Wide Application of Minimally Processed Saliva on Multiple RT-qPCR Kits for SARS-CoV-2 Detection in Indonesia

Abstract

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, evaluating a proposed workflow amongst the local population is recommended. Here, we aim to validate the collection and treatment of human saliva as a direct specimen for RT-qPCR-based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimens and remained stable for five days either refrigerated or stored at room temperature. The method of processing saliva specimens described in this report bypasses the need for an RNA-extraction process, thereby reducing the cost, time, and manpower required for processing samples. The developed method was tested across nine commercial kits, including the benchmark, to demonstrate its wide applicability on multiple existing workflows. Our developed method achieved an 86% overall agreement rate compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a saliva sampling device, the collection was found to be more convenient for individuals and improved the overall agreement rate to 97%.

Accelerating the Laboratory Testing Capacity through Saliva Pooling Prior to Direct RT-qPCR for SARS-CoV-2 Detection

“…In this study, we utilized saliva specimens, which allows for a more convenient sampling process. We validated the pooling-of-four method toward heat-treated saliva specimens, an RNA-extraction-free method previously described by Mahendra et al [ 6 ]. A study by Watkins et al pooled 5, 10, and 20 saliva specimens prior to RNA extraction and RT-qPCR detection of SARS-CoV-2 and reported a decrease in sensitivity of 7.41%, 11.11%, and 14.81%, respectively [ 31 ].…”

“…Some studies indicate that the addition of Proteinase K may help to reduce the viscosity of saliva specimens [8,34,35]. However, this is still equivocal, as other prior studies also indicated that the addition of Proteinase K did not improve the outcome of testing using saliva specimens [6,36]. Further study might be needed to find a solution to this problem.…”

“…A total of 52 saliva specimens were used in this study, consisting of five freshly collected specimens and 47 stored specimens obtained from the previous study [ 6 ]. As many as 27 positive and 20 negative stored saliva specimens were kept for approximately seven months at −80 °C in the COVID-19 Laboratory Center, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia (AJCUI).…”

“…Viability tests were performed on 47 specimens to determine the quality of the specimens’ post-storage. The evaluation used a direct RT-qPCR of the heat-treated saliva (at 95 °C for 10 min) method as developed by Mahendra et al [ 6 ]. Specimens that exhibited concordance results with pre-storage testing were regarded as viable.…”

“…The FDA allows for the self-collection of saliva; aside from being cheaper, it reduces the risk of infecting front-line healthcare workers; making saliva an excellent alternative specimen [ 5 ]. Previous studies have proven that heat-treated saliva as a direct RT-qPCR specimen bypasses the need for saliva specimens to undergo RNA extraction, thereby reducing cost and processing time while increasing testing capability [ 6 , 7 , 8 , 9 ]. The high agreement rates with NPOP specimens demonstrate the robustness of the developed method.…”