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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 23  |  Issue : 2  |  Page : 155-158

Self-collected saliva: Diagnostic potential for severe acute respiratory syndrome coronavirus-2 testing in a resource-limited setting


1 Department of Microbiology, Armed Forces Medical College, Pune, Maharashtra, India
2 Classified Specialist Microbiology, Army Institute of Cardio Thoracis Sciences, Pune, Maharashtra, India
3 Microbiology and Former Dean, Armed Forces Medical College, Pune, Maharashtra, India

Date of Submission29-Dec-2020
Date of Decision24-Jan-2021
Date of Acceptance08-Feb-2021
Date of Web Publication21-Sep-2021

Correspondence Address:
Lt Col (Dr) Santosh Karade
Department of Microbiology, AFMC Pune - 411040, Pune, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jmms.jmms_198_20

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  Abstract 


Background: The gold standard for the diagnosis of coronavirus (CoV) disease 2019 in the hospital and community setting is by the real-time reverse transcription-based polymerase chain reaction (RT-PCR) using the respiratory samples. The collection of nasopharyngeal or oropharyngeal swab (NP/OP) needs experienced medical personal and appropriate biosafety conditions. There is need for a simpler strategy for the sample collection without compromising the sensitivity. Saliva sample collection is noninvasive. Therefore, the primary objective of the study was to evaluate the efficacy of severe acute respiratory syndrome CoV-2 (SARS-CoV-2) RT-PCR using self-collected saliva specimen. Materials and Methods: This pilot study was carried out at an Indian Council of Medical Research (ICMR) approved molecular laboratory. Self-collected saliva and OP/NP swab in the viral-transported medium (VTM) were collected in parallel from the study participants and processed for SARS-CoV-2 RT-PCR. Cycle threshold (Ct) value of envelope gene (“E”), HKU Orf 1b gene (“HKU”), and Actin (internal control) obtained for saliva and NP/OP swab were tabulated. Results: Saliva and OP/NP swab in parallel were collected from 85 individuals. The mean age of the study population was 35.2 years, and 81% were male. The overall sensitivity and specificity of RT PCR on the saliva sample were 95.6% and 92.5%. The concordance between 'E” and “HKU” Ct value obtained by NP/OP and saliva RT-PCR was 97.64% and 95.29%. The positive and negative predictive value of the novel method was 93.5% and 94.9%, respectively. Conclusion: Self-collected saliva is a reliable biological sample and promising tool for SARS-CoV-2 testing in the resource-limited setting.

Keywords: Coronavirus disease 2019, nasopharyngeal swab, RT-PCR, viral-transported medium


How to cite this article:
Anand KB, Karade S, Jindamwar P, Sen S, Babu B, Bobdey S, Gupta R M. Self-collected saliva: Diagnostic potential for severe acute respiratory syndrome coronavirus-2 testing in a resource-limited setting. J Mar Med Soc 2021;23:155-8

How to cite this URL:
Anand KB, Karade S, Jindamwar P, Sen S, Babu B, Bobdey S, Gupta R M. Self-collected saliva: Diagnostic potential for severe acute respiratory syndrome coronavirus-2 testing in a resource-limited setting. J Mar Med Soc [serial online] 2021 [cited 2021 Dec 9];23:155-8. Available from: https://www.marinemedicalsociety.in/text.asp?2021/23/2/155/326274




  Introduction Top


In late December 2019, the outbreak of unknown viral pneumonia cases in the city of Wuhan and Chinese province of Hubei shook the world.[1] A week later, whole genome sequence analysis of the causative viral agent indicated the emergence of a novel coronavirus, currently known as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).[2] Considering the alarming level of spread globally and severity of cases, the Director-General, World Health Organization declared coronavirus disease 2019 (COVID-19), a pandemic.[3]

The disease has affected over 80 million individuals across 205 countries till the end of December 2020.[4] The laboratory plays a crucial role in diagnosing infection, thereby allowing early isolation and initiating contact tracing protocol. Conventionally, the diagnosis of SARS-CoV-2 infection in a suspected individual is made by the real-time reverse transcription-based polymerase chain reaction (RT-PCR) on nasopharyngeal swab (NP) or oropharyngeal swab (OP) collected in viral-transported medium (VTM).

