P-14 OPTIMIZATION OF MOLECULAR METHODS FOR SARS-CoV-2 QUALITATIVE DETECTION AND GENOTYPING IN RESPIRATORY SPECIMENS FROM PATIENTS WITH LIVER DISEASE

Introduction and Objectives

SARS-CoV-2 active infection diagnosis is currently performed through RT-qPCR. Despite the fact that PCR-based assays can provide results relatively fast, these techniques require capable professionals, specific equipment and adequate infrastructure. In order to facilitate COVID-19 diagnosis in remote areas, an alternative to RT-qPCR would be loop-mediated isothermal (RT-LAMP) amplification. SARS-CoV-2 variant genotyping through high-throughput sequencing (HTS) allows SARS-CoV-2 genomic surveillance, especially for patients with a higher vulnerability. This study aimed to optimize RT-LAMP and HTS methods for SARS-CoV-2 RNA detection and genotyping, respectively, in respiratory samples from patients with liver disease.

Materials and Methods

A total of 142 respiratory secretions were obtained from individuals with SARS-CoV-2 RNA detectable by RT-qPCR (N1 Ct ≤ 30), divided into groups with (n=18) or without (n=124) liver disease. The study also enrolled 55 individuals who had SARS-CoV-2 RNA undetectable at RT-qPCR. For RT-LAMP methodology, primers were used for ORF1 gene amplification. As for HTS genotyping, the steps of cDNA synthesis, complete SARS-CoV-2 genome PCR amplification, preparation of genomic libraries and sequencing in MinION device were performed for 26 swab samples.

Results

Samples with viral RNA detectable by RT-qPCR had a mean Ct value of 24.3 ± 3.75. Referring to RT-LAMP, it was observed a sensitivity of 71.1% (101/142). When considering RT-qPCR mean Ct value, RT-LAMP sensitivity was 88.9% (16/18), associated with a mean Ct of 23.3 ± 3.5 for patients with COVID and hepatitis. A specificity of 100% (55/55) was observed since all negative swabs tested by RT-qPCR were negative at RT-LAMP. Through sequencing by MinION, SARS-CoV-2 lineages gamma (7/26; 27%), zeta (1/26; 3.9%), delta (6/26; 23%) and omicron (12/26; 46.1%) were genotyped and detected by RT-LAMP.

Conclusions

RT-LAMP demonstrated high sensitivity for molecular detection of SARS-CoV-2 RNA for patients with high viral load. Besides, RT-LAMP was capable of detecting all SARS-CoV-2 lineages genotyped by MinION in both groups.