SARS-CoV-2 variants in Paraguay: Detection and surveillance with a readily modifiable, multiplex real-time RT-PCR

Martinez, M.; Nguyen, P.-V.; Su, M.; Cardozo, F.; Valenzuela, A.; Franco, L.; Galeano, M. E.; Rojas, L. E.; Diaz Acosta, C. C.; Fernandez, J.; Ortiz, J.; del Puerto, F.; Mendoza, L.; Nara, E.; Rojas, A.; Waggoner, J.

Objectives The objective of the current study was to develop a lower-cost and scalable protocol to identify and monitor SARS-CoV-2 variants in Paraguay by pairing real-time RT-PCR detection of spike mutations with amplicon Sanger sequencing and whole-genome Nanopore sequencing. Methods 201 acute-phase nasopharyngeal samples from SARS-CoV-2-positive individuals were tested with two rRT-PCRs: 1) N2RP assay to confirm SARS-CoV-2 RNA detection (CDC N2 target), and 2) the Spike SNP assay to detect mutations in the spike receptor binding domain. The assay was performed with probes to identify mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Results All samples were positive for SARS-CoV-2 in the N2RP assay (mean Ct, 20.8; SD 5.6); 198/201 (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%) and most consistent with P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%); and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). Results were confirmed by Sanger sequencing in 181/181 samples (100%) with high-quality amplicon sequences, and variant calls were consistent with Nanopore sequencing in 29/29 samples. Conclusions The Spike SNP assay provides accurate detection of mutations associated with SARS-CoV-2 variants. This can be implemented in laboratories performing rRT-PCR to improve population-level surveillance for these mutations and inform the judicious use of scarce sequencing resources.