlate with occurrence of acute gastroenteritis occurring from the same region. Drinking water sample were collected from various sources from the defined area. Samples were tested for fecal coliforms and E.coli count by multiple tube method and pathogens by membrane filtration technique. E. coli were screened for heat labile toxin (LT) by the reverse passive agglutination method and heat stable toxin (ST) by ELISA. Stool samples from cases of acute gastroenteritis from the same region and time were collected and processed for V. cholerae, Enterotoxigenic E coli (ETEC) and others like Salmonella, Shigella and Aeromonas spp. Of 364 water samples examined, 116 (31.8%) samples were contaminated with fecal coliforms (58.5% rural, 33.4% semi-urban and 11.1% from urban areas). E. coli were grown from 58 samples. Ninety-two isolates of E. coli were tested for enterotoxins of which 8 and 24 were positive for LT and ST respectively. V. cholerae were isolated from 2 samples during the outbreak investigation. Stored water samples showed a significantly higher level of contamination and most of Enterotoxigenic E. coli were isolated from stored water samples. A total of 780 acute gastroenteritis cases occurred; 445 from semiurban, 265 rural and 70 from urban areas. Out of 189 stool samples submitted, ETEC were the commonest (30%) followed by V. cholerae (19%), Shigellae (8.4%), Salmonellae (2.1%) and Aeromonas (2.6%). ST-ETEC (40/57) were commoner than LT- ETEC(17/57). In the present study, high levels of contamination of drinking water supplies (32.1%) correlated well with cases of acute gastroenteritis. Majority of cases of acute gastroenteritis occurred in the semi-urban area corresponding with high level of contamination (33.4%). The highest level of water contamination was seen in rural areas (58.5%) but the number of acute gastroenteritis cases were lesser (33.9%) as ponds were infrequently used for drinking purpose. Safer household water storage and treatment is recommended to prevent acute gastroenteritis, together with point-of-use water quality monitoring.

Re-emergence of multi-drug resistant Shigella dysenteriae with added resistance to ciprofloxacin in north India & their plasmid profiles.

BACKGROUND & OBJECTIVES: Multi drug resistant Shigellae pose a major threat in control of shigellosis with. Shigella dysenteriae being the most prevalent species at our centre before 1994. A gradual decrease in S. dysenteriae occurred over the years with a corresponding increase in S. flexneri which became the predominant serotype. From May to November 2003, an increase in number of patients admitted with clinical diagnosis of dysentery was noted in comparison to previous years, with a corresponding increase in the isolation of multi drug resistant S. dysenteriae. We report here the re-emergence of multi drug resistant S. dysenteriae at our tertiary care centre in north India after a gap of about 10 yr. Plasmid analysis of S. dysenteriae was also performed to study the origin and clonality of the isolates. METHODS: Stool samples were collected in Cary-Blair medium and processed by standard methods. Shigellae were confirmed by serotyping. Minimum inhibitory concentration was done by agar dilution method and E-test. Plasmid profiling of 18 isolates (16 S. dysenteriae 1 and 2 S. dysenteriae 2) was performed by modified alkali lysis method. Clinical details of patients were noted. RESULTS: A total of 64 patients with dysentery were admitted during the study period. Patients presented with unusually severe symptoms and six developed complications. Treatment failure with ciprofloxacin occurred in six patients who received cefotaxime and amikacin. There were 38 children below 5 yr of age. S. dysenteriae (18 isolates of S. dysenteriae 1 and 2 isolates of S. dysenteriae 2) were isolated from 20 of the 64 (31.2%) stool samples. S. dysenteriae re-emerged as the commonest isolate after a gap of nearly 10 yr. Fourteen of the 20 S. dysenteriae isolates were multi drug resistant; 12 were resistant to ciprofloxacin with MIC of 8-32 mug/ml. Plasmid profile analysis revealed that 6 of 11 ciprofloxacin resistant S. dysenteriae 1 had similar profiles. INTERPRETATION & CONCLUSION: Emergence of a clone of ciprofloxacin resistant S. dysenteriae 1 in north India is disturbing as treatment options in our geographic area are limited in view of already existing high drug resistance to nalidixic acid, co-trimoxazole and amoxycillin. A close monitoring of shifts in serogroup distribution and antibiotic resistance is required to guide clinicians for treatment of shigellosis.

A recent outbreak of cholera due to Vibrio cholerae O1 Ogawa in & around Chandigarh, North India.

An outbreak of cholera caused by Vibrio cholerae O1 Ogawa occurred in and around Chandigarh during July 22-31, 2002. Of the 303 patients admitted to two hospitals, 82 were confirmed by culture. Two rehabilitation colonies located at the periphery of Chandigarh were mainly affected. The isolates were biotyped as Eltor and were susceptible to many antibiotics. Thirty one (35.2%) of 88 water samples showed evidence of faecal contamination. The survey of the area revealed sewage contamination of the drinking water supply. The outbreak was controlled by providing safe drinking water to the people and correcting the defects in the sewage and water pipelines.