SARS-CoV-2 Variants in Paraguay: Detection and Surveillance with an Economical and Scalable Molecular Protocol

Abstract

SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the 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). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.

Concordance of SARS-CoV-2 Results in Self-collected Nasal Swabs vs Swabs Collected by Health Care Workers in Children and Adolescents

“…The primary outcome was SARS-CoV-2 detection and relative quantitation by cycle threshold (Ct) in self-vs health care worker-collected nasal swabs when tested with a real-time reverse transcriptase-polymerase chain reaction test with Emergency Use Authorization. RESULTS Among the study participants, 108 of 194 (55.7%) were male and the median age was 9 years (IQR,(6)(7)(8)(9)(10)(11). Of the 196 participants, 87 (44.4%) tested positive for SARS-CoV-2 and 105 (53.6%) tested negative by both self-and health care worker-collected swabs.…”

“…This assay was performed as described. 6,7 Usability Evaluation A set of usability questions was developed to determine whether all tasks associated with self-swabbing were completed correctly, whether participants required any assistance, how participants felt about self-swabbing, and whether participants understood the instructions (eAppendix in the Supplement). A portion of the questions were HCW facing; that is, the HCW conducting the study observed participant actions and responded to questions accordingly.…”

Subgenomic RNA Abundance Relative to Total Viral RNA Among Severe Acute Respiratory Syndrome Coronavirus 2 Variants

“…Specimens included 169 SARS-CoV-2–positive acute-phase samples collected as part of 3 ongoing studies to characterize SARS-CoV-2 variants in distinct patient populations: (1) acute infections in Paraguay (75 samples); (2) hospitalized adults in the Emory Healthcare System as part of the ongoing RSV in Older Adults and Pregnant women Study (ROAPS, 65 samples); and (3) individuals in the Atlanta area tested at the Emory/Children’s Laboratory for Innovative Assay Development (ELIAD) to confirm diagnostic test performance (29 samples). The Paraguay sample set has been described previously [ 9 ]. ROAPS samples included a mixture of nasopharyngeal swabs obtained at the time of hospitalization for standard-of-care clinical testing and nasal midturbinate swabs collected at enrollment.…”

“…Nucleic acids were eluted from 400 µL of sample into 50 µL of elution buffer. All eluates had previously been tested in the N2RP rRT-PCR, a duplex assay targeting the N2 target in the SARS-CoV-2 genome and RNase P as an internal control [ 6 , 9 ]. Each sample was also tested in 2 different rRT-PCR assays of the spike single nucleotide polymorphism (Spike SNP) assay, including probes for (1) K417, 452R, 484K, and 501Y and (2) 452Q, 478K, and 490S, as previously described [ 9 , 10 ].…”

“…All eluates had previously been tested in the N2RP rRT-PCR, a duplex assay targeting the N2 target in the SARS-CoV-2 genome and RNase P as an internal control [ 6 , 9 ]. Each sample was also tested in 2 different rRT-PCR assays of the spike single nucleotide polymorphism (Spike SNP) assay, including probes for (1) K417, 452R, 484K, and 501Y and (2) 452Q, 478K, and 490S, as previously described [ 9 , 10 ]. Variant calls shown in Table 1 were made by comparing spike SNP assay results to the expected profile of known variants.…”