Background

The coronavirus disease (COVID-19) pandemic has resulted in significant socioeconomic and public health burden in affected countries worldwide. The unprecedented spread of the disease has placed healthcare systems under considerable pressure, particularly in efforts to detect cases as efficiently as possible, contain the disease, and treat cases in a timely manner. Disease surveillance and the confirmation of positive COVID-19 cases has also mostly relied upon quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) of nasopharyngeal/oropharyngeal (NP/OP) swab specimens for the detection of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) following WHO guidelines (Corman et al., 2020). However, the RT-qPCR testing capacity can be highly variable, particularly in rural areas, where primary healthcare facilities may not have trained personnel or the necessary equipment and facilities to carry out confirmatory testing. Hence, samples are sent to centralized facilities, which in turn are met with an overwhelmingly high demand for testing. As COVID-19 cases continue to rise, these laboratories have begun to face a backlog of pending tests and rising turnaround times that affect the clinical utility of results and timely surveillance of populations at the greatest risk.

In efforts to increase the cost-effectiveness and capacity of COVID-19 screening, we developed a pooling strategy during nucleic acid extraction without a reduction in the sensitivity of RT-qPCR (Lim et al., 2020). The pooling of clinical samples has previously been employed for the screening of the human immunodeficiency virus (HIV), hepatitits C virus (HCV) (Hourfar et al., 2008), influenza viruses (Pilcher et al., 2005; Sullivan et al., 2011), and during the COVID-19 pandemic, SARS-CoV-2 (Lim et al., 2020). This method has been demonstrated to be cost-effective by allowing an increase in the viral detection capacity for mass screening while maintaining testing accuracy (Abdalhamid et al., 2020). Pooled testing for RT-qPCR has been frequently utilized (Hogan et al., 2020; Lohse et al., 2020), unlike the strategy described in this paper, where we pool samples during nucleic acid extraction prior to performing RT-qPCR. This method has been evaluated as an effective approach in recent studies (Garg et al., 2020; Wacharapluesadee et al., 2020), where in a similar fashion, NP/OP swab samples were pooled in Viral Transport Medium (VTM) for extraction. Findings by Yelin et al., (2020) suggest that even in pools of up to 32 samples, one positive sample could be detected.

Pooling during the extraction stage provides additional reductions in testing costs and workload. Using the strategy described in this protocol leads to a 9-fold reduction in the number of nucleic acid extractions and RT-qPCR reactions required (Lim et al., 2020). For laboratories conducting screening for SARS-CoV-2, the pooled testing strategy used in this protocol does not introduce any significant changes to the current testing workflow and may be incorporated into routine procedures. We note that the optimal pool size will be dependent upon, among others, the prevalence of COVID-19, the objective of the testing strategy, and the resources available to the testing facility; therefore, it is recommended that laboratories account for these differences when adopting this strategy.

Thus, in this paper, we aim to provide a step-by-step protocol that details the pooling strategy for the screening of SARS-CoV-2. The strategy involves the pooling of 10 NP/OP swabs for extraction and subsequent testing by RT-qPCR. Implementing such a pooling strategy can minimize the costs incurred and the reagents utilized, while simultaneously reducing turnaround times for the rapid identification and isolation of positive COVID-19 cases. This protocol may also be applied to other pathogens that are screened using similar techniques, and would be particularly useful for the community screening of large groups in resource-poor settings and/or where the prevalence of infection is low.