Evaluating the efficiency of specimen (sample) pooling for real-time PCR based diagnosis of COVID-19

Abstract

Purpose This study is aims at evaluating the efficacy and sensitivity of specimen pooling for testing of SARS-CoV-2 virus to determine the accuracy, resource savings, and identification of borderline positive cases without impacting the accuracy of the testing. Method This study was conducted between August and October 2020, we performed COVID-19 testing by RT-PCR on the samples from varying prevalence of rural population (non-hot spot) referred to COVID laboratory, in the first step, the samples were collated into pools of 5 or 10. These pools were tested by RT-PCR. Negative pools were reported as negative whereas positive pools of 5 and 10 were then de-convoluted and each sample was tested individually. Results In the present study, we tested 1580 samples in 158 pools of 10 and 17,515 samples in 3503 pools of 5. Among 10 samples pool, 11 (13%) pools flagged positive in the first step. In the second step, among 11 pools (110 samples) de-convoluted strategy was followed in which 10 individual samples came positive. Among 5 samples pool, 164 (13%) pools flagged positive in the first step. In the second step, among 164 pools (820 samples) de-convoluted strategy was followed in which 171 individual samples came positive. The pooled sample testing strategy saves substantial resources and time during surge testing and enhanced pandemic surveillance. This approach requires around 76%–93% fewer tests in low to moderate prevalence settings and group sizes up to 5–10 in a population, compared to individual testing. Conclusion Pooled sample RT- PCR analysis strategies can save substantial resources and time for COVID-19 mass testing in comparison with individual testing without compromising the quality of outcome of the test. In particular, the pooled sample approach can facilitate mass screening in the early asymptomatic stages of COVID-19 infections.

Pooled sample testing for COVID-19 diagnosis: Evaluation of bi-directional matrix pooling strategies

“…In the present study, for a given positive sample the Ct value in row and column pool had considerable variation suggesting the effect of co-extracted samples. Similar observations in varying Ct differences have been reported from different laboratories when pooling is attempted from VTM samples ( Praharaj et al, 2020 , Prakash et al, 2021 , Shukla et al, 2021 Jul , Volpato et al, 2021 Apr ).…”

“…For reagent use and time required, the RNA extraction step remains one of the most rate limiting stages for SARS-CoV-2 RT-PCR ( Grant et al, 2020 ). At this point in time, number of studies have been published on conventional pooling strategies for COVID-19 RT-PCR testing ( Praharaj et al, 2020 , Barak et al, 2021 , Prakash et al, 2021 , Sawicki et al, 2021 , Shukla et al, 2021 Jul ).…”

Diagnostic performance of RT-PCR-based sample pooling strategy for the detection of SARS-CoV-2

“…First of all, this strategy would be helpful in populations where the positivity rate is low (less than 10%) [ 14 ], such as for routine monitoring of workgroups (health personnel, military units, and factory workers), where diagnosing even one positive person requires quarantine of the entire group to prevent further spread in the community [ 19 ]. On the other hand, in populations where the positivity rate is high, it may not be adequate since it would give more positive groups that will affect the response time and require a more significant amount of tests, consuming more resources [ 12 ]. Another point to consider is the time of illness of the patients, which is probably more helpful in patients in the first days of illness since a higher viral load could be found [ 15 ].…”

“…Clustering combines individual patient samples (e.g., ten) into one block and analyses the group as a single sample. If the cluster evaluation is negative, all individual samples are negative; Likewise, if it presents a positive result, at least one of the individual samples is positive, so they must be individually analyzed to determine which samples are positive [ 11 , 12 ].…”

Diagnostic Performance of RT-PCR-Based Sample Pooling Strategy for the Detection of SARS-CoV-2

“…First of all, this strategy would be helpful in populations where the positivity rate is low (less than 10%) (14) , such as for routine monitoring of workgroups (health personnel, military units, and factory workers), where diagnosing even one positive person requires quarantine of the entire group to prevent further spread in the community (19) . On the other hand, in populations where the positivity rate is high, it may not be adequate since it would give more positive groups that will affect the response time and require a more signi cant amount of tests, consuming more resources (12) . Another point to consider is the time of illness of the patients, which is probably more helpful in patients in the rst days of illness since a higher viral load could be found (15) .…”