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Multiplex PCR for the Detection of 10 Viruses Causing Encephalitis/Encephalopathy and its Application to Clinical Samples Collected from Japanese Children with Suspected Viral by Ngan T. K. Pham, Hiroshi Ushijima, Aksara Thongprachum, Quang D. Trinh, Pattara Khamrin, Chikako Arakawa, Wakako Ishii, Shoko Okitsu, Shihoko Komine-Aizawa, Satoshi Hayakawa

Background: Acute encephalitis is a serious neurological condition having a high mortality rate and affecting both children and adults. This study aimed to develop a multiplex PCR method for the simultaneous screening of clinical samples for the presence of the 10 viruses presently considered as the major viral causes of acute encephalitis/ encephalopathy in Asia.
Methods: Using previously published primers that have been widely used to screen for herpes virus-6, influenza A virus, human parechovirus, herpes simplex viruses 1 and 2, Japanese encephalitis virus, group A rotavirus, enterovirus, adenovirus, and dengue virus in clinical samples, a single-tube multiplex PCR assay was developed and was tested for its sensitivity and specificity. The method was then applied to screen 57 clinical samples, consisting of 13 fecal samples, 5 throat swabs, 3 post-nasal swabs, 18 serum samples, and 18 cerebrospinal fluid (CSF) samples, collected from 18 hospitalized Japanese children with suspected viral encephalitis/encephalopathy for the target viruses, and the results were compared with those of a monoplex PCR method.
Results: Positive viral controls of the 10 viruses were correctly typed using this multiplex PCR method. The multiplex PCR method showed high specificity with no unspecific amplification to non-target viruses. The results of applying this PCR method for screening clinical samples showed that 6 fecal samples, 2 serum samples, and 1 CSF sample collected from 7 patients were positive for a virus, specifically group A rotavirus (4 patients, 22.2%), enterovirus (2 patients, 11.1%), or adenovirus (1 patient, 5.6%). In comparison with monoplex PCR, for group A rotavirus, enterovirus, and adenovirus, the sensitivity of this multiplex PCR method decreased for serum, cerebrospinal fluid, and throat swab samples.
Conclusions: This newly developed multiplex PCR method is a simple, rapid diagnostic tool and can be used to screen clinical samples for viruses causing acute encephalitis/encephalopathy in children in Asian countries.

DOI: 10.7754/Clin.Lab.2016.160630