用于临床呼吸道标本扩展病毒检测的多重PCR靶向NGS检测方法的开发及初步分析评估

文献信息

中文摘要

背景： 感染性疾病的诊断目前主要依赖聚合酶链反应（PCR）检测。然而，这些方法可同时检测的病原体范围有限，其有效使用取决于先验的病原学假设。基于多重PCR的靶向下一代测序（mp-tNGS）被认为是一种有前景的扩展病毒检测补充方法。本研究旨在开发一种内部mp-tNGS方法，对其分析灵敏度进行初步评估，并利用临床呼吸道标本探索其性能。
方法： 该方法包括一个靶向28种病毒的panel以及用于mp-tNGS数据处理的生物信息学流程。分析灵敏度使用含有包封RNA或DNA的阳性对照样本（PCS）进行评估。利用910份鼻咽和口咽拭子标本探索其在临床标本中的性能，这些标本根据既往PCR检测结果分为三组。
结果： 在28个病毒靶标中，23个进行了实验性分析评估，而5个仅进行了计算机模拟评估。在测试的23个PCS中，有19个的估计检出限（LoD）范围为6.3 × 10²至9.4 × 10³ copies/mL。在216份PCR阳性标本组中，PCR与mp-tNGS结果在139例中完全一致；在28份标本中，mp-tNGS额外检测到其他病毒。在394份PCR阴性标本中，mp-tNGS在81份中检测到病毒。在300份医院来源标本组中，mp-tNGS在131份标本中检测到至少一个病毒靶标，包括初始检测未发现的额外病毒检出和合并感染。额外发现中相当一部分涉及EB病毒（EBV）和巨细胞病毒（CMV），需要谨慎的临床解读。
结论： 所开发的mp-tNGS方法显示出同时检测多种已知病毒、提供病毒分型并识别合并感染的潜力。该方法可作为辅助工具拓宽呼吸道感染的实验室诊断；然而，仍需进一步优化、验证并标准化结果解读标准。

英文摘要

Background: The diagnosis of infectious diseases currently relies predominantly on polymerase chain reaction (PCR) assays. However, the range of pathogens that can be detected simultaneously using these methods is limited, and their effective use depends on an a priori etiological hypothesis. Multiplex PCR-based targeted next-generation sequencing (mp-tNGS) is considered a promising complementary approach for expanded viral testing. This study aimed to develop an in-house mp-tNGS method, perform an initial evaluation of its analytical sensitivity, and explore its performance using clinical respiratory specimens. Methods: The method comprised a panel targeting 28 viruses and bioinformatics pipelines for mp-tNGS data processing. Analytical sensitivity was evaluated using positive control samples (PCSs) containing encapsulated RNA or DNA. Performance with clinical specimens was explored using 910 nasopharyngeal and oropharyngeal swab specimens, which were divided into three groups based on the results of prior PCR testing. Results: Of the 28 viral targets, 23 underwent experimental analytical evaluation, whereas five were assessed in silico only. For 19 of the 23 PCSs tested, the estimated limit of detection (LoD) ranged from 6.3 × 102 to 9.4 × 103 copies/mL. In the group of 216 PCR-positive specimens, PCR and mp-tNGS results were fully concordant in 139 cases; in 28 specimens, mp-tNGS additionally detected other viruses. Among 394 PCR-negative specimens, mp-tNGS detected viruses in 81. In the group of 300 hospital-derived specimens, mp-tNGS detected at least one viral target in 131 specimens, including additional viral detections and coinfections not identified during the initial testing. A substantial proportion of the additional findings involved Epstein-Barr virus (EBV) and cytomegalovirus (CMV), warranting cautious clinical interpretation. Conclusions: The developed mp-tNGS approach demonstrated the potential to simultaneously detect a broad range of known viruses, provide viral typing, and identify coinfections. The method may be used as an adjunctive tool to broaden the laboratory diagnosis of respiratory infections; however, further optimization, validation, and standardization of criteria for result interpretation are required.