Svraka Sanela, van der Veer Bas, Duizer Erwin, Dekkers Jojanneke, Koopmans Marion, Vennema Harry
Centre for Infectious Disease Control, National Institute for Public Health and the Environment, P.O. Box 1, Bilthoven 3720 BA, The Netherlands.
J Clin Microbiol. 2009 Jun;47(6):1674-9. doi: 10.1128/JCM.00307-09. Epub 2009 Apr 1.
Acute gastroenteritis is one of the most common diseases worldwide, with viruses, particularly noroviruses, being the leading cause in developed countries. In The Netherlands, systematic surveillance of gastroenteritis outbreaks of suspected viral etiology was established by the National Institute for Public Health and the Environment in 1994. Since 2002, the total number of outbreaks reported has been increasing, and with that comes the need for sensitive assays that can be performed quickly. In addition, the diagnostic demand changed so that now the proportion of samples from hospitals is higher and there is a need for patient-based test results. In order to target the diagnosis of acute gastroenteritis, we reviewed our data on outbreaks of gastroenteritis and the prevalence of individual viruses to provide a priority list of viruses for which samples should be evaluated. Random primers were used to replace the separate specific primers for each virus used in the reverse transcription steps. The individual PCR assays were replaced by multiplex PCR assays. We employed a two-step method in which in the first step we screened for the most common causes of viral gastroenteritis, noroviruses of genogroup II and rotaviruses of group A, with equine arteritis virus used as the internal control. Subsequently, in the second step, two parallel PCR assays were developed for the detection of noroviruses of genogroup I and equine arteritis virus in one run and adenoviruses, sapoviruses, and astroviruses in the other run. The specificities of the assays were calculated to be 92.5% for the assay for noroviruses of genogroup I and 100% for the assays for all other viruses, the detection limits were equal for all viruses, and the turnaround time was reduced to 1 day compared to the at least 3 days required for the methods used previously. This approach allows the targeted, rapid, and cost-effective elucidation of the causes of acute gastroenteritis outbreaks.
急性肠胃炎是全球最常见的疾病之一，在发达国家，病毒尤其是诺如病毒是主要病因。1994年，荷兰国家公共卫生与环境研究所建立了对疑似病毒病因的肠胃炎暴发的系统监测。自2002年以来，报告的暴发总数一直在增加，因此需要能够快速进行的灵敏检测方法。此外，诊断需求发生了变化，现在来自医院的样本比例更高，并且需要基于患者的检测结果。为了针对急性肠胃炎的诊断，我们回顾了肠胃炎暴发数据和各病毒的流行情况，以提供应评估样本的病毒优先清单。随机引物被用于替代逆转录步骤中针对每种病毒使用的单独特异性引物。单个聚合酶链反应（PCR）检测被多重PCR检测所取代。我们采用了两步法，第一步筛查病毒性肠胃炎最常见的病因，即II基因组诺如病毒和A组轮状病毒，使用马动脉炎病毒作为内部对照。随后，在第二步中，开发了两个平行的PCR检测，一次检测I基因组诺如病毒和马动脉炎病毒，另一次检测腺病毒、札幌病毒和星状病毒。I基因组诺如病毒检测的特异性计算为92.5%，所有其他病毒检测的特异性为100%，所有病毒的检测限相同，周转时间缩短至1天，而之前使用的方法至少需要3天。这种方法能够有针对性、快速且经济高效地查明急性肠胃炎暴发的病因。
