Estimation of faecal carriage of Clostridium difficile in patients with ulcerative colitis using real time polymerase chain reaction.

ABSTRACT

BACKGROUND & OBJECTIVE: Ulcerative colitis (UC) is a disease of unknown aetiology in which exacerbations are sometimes linked to intestinal colonization by toxin-producing Clostridium difficile. We undertook this study to detect and quantitatively assess C. difficile in the stool of patients with UC using real time polymerase chain reaction (RT-PCR), and to compare it with healthy individuals. METHODS: A total of 37 consecutive patients with UC (26 male, mean age 41.3 yr) and 36 healthy adult volunteers (20 male, mean age 36.4), none of whom had received antibiotics within two months prior to faecal collection, were included in the study. Faecal DNA was extracted, quantitative PCR (qPCR) carried out using primers to amplify species-specific segments of 16S rDNA of C. difficile, and expressed as relative fold difference against amplification of highly conserved (universal) segments. Toxins A and B were assayed by ELISA. RESULTS: Quantitative PCR detected C. difficile sensitively, and spiking with increasing numbers of the organism resulted in linear increase in amplification (R(2)=0.974). C. difficile was detected by qPCR in faeces of 20 of 36 healthy volunteers and 34 of 37 patients with UC. Relatively greater amplification of C. difficile (fold difference) was noted in UC compared to controls (P<0.0001). There was no significant difference in C. difficile amplification between patients with proctitis, left sided colitis and pancolitis, or between active and quiescent colitis. Toxin was detected in the faeces of 8 of 37 patients with UC compared to 2 of 36 healthy volunteers. INTERPRETATION & CONCLUSION: Findings of this study showed overgrowth of C. difficile in the stool of Indian patients with UC. However, its relevance to disease pathogenesis and severity in a tropical country like India needs to be investigated further.