Detection of herpesvirus DNA in the large intestine of patients with ulcerative colitis and Crohn's disease using the nested polymerase chain reaction

Abstract

The prevalence of herpesvirus DNA was examined in inflammatory bowel disease tissue. DNA was extracted from resection and biopsy specimens of the large intestine from patients with ulcerative colitis (n = 21), patients with Crohn's disease (n = 29), and patients with noninflammatory bowel disease (controls) (n = 21). The nested polymerase chain reaction was used to detect viral DNA using primer pairs specific for either cytomegalovirus (CMV), herpes simplex virus 1 (HSV1), human herpesvirus 6 (HHV6), varicella zoster virus (VZV), or Epstein Barr virus (EBV). HSV1 and VZV DNA were not detected in any of tissue samples. There was a high prevalence of CMV (81%), HHV6 (76%), and EBV (76%) DNA in ulcerative colitis tissue compared to Crohn's disease tissues (CMV 66%, HHV6 45%, EBV 55%). Control tissue had a relatively low frequency of CMV (29%) and EBV (19%) DNA but a prevalence of HHV6 DNA similar to that of ulcerative colitis (86%). However, the simultaneous presence of HHV6 and CMV and/or EBV DNA in ulcerative colitis tissue (76%) was much greater than in either Crohn's disease tissues (38%) or control tissue (29%) (P < 0.05). There was a low prevalence of CMV, HHV6, and EBV DNA in peripheral blood mononuclear cells from all patient groups. CMV and EBV are capable of reactivating HHV6: the high prevalence of coexistent HHV6 infection with either or both of these two viruses in ulcerative colitis tissue suggests that they may play a synergistic role in the pathogenesis of this disease.

Cytomegalovirus and inflammatory bowel disease: Is there a link?

“…A study that used PCR showed positive detection in about 66% of CD patients, 81% in UC patients, and 29% in controls [9] .…”

“…The first report about the possible role of cytomegalovirus in IBD dates to 1961 when Powell [31] described a case of ulcerative colitis and cytomegalic inclusion disease. After many sporadic reports over the last decade, the topic has regained attention due to more frequent publications of case-reports or small series studies in which the virus provided a worsening prognosis influence, sometimes promoting disease initiation or otherwise acting as a bystander [9] . The coincidental detection of primary CMV infection at the first appearance of IBD is reported in some casereports [32,33] , underlining the ability of CMV proteins to enhance pro-inflammatory cytokines that are able to maintain a local colonic inflammation with an immune response.…”

“…Although clinically significant CMV infection occurs in individuals with acquired immunodeficiency syndrome or due to immunosuppressive therapy, the same inflammatory disease could be considered a booster of viral infection with local factors, even without previous immunosuppressive therapy. Antiviral treatment: A review of the literature does not affirm that antiviral treatment is mandatory when CMV is detected in biopsy specimens or in peripheral blood, however, some authors are favourable to the use of antiviral treatment [2][3][4][5][6][7][8][9] . Eddleston [39] proposes the consideration of antiviral therapy in immunocompetent patients with multiple organ disease, taking into consideration the poor prognosis with widespread CMV disease.…”

“…The authors demonstrated that a specific primer directed to the HIND III-X fragment region is the most sensitive primer for the early detection of CMV DNA in peripheral blood leukocytes (PBLs), providing detection 17 d before the onset of the symptoms. In situ hybridization and qualitative PCR in colonic biopsies seem to offer the greatest accuracy [7][8][9] in detecting the virus. Quantitative PCR (viral-load) in cells from the colon [10] showed a high positive-predictive value for detecting disease and for monitoring therapeutic response.…”

Prevalence of Congenital Cytomegalovirus Infection Assessed Through Viral Genome Detection in Dried Blood Spots in Children with Autism Spectrum Disorders

“…Stringent control measures were applied to prevent both carryover and contamination (65). In brief, DNA was extracted from the circle of DBS by adding 35 μl of cell culture medium (minimum essential medium) followed by thermal shock (55˚C for 60 min and 100˚C for 7 min, centrifugation at 11,200 rcf, and the supernatant was frozen at −80˚C overnight) and amplified using a nested polymerase chain reaction (PCR) designed to amplify one region in the GP58 gene (66). The first round of CMV-DNA amplification was carried out starting with 5 μl extract in 45 μl PCR mixture (10 mM TrisHCl, 1.5 mM MgCl2,50 mM KCl, 0.1% triton X-100, 200 mM dNTP) containing 1U Taq-polymerase (DyNAzyme™ II DNA Polymerase; Finnzymes, Thermo Fisher Scientific Inc. Ratastie, Vantaa, Finland) and the primers (1 mM) necessary for amplification.…”