|Year : 2021 | Volume
| Issue : 2 | Page : 206-210
|Comparative analysis of the naso/oropharyngeal swab and oral bio-fluid (whole saliva) samples for the detection of SARS-CoV-2 using RT-qPCR
Parmar Girish1, Pillai Jayasankar2, Parmar Abhishek3, Soni Sumeeta4, Parmar Gunvant5, Parmar Shalin6
1 Dean and Additional Director (Dental), Govt. Dental College and Hospital, Ahmedabad, Gujarat, India
2 Department of Oral Pathology, Govt. Dental College and Hospital, Ahmedabad, Gujarat, India
3 Department of Conservative Dentistry and Endodontics, Govt. Dental College and Hospital, Ahmedabad, Gujarat, India
4 Department of Microbiology, BJ Medical College, Ahmedabad, Gujarat, India
5 Department of Dentistry, Govt. Medical College, Surat, Gujarat, India
6 Department of Pediatrics, MGM Medical College, Jaipur, Rajasthan, India
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|Date of Submission||18-May-2021|
|Date of Decision||23-Jun-2021|
|Date of Acceptance||30-Aug-2021|
|Date of Web Publication||22-Nov-2021|
| Abstract|| |
Context: The Corona Virus Disease 2019 (COVID-19) is a contagious disease caused by the novel Coronavirus (2019-nCoV) and was declared a pandemic disease by the World Health Organization (WHO) in March 2020. The nasopharyngeal and the oropharyngeal swabs are being taken during the screening procedure. However, the virus is also present in the oral bio-fluid and hence it could be a potential tool for screening COVID-19 cases. Aim: The aim of the present study was to test the accuracy of whole saliva as a diagnostic specimen in COVID-19. Settings and Design: This cross-sectional, analytical study was conducted on out-patients visiting the COVID-19 hospital. Methods and Material: The whole saliva and the nasopharyngeal/oropharyngeal samples from 309 COVID-19 suspected patients were collected and subjected to RT-PCR analysis. Statistical Analysis Used: The paired t test was used to compare the measured variables (CT values) between the saliva and the swab samples. The positive predictive value (PPV), negative predictive value (NPV), the sensitivity, and the specificity of the tests were calculated for the saliva sample. Results: The saliva and swab results revealed a similar result (ties) in 86.73% of the samples. The sensitivity and the specificity between the swab and saliva samples were 40% and 96.85%, respectively. The positive predictive value of the saliva sample was 73.3%. Conclusions: The sensitivity of whole saliva when compared to the swab samples is low. Large sample studies are needed to validate the role of saliva as a diagnostic tool in COVID-19.
Keywords: Biological marker, COVID-19, oral bio-fluid, SARS-CoV-2, whole saliva
|How to cite this article:|
Girish P, Jayasankar P, Abhishek P, Sumeeta S, Gunvant P, Shalin P. Comparative analysis of the naso/oropharyngeal swab and oral bio-fluid (whole saliva) samples for the detection of SARS-CoV-2 using RT-qPCR. Indian J Dent Res 2021;32:206-10
|How to cite this URL:|
Girish P, Jayasankar P, Abhishek P, Sumeeta S, Gunvant P, Shalin P. Comparative analysis of the naso/oropharyngeal swab and oral bio-fluid (whole saliva) samples for the detection of SARS-CoV-2 using RT-qPCR. Indian J Dent Res [serial online] 2021 [cited 2021 Dec 8];32:206-10. Available from: https://www.ijdr.in/text.asp?2021/32/2/206/330853
| Introduction|| |
The pandemic outbreak of COrona VIrus Disease 2019 (COVID-19) is a serious public health concern worldwide. The aetiological agent responsible for this disease is a novel single-stranded RNA virus that belongs to the Coronaviridae family. The new virus shares 80% sequence similarity with the Sub Acute Respiratory Syndrome (SARS) virus, which was the agent for the 2003 SARS epidemic. Thus, the new virus was scientifically designated as SARS-CoV-2 by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses. Since its outbreak in December 2019, there has been a steep worldwide increase in the spread of COVID-19 infection. The World Health Organization (WHO) declared COVID-19 as a global pandemic in March 2020. As on February 13, 2021, there