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

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

    Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays Akane Sueki, Kazuyuki Matsuda, Akemi Y...

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    Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays Akane Sueki, Kazuyuki Matsuda, Akemi Yamaguchi, Masayuki Uehara, Mitsutoshi Sugano, Takeshi Uehara, Takayuki Honda PII: DOI: Reference:

S0009-8981(15)30075-9 doi: 10.1016/j.cca.2015.12.006 CCA 14197

To appear in:

Clinica Chimica Acta

Received date: Revised date: Accepted date:

29 September 2015 3 December 2015 4 December 2015

Please cite this article as: Sueki Akane, Matsuda Kazuyuki, Yamaguchi Akemi, Uehara Masayuki, Sugano Mitsutoshi, Uehara Takeshi, Honda Takayuki, Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays, Clinica Chimica Acta (2015), doi: 10.1016/j.cca.2015.12.006

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

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assays

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Akane Sueki,a Kazuyuki Matsuda,a# Akemi Yamaguchi,b Masayuki Uehara,b Mitsutoshi

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Sugano,a Takeshi Uehara,a Takayuki Hondaa

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Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japana; Core

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Technology Development Center, Seiko Epson Corporation, 281 Fujimi, Suwa, Japanb

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# Correspondence: Kazuyuki Matsuda, PhD, Department of Laboratory Medicine, Shinshu

University Hospital, 3-1-1, Asahi, Matsumoto, 390-8621, Japan.

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TEL: +81-263-37-2802; FAX: +81-263-34-5316; E-mail: [email protected]

Keywords: Influenza virus, saliva, droplet-RT-PCR, rapid detection, one-step real-time RT-PCR

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ACCEPTED MANUSCRIPT ABSTRACT

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Background: Immunochromatographic antigen tests have been widely used for detection of

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influenza virus; however its low sensitivity restricts the use of clinical materials other than

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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

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time consuming. We evaluated the utility of saliva as diagnostic materials for influenza virus

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infection by PCR-based assays.

Methods: Nasopharyngeal swabs and saliva were simultaneously collected from 144 patients

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and investigated by reverse transcription-quantitative PCR (RT-qPCR) and droplet-RT-PCR.

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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

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well as nasopharyngeal swabs contained more than 1 × 102 copies/l of the influenza gene. About half of the patients provided positive results in nasopharyngeal swabs and saliva within 24 hours 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.

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ACCEPTED MANUSCRIPT 1. Introduction

complications,

especially in

young

children,

elderly

adults,

and

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life-threatening

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Influenza virus causes acute febrile respiratory infection with severe illness and

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immunocompromised patients [1, 2]. Even outside these vulnerable populations, the extent of the infection during epidemic outbreaks leads to increased workplace absenteeism, thereby

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leading to a dramatic impact on economies [3]. The ability to rapidly diagnose influenza

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infections is critical for early clinical treatment and isolation of patients. Immunochromatographic antigen (IC) tests are widely used in clinical laboratories to detect

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the influenza viral nucleoprotein; however, the low sensitivity of the IC test is a major

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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

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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 [9-11], though saliva can be sampled more easily than nasonasopharyngeal 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) [12-14]. RT-PCR is one of the most sensitive methods for detecting the presence of RNA, and 3

ACCEPTED MANUSCRIPT various samples, including saliva, can be subjected to RT-PCR analysis. This makes RT-PCR

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a valuable tool for the diagnosis of influenza virus infections if the turnaround time of the

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PCR-based assay is improved. We previously reported the sensitivity of the droplet-RT-PCR

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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],

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making droplet-RT-PCR potentially one of the most reliable methods for the detection of

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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

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compartmentalized [18].

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In this study, we evaluated the utility of saliva as diagnostic materials for influenza virus

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infection using the conventional RT-qPCR and the high-speed droplet-RT-PCR.

2. Materials and Methods 2.1. 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, 4

ACCEPTED MANUSCRIPT malaise, and chills and were subjected to the immunochromatographic antigen (IC) tests

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(ESPLINE Influenza A & B-N, Fujirebio Inc, Tokyo, Japan) of nasopharyngeal swabs in

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Shinshu University Hospital from January 2012 through March 2014. The IC tests using the

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nasopharyngeal swabs diagnosed 24 patients as having influenza A or B virus. Five control samples were obtained from normal individuals without influenza-like symptoms. This study

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was approved by the Institutional Review Board of Shinshu University (no. 1785).

