Reliable detection and quantitation of viral nucleic acids in oral fluid: Liquid phase‐based sample collection in conjunction with automated and standardized molecular assays

Abstract

Oral fluid has been used widely as sample matrix for the detection and quantitation of viral nucleic acids. However, in the vast majority of previous studies, various methods for collection of oral fluid and molecular assays lacking automation and standardization were used. In this study, a new standardized liquid phase-based saliva collection system was employed followed by a fully automated viral nucleic acid extraction and real-time PCR using commercially available in vitro diagnostics (IVD)/Conformité Européene (CE) labeled molecular assays. When the lower limit of detection of herpes simplex virus (HSV)-1/2 DNA, varicella zoster virus (VZV) DNA, and hepatitis C virus (HCV) RNA in spiked oral fluid was tested, the results were found to be comparable to those with defined sample materials recommended by the assay manufacturers. When clinical specimens were investigated, 21 of 25 (84%) oral fluids obtained from patients with clinically apparent herpetic lesions tested positive for HSV DNA, 7 of 10 (70%) oral fluids obtained from patients with Ramsay Hunt Syndrome tested positive for VZV DNA, and 19 of 40 (48%) oral fluids collected from patients with chronic HCV infection tested positive for HCV RNA. The automated extraction instruments completed all extractions without malfunction and no inhibitions were observed throughout the entire study. Liquid phase-based saliva collection in conjunction with automated and standardized commercially available molecular assays allows reliable quantitation of viral nucleic acids in oral fluid samples and may contribute to improved comparable and interpretable test results.

Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid: a systematic review

“…This collection system has been recently tested using routine laboratory methods. It has demonstrated reliable and reproducible results for measurement of calcium, magnesium ( 19 ), salivary alpha-amylase (SAA), UA ( 2 ), IgA, cortisol, drug and medication monitoring ( 33 ) and quantification of viral RNA ( 7 ). However, some analytes may suffer interference due to low pH and additives present in the extraction solution ( 2 , 33 ).…”

“…Salivary DNA is being routinely used in many clinical laboratories for assessing the genetic susceptibility to various diseases (Table 1). Saliva based tests have been successfully used in human immunodeficiency virus (HIV) infection diagnosis ( 4 ), monitoring renal disease ( 5 ), prevention of cardiometabolic risk ( 6 ), detection and quantification of viral nucleic acids ( 7 ), forensic medicine investigations ( 8 ), dental studies ( 9 , 10 ) and drug abuse monitoring ( 8 ). There are also some studies proposing the use of saliva in monitoring physically active individuals, incremental effort test ( 2 , 11 , 12 ) and psychological stress ( 13 ).…”

Salivary glucose in monitoring glycaemia in patients with type 1 diabetes mellitus: a systematic review

“…Published studies have indicated the possible utility of saliva in monitoring of uric acid for cardiometabolic risk [7], drugs such as theophylline and steroids, quantitation of viral nucleic acids in herpes simplex virus DNA, varicella zoster virus DNA, and hepatitis C virus RNA [8], identification of biomarkers (IL-1β, -6, -8, TNF-α, lysozyme, MMP-8, TIMP-1) and total protein concentration in systemic diseases [9], inter alia . The non-invasiveness, ease and low-cost in collection have made saliva an attractive sample.…”

Ramsay Hunt syndrome

“…When cutaneous signs and symptoms are lacking, the diagnosis can only be made using serological or molecular genetic methods. In some cases the antigen could be detected by a swab of the oral mucosa and subsequent polymerase chain reaction [25]. Experience shows that affected patients consult ENT specialists and neurologists due to the pain and the cranial nerve lesions [26].…”