Abstract
Deep sequencing allows for a rapid, accurate characterization of microbial DNA and RNA sequences in many types of samples. Deep sequencing (also called next generation sequencing or NGS) is being developed to assist with the diagnosis of a wide variety of infectious diseases. In this study, seven frozen brain samples from deceased subjects with recent encephalitis were investigated. RNA from each sample was extracted, randomly reverse transcribed and sequenced. The sequence analysis was performed in a blinded fashion and confirmed with pathogen-specific PCR. This analysis successfully identified measles virus sequences in two brain samples and herpes simplex virus type-1 sequences in three brain samples. No pathogen was identified in the other two brain specimens. These results were concordant with pathogen-specific PCR and partially concordant with prior neuropathological examinations, demonstrating that deep sequencing can accurately identify viral infections in frozen brain tissue.
Highlights
Current diagnostic methods used in cases of infectious encephalitis successfully identify a specific microbiologic cause of the disease in,40% of cases.[1,2] Recent work suggests that a larger number of cases have an infectious etiology but are misdiagnosed.[3]
PCR of CSF can be very helpful for identifying DNA viruses though it is less effective for the detection of RNA viruses (e.g. West Nile Virus).[4]
The remaining 36 taxon-sample pairs were distributed among the 7 encephalitis brain samples (Table 2)
Summary
Current diagnostic methods used in cases of infectious encephalitis successfully identify a specific microbiologic cause of the disease in ,40% of cases.[1,2] Recent work suggests that a larger number of cases have an infectious etiology but are misdiagnosed.[3]. PCR of CSF can be very helpful for identifying DNA viruses (e.g. herpes simplex virus type 1, HSV1) though it is less effective for the detection of RNA viruses (e.g. West Nile Virus).[4] Further limiting the efficacy of all PCR, culture, and antibody-dependent diagnostic methods are the requirements of specialized reagents and a priori knowledge of pathogens to be tested. An incomplete panel of microbial candidates for specific testing can lead to false-negative test results with missed opportunities for effective therapy.[5] validated PCR primers and protocols sometimes fail to identify known pathogens due to mutations in the primer-binding region, an issue previously addressed by our group in the detection of GB Virus C (GBV-C) in demyelinated human brain.[6]
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