2.2. RNA extraction

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RNA was extracted from the nasopharyngeal swabs suspended into sterile PBS (70 L) and

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saliva (140 L) according to the collection volume of each sample using the QIAamp viral

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RNA Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions.

2.3. One-step high speed droplet-RT-PCR The primers and TaqMan probes for universal influenza A (H1N1) and B virus were used according to the Centers for Disease Control and Prevention (CDC) protocol [5, 6] as follows: influenza A: forward primer, 5'-GACCRATCCTGTCACCTCTGAC-3', reverse primer, AGG 5'-GCATTYTGGACAAAKCGTCTA-3', and probe, 5'-FAM-TGCAGTCCTCGCTCACTG GGCACG-BHQ1-3'; influenza B: forward primer, 5'-TCCTCAACTCACTCTTCGAGCG-3', reverse

primer,

5'-CGGTGCTCTTGACCAAATTGG-3', 5

and

probe,

ACCEPTED MANUSCRIPT 5'-FAM-CCAATTCGAGCAGCTGAAACTGCGGTG-BHQ1-3'. One-step real-time RT-PCR

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was performed using the droplet-PCR machine (Seiko Epson, Matsumoto, Japan) [16]. The

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RT-PCR mixture contained template RNA, SuperScript III/Platinum Taq Mix (Life

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Technologies, Grand Island, NY), 800 nmol/L of each primer, designed as above, 200 nmol/L TaqMan probe, and reaction buffer composed of Tris–HCl, pH 9.0, KCl, and MgCl2, in a total

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volume of 5 μL. One microliter of the reaction mixtures was used for the one-step

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droplet-RT-PCR assay. The reaction conditions used for Influenza A were: RT at 50 °C for 1 min, RT inactivation at 98 °C for 10 s, and 50 cycles of 98 °C for 3 s and 58 °C for 6 s. The

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reaction conditions used for Influenza B were: RT at 50 °C for 1 min, RT inactivation at 98 °C

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for 10 s, and 50 cycles of 98 °C for 5 s and 60 °C for 8 s. All samples were analyzed in duplicate. We determined that the influenza gene was present when the amplification

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exceeded 101 copies per reaction at 40 cycles.

2.4. Conventional RT-qPCR The conventional RT-qPCR was performed with QuantStudio 12K flex systems (Life Technologies, Carlsbad, CA) using the Superscript III One-Step qRT-PCR kit (Life Technologies, Grand Island, NY). The RT-qPCR reaction mixture (25 L) comprised 5 L template RNA, 0.5 L SuperScript III/Platinum Taq Mix, 12.5 L 2× Reaction Mix with ROX, 800 nmol/L of each primer, and 200 nmol/L of TaqMan probe. The reaction conditions were 6

ACCEPTED MANUSCRIPT as follows: initial denaturation at 94 °C for 5 min, followed by RT at 50 °C for 30 min, 95 °C

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for 2 s for inactivation of RT, and 50 cycles of 95 °C for 15 s and 55 °C for 30 s.

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The inputs for the standards were known concentrations (copies/L) of plasmids (101 –

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106) carrying the relevant 100 bp-regions of the influenza A (H1N1) and B genome for PCR amplification. The standard curves were calculated as equivalent numbers of influenza A or B

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amplicons. A standard curve was generated for each assay to validate the reaction conditions

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and calculate the number of amplicoms. All samples were analyzed in triplicate.

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3. Results

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3.1. 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

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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 influenza A within 8.6 min at 40 cycles and influenza B within 11.3 min at 40 cycles (Fig. 1). On the other hand, no amplification was detected in 5 control samples obtained from normal individuals without influenza-like symptoms. 7

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3.2. Quantitative evaluation of influenza gene in the nasopharyngeal swabs and saliva

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The quantity of the influenza gene in the nasopharyngeal swabs and saliva was evaluated

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by the conventional RT-qPCR. Nasopharyngeal swabs or saliva positive for influenza virus infection contained more than 1 × 102 copies/l of the influenza A or B gene (Fig. 2). The 2

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patients negative for the virus in nasopharyngeal swabs had 6 × 103 and 8 × 103 copies/µl in

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their saliva, respectively, whereas the 4 patients with saliva negative for the virus had 2 × 103

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- 8 × 104 copies/µl in their nasopharyngeal swabs (Fig. 2).

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3.3. Early detection of influenza A virus in the nasopharyngeal swabs and saliva We determined the positive rate of influenza A detection in nasopharyngeal swabs and

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saliva from 23 patients available for information on the time from the onset of clinical symptoms. Within 24 hours, 50% of the nasopharyngeal swabs and 55% of saliva were found positive for influenza gene, which were confirmed by either the RT-qPCR or droplet-RT-PCR. (Fig. 3).

