以次世代定序評估台灣人類免疫缺乏病毒基因型之抗藥性與偵測接受過治療病患體內之多重感染

Abstract

在台灣的HIV-1感染者以男同性戀者為主，感染之病毒亞型主要為B亞型；靜脈注射毒癮者次之，感染之病毒亞型以CRF07_BC為主；而異性戀者感染之病毒亞型有B亞型、CRF07_BC和CRF01_AE。由於有多種病毒亞型在台灣同時流行，感染者體內存在不同之病毒株或多重感染的機會也相對提高。過去由於技術上的限制，當HIV-1感染者體內存在兩種或以上病毒時，很難以傳統桑格氏定序偵測。本研究期以具更高敏感性之次世代定序系統，來了解台灣是否有多重感染發生及其在抗藥性基因型檢測的應用性。 本研究首先評估次世代定序系統之引子在不同亞型與不同循環重組模式中是否適用，再評估引子在台大醫院HIV抗藥性篩檢資料庫中不同亞型是否適用，最後以臨床病人檢體進行實際測試。當病人之HIV蛋白酶和反轉錄酶區域的桑格氏定序結果，分別被定義為不同的基因型，便被認為可能是共感染或多重感染或經過重組。我們提供三位疑似多重感染患者與九名其餘亞型之患者，並使用羅氏454次世代定序系統調查他們是否是共感染或多重感染，接著在針對一多重感染患者做長達6年之回溯性研究。 結果顯示次世代系統引子對於全球部份亞型及CRF可能不適用，而對台灣的B亞型與CRF07_BC適用，對CRF01_AE有較差適用性。不論是否懷疑為多重感染，部份HIV病患確實同時感染不只一種的HIV亞型。因為共同感染，一些重組病毒株確實存在於部分檢體中，證實重組確實會發生在這些感染了不同病毒株的病人體中。有部份檢體之次族群被分析出具有與主要族群不同之抗藥性變化。而在長時間的回溯性研究，可以看到病人體內不同亞型族群的改變，重組病毒株的消長，及抗藥性族群的出現與置換。本研究顯示此次世代系統適用於台灣不同亞型之抗藥性檢測與多重感染之偵測，而台灣多重感染的發生率可能被低估，需要進一步評估。

In Taiwan, the major transmisstion routes for HIV infection are men who have sex with men (MSM), injecting drug use (IDU), and hetersexual sexual contact. The major HIV subtypes in these three populations are subtype B, CRF07_BC, and subtypeB/CRF07_BC/CRF01_AE, respectively. The cocirculation of different subtypes in Taiwan has raised the concern of super-infection or co-infection by different HIV subtypes. In the past, due to the technical limitations of Sanger sequencing, co-infection or super-infection patients were difficult to be identified. Here we used the deep sequencing to investigate the presence of super-infection or co-infection in Taiwan and to evaluate its application in the genotypic drug resistance testing. First, we evaluated the applicability of next generation sequencing (NGS) primers in different subtypes from the database and the epidemic strains amplified in Taiwan. We selected three patients whose protease and reverse transcriptase sequences were previously defined as different genotypes to evaluate whether they were infected by different HIV subtypes. Nine patients infected with dominant HIV-1 subtypes in Taiwan were selected for comparison. We used Roche 454 NGS to investigate whether these patients had super-infection, and we determined the genotypic resistance of a patient with super-infection in a longitudinal follow-up study. Our data showed that the primers designed for NGS might not be suitable for all HIV subtypes. Super-infection (or co-infection) was observed in some cases. Because of multiple infections with HIV, recombinant strains did exist. Some undetected drug resistance mutants appeared in a minor viral population. In the longitudinal study, we observed the shift of different virus population, the rise and fall of recombinant strains, and the replacement of wild type by resistance strains. In conclusion, the NGS can be applied to detect the genetypic drug resistance and super-infection. Furthermore, the rate of super-infection in Taiwan may have been underestimated.