MOLECULAR DETECTION PROTOCOL OF SARS-COV-2 THROUGH SELF-COLLECTED SALIVA SPECIMENS VERSUS NASOPHARYNGEAL SWABS

Abstract

Background: Several reports have shown that saliva specimen is an excellent alternative biofluid sample for SARS-CoV-2 detection. We conducted this study, in order to assess the sensitivity and specificity of using saliva self-collected by adult and pediatric patients, as a biological sample for RT-PCR diagnosis.

Aims: The present study was carried out to assess the sensitivity and specificity of using saliva self-collected from adult and pediatric patients, as a biological sample for RT-qPCR diagnosis.

Methods: In this study, 50 symptomatic patients and 40 asymptomatic subjects (adult and pediatric) were enrolled between September 2020 and November 2020 at the Department of Infectious Diseases, Bejaia University Hospital (CHU), and tested simultaneously for the sensitivity and specificity of the SARS-CoV-2 viral genome by RT-PCR on both nasopharyngeal swabs NP swab and saliva samples.

Results: Our RT-qPCR results revealed that saliva samples showed the highest sensitivity (95% CI [27.67, 29.82]) followed by a nasopharyngeal swab for symptomatic (95% CI [29.64, 31.49]) as well as for asymptomatic adult patients. Moreover, the saliva of symptomatic and asymptomatic patients was monitored, and the presence of viral RNA was detected in >95% of the asymptomatic patients as well as the symptomatic patients. Surprisingly, the Ct values of ORF1ab and N genes are highly lower in nasopharyngeal swabs compared to saliva. Indeed, the mean difference note that for the ORF1ab gene and N gene, the mean of difference in ΔCt value were respectively 3.683 and 3.578. Together, including symptomatic and asymptomatic subjects, the overall agreement between the saliva sample and the nasopharyngeal is about 84%.

Conclusion: The sensitivity of saliva samples remains acceptable; it may still be a viable option in locations where laboratory facilities are lacking for diagnostic purposes in the early phase of the disease.

Keywords: Diagnosis; Nasopharyngeal swab; Reverse Transcriptase Polymerase Chain Reaction; SARS-CoV-2; Saliva.

Figure 1

Quality of sampling according to Rnase P gene Cycle threshold value by RT-PCR. A: Comparison between Ct value of Rnase P for adult subjects (n=75) and children group (n=15). B: Comparison between Rnase P Ct values of Nasopharyngeal swab (n=75) and Saliva from adult subjects (n=75)***: p-value=0.0003. C: Comparison between Rnase P Ct values of Nasopharyngeal swab (n=75) and Saliva from symptomatic adult subjects (n=50) (p-value :0.0173) and Asymptomatic subjects (n=25) (p-value :0.0054). Noted that, we performed two RNA extraction for each patient. Error bars denote mean ± standard error of the mean (SEM) for two technical replicates. P values were determined by using paired and unpaired t- tests.

Figure 2

Cycle threshold value of SARS-Cov2 target genes (ORF1ab and N gene) in adults NPS compared with saliva samples. A: Cycle threshold value distribution of Orf1ab gene for Nasophargeal Swab (n= 50) vs Saliva Symptomatic adult (n=50) B: Cycle threshold value distribution of N gene for Nasophargeal Swab & Saliva of Symptomat adult (n=50). The dotted line represents the Ct positivity threshold according to the manufacturer of RT-qPCR SARS-CoV-2 detection kit. Noted that, we performed two RNA extraction for each patient. Error bars denote mean ± standard error of the mean (SEM) for two technical replicates. P values were determined by using paired t- test.

Figure 3

Evolution of target genes Cycle threshold values in saliva samples, according to time. The monitoring of Ct values of Orf1ab & N genes for SARS-CoV-2 is carried out by sampling patients saliva at an interval of two days. D₀ represents the first sampling, A: 37-year-old male patient is. B: 67-year-old female patient. C: 42-year-old female patient. Noted that, we performed two RNA extraction for each patient.