were 10,78,38,255 confirmed cases of COVID-19 including 23,73,398 deaths globally. Several measures like social distancing, hand hygiene, quarantine, and lockdown are also being taken by the governments to curtail the spread of this disease. The screening process involves the collection of nasopharyngeal and oropharyngeal swabs from the suspected cases. This is based on the guidelines published by WHO for the detection of SARS-CoV-2 using respiratory materials (nasopharyngeal and oropharyngeal swab in ambulatory patients and sputum (if produced) and endotracheal aspirate or bronchoalveolar lavage in patients with more severe respiratory disease) and serum for serological testing. The swabs are subjected to nucleic acid amplification testing (NAAT) through real-time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR), which is considered as a Gold standard testing protocol by the WHO., However, during the collection of nasopharyngeal and oropharyngeal swabs, higher risk for disease transmission exists as the procedures might stimulate sneezing and cough reflex, respectively. Each cough can produce around 3000 saliva droplets nuclei and each sneeze can produce roughly around 40,000 droplets of saliva covering several metres in the air. This might eventually put healthcare workers collecting those samples more susceptible to the infection., The other biological samples investigated in COVID-19 patients were sputum, blood, faeces, anal swabs, and saliva. It was found that the positive rate of coronavirus nucleic acid in sputum is higher than that of the other biological specimens. The whole mouth saliva samples were also used in the laboratory diagnosis of both the DNA and RNA virus infections.,, During the previous SARS epidemic, it was reported that SARS-CoV RNA can be detected in saliva and throat wash of the infected patients. The saliva sample has shown a high concordance rate of more than 90% with the nasopharyngeal swab sample in the detection of the coronavirus., The present study was designed to investigate the efficacy of whole saliva as a diagnostic marker in comparison with the nasopharyngeal smear in COVID-19 cases.
| Methods|| |
The whole saliva samples were collected from the 309 suspected patients in the OPD of COVID-19 hospital in Gujarat. The permission from the Health and Family Welfare department of Govt. of Gujarat was obtained for this study. The Institutional Ethical Committee's approval was also obtained from BJ Medical College and Civil Hospital, Ahmedabad prior to the start of the study.(Ref. No. EC/approval/80/2020/17/8/2020) The purpose of the study was explained to the patients in the language they understand and written consents were obtained from the patients who were willing to participate in the study. The saliva samples were collected into the sterile vials containing viral transport medium using the spit method and the samples were labelled and kept in polythene covers. The standard aseptic precautions were followed during the sample-collection procedures. For the saliva sample, the patients were asked to pool saliva in their mouth for 1 to 2 minutes prior to collection and then asked to gently spit the content into a sterile, plastic container containing the viral transport medium. The nasal and the oropharyngeal swabs from the same patients were collected by trained medical residents. Both the nasal and the oropharyngeal swabs were placed in the same container to increase the viral load, as per the ICMR guidelines. The saliva and the swab samples were paired and transferred to the laboratory for analyses.
Nucleic acid extraction
Nucleic acids were recovered from clinical specimens- nasopharyngeal and oropharyngeal swab in viral transport medium and saliva in viral transport medium by using the ICMR-approved RNA extraction kits- Qiagen, Zybio, Gsure, and so on. Following manufacturer's instructions, specimens received were added and mixed with those provided in the extraction kit. The extracted RNA in the Eppendorf tubes were subjected to further processing for the nucleic acid amplification.
