Development and Initial Analytical Evaluation of a Multiplex PCR-Based Targeted NGS Assay for Expanded Viral...

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.