The collection of suitable respiratory sample is essential for the laboratory diagnosis of SARS-CoV-2 infection. In a pandemic setting collection of NP and OP swabs have several limitations. First, the collection of NP/OP swab requires a trained health-care worker (HCW), who understands the anatomy of NP tract. Furthermore, the collection requires the use of personal protective equipment (PPE) and appropriate biosafety conditions.[5] The NP/OP swab collection technique is uncomfortable. The nylon flocked swab in the oropharynx or nasopharynx and can stimulate coughing, sneezing, or vomiting, which can generate aerosols. The proximity to the patient during the sample collection places the HCW at risk of acquiring iatrogenic SARS-CoV-2 infection. The collection of NP/OP swab is at time troublesome in nonco-operative individuals, especially children. The procedure is invasive that may cause mucosal bleed in a patient with bleeding disorder. Thus, there is a need for an alternate strategy for sample collection during the mass screening program without compromising the sensitivity.

Salivary fluid contains the high concentration of SARS-CoV-2 and is responsible for droplet transmission. Angiotensin-converting enzyme-2 (ACE-2) receptors, the functional receptors for SARS CoV-2 are present in the lungs, salivary glands, and tongue. In a Chinese study on rhesus monkey, Liu et al. demonstrated the presence of ACE-2 receptors in the epithelial lining of salivary gland duct.[6] The present study indicated the salivary gland as an early target for SARS-CoV-2 infection following intranasal inoculation of the virus. Therefore, saliva can be promising specimen for SARS-CoV-2 diagnostic testing. There are other advantages of salivary fluid specimen. First, saliva sample can be collected in a universal sterile container without any intervention of HCW. Saliva collection is noninvasive and can be performed by the individual himself at home. Such procedure not only reduces the patient waiting period outside the sample collection center but also decreases the exposure of HCW to the infective aerosols. After education of clientele by instructional leaflets or videos, self-collected saliva protocol can be implemented for mass screening program in a pandemic setting. Recently, Chenyao Lin et al, compared the SARS-CoV-2 detection by RT-PCR using saliva samples and throat swab.[7] The study reported significantly higher detection with salivary specimen.[7] There is a lack of such studies from India in the current pandemic setting. Thus, our primary objective of this study was to evaluate the efficacy of SARS-CoV-2 RT-PCR using self-collected saliva specimen in the resource-limited setting.


  Materials and Methods Top


This pilot study was carried out at an ICMR approved molecular biology laboratory. The study population consisted of adults reporting to flu outpatient department and hospitalized patients suspected of COVID-19. The present study was carried out in the month of September to October 2020 after the approval of Institutional Ethics Committee. Specimens for RT-PCR were collected after explaining the saliva collection method and obtaining written informed consent from study participants.

Saliva was collected by the passive drooling method. The individual was asked to clear his/her throat and sit on a chair or edge of a bed. Approximately 2–3 ml of the drooling saliva was collected over 1 min by bending forward and spitting in a universal container. The participants were asked to place the universal container in a zip lock after tightly securing the screw-cap.

Parallel NP/OP swab in VTM was also collected by a HCW under appropriate biosafety precautions as per the national guidelines. Samples were transported to molecular laboratory within an hour of collection. Both type of specimens were subjected to RNA extraction and SARS-CoV-2 RT PCR using ICMR approved kit.[8] Cycle threshold (Ct) value of Envelope_sarbeco gene (“E”), HKU Orf 1b gene (“HKU”), and Actin (Internal control) obtained for saliva and NP/OP swab specimen were measured and tabulated in Microsoft Excel spreadsheet. In this study, specimen with Ct value of 35 or lesser for both E and HKU targets were considered positive. Sample with only one of the targets (out of E and HKU genes) showing Ct value of 35 or lesser was regarded inconclusive for the analysis.