4. 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 8

ACCEPTED MANUSCRIPT and saliva. The droplet-RT-PCR assay could amplify influenza A or B virus in the saliva as

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well as in nasopharyngeal swabs in less than 12 min. Nasopharyngeal swabs or throat swabs

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samples have been commonly used for IC tests because they have a higher concentration of

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influenza virus than is found in other samples [9-11]. The previous study suggested that the saliva was potentially valuable for diagnosis of the respiratory virus infection disease because

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the viruses exist in saliva [13, 19, 20]. Our study demonstrated that saliva provided 102-106

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copies/l of influenza A or B gene which was almost equivalent to that of nasopharyngeal swabs. Saliva can be collected safely and easily without pain, which are preferable to

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nasopharyngeal swabs especially for children and older adults [12].

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Using either nasopharyngeal swabs or saliva, about half of the patients provided the positive results within 24 hours from the onset of flu-like symptoms by the conventional

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RT-qPCR and the droplet-RT-PCR assays. The amount of viral shedding increased during the first day after inoculation, in parallel with the time of symptom onset [21]. Reports from recent pandemics suggest that treatment administered within 48 hours after symptom onset reduced the likelihood of hospitalization or the requirement for critical care, compared with late treatment administered more than 48 hours after the onset of symptoms [22, 23]. Another study showed that the treatment benefits of neuraminidase inhibitors increase by early therapeutic intervention [24, 25]. In this study, we could not evaluate the correlation between viral load with severity of clinical symptoms or with time to resolution of symptoms due to 9

ACCEPTED MANUSCRIPT lack of these information. The correlation between viral load with these factors need to be

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evaluated in future.

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Because the IC tests for influenza are rapid and easy procedure, they are widely performed

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in clinical laboratories. However the low sensitivity of the IC tests makes them insufficiently reliable for widespread use [11, 17]. The most significant feature of PCR is its high sensitivity

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to detect the target; however, traditionally PCR-based assays have been considered to be

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time-consuming. Recently, remarkable advances have been made in increase of PCR speed [26-30]. In this study, all of the RT-PCR steps in the droplet-RT-PCR were completed within

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14 min. Extraction of nucleic acid is one of the important steps in the workflow of the PCR

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analysis. The process of nucleic acid extraction also has been improved to be rapid and automatic [31-35]. Although the present study utilized the conventional RNA extraction

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procedure required 20 min, the turn-around time is improved when the rapid RNA extraction methods are available routinely. In this study, we demonstrated that saliva can be used as a clinical specimen to detect influenza virus as well as nasopharyngeal swabs. If rapid PCR assay including RNA extraction and subsequent RT-PCR can be fully automated in a miniaturized instrument, point-of-care test based on PCR assay using not only nasopharyngeal swabs but also saliva may be put into practice and can be used in place of IC tests for detection of influenza virus.

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ACCEPTED MANUSCRIPT Acknowledgments

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We acknowledge staffs assisted with sample collection.

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References

[1] Hall CB, Weinberg GA, Iwane MK, Blumkin AK, Edwards KM, Staat MA,et al. The

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burden of respiratory syncytial virus infection in young children. N Engl J Med

MA

2009;360:588-98.

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Med 2008;359:2579-85.

D

[2] Glezen WP. Clinical practice. Prevention and treatment of seasonal influenza. N Engl J

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[3] Akazawa M, Sindelar JL, Paltiel AD. Economic costs of influenza-related work absenteeism. Value Health 2003;6:107-15.

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[4] Kok J, Blyth CC, Foo H, Patterson J, Taylor J, McPhie K,et al. Comparison of a rapid antigen test with nucleic acid testing during cocirculation of pandemic influenza A/H1N1 2009 and seasonal influenza A/H3N2. J Clin Microbiol 2010;48:290-1. [5] Selvaraju SB, Selvarangan R. Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses. J Clin Microbiol 2010;48:3870-5. [6] World Health Organization. CDC protocol of real-time RT-PCR for swine influenza (H1N1).

Geneva,

Switzerland:

World 11

Health

Organization;

2009.

Available

ACCEPTED MANUSCRIPT at:http://www.who.int/csr/resources/publications/swineflu/CDCrealtimeRTPCRprotocol_

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20090428.pdf (Accessed June 2010).

