Indian Journal of Pathology and Microbiology
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Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 907-910
Diagnostic accuracy of saliva as a specimen for detection of SARS-CoV-2 by RT-PCR


Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India

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Date of Submission03-Apr-2021
Date of Acceptance17-Aug-2021
Date of Web Publication06-Jun-2022
 

   Abstract 


Context: COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an emerging pandemic that is rapidly spreading with more than 114 million confirmed cases and 2.5 million deaths by far. Nasopharyngeal swab (NPS) in VTM has been used as the gold standard respiratory specimen for SARS-CoV-2 reverse-transcriptase real-time PCR (rRT-PCR) tests. But now the virus can also be detected in other clinical specimens like bronchoalveolar lavage, sputum, saliva, throat swab, blood, and stool specimens. Aims: The aim of this study was to determine the diagnostic potential of saliva as a sample in comparison to NPS for detection of SARS-CoV-2 by rRT-PCR. Settings and Design: A cross-sectional study was conducted among 256 paired samples (NPS and Saliva) received in the Department of Microbiology, SMS Medical College, Jaipur over a period of 2 months Methods and Material: NPS from individuals were collected in a sterile tube containing Viral Transport Medium™. Before swab collection, whole saliva was collected by spitting from the suspected patient into a sterile container. Both were stored at room temperature and transferred to the diagnostic laboratory within four hours of collection where extraction was done using Perkin Elmer chemagic extractor and rRT- PCR was performed using NIV, Pune mastermix. Results: Sensitivity, specificity, PPV, and NPV of RT-PCR for the diagnosis of COVID-19 in saliva were 84.26%, 100%, 100%, and 54.05%, respectively. The accuracy of detection of COVID-19 by saliva samples compared to the routinely used NPS samples (considered as the standard reference) for RT PCR was 86.72%. Conclusions: Our results show that saliva as a reliable sample type for SARS-CoV-2 detection.

Keywords: Nasopharyngeal swab, rRT-PCR, saliva, SARS-CoV-2

How to cite this article:
Gupta M, Sinha P, Gupta S, Jain DK, Hooja S, Vyas N. Diagnostic accuracy of saliva as a specimen for detection of SARS-CoV-2 by RT-PCR. Indian J Pathol Microbiol 2022;65:907-10

How to cite this URL:
Gupta M, Sinha P, Gupta S, Jain DK, Hooja S, Vyas N. Diagnostic accuracy of saliva as a specimen for detection of SARS-CoV-2 by RT-PCR. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Nov 30];65:907-10. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/907/346693





   Introduction Top


Coronavirus disease-2019 (COVID-19)––a global pandemic––caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is rapidly spreading with more than 114 million confirmed cases of COVID-19, including 2.5 million deaths by far.[1] It was first detected and described in December 2019 in the Wuhan City of China and has led to rapid spread causing global chaos.[2]

Detection of SARS-CoV-2 in patient's samples is the first crucial step guiding towards treatment and infection control in the community. World Health Organization (WHO) recommends screening of infection in suspected cases with a nucleic acid amplification test, such as real-time reverse transcriptase-polymerase chain reaction (rRT-PCR), in respiratory specimens.[3]

Nasopharyngeal swab (NPS) in the VTM has been used as the referred sample type (respiratory specimen) for SARS-CoV-2 reverse-transcriptase real-time PCR (RT-PCR) tests.[4] The virus can also be detected in other clinical specimens such as bronchoalveolar-lavage,[5] sputum,[6] throat swab,[7] stool, and blood.[8]

For the molecular diagnosis of SARS-CoV-2, use of oropharyngeal secretions has recently been suggested.[9],[10] The detection of virus in the oral cavity may be due to a high expression of that angiotensin-converting enzyme II (ACE2) receptors on the epithelial cells of the oral mucosa which might play an active role in the pathogenesis of COVID-19. It was seen that ACE-2 which is a SARS-CoV-2 host-cell receptor is expressed in the salivary glands so SARS-CoV-2 can be detected in saliva.[11]

In clinical practice, the use of saliva samples to detect SARS-CoV-2 has many advantages. Firstly, collecting saliva rather than nasopharyngeal and oropharyngeal swabs prevents undue discomfort to patient. Second, saliva sample is of advantage in scenarios where collection of nasopharyngeal specimens is contraindicated, such as patients with severe bleeding tendency and with ulceration of nasal mucosal layer. Third, saliva can be self-collected following simple instructions, whereas the collection of NPS needs presence of a healthcare personnel for collection.[12]

The aim of this study was to determine the diagnostic potential of saliva as a sample in comparison to NPS for detection of SARS-CoV-2 by rRT-PCR.


