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Clostridium difficile is a Gram positive spore bearing anaerobic bacillu s ncreasingly associated with both community and hospital acquired colitis and diarrho ea. Among all the risk factors, inclusive of the ho st and the environmental factors, antibiotics are the most imp ortant ones, as validated by various studies. Patie nts receiving antibiotics and other drugs such as immunosuppressi ves, chemotherapeutics and proton pump inhibitors m ay also be important risk factors. The present study was plann ed to find out the prevalence and risk factors for Clostridium difficile associated diarrhoea (CDAD). Material and Methods: After taking approval from ethics committee ,150 pa tients with antibiotic associated diarrhoea were taken as study group and 50 patients with exposure to antibiotics but who did not develop diarrhoea were taken as controls. Stool spe cimens were processed for both culture on Cycloseri ne Cefoxitin Fructose Agar (CCFA) and toxin detection by IVD Tox A+B ELISA. Risk factor analysis was done by calcul ating odds ratio and significance of p value among various par ameters related to drugs and other factors. Result: Prevalence of CDAD in the present study was 8.67%.Third generatio n cephalosporins, clindamycin, aminoglycosides, qui nolones and trimethoprim sulfamethoxazole were significant risk factors for both antibiotic associated diarrhoea ( AAD) and Clostridium difficile associated diarrhoea (CDAD).U se of proton pump inhibitors, immunosuppressants an d prolonged stay in the hospital were other significant risk fa ctors associated with CDAD. Conclusion: Although CDAD occurs at a lower frequency in this setting, rational antibioti c policy and infection control measures should be f ollowed to prevent its occurrence and nosocomial spread. KeywordsAntibiotics, Diarrhoea, Clostridium difficile  Introduction Clostridium difficile is a Gram positive spore bearing anaerobic bacillus increasingly associated with bot h community and hospital acquired colitis and diarrhe a. It is the most common identifiable bacterial cause of nosocomial diarrhoea associated with antibiotic use and one of the most common anaerobic infections [1]. CDAD (Clostridium difficile associated diarrhoea) is a life threatening disease with an attributable morta lity of 6-15% and up to 25% in frail elderly people [2]. The clinical presentations in increasing order of s everity include asymptomatic carriage, colitis without pseu do membrane formation, pseudomembranous colitis Manuscript received: 10 th March 2017 Reviewed: 18 March 2017 Author Corrected: 25 th March 2017 Accepted for Publication: 31 st March 2017 (PMC) and fulminant colitis [3, 4]. Among all the r isk factors, inclusive of the host and the environmenta l factors, antibiotics are the most important ones, a s validated by various studies. Patients receiving antibiotics and other drugs such as immune-suppressives, chemotherapeutics and proton pump inhibitors may also be important risk factors [5]. Outbreaks in various parts of the world have been reported including the mutant hypervirulent strain, NAP1/BI/027 (North American Pulse-field gel electrophoresis type1 / restriction endonuclease an alysis BI / ribotype 027) [6] has finally put the spotlight on this pathogen. March, 2017/ Vol 5/Issue 03 ISSN2321-127X Original Research Article International Journal of Medical Research and Review Available online at: www.ijmrr.in 299 | P a g e Materials and Methods Study designProspective case control study carried out after ob taining ethics committee permission SettingFrom January 2012 to December 2013 in a tertiary ca re hospital Inclusion Criteria 1) Diarrhoea and history of antibiotic use either i n the previous month or recently since 5 days. 2) Pseudomembranous colitis detected on lower gastr ointestinal endoscopy referred for C .difficile detection and no other recognized aetiology of diarrhoea. Exclusion criteria 1) Diarrhoea during the first 72 hours of admission in a hospital 2) Neonates and psychiatric patients Study size150 cases and 50 controls of any age and gender. ParticipantsMedicine , allied medicine and paediatric departmen ts were informed to send stool samples from patient s who satisfied inclusion criteria mentioned above af ter taking written, informed consent. Also the stud y investigator visited different wards to identify patients. Diarr hoea was defined as six watery stools over 36 hours r three unformed stools in 24 hours for 2 days or eight unformed sto ol ver 48 hours. Controls were those patients adm itted during the study period who had taken antimicrobials for at le st 5 days but did not develop diarrhoea. VariablesA detailed study Performa was filled up for each on e f them, which included various parameters like a g , sex, severity of diarrhoea with duration ward and u it of admission, ICU stay, association with other symptoms like abdominal pain, fever, antibiotics used and their d uration, other