  Results Top


Parallel saliva and NP/OP swab were collected from a total of 85 individuals. The mean (interquartile range) age of the study population was 35.2 (16) years and 81% of them were male [Figure 1]. RT-PCR study on 85 NP/OP specimens showed 45 positive, 39 negative, and 01 inconclusive result for SARS-CoV-2 infection, whereas saliva RT-PCR indicated 46 positive, 37 negative, and 2 inconclusive result. The sensitivity and specificity of RT PCR on the saliva sample were 95.6% and 92.5%. Overall concordance in the result obtained from OP/NP swab RT PCR and saliva RT PCR was 94.11% (80/85). Importantly, 1 sample inconclusive by OP/NP swab RT PCR was detected positive by saliva PCR. The positive and negative predictive value of the saliva RT-PCR was 93.5% and 94.9%, respectively. The overall concordance between Ct value for “E” and “HKU Orf1b” gene obtained by both methods was 97.64% and 95.29%, respectively.
Figure 1: Age distribution of the study participants (69 male and 16 female)

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  Discussion Top


Appropriate specimen collection is important for the diagnosis of respiratory viral infections. At present, RT-PCR remains the gold standard for the diagnosis of SARS-CoV-2 infection. The specimen for PCR-based detection of SARS-CoV-2 gene targets includes the upper respiratory tract samples such as NP swab, OP swab, or bronchoalveolar lavage.[9] Mass screening of individuals for SARS-CoV-2 infection is essential to institute isolation measures and break the chain of infection. Hence, there is a need to strengthen the laboratory capacity for SARS-CoV-2 diagnostic testing as well as decrease the turn-around time.

The collection process of OP and NP swab needs the requirement of PPE and a biosafety trained HCW. To overcome the drawbacks of OP/NP swab collection, in this study, we compared the detection rates of SARS-CoV-2 RNA using conventional OP/NP swabs and saliva specimens.[10] The overall sensitivity and specificity of RT-PCR were saliva sample 95.6% and 92.5%. The concordance between “E” and “HKU” Ct value obtained by OP/NP and saliva RT-PCR was 97.64% and 95.29%. The results are indicative of saliva as the good biological specimen for SARS-CoV-2 RT PCR. Prior studies from other countries have also elucidated the utility of saliva PCR in terms of sensitivity and ease of use.[11],[12] Recently, Nagura-Ikeda et al. improvised the method by diluting saliva specimen with phosphate-buffered saline to reduce viscosity and aid further processing.[13] For sake of simplicity, we directly included saliva for RNA extraction and showed similar results.

To break the chain of SARS-CoV-2 infection, mass screening, especially testing of asymptomatic infected individuals is important. As soon as these asymptomatic carriers are diagnosed, the surveillance machinery is triggered, and nonpharmacological interventions are initiated to break the chain of infection. Self-collected saliva specimen offers distinct advantage in cases of mass screening program. As saliva RT-PCR is noninvasive, this technique can be used for routine screening of HCWs also. Recently, Zhang et al. explored efficacy of every 5th day saliva RT-PCR-based screening of HCW for the identification of silent infection and prevention of outbreaks in the hospital setting.[14] The saliva specimen can be collected from multiple members of a group or a family conveniently without any intervention by HCW. This also prevents over-crowding at sample collection centers of a health care setting. As the COVID-19 cases are achieving a plateau, the educational institutions are destined to be functional soon. We foresee utility of saliva specimen during mass testing of students in schools, colleges, and hostels.

Being a pilot study, the study results are limited by a sample size of 85. The positive and negative predictive value of the novel method was 93.5% and 94.9%, respectively. The study results can facilitate the selection of saliva specimen in resource-limited setting. Saliva collection is noninvasive, requires lesser logistic requirement, saves viral transport medium and a cost-effective alternative to OP/NP swab collection in the pandemic setting.[15] The initial phase of COVID-19 pandemic was concentrated in the urban region. With community spread and heard immunity, the possibility of the second wave cannot be ruled out in the rural areas. Thus, at present, the focus is shifted to the rural areas, where health resources are scarce. In the absence of trained workforce, SARS-CoV-2 RT-PCR on self-collected saliva could be cost-effective strategy in the rural villages. To conclude, self-collected saliva is a reliable biological sample and promising tool for SARS-CoV-2 testing in resource-limited setting.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Wuhan Municipal Health Commission. Wuhan Municipal Health Commission Briefing on the Pneumonia Epidemic Situation 31 December, 2019 [in Chinese]; 2020. Available from: http://wjw.wuhan. gov.cn/front/web/showDetail/2019123108989. [Last accessed on 2020 Feb 20].  Back to cited text no. 1
    
