Boosting SARS-CoV-2 detection combining pooling and multiplex strategies

Abstract

RT-qPCR is the gold standard technique available for SARS-CoV-2 detection. However, the long test run time and costs associated with this type of molecular testing are a challenge in a pandemic scenario. Due to high testing demand, especially for monitoring highly vaccinated populations facing the emergence of new SARS-CoV-2 variants, strategies that allow the increase in testing capacity and cost savings are needed. We evaluated a RT-qPCR pooling strategy either as a simplex and multiplex assay, as well as performed in-silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). Although the sensitivity reduction in samples pooled with 32 individuals in a simplex assay was observed, the high-test sensitivity was maintained even when 16 and 8 samples were pooled. This data was validated with the results obtained in our mass testing program with a cost saving of 51.5% already considering the expenditures with pool sampling that were analyzed individually. We also demonstrated that the pooling approach using 4 or 8 samples tested with a triplex combination in RT-qPCR is feasible to be applied without sensitivity loss, mainly combining Nucleocapsid (N) and Envelope (E) gene targets. Our data shows that the combination of pooling in a RT-qPCR multiplex assay could strongly contribute to mass testing programs with high-cost savings and low-reagent consumption while maintaining test sensitivity. In addition, the test capacity is predicted to be considerably increased which is fundamental for the control of the virus spread in the actual pandemic scenario.

Efficient large-scale screening of viral pathogens by fragment length identification of pooled nucleic acid samples (FLIPNAS)

“…The cost of Dorfman-based pool testing was calculated from one large-scale real-practice screening. 18 The model showed that the cost-effectiveness of FLIPNAS is comparable with that of Dorfman group testing at a low prevalence, such as 1%. The Dorfman-based group testing (ESI Fig.…”

“…To assess the economy of FLIPNAS, we calculated the hypothetical cost savings with a similar prevalence of around 7% found in one practical large-scale screening. 18 We found that utilizing FLIPNAS would represent savings of 86.97% and 73.37% compared with individual PCR tests and the Dorfman group testing, respectively (ESI Table S6 †). These savings were attributed to the removal of the retesting step for positive pools, the further enlarged pool size, and as there is no requirement for expensive probe modification.…”

“…17 The optimal pool size was evaluated under different prevalence values based on the current benefit model of the Dorfman group testing method under reasonable estimation (ESI Text S2 †). 18…”

Implemented occupational health surveillance limits the spread of SARS-CoV-2 Omicron at the workplace

“…One such solution, in which these factors are fed to an AI algorithm, has already been developed and successfully implemented by us to support the mass testing program in companies of the state of Rio de Janeiro, increasing the throughput and reducing costs. A straight-forward add-on was the pooling of asymptomatic samples, with individual re-testing restricted to positive pools ( 19 , 20 ). Here, statistical modeling predicted a cost saving of 48% when prevalence rates reach around 7.5% and a four-samples pooling strategy is adopted.…”

“…Here, statistical modeling predicted a cost saving of 48% when prevalence rates reach around 7.5% and a four-samples pooling strategy is adopted. Indeed, in our mass-testing routine, this strategy accounts for a cost saving of about 51%, already considering subsequent re-testing of individual samples and false-negative rates ( 20 ).…”