qPCR is a sensitive and rapid method for detection of cytomegaloviral DNA in formalin-fixed, paraffin-embedded biopsy tissue.

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

qPCR increases sensitivity to detect cytomegalovirus in formalin-fixed, paraffin-embedded tissue of gastrointestinal biopsies.

Histopathologic diagnosis of gastrointestinal (GI) tract cytomegaloviral (CMV) infection relies on hematoxylin and eosin (H&E)-stained tissue, along with the aid of immunohistochemistry (IHC). However, non-classic appearing inclusions or atypical IHC staining patterns remain an ongoing concern for pathologists. We reported the use of real-time polymerase chain reaction (qPCR) on nucleic acid extracted from paraffin-embedded, formalin-fixed tissue of GI biopsies from cases of CMV infection (n = 91) diagnosed by H&E and IHC. Seventy-nine biopsies, including normal colon biopsies (n = 35), active colitis (n = 25), and active duodenitis (n = 19), were used as negative controls. Of 91 CMV-positive biopsies diagnosed by histology, 88 tested positive by qPCR, with a sensitivity of 96.7%. Of 79 negative controls, 78 were negative and 1 positive by qPCR, resulting in a specificity of 98.7%. Of the cases that were positive for CMV by histopathology, there were an additional 40 biopsies taken from these patients either during the same or previous procedures, some taken just days prior, which were negative for CMV by histology. Interestingly, 22 (55%) of these biopsies tested positive by qPCR, which correlated well with additional clinical CMV results. By defining qPCR as the "gold standard" for a CMV result, histology (H&E and/or IHC) had a sensitivity and specificity of 79% and 97%, respectively. Eighteen biopsies were found negative by H&E and equivocal by IHC. Among them, 14 (78%) tested positive for CMV by qPCR, which also correlated well with additional clinical results. qPCR is a sensitive, specific, and rapid molecular tool that may be helpful to aid in early diagnosis of CMV infection on equivocal or clinically highly suspicious small GI biopsies.