Real Time RT-PCR assay
The real-time RT-PCR assay was performed using the Real-Time RT-PCR Master Mix multiple primer and probe sets designed commercially for SARS CoV-2 (Meril Diagnostics, Lab genomics Labgun, ICMR NIV protocols). Each run included a SARS-CoV-2 genomic positive control and a negative control. As a control for PCR inhibitors and to monitor nucleic acid extraction efficiency, each sample was tested by real-time RT-PCR for the presence of the human ribonuclease (RNase) P gene. Fluorescence measurements were taken and the threshold cycle (CT) value for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit set above the baseline. Clinical samples were considered positive if two or more of the SARS genomic targets showed positive results (CT ≤38-40 cycles) as per kit literature, and all positive and negative control reactions gave expected values. The thermocyclers used are Applied Biosystems-Step One, 7300, 7500, Rotor- Gene Q MD × 5 Plex, Light Cycler 480. The result of the RT-PCR from both the above two specimen sources were compared and analysed statistically.
The paired t-test and the Wilcoxon signed-rank test were used to compare the measured variables (CT values) between the saliva and the swab samples. The cross tabulation of the results from both the samples was done in order to calculate the positive predictive value (PPV), negative predictive value (NPV), the sensitivity, and the specificity of the tests.
| Results|| |
A total of 309 paired saliva and swab samples were analysed. Nearly 18% of the patients were tested positive in swab samples and 9.7% were tested positive in saliva samples. Twenty-two samples (7%) tested positive from both swab and saliva, and 79.8% tested negative from both of them. From the overall samples, 10.7% tested positive from swab samples but negative from saliva samples. However, 2.6% tested positive from saliva samples but negative from swab samples. [Table 1] shows the cross tabulation of the results from both the samples. In the overall samples, the results of the RT-PCR analysis showed a significant difference in both the swab and the saliva samples (P < 0.001). The Wilcoxon signed-rank test between the saliva and the swab results revealed a similar result (ties) in 268 samples (86.73%). The positive percentage agreement (sensitivity) and the negative percentage agreement (specificity) between the swab and saliva samples were 40% and 96.85%, respectively. The overall agreement in the test results between the two samples was 86.7%. The positive predictive value of the saliva sample was 73.3%. The mean CT value of saliva sample was significantly higher than the CT value of swab sample [Figure 1] and [Figure 2]; [Table 2]. [Figure 2] shows the case-wise CT values of the n gene and orf gene in both the samples.
|Figure 1: Chart showing the mean cycle threshold (CT) value in saliva and swab samples|
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|Figure 2: Chart showing the case-wise CT values of the n gene and orf gene in saliva and swab samples|
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|Table 1: Table showing the results of cross tabulation of the results from saliva and swab samples|
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|Table 2: Table showing the descriptive statistics of the CT values in swab and saliva samples|
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| Discussion|| |
The SARS-CoV-2 virus was first reported in saliva by To et al. in laboratory confirmed COVID-19 patients in Hong Kong. Following this, the efficacy of saliva as a diagnostic tool in comparison with the gold standard diagnostic tools like nasopharyngeal and oropharyngeal swabs was tested by several investigators.,,,,, It was found that the diagnostic efficiency of saliva in COVID-19 cases ranges from 30.7% to 100%. In the present study, it was observed that the saliva sample had a positive predictive value of 73.3% and an overall agreement of 86.7% with the swab samples taken from the suspected COVID-19 patients. The PPA for saliva test was 66.7% and the overall agreement between saliva and swab sample results was around 74% according to a study by Ku et al. In the present study, the overall agreement was slightly higher than their study. In the present study, the sensitivity of saliva samples is only 40% whereas the specificity is around 97%. Studies have shown the sensitivity and the specificity of RT-qPCR analysed saliva samples as compared to the NPS as ranging from 66% to 91.7% and from 97% to 100%, respectively., Such validation studies with larger sample size are required to support the use of saliva samples as a diagnostic tool in COVID-19, which may substantially reduce the risk of contamination and cross infection associated with the nasal and oropharyngeal swab specimen collections. Moreover, the collection of saliva samples for