significant laboratory investigation s, duration of hospital stay ,presence of nasogastric feed and provisional diagnosis. Associated and/or underlying illnesses ( inflammatory bowel disease, prior abdominal surgery, malignancy, prior h spitalization, immunosuppressive state), and add ictions were recorded. Exposure to immunosuppressive agents, can cer chemotherapy, and Proton pump inhibitors (PPI) was noted. Methodology Microbiological Method Specimen collection: Faecal samples were collected from antibiotic assoc iated diarrhoea cases in sterile wide mouthed screw capped containers and immediately transferred to the laboratory. Specimens were immediately proc essed for microscopy, anaerobic culture and ELISA. For ELISA, unpreserved specimens were kept at 2 -8 ͦ C and tested within 24 hours of collection. Specimens that could not be te sted within this time were frozen at -20 ͦ C or lower until used. MICROSCOPY: A direct wet mount for faecal leucocytes and a Gram ’s stain for detecting organisms with characteristi c morphology as that of C. difficile which appears as a gram positive bacillus with sub terminal spore were carried out. (Figure 1) CultureFor C. difficile isolation, stool samples were inoculated into Robertson’s cooked me at (RCM) broth for enrichment, and incubated at 37°C for 24-48 hours. Samples were also directly plated on Cycloserine Ce foxitin Fructose agar (CCFA). RCM was subcultured after 48 hours on CCFA. All the plates were incubated anaerobically in McIn tosh Filde’s jar for 48-72 hours. Anaerobiosis was monitored as per standard protocol by keeping a known strain of pseudomonas aeruginosa inoculated in a citrate slant in the jar. Validation of the method of isolation of C. difficile by culture was done by subculture of a known stand ard strain of C.difficile (ATCC 9689) on (CCFA), HiMedia and incubating anae robically. March, 2017/ Vol 5/Issue 03 ISSN2321-127X Original Research Article International Journal of Medical Research and Review Available online at: www.ijmrr.in 300 | P a g e FIG-1: Culture of Clostridium difficile on CCFA After 48 hours of incubation on CCFA, colonies of C .difficile were 4 mm or larger, flat to slightly raised rhizo id colonies which had a speckled opalescence and strong horse m anure like odour (Figure 2). Colonies of distinctiv e morphology were Gram’s stained and subcultured in Robertson’s cooked meat medium. A test for aero tolerance was d one to confirm that each colony type is an obligate anaerobe. Posi tive cultures were identified by gross colonial mor phology, gram’s stain characteristics and standard biochemical test s .glucose, fructose, and mannose were fermented, a nd lactose and sucrose were not fermented .Gelatin was liquefied a nd lecithinase was not produced. FIG-2: Morphology of Clostridium difficile in Gram’s stain FIG-3: ELISA for TOX A+B detection of C.difficile March, 2017/ Vol 5/Issue 03 ISSN2321-127X Original Research Article International Journal of Medical Research and Review Available online at: www.ijmrr.in 301 | P a g e Toxin Assay ElisaFor toxin assay C. difficile Toxin A+B Stool Antigen Microwell ELISA Kit manufac tured by IVD Research Inc. Carlsbad, USA was used. (Figure3). Th e test was carried out as per manufacturer’s instru ctions. Any sample well that was obviously more yellow than the negative control well or gave an absorbance re ading of 0.15 OD units and above indicated that the sample contai ned C. difficile toxin and vice versa. Selecting CasesThe samples giving positive reaction in ELISA and w hich had grown C.difficile in culture were considered as cases. All the patients were followed up for their response to discontinuation of antibi o c therapy and/or treatment with metronidazole and treatment with van comycin. Statistical AnalysisA case control study was carried out. The study sub jects were divided into two groups, group A were those with AAD in whose stool specimen C. difficile was detected and Group B were those with AAD in wh ose stool specimens C. difficile was not detected. Data was analysed by frequency p er entage. Odds ratio was calculated for risk factors which were taken as parameters in a ca se record form during specimen collection from pati ents. To determine the significance of the value obtained, Chi-square test and fisher exact test was used, p value </= 0. 05 was considered significant. Result The total sample size of the study was 150 patients who were having antibiotic associated diarrhoea. O f these, 31 were children and 119 were adults.50 age and gender (for adult group) matched controls were also taken from the same hospital setting. Out of 150 patients in the study group, Clostridium difficile was isolated from the stool of 4 patients (3adults and one child). 13 faecal samples tested positive for Toxin A+B by Enzyme Immunoassay (11 adults and 2 childre n). All the

Estimation of prevalence and risk factors for clostridium difficile infection: a neglected pathogen in a tertiary care setting in India