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Background Human Cytomegalovirus (HCMV) infection has been reported to be a cause of refractory ulcerative colitis (UC). Toxic megacolon (TM) is a rare but severe complication of an acute attack of UC. Objectives Aim of this study is to evaluate in a case-control study the association between HCMV and TM. Study design All patients who were admitted at Medicine Department of V. Cervello Hospital in Palermo (tertiary referral center) for a severe UC flare-up complicated by the onset of TM (diameter of the transverse colon > 6 cm) between January 1990 and November 2011 were identified through the electronic database. A total of 24 consecutive patients (16 male/8 female) with TM were identified. Each case of TM were individually matched by sex, age, extent of the underlying disease to 24 severe UC controls who did not develop TM. A further non matched control population of 48 severe UC was included. Haematoxilin and eosin stain, immunohistochemical procedure and nested polymerase chain reaction were performed to detect HCMV genes and proteins on rectal biopsies or surgical specimens. Pp65 antigenemia was performed in order to diagnose any possible systemic infection. HCMV frequency was compared between patients with and without TM during follow-up, using Fisher’s Exact test. Results and conclusions HCMV was detected in histological specimens of 11 patients (46%) with TM compared to 2 (9%) severe UC matched controls ( P = 0.0078) and 7 (14%) unmatched controls ( p = 0,003). In severe colitis the presence of HCMV is more frequently associated with TM. [ABSTRACT FROM AUTHOR]