Elsevier

Clinica Chimica Acta

Volume 453, 30 January 2016, Pages 71-74
Clinica Chimica Acta

Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays

https://doi.org/10.1016/j.cca.2015.12.006Get rights and content

Highlights

  • High degree of result concordance was obtained from nasopharyngeal swabs and saliva.

  • The droplet-RT-PCR assay could amplify influenza gene in the saliva within 14 min.

  • Saliva contained influenza gene more than 1 × 102 copies/μl of influenza gene.

  • About 50% of saliva was positive within 24 h after the onset of symptoms.

Abstract

Background

Immunochromatographic antigen tests have been widely used for detection of influenza virus; however its low sensitivity restricts the use of clinical materials other than nasopharyngeal swabs. Saliva is obtained non-invasively and has utility for diagnosis of influenza. Polymerase chain reaction (PCR) is not typically used for rapid testing because it is time consuming. We evaluated the utility of saliva as diagnostic materials for influenza virus infection by PCR-based assays.

Methods

Nasopharyngeal swabs and saliva were simultaneously collected from 144 patients and investigated by reverse transcription-quantitative PCR (RT-qPCR) and droplet-RT-PCR.

Results

Overall concordance of results from nasopharyngeal swabs and saliva were 95.8%. Influenza gene was detectable in less than 12 min in saliva by the droplet-RT-PCR. Saliva as well as nasopharyngeal swabs contained more than 1 × 102 copies/μl of the influenza gene. About half of the patients provided positive results in nasopharyngeal swabs and saliva within 24 h from the onset of the symptoms.

Conclusion

The study demonstrates that saliva can be used as an alternative specimen source to nasopharyngeal swabs. When rapid PCR assay including RNA extraction to be full-automation in a miniaturized machine, point-of-care test based on PCR may be realized using saliva without restriction of materials.

Introduction

Influenza virus causes acute febrile respiratory infection with severe illness and life-threatening complications, especially in young children, elderly adults, and immunocompromised patients [1], [2]. Even outside these vulnerable populations, the extent of the infection during epidemic outbreaks leads to increased workplace absenteeism, thereby leading to a dramatic impact on economies [3]. The ability to rapidly diagnose influenza infections is critical for early clinical treatment and isolation of patients.

Immunochromatographic antigen (IC) tests are widely used in clinical laboratories to detect the influenza viral nucleoprotein; however, the low sensitivity of the IC test is a major problem for influenza diagnosis in the early stages of infection [4]. On the other hand, detection of genomic RNA by polymerase chain reaction (PCR) analyses is the gold standard for identifying and classifying influenza virus [5], [6].

Most influenza viruses infect the respiratory tract and replicate productively in the airway epithelial cells, including the nasopharynx [7], [8]. Nasopharyngeal specimens are generally used for isolation of influenza virus [9], [10], [11], though saliva can be sampled more easily than nasopharyngeal swabs. Such a non-invasive test, particularly for children, would provide potentially valuable materials for detection of the influenza virus by reverse transcription-quantitative PCR (RT-PCR) [12], [13], [14].

RT-PCR is one of the most sensitive methods for detecting the presence of RNA, and various samples, including saliva, can be subjected to RT-PCR analysis. This makes RT-PCR a valuable tool for the diagnosis of influenza virus infections if the turnaround time of the PCR-based assay is improved. We previously reported the sensitivity of the droplet-RT-PCR for influenza virus detection was similar to the conventional RT- quantitative PCR (RT-qPCR) [15]. RT-qPCR is as sensitive for influenza detection as viral culture isolation is [16], [17], making droplet-RT-PCR potentially one of the most reliable methods for the detection of influenza virus. PCR performed in a small volume can achieve efficient amplification while retaining specificity, as exemplified by emulsion PCR, in which the reaction mixtures are compartmentalized [18].

In this study, we evaluated the utility of saliva as diagnostic materials for influenza virus infection using the conventional RT-qPCR and the high-speed droplet-RT-PCR.

Section snippets

Sample collection

Nasopharyngeal swabs and saliva were obtained simultaneously from 144 patients who had provided informed consent. The saliva was collected via dropper. The study population included 64 female (mean age: 39.5 years old, range 24–62) and 80 male (mean age: 41.6 years old, range 27–63) individuals. Patients enrolled in this study were selected based-on the following influenza-like symptoms; fever, cough, headache, sore throat, myalgia, congestion, malaise, and chills and were subjected to the

Influenza gene in the nasopharyngeal swabs and saliva by the PCR-based assays

The conventional RT-qPCR and droplet-RT-PCR provided the completely-consistent results from the nasopharyngeal swabs and saliva (Table 1). Among the 144 patients, 28 and 110 were positive or negative in both samples, while 4 and 2 patients were only positive nasopharyngeal swabs or saliva, respectively (Table 1). The overall concordance of the results from both samples was 95.8% (Table 1). In both nasopharyngeal swabs and saliva from patients, the droplet-RT-PCR method was able to detect

Discussion

In this study, we showed that saliva could be used for the diagnosis of influenza virus infection. High degree of result concordance was obtained from the nasopharyngeal swabs and saliva. The droplet-RT-PCR assay could amplify influenza A or B virus in the saliva as well as in nasopharyngeal swabs in less than 12 min. Nasopharyngeal swabs or throat swabs samples have been commonly used for IC tests because they have a higher concentration of influenza virus than is found in other samples [9],

Acknowledgments

We acknowledge staffs assisted with sample collection.

References (35)

  • H. Zeng et al.

    The 2009 pandemic H1N1 and triple-reassortant swine H1N1 influenza viruses replicate efficiently but elicit an attenuated inflammatory response in polarized human bronchial epithelial cells.

    J. Virol

    (2011)
  • I.N. Kandun et al.

    Three Indonesian clusters of H5N1 virus infection in 2005

    N. Engl. J. Med.

    (2006)
  • V.O. e la Tabla et al.

    Comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza A/H1N1 2009 vvirus by real-time reverse transcriptase PCR

    J. Clin. Microbiol

    (2010)
  • S.A. Irving et al.

    Comparison of nasal and nasonasopharyngeal swabs for influenza detection in adults

    Clin. Med. Res.

    (2012)
  • Y. Sakai-Tagawa et al.

    Sensitivity of influenza rapid diagnostic tests to H5N1 and 2009 pandemic H1N1 viruses

    J. Clin. Microbiol.

    (2010)
  • E. Kaufman et al.

    The diagnostic applications of saliva—a review

    Crit. Rev. Oral Biol. Med.

    (2002)
  • S.E. Detmer et al.

    Detection of influenza A virus in porcine oral fluid samples

    J. Vet. Diagn. Investig.

    (2011)
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