The unprecedented demand for testing for the ongoing coronavirus disease 2019 (COVID‐19) pandemic caused by severe acute respiratory syndrome coronavirus 2 has led to an acute shortage and limited availability of test reagents for which pooling of samples has been recommended in areas with low prevalence. Considering the possibility of dilution factor in pool testing, an attempt was made to find out possibility of any true positive samples in pools with late amplification. The study was conducted on samples received from various collection centers in different districts of Odisha as well as from patients attending the screening clinic or admitted in COVID ward of the hospital. Nasal/nasopharyngeal/throat swabs received in viral transport media in cold chain were subjected to Real‐time polymerase chain reaction (RT‐PCR) testing in a Biosafety Laboratory level‐2 by including uniform volume of four units (samples) per pool. All confirmed and probable positive pools in screening assay were de‐convoluted and individual samples tested for confirmatory assay. Inclusion of an additional criteria of probable positive pool (C_t value >35 with non‐sigmoid amplification curve or showing a line of amplification towards the end of the cycle) yielded 39 (15.5%) more true positive samples out of a total of 251 positive samples that would otherwise have been missed if only the classical criteria of positive (C_t within 35 with proper sigmoid curve) had been considered. The study highlights the importance of considering any indication of late amplification in the RT‐PCR test to label a pool as positive to avoid missing any true positive sample in the pool.

中文翻译：

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SARS-CoV-2合并样本测试中表征阳性的复杂性

对由严重急性呼吸系统综合症冠状病毒2引起的进行中冠状病毒病2019（COVID-19）大流行的测试需求空前高涨，导致严重短缺且检测试剂的可用性有限，建议在低流行地区汇集样本。考虑到池测试中稀释因子的可能性，试图找出在后期扩增中池中任何真实阳性样品的可能性。该研究是从奥里萨邦不同地区的各个采集中心以及从筛查诊所就诊或在医院的COVID病房接受治疗的患者所采集的样本中进行的。通过将每个池中四个单位的均匀体积（样本）包括在内，在冷链病毒运输介质中接收的鼻/鼻咽/咽拭子在生物安全实验室级别2中进行了实时聚合酶链反应（RT-PCR）测试。对筛选测定中所有已确认和可能的阳性池进行反卷积，并对单个样品进行检验以进行确定性测定。包括可能的阳性合并量的其他标准（C _t值> 35（具有非S型曲线）或在周期结束时显示一条扩增线）在总共251个阳性样品中产生了39个（15.5％）更多的真实阳性样品，否则仅在这些样品中会漏掉考虑了经典的阳性标准（C _t在35以内，并具有适当的S型曲线）。这项研究强调了在RT-PCR测试中考虑任何后期扩增迹象的重要性，以将池标记为阳性以避免避免池中任何真实阳性样品的丢失。