2.
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. 2
    
3.
World Health Organization. Statement on the Second Meeting of the International Health Regulations (2005) Emergency Committee Regarding the Outbreak of Novel Coronavirus (2019-nCoV). Geneva: The Organization; 2020. Available from: https://www.who.int/news-room/detail/30-01-2020-statement-on-the-second-meeting-of-theinternational-health-regulations-(2005)-emergency committee regarding- the-outbreak-of-novel-coronavirus-(2019-ncov). [Last accessed on 2020 Feb 20].  Back to cited text no. 3
    
4.
World Health Organization. Coronavirus Disease 2019 (COVID-19) Situation Report – 67; 2020. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/. [Last accessed on 2020 Mar 28].  Back to cited text no. 4
    
5.
Jeong JH, Kim KH, Jeong SH, Park JW, Lee SM, Seo YH. Comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses. J Med Virol 2014;86:2122-7.  Back to cited text no. 5
    
6.
Liu L, Wei Q, Alvarez X, Wang H, Du Y, Zhu H, et al. Epithelial cells lining salivary gland ducts are early target cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory tracts of rhesus macaques. J Virol 2011;85:4025-30.  Back to cited text no. 6
    
7.
Lin C, Xiang J, Yan M, Li H, Huang S, Shen C. Comparison of throat swabs and sputum specimens for viral nucleic acid detection in 52 cases of novel coronavirus (SARS-Cov-2)-infected pneumonia (COVID-19). Clin Chem Lab Med 2020;58:1089-94.  Back to cited text no. 7
    
8.
Gupta N, Potdar V, Praharaj I, Giri S, Sapkal G, Yadav P, et al. Laboratory preparedness for SARS-CoV-2 testing in India: Harnessing a network of Virus Research & Diagnostic Laboratories. Indian J Med Res 2020;151:216-25.  Back to cited text no. 8
[PUBMED]  [Full text]  
9.
Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, et al. Detection of SARSCoV-2 in different types of clinical specimens. JAMA 2020;323:1843-4.  Back to cited text no. 9
    
10.
Winichakoon P, Chaiwarith R, Liwsrisakun C, Salee P, Goonna A, Limsukon A, et al. Negative nasopharyngeal and oropharyngeal swabs do not rule out COVID-19. J Clin Microbiol 2020;58. doi: 10.1128/JCM.00297-20.  Back to cited text no. 10
    
11.
Azzi L, Carcano G, Gianfagna F, Grossi P, Gasperina DD, Genoni A, et al. Saliva is a reliable tool to detect SARS-CoV-2. J Infect 2020;81:e45-e50.  Back to cited text no. 11
    
12.
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. 12
    
13.
Nagura-Ikeda M, Imai K, Tabata S, Miyoshi K, Murahara N, Mizuno T, et al. Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19. J Clin Microbiol. 2020 Aug 24;58(9):e01438-20. doi: 10.1128/JCM.01438-20.  Back to cited text no. 13
    
14.
Zhang K, Shoukat A, Crystal W, Langley JM, Galvani AP, Moghadas SM. Routine saliva testing for the identification of silent COVID-19 infections in healthcare workers. Infect Control Hosp Epidemiol. 2021;1-17. doi: 10.1017/ice.2020.1413.  Back to cited text no. 14
    
15.
Vaz SN, Santana DS, Netto EM, Pedroso C, Wang WK, Santos FDA, et al. Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection. Braz J Infect Dis 2020;24:422-7.  Back to cited text no. 15
    


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