Pham Ngan T K, Ushijima Hiroshi, Thongprachum Aksara, Trinh Quang D, Khamrin Pattara, Arakawa Chikako, Ishii Wakako, Okitsu Shoko, Komine-Aizawa Shihoko, Hayakawa Satoshi
Clin Lab. 2017 Jan 1;63(1):91-100. doi: 10.7754/Clin.Lab.2016.160630.
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.
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.
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.
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.
急性脑炎是一种严重的神经系统疾病，死亡率高，影响儿童和成人。本研究旨在开发一种多重PCR方法，用于同时筛查临床样本中目前被认为是亚洲急性脑炎/脑病主要病毒病因的10种病毒。
使用先前发表的引物，这些引物已广泛用于临床样本中筛查疱疹病毒6型、甲型流感病毒、人细小病毒、单纯疱疹病毒1型和2型、日本脑炎病毒、A组轮状病毒、肠道病毒、腺病毒和登革病毒，开发了一种单管多重PCR检测方法，并对其敏感性和特异性进行了测试。然后将该方法应用于筛查57份临床样本，这些样本包括13份粪便样本、5份咽拭子、3份鼻后拭子、18份血清样本和18份脑脊液（CSF）样本，这些样本来自18名住院的疑似病毒性脑炎/脑病的日本儿童，用于检测目标病毒，并将结果与单重PCR方法的结果进行比较。
使用这种多重PCR方法对10种病毒的阳性病毒对照进行了正确分型。多重PCR方法显示出高特异性，对非靶病毒无非特异性扩增。应用该PCR方法筛查临床样本的结果表明，从7名患者收集的6份粪便样本、2份血清样本和1份脑脊液样本中一种病毒呈阳性，具体为A组轮状病毒（4名患者，22.2%）、肠道病毒（2名患者，11.1%）或腺病毒（1名患者，5.6%）。与单重PCR相比，对于A组轮状病毒、肠道病毒和腺病毒，这种多重PCR方法对血清、脑脊液和咽拭子样本的敏感性降低。
这种新开发的多重PCR方法是一种简单、快速的诊断工具，可用于筛查亚洲国家儿童中引起急性脑炎/脑病的病毒的临床样本。
