Effect of sample pooling on the Cycle threshold value of RT-PCR testing for Covid-19 and logistic savings

ABSTRACT

Background:This ongoing coronavirus disease 2019 (Covid-19) pandemic is a great challenge for our health-care systems and their infrastructure. Diagnostic confirmation of infected individuals based on RT-PCR is important for the contain- ment of viral spread because despite high viral loads, infection can be asymptomatic. To come up with the limited sup- plies in low socio economic countries like ours, pooling of specimens was proposed as a method to screen large number of patients. To analyse the effect of pooling of samples on the sensitivity of RT-PCR, we compared Cycle threshold (Ct) values of pools with those of the deconvulated (individual) samples. Methods:From March 2020 to October 2020, we performed Covid-19 testing by RT-PCR from areas with varying preva- lence of population referred to VRDL, JNMCH, Aligarh. To circumvent the limited availability of RNA extraction and PCR reagents, pool testing was started. 5 samples were pooled together and these pools were tested by RT-PCR. Negative pools were reported as negative whereas positive pools were deconvoluted and each sample was tested individually. In this study, 408 pools i.e 2040 samples were analysed. Results:In our study, a ΔCt value of 0.96 (i.e. an increase in Ct value in the pooled sample as compared to individual sample) was found that shows a slight loss of PCR sensitivity in pooled samples which is relatively small compared to the inherent clinical sensitivity of the standard assay. But the gains in cost effectivity and savings of resources are considera- bly high. Conclusions: As pooling of samples is a cost effective way for Covid-19 testing, the slight loss in the sensitivity of PCR can be overcomed by considering the Ct cut-off value for positive pools slightly above the kit cut off value to circumvent the dilution effect.