Background Inadequate sampling poses challenges in the COVID-19 diagnostic cycle. Nasopharyngeal swabs are gold-standard but often associated with patient discomfort, require trained healthcare workers (HCW), and are resource intensive. The saline gargle (SG) method has proven to be acceptable for respiratory pathogen detection. We performed a prospective cross-sectional study to evaluate the SG method against the nasopharyngeal and oropharyngeal (NO/OP) method in the molecular detection and next generation sequencing (NGS) of SARS-CoV-2 in Botswana.

Methods Eligible participants aged ≥5 years, who were close contacts of a positive case, and/or presented with clinical symptoms of COVID-19, were recruited December 2021- January 2022, and July-September 2022. NP/OP samples were HCW-collected followed by SG collection where participants swished and gargled 5ml sterile 0.9% saline for 20 seconds. Samples collected December 2021-January 2022 underwent nucleic acid extraction and RT-PCR while samples collected July-September 2022 were tested with GeneXpert SARS-CoV-2 Assay. McNemar exact test was used to analyze comparability of testing with significance set as P<0.05.

Post-recruitment, random sampling of 10 lab-confirmed SARS-Cov-2 positive stored sample pairs underwent NGS.

Results Of 127 pairs, 25 matched samples tested positive for SARS-CoV-2 on both sampling methods. Additionally, SG had 6 false negatives and one sample which was positive but negative with NP/OP. Statistical analysis revealed some evidence of a difference in the detection of SARS-CoV-2 between SG and NP/OP samples (p=0.031). SG showed an overall sensitivity of 81.25% (95%CI 68.8%-96.0%).

NGS was successful in 16 samples, 10 SG and 6 NP/OP. The 5 matched successful pairs revealed similar genomic strains (73–100% relatedness). All samples had mutations of high affinity to ACE2 receptor in the Spike gene suggesting circulation of Omicron variant.

Conclusion The SG method is a reliable and logistically easier alternative for SAR-CoV-2 detection and NGS to contribute toward efforts of COVID-19 surveillance in Botswana.