histolytica infected individuals compared to healthy individuals. In the present study we used Real Time PCR for absolute PD0332991 mouse quantification of predominant gut bacterial population in E. histolytica patients BAY 57-1293 chemical structure suffering from

dysentery for 5–7 days. We also quantified the copy number of nim gene in stool sample of healthy vs E. histolytica patients. Methods Study subjects & fecal sample collection Stool samples of healthy person (without any enteric disease) were collected as controls from volunteers of a community in Delhi. Initial survey involved discussion with the focus group and informed consent was taken from participating volunteers for the study. Volunteers in age group of 21–40 year (mean age 31 year) were randomly recruited. Subjects who have taken any antibiotic/antiamoebic drug or suffered from any gastrointestinal disorder in past one Z-IETD-FMK molecular weight month before sample collection were not included in

the study. Twenty two stool samples were collected from healthy volunteers. Clinical diagnosis of amoebic colitis was based on standard criteria: patients experiencing days to weeks of dysentery (stool with blood and mucus) or diarrhea with cramps followed by abdominal pain and/or weight loss. The sub acute onset of the disease was a helpful clue in the differential diagnosis because bacillary dysentery caused by Shigella, Salmonella, Campylobacter and EHEC E. coli mostly lead to a abrupt onset of the disease [15]. Since we did not take samples from individuals administered with any antibiotic, therefore cases of antibiotic associated diarrhea were excluded. Stool samples of chronic/acute diarrhea as diagnosed by Gastroenterologist

were collected from Gastroenterology department of All India Institute of Medical Sciences & Safdarjung hospitals, New Delhi. The samples were transported to the laboratory unless at 4°C within 2 hrs and stored at -20°C until processed. The study was approved by the research ethics board of respective institutes. The samples (n = 550) were collected with the informed consent of the patients. Enrichment of entamoeba cysts Cysts were enriched following the protocol of Knight et al., 1976 [16] with slight modifications. Briefly, fecal samples (1gm) were homogenized in 10 ml of autoclaved distilled water, strained through cheesecloth in 50 ml falcon tube. This suspension was centrifuged at 2000 rpm for 5 min and pellet was re-dissolved in 10 ml of 10% formaldehyde. 3 ml of diethyl ether was added to the tube and this mixture was vortexed and incubated at RT for 30 min. The mixture was subjected to centrifugation at 2000 rpm for 5 min, supernatant was removed and pellet was washed with double distilled water. The Pellet containing concentrated cyst was re-dissolved in 400 μl T10E1 buffer. Cysts in T10E1 buffer was subjected to freeze-thaw cycle and thereafter to sonication in order to obtain crude DNA for Dot-blot hybridization experiment.