   Subjects and Methods Top


Study population

A cross-sectional study was conducted among 256 paired samples (NPS and saliva) received in Department of Microbiology, SMS Medical College, Jaipur over a period of 2 months between 1st January 2021 and 1st March 2021. Fresh NPS and saliva sample from known positive samples were collected which were reported positive a day before by the routine rRT-PCR.

As a standard protocol, nasopharyngeal samples from individuals were collected using flocked swabs in a sterile tube containing Viral Transport Medium™ (Vitromed healthcare, Biotech Park, Jaipur, Rajasthan). Saliva collection-Whole saliva (≈1.5 mL) was collected by spitting from the suspected patients into a sterile container.

NPS collection-To collect NPS, the swab was passed through the nostril until it reached the posterior nasopharynx and removed while rotating and was placed immediately into a sterile tube with viral transport medium. Paired swab and saliva samples were stored at room temperature and transferred to the diagnostic laboratory within four hours of collection.

In Biosafety level 2, viscous saliva was added to 300 μL of viral transport media (VTM), mixed vigorously, and then 200 μL of sample was used for RNA isolation. Patients' informed consent was taken at the time of sample collection. All those samples for which quantity was not sufficient and those which showed invalid results on PCR by standard NPS as sample were excluded from the study.

Specimen processing

Extraction

After thorough vortexing and brief centrifugation of the VTM, 300 μl of the sample was transferred to a 96 deep well processing plate to which 4 μl Poly (A) RNA,10 μl of proteinase K, 300 μl lysis buffer along with 150μl magnetic beads and 900 μl of RNA binding buffer were already been added.

The beads/RNA mixture was washed with washing buffer and elutes were obtained in elution buffer in the automated system (Perkin Elmer Chemagic 360).

Real time PCR (NIV, Pune master mix)

The primers used in NIV Pune RT PCR kit are designed to target E, Orf, RdRp, and RNase P genes. For PCR, 7 μL RNA and 13 μL PCR master mix solution containing Taq Man Fast Viral 1-Step Master Mix (4X) 5.0 μL, 3.3 μL of Primer probe mix, RT-qPCR Grade Water 4.7 μL was used. Cyclic conditions used as per the manufacturer's instructions were 50°C for 5 mins, 95°C for 20 secs, then 40 repeat cycles of 95°C for 5 secs and 60°C for 30 secs using Quant Studio platform. Sample was considered positive if Ct values for E, Orf and Rdrp was ≤35.

Statistical analysis

Data was entered and analyzed using SPSS statistical software version 24. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and a 95% confidence interval (CI) were calculated. Kappa coefficient was used to estimate agreement between NPS and saliva RT-PCR test results.


   Results Top


256 sample pairs of NPS and saliva were collected from known positives [Table 1]. Of these subjects, 84.37% (216/256) were positive for SARS-CoV-2 and 15.62% (40/256) were negative with NPS by RT-PCR. For saliva, 71.09% (182/256) were positive for SARS-CoV-2 and 28.90% (74/256) were negative [Table 1]. 13.28% (34/256) saliva samples showed invalid results but a repeat extraction was performed and they showed a negative result by RT-PCR.
Table 1: Detection of SARS-CoV-2 by RT-PCR in Nasopharyngeal swabs and saliva samples

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A comprehensive assessment of the diagnostic accuracy of COVID-19 using nucleic acid detection in saliva specimens were performed [Table 2].
Table 2: Comprehensive assessment of the diagnostic accuracy of COVID-19 using saliva as a sample for rRT-PCR

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Sensitivity and specificity of RT-PCR for the diagnosis of COVID-19 in saliva were 84.26% (95% CI 78.70% to 88.85%) and 100.00% (95% CI -91.19% to 100.00%), respectively. The PPV and NPV were 100% (95% CI 91.19% to 100.00%) and 54.05% (95% CI -46.36% to 61.56%), respectively. The accuracy of detection of COVID-19 by saliva samples compared to the routinely used NPS samples (considered as the standard reference) for RT PCR was 86.72% (95% CI-81.94% to 90.62%) [Table 2].

