Performance of novel digital real-time Novel Digital Real-Time PCR for detection Detection of SARS-CoV-2, respiratory syncytial viruses, and influenza virusesRespiratory Syncytial Viruses, and Influenza Viruses in Ghana

Digital PCR (dPCR) systems offer high sensitivity and reproducibility without requiring external control standards. However, their performance against real-time reverse transcription-PCR (rRT-PCR) for detecting respiratory viruses remains unexplored in Ghana. We therefore evaluated the performance of a novel dPCR, Lab-On-An-Array (LOAA), for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), and influenza viruses type A (Flu A) and B (Flu B). A cross-sectional hospital-based study was conducted between August- 2022- and January- 2023 in Ghana’s Ashanti and Savannah Regions. Oropharyngeal swabs from 356 participants with a median age of 19 years, presenting with suspected respiratory illness, were tested using LOAA and rRT-PCR. Viral RNA was extracted using a Qiagen Viral Mini Kkit (Qiagen Diagnostics GmbH, Germany). LOAA and rRT-PCR tests were performed using Genoplexor^TM COVID-19/Flu/RSV Detection Kit (Optolane Technologies Inc, South Korea) and FluoroType^® SARS-CoV-2/Flu/RSV kits (Hain Lifescience GmbH, Germany), respectively. LOAA’s performance metrics were assessed using rRT-PCR as the gold standard. Overall positivity rates were 29.78% and 30.90% for LOAA and rRT-PCR, respectively. Compared to rRT-PCR, LOAA’s sensitivity was: 87.76% for RSV;, 91.30% for SARS-CoV-2;, 86.21% for Flu B;, and 88.89% for Flu A. Positive predictive value (PPV) was the highest for RSV (97.73%) and lowest for Flu A (61.54%); negative predictive values (NPV) were ≥98.00% for all respiratory viruses. LOAA recorded an “‘almost perfect”’ agreement (κ ≥0.88) with rRT-PCR for RSV, SARS-CoV-2, and Flu B and good agreement for Flu A (κ = 0.72). LOAA is sensitive in detecting SARS-CoV-2, RSV, and Flu B infections,; however, minor improvements for Flu A are required.

数字聚合酶链式反应（Digital PCR, dPCR）系统无需外部对照标准即可实现高灵敏度与良好重复性。然而，在加纳地区，其针对呼吸道病毒检测的性能表现相较于实时逆转录聚合酶链式反应（real-time reverse transcription-PCR, rRT-PCR）的优劣尚未得到探索。因此，本研究对一款新型阵列式微流控芯片实验室（Lab-On-An-Array, LOAA）的检测性能进行评估，旨在检测严重急性呼吸综合征冠状病毒2型（SARS-CoV-2）、呼吸道合胞病毒（RSV）以及甲型流感病毒（Flu A）与乙型流感病毒（Flu B）。

本研究于2022年8月至2023年1月期间，在加纳阿散蒂大区与萨瓦纳大区开展了一项基于医院的横断面研究。研究共纳入356名疑似呼吸道感染患者的口咽拭子样本，受试者年龄中位数为19岁，分别采用LOAA与rRT-PCR进行检测。

病毒RNA提取采用德国Qiagen诊断有限公司的Qiagen病毒微量提取试剂盒。LOAA检测使用韩国Optolane技术公司的Genoplexor™ 新型冠状病毒/流感/呼吸道合胞病毒检测试剂盒，rRT-PCR检测则采用德国Hain生命科学有限公司的FluoroType® 新型冠状病毒/流感/呼吸道合胞病毒检测试剂盒。

本研究以rRT-PCR作为金标准，对LOAA的各项性能指标进行评估。LOAA与rRT-PCR的总体阳性率分别为29.78%与30.90%。相较于rRT-PCR，LOAA对各病毒的检测灵敏度分别为：呼吸道合胞病毒87.76%、严重急性呼吸综合征冠状病毒2型91.30%、乙型流感病毒86.21%以及甲型流感病毒88.89%。阳性预测值（PPV）以呼吸道合胞病毒最高（97.73%），甲型流感病毒最低（61.54%）；所有呼吸道病毒的阴性预测值（NPV）均≥98.00%。LOAA与rRT-PCR的检测一致性方面，呼吸道合胞病毒、严重急性呼吸综合征冠状病毒2型及乙型流感病毒达到“近乎完美”的一致性（κ≥0.88），甲型流感病毒则为良好一致性（κ=0.72）。

LOAA可有效检测严重急性呼吸综合征冠状病毒2型、呼吸道合胞病毒与乙型流感病毒感染，但针对甲型流感病毒的检测仍需小幅优化。