Oskar Skog1, Sofie Ingvast2, Olle Korsgren2. 1. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Electronic address: oskar.skog@igp.uu.se. 2. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Abstract
BACKGROUND: Enteroviruses have been implicated in the etiology of type 1 diabetes, supported by immunoreactivity of enteroviral protein in islets, but presence of enteroviral genome has rarely been reported. Failure to detect enterovirus with RT-PCR has been attributed to the possible presence of PCR inhibitors and that only few cells are infected. OBJECTIVES: The aim of this study was to evaluate strategies for detection of enterovirus in human islets. STUDY DESIGN: A scenario was modeled with defined infected islets among a large number of uninfected pancreatic cells and the sensitivity of immunohistochemistry and PCR for detection of enterovirus was evaluated. RESULTS: Enterovirus was detected with PCR when only one single human islet, infected in vitro with a low dose of virus, was mixed with an uninfected pancreatic biopsy. Enterovirus could not be detected by immunohistochemistry under the same conditions, demonstrating the superior sensitivity of PCR also in pancreatic tissue with only a small fraction of infected cells. In addition, we demonstrate that pancreatic cell culture supernatant does not cause degradation of enterovirus at 37°C, indicating that under normal culture conditions released virus is readily detectable. Utilizing PCR, the pancreases of two organ donors that died at onset of type 1 diabetes were found negative for enterovirus genome despite islet cells being positive using immunohistochemistry. CONCLUSIONS: These data suggest that PCR should be the preferred screening method for enterovirus in the pancreas and suggest cautious interpretation of immunostaining for enterovirus that cannot be confirmed with PCR.
BACKGROUND: Enteroviruses have been implicated in the etiology of type 1 diabetes, supported by immunoreactivity of enteroviral protein in islets, but presence of enteroviral genome has rarely been reported. Failure to detect enterovirus with RT-PCR has been attributed to the possible presence of PCR inhibitors and that only few cells are infected. OBJECTIVES: The aim of this study was to evaluate strategies for detection of enterovirus in human islets. STUDY DESIGN: A scenario was modeled with defined infected islets among a large number of uninfected pancreatic cells and the sensitivity of immunohistochemistry and PCR for detection of enterovirus was evaluated. RESULTS: Enterovirus was detected with PCR when only one single human islet, infected in vitro with a low dose of virus, was mixed with an uninfected pancreatic biopsy. Enterovirus could not be detected by immunohistochemistry under the same conditions, demonstrating the superior sensitivity of PCR also in pancreatic tissue with only a small fraction of infected cells. In addition, we demonstrate that pancreatic cell culture supernatant does not cause degradation of enterovirus at 37°C, indicating that under normal culture conditions released virus is readily detectable. Utilizing PCR, the pancreases of two organ donors that died at onset of type 1 diabetes were found negative for enterovirus genome despite islet cells being positive using immunohistochemistry. CONCLUSIONS: These data suggest that PCR should be the preferred screening method for enterovirus in the pancreas and suggest cautious interpretation of immunostaining for enterovirus that cannot be confirmed with PCR.
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