The overall agreement between the two sampling methods was 86.72% and the kappa (κ) coefficient was 0.626 with 95% confidence interval: of 0.517-0.734 indicating moderate agreement between the two sample types.

The median Ct values were not significantly different between NPS and saliva (P > 0.05). Median Ct value for E gene in NPS and Saliva samples was 22 and 24, respectively, for Orf gene in NPS and Saliva samples was 26 and 28, respectively, whereas for Rdrp gene for in NPS and Saliva samples was 26 and 28, respectively.


   Discussion Top


Rapid and accurate screening of suspected SARS-CoV-2 cases is of great significance for epidemic prevention and control. NPS swabs are the referred sample type to diagnose COVID-19. However, NPS is relatively invasive, induce coughing and cause bleeding and mucosal erosion and damage which may increase risks of healthcare workers infection due to more shedding of virus. The use of saliva samples for diagnosis of COVID-19 has advantages of being noninvasive, inexpensive, and has ease in collection in clinical practice.

In this study, we found a comparable detection rate of SARS- CoV-2 between saliva and NPSs as recommended by interim guidelines for clinical specimens of COVID-19 testing. It also showed the value of testing a saliva sample as a non-invasive method of detection of SARS-CoV-2.

We found that the detection rate of SARS-CoV-2 by saliva RT-PCR was 86.72%. This result was in accordance with the previous studies by Eloise Williams et al.[13] which showed an 84.6% detection rate of SARS-CoV-2 in saliva samples. Another study by Shufa Zheng et al. showed that the detection rate of SARS-CoV-2 as 88.09% from saliva samples.[14]

Results from our study showed that the saliva RT-PCR test showed high sensitivity (83.26%) and specificity (100%) and substantial agreement to the current standard of NPS. This was in agreement with a cross-sectional study by E. Pasomsub et al.[15] who reported sensitivity and specificity of the saliva sample RT-PCR are 84.2% and 98.9%, respectively.

There was a significant difference in the Ct values in NPS and saliva samples in our study (P > 0.05) but in a study by Fakheran et al.[16] there was no statistically significant difference between nasopharyngeal and saliva samples in terms of Ct value.

As the collection of NPS is invasive and requires close contact between healthcare workers (HCWs) and patients, HCWs are required to wear full PPE for prevention of viral transmission, during sample collection. The patients also experience discomfort during sample collection of the NPS specimens, especially when the patient is a child or when serial sampling is required.[9] Moreover, saliva samples require no special media and special transportation requirements and less specimen degradation from delay in processing is seen. Saliva collection minimizes the aerosol generation too which is seen more in NPS collection.

Saliva can be easily obtained, as collection is non-invasive and can be done by self or under guidance so it's a boon for pediatric age group population and critically ill patients. As the saliva specimen collection is easy, shortage of PPE and the transmissibility of the virus, saliva could enable self-collection for accurate large-scale SARS-CoV-2 surveillance testing.

This can create a possibility of using this simpler technique in detecting SARS-CoV-2 in non-hospital setups which will reduce the waiting period on reporting due to transportation to tertiary testing centers and can allow immediate intervention based on positive results.

Limitations

The sample size of this study is small, so the influence of severity of disease and disease progression on detection rate cannot be evaluated. Our study did not include unconscious critically ill patients who would be unable to produce spit. Finally, as this study only included known positives where the viral load may be higher, further studies need to be conducted with milder symptoms in the outpatient setups. Moreover, the patients who were comatose and on ventilator support were unable to produce spit so couldn't be included in the study.