virus detection is convenient and has a better patient compliance when compared to the nasal and oropharyngeal swab collections. The nasal and the oropharyngeal swabs taken independently from the patients were kept together in the same vial and was transported to the lab. The saliva sample was collected in a separate vial containing the same viral transport medium as in the other vial. In the present study, the saliva specimens were collected from the patients using the spit technique. Though there are other methods of saliva collection for viral studies, the spit saliva method has shown an efficiency rate of 85% in detecting the SARS-CoV-2 virus. The RT-PCR analysis of saliva samples has reported a positive rate of 78.1%. A longitudinal analysis of serial saliva specimens showed a decline in the viral load after hospitalization and during patients' recovery. A study also showed that the overall detection rate from saliva samples was comparable to that from NPS specimens when multiplex RT-PCR was used for RV detection. In the present study, the ICMR recommended RT-PCR method to be applied. The CT value was significantly higher in the saliva sample than in the swab sample. This means that the viral load is comparatively lesser in the saliva samples. This finding was similar to a study by Kojima et al. A study by Yoon et al. also reported higher viral load in the nasopharyngeal swab in comparison with saliva sample. However, in a study by Rao et al., cycle threshold (CT) values for E and RdRp genes were significantly lower in saliva specimens compared to NP swab specimens, and concluded that saliva is a better alternative to NPs for detection of SARS-CoV-2. Similarly, Wyllie et al. reported that saliva is more sensitive for SARS-CoV-2 than NPS. However, a study by Azzi et al. showed no significant difference in the viral load between the saliva and NPS samples. A low sensitivity or positive percent agreement (40%) of saliva sample in the present study may limit the use of saliva as a diagnostic tool to detect SARS-CoV-2. This was similar to the results of studies by Williams et al. and Becker et al., Studies have also shown wide range of PPA in saliva samples., A systematic review and meta-analysis of the data from several studies showed that the nasopharyngeal swab is the gold standard technique for SARS-CoV-2 detection. However, there are other specimens that could prove to be promising. Based on the analysis of the study, it was found that saliva had a % positivity ranging from 81% to 93%. In the present study, the positive predictive value was around 73%. The amount of saliva sample collected, the time of saliva collections, the technique used for saliva collection, and the overall sample size of the study are some of the reasons believed to be responsible for such variations. The collected saliva samples are a potential source of transmission of SARS-CoV-2 and hence they must be handled with care to avoid spilling and spreading of live viruses during the collection, the storage, and the analytical phases. Perhaps, certain factors need to be considered before, during, and after collection of the whole saliva specimens. Also, the protocol for the saliva collection like the deep throat coughing out and drooling of the whole saliva plays a role in the diagnostic sensitivity of the saliva sample.
| Conclusion|| |
The present study revealed a low sensitivity rate (40%) of saliva when compared to the nasal and oropharyngeal swabs in detecting the novel Corona virus. Though there are other studies that recommend saliva as a potential diagnostic marker for COVID-19, studies with larger cohorts are necessary to confirm the effectiveness or the ineffectiveness of saliva as a diagnostic tool in COVID-19.
The authors acknowledge the Health and Family welfare Department Govt. of Gujarat for granting the permission and support to conduct this study. The authors wish to acknowledge the support of Dr. Karthikey Parmar, Nodal officer, 1200 bedded hospital during the sample collection from COVID-19 OPD. The authors also acknowledge the support and efforts of the PG students Dr. Shreejit S.U, Dr. Vardesh Sail, Dr. Harita Paghadal, Dr. Namrata Jaysheel, and the Interns Mr. Dhruv Sinhal, Miss Sandhya Patel, Mr. Chirag Patel and Miss Divyani Patel of Govt. Dental College and Hospital, Ahmedabad and the technical teams involved in the COVID-19 testing laboratory at BJ Medical College, Ahmedabad.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Dept. of Health and Family Welfare, Govt. of Gujarat.
Conflicts of interest
There are no conflicts of interest.
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Dr. Pillai Jayasankar
Department of Oral Pathology, Govt. Dental College and Hospital, Ahmedabad - 380 016, Gujarat
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]
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