優化分子檢測流程在腸病毒的應用

Optimization of Molecular Procedure Applied to Enteroviruses Detection

臺灣腸病毒在臨床表徵主要是手足口症（hand-foot-and-mouth disease；HFMD）及疱疹性咽唊炎（herpengina），部分重症者會造成嚴重的全身性症狀或侵犯中樞神經系統，甚至死亡。腸病毒在國內已成為一年四季皆流行的傳染病病原體之一，一般常規的檢驗依賴3~4種細胞株進行腸病毒培養，當培養細胞出現病變時，再以螢光染色進行鑑定。由於腸病毒血清型別非常複雜，目前林口長庚醫院常規螢光染色可以鑑定24種腸病毒血清型，但還有6.7%（33/492）為無法分型的腸病毒（untypable enterovirus）。CODEHOP（Consensus DEgenerate Hybrid Oligonucleotide Primer）RT-PCR可以擴增腸病毒序列中具有高度變異性（VP1）的基因，近年來廣泛用來檢測無法分型腸病毒；林口長庚院病毒合約實驗室2018年間，共有492件腸病毒分離株，其中33件利用目前常規間接免疫螢光染色法（IFA）無法鑑定（NPEV；Non-Polio Enterovirus）。這33例中，利用CODEHOP技術鑑定出24例腸病毒，分型結果分別為：14例HRV-A、5例CB3、2例E18、1例CB5、1例CoxA8、1例EV-D68。另外的9例我們利用最佳化HPeV及HRV這2套系統進行RT-PCR及基因定序進行分型。結果顯示，其中6例基因型皆為HPeV-1；3例為HRV，基因型包括2例HRV-A19及1例HRV-B84。HPeV腸道病毒基因序列親緣性演化樹分析結果顯示，其中4例基因序列分析結果和我們先前發表的HPeV-1病毒株相似性極高。建立一套優化腸病毒的監測系統，讓常規病毒培養及分生實驗室可以常態化運作，讓我們確實掌握不同腸病毒基因型在臺灣地區的流行趨勢，及對疾病嚴重程度的影響。持續進行優化檢驗技術，提升報告品質，將有助於過去出現過的新興腸病毒或再浮現病原體的監控。 In Taiwan, enterovirus infection mainly causes Hand-foot-and-mouth disease (HFMD) and Herpengina; severe infection can lead to systemic symptoms, the central nervous system disease, or even death. Enterovirus is one of the pathogens causing epidemics every year. The general routine tests rely on the viral culture using 3–4 cell lines, and when the cultured cells exhibit cytopathic effect (CPE), they are subjected to fluorescent staining for identification. The enteroviruses are highly diverse antigenically, comprising over 100 serotypes. Nowadays, the fluorescent staining only identified 24 serotypes, but there are 6.7% (33/492) of the enteroviruses that are not typable in regular screening. Consensus degenerate Hybrid oligonucleotide Primer (CODEHOP) RT-PCR could amplify the highly variable VP1 region, and recently, has been widely used to detect the untypable enteroviruses. Out of the 492 enterovirus isolates, 33 could not be identified by conventional indirect immunofluorescence staining. Among the 33 cases, 24 were identified by CODEHOP, in which 14 had HRV-A, 5 had CB3, 2 had E18, 1 had CB5, 1 had CoxA8, and 1 had EV-D68. RT-PCR and Sanger sequencing were used for classifying the remaining 9 isolates as HPeV or HRV. The results showed that 6 cases were HPeV and 3 cases were HRV. From the positive specimens that were sequenced, 2 had HRV-A19, 1 had HRV-B84, 6 had HPeV-1. By the application of molecular technology in clinical virology, we could compare the enterovirus genotypes and the disease severity in Taiwan. Continuous optimization of testing techniques and improved reporting quality will contribute to the monitoring of emerging enterovirus or reemerging pathogens that have been detected in the past.