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