   Conclusion Top


The detection rate of SARS-CoV-2 in saliva was comparable to other respiratory samples. Our results show that saliva is a reliable sample type for SARS-CoV-2 detection, and worthy of diagnostic promotion. Saliva collection does not require specialized consumables, causes less patient discomfort and the convenience in sampling saliva may also significantly reduce the risk of infection of medical staff. It may be considered a suitable alternative first-line screening test in low-resource settings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
WHO, COVID-19 Weekly Epidemiological Update, Feb 2021.  Back to cited text no. 1
    
2.
Klein S, Müller TG, Khalid D, Sonntag-Buck V, Heuser AM, Glass B, et al. SARS-CoV-2 RNA extraction using magnetic beads for rapid large-scale testing by RT-qPCR and RT-LAMP. Viruses 2020;12:863.  Back to cited text no. 2
    
3.
World Health Organization. Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases. 2020. Available from: https://www.who.int/publications-detail/laboratory-testing-for-2019-novel-coronavirusin-suspected-human-cases-20200117.  Back to cited text no. 3
    
4.
Zou L, Ruan F, Huang M, Liang L, Huang H, Hong Z, et al. SARS-CoV-2 viral load in upper respiratory specimens of infected patients. N Engl J Med 2020;382:1177-9.  Back to cited text no. 4
    
5.
Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al., A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 2020;382:727-33.  Back to cited text no. 5
    
6.
Lin X, Gong Z, Xiao Z, Xiong J, Fan B, Liu J. Novel coronavirus pneumonia outbreak in 2019: Computed tomographic findings in two cases. Korean J Radiol 2020;21:365-8.  Back to cited text no. 6
    
7.
Bastola A, Sah R, Rodriguez-Morales AJ, Lal BK, Jha R, Ojha HC, et al. The first 2019 novel coronavirus case in Nepal. Lancet Infect Dis 2020;20:279-80.  Back to cited text no. 7
    
8.
Zhang W, Du RH, Li B, Zheng XS, Yang XL, Hu B, et al. Molecular and serological investigation of 2019-nCoV infected patients: Implication of multiple shedding routes. Emerg Microbes Infect 2020;9:386-9.  Back to cited text no. 8
    
9.
To KK, Tsang OT, Yip CC, Chan KH, Wu TC, Chan JM, et al. Consistent detection of 2019 novel coronavirus in Saliva. Clin Infect Dis 2020;71:841-3.  Back to cited text no. 9
    
10.
Tian HY. [2019-nCoV: New challenges from coronavirus]. Zhonghua Yu Fang Yi Xue Za Zhi 2020;54:E001. Chinese. doi: 10.3760/cma.j.issn. 0253-9624.2020.0001.  Back to cited text no. 10
    
11.
Xu H, Zhong L, Deng J, Peng J, Dan H, Zeng X, et al. High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci 2020;12:8.  Back to cited text no. 11
    
12.
To KK, Lu L, Yip CC, Poon RW, Fung AM, Cheng A, et al. Additional molecular testing of saliva specimens improves the detection of respiratory viruses. Emerg Microbes Infect 2017;6:e49.  Back to cited text no. 12
    
13.
Williams E, Bond K, Zhang B, Putland M, Williamson DA, McAdam AJ. Saliva as a non-invasive specimen for detection of SARS-CoV-2. JCM 2020;58:e00776-20.  Back to cited text no. 13
    
14.
Fan J, Yu F, Wang X, Zou Q, Lou B, Xie G, et al. Saliva as a diagnostic specimen for SARS-CoV-2 by a PCR-based assay: A diagnostic validity study. Clinica Chimica Acta 2020;511:177-80.  Back to cited text no. 14
    
15.
Pasomsub E, Watcharananan SP, Boonyawat K, Janchompoo P, Wongtabtim G, Suksuwan W, et al. Saliva sample as a non-invasive specimen for the diagnosis of coronavirus disease 2019: A cross-sectional study. Clin Microbiol Infect 2021;27:285.e1-4.  Back to cited text no. 15
    
16.
Fakheran O, Dehghannejad M, Khademi A. Saliva as a diagnostic specimen for detection of SARS-CoV-2 in suspected patients: A scoping review. Infect Dis Poverty 2020;9;100.  Back to cited text no. 16
    

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Correspondence Address:
Nitya Vyas
Department of Microbiology, SMS Medical College, Jaipur 302 004, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_292_21

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