IP

[7] Zeng H, Pappas C, Katz JM, Tumpey TM. The 2009 pandemic H1N1 and

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triple-reassortant swine H1N1 influenza viruses replicate efficiently but elicit an attenuated inflammatory response in polarized human bronchial epithelial cells. J Virol

NU

2011.85:686-96.

MA

[8] Kandun IN, Wibisono H, Sedyaningsih ER, Yusharmen, Hadisoedarsuno W, Purba W,et al. Three Indonesian clusters of H5N1 virus infection in 2005. N Engl J Med

TE

D

2006;355:2186-94.

CE P

[9] de la Tabla VO, Masiá M, Antequera P, Martin C, Gazquez G, Buñuel F,et al. Comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection

AC

of pandemic influenza A/H1N1 2009 virus by real-time reverse transcriptase PCR. J Clin Microbiol 2010.48:3492-5. [10] Irving SA, Vandermause MF, Shay DK, Belongia EA. Comparison of nasal and nasonasopharyngeal swabs for influenza detection in adults. Clin Med Res 2012.10:215-8. [11] Sakai-Tagawa Y, Ozawa M, Tamura D, Le Mt, Nidom CA, Sugaya N,et al. Sensitivity of influenza rapid diagnostic tests to H5N1 and 2009 pandemic H1N1 viruses. J Clin Microbiol 2010;48:2872-7. 12

ACCEPTED MANUSCRIPT [12] Kaufman E, Lamster IB. The diagnostic applications of saliva--a review. Crit Rev Oral

T

Biol Med 200213:197-212.

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[13] Detmer SE, Patnayak DP, Jiang Y, Gramer MR, Goyal SM. Detection of influenza A

SC R

virus in porcine oral fluid samples. J Vet Diagn Invest 2011;23:241-7. [14] Decorte I, Van der Stede Y, Nauwynck H, De Regge N, Cay AB. Effect of saliva

NU

stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral

MA

fluid by quantitative reverse transcriptase real-time PCR. Vet J 2013;197:224-8. [15] Matsuda K, Yamaguchi A, Taira C, Sueki A, Koeda H, Takagi F,et al. A novel

TE

D

high-speed droplet-polymerase chain reaction can detect human influenza virus in less

CE P

than 30 min. Clin Chim Acta 2012;413:1742-5. [16] Cheng PK, Wong KK, Mak GC, Wong AH, Ng AY, Chow SY,et al. Performance of

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laboratory diagnostics for the detection of influenza A(H1N1)v virus as correlated with the time after symptom onset and viral load. J Clin Virol 2010;47:182-5. [17] Ruest A, Michaud S, Deslandes S, Frost EH. Comparison of the Directigen flu A+B test, the QuickVue influenza test, and clinical case definition to viral culture and reverse transcription-PCR for rapid diagnosis of influenza virus infection. J Clin Microbiol 2003;41:3487-93. [18] Zhu Z, Jenkins G, Zhang W, Zhang M, Guan Z, Yang CJ. Single-molecule emulsion PCR in microfluidic droplets. Anal Bioanal Chem 2012;403:2127-43. 13

ACCEPTED MANUSCRIPT [19] Robinson JL, Lee BE, Kothapalli S, Craig WR, Fox JD. Use of throat swab or saliva

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specimens for detection of respiratory viruses in children. Clin Infect Dis 2008;46:e61-4.

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[20] Wang WK, Chen SY, Liu IJ, Chen YC, Chen HL, Yang CF,et al. Detection of

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SARS-associated coronavirus in throat wash and saliva in early diagnosis. Emerg Infect Dis 2004;10:1213-9.

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[21] Carrat F, Vergu E, Ferguson NM, Lemaitre M, Cauchemez S, Leach S,et al. Time lines

J Epidemiol 2008;167:775-85.

MA

of infection and disease in human influenza: a review of volunteer challenge studies. Am

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[22] Louie JK, Acosta M, Jamieson DJ, Honein MA; California Pandemic (H1N1) Working

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Group. Severe 2009 H1N1 influenza in pregnant and postpartum women in California. N Engl J Med 2010;362:27-35.

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[23] Siston AM, Rasmussen SA, Honein MA, Fry AM, Seib K, Callaghan WM,et al. Pandemic 2009 influenza A (H1N1) virus illness among pregnant women in the United States. JAMA 2010;303:1517-25. [24] Muthuri SG, Myles PR, Venkatesan S, Leonardi-Bee J, Nguyen-Van-Tam JS. Impact of neuraminidase inhibitor treatment on outcomes of public health importance during the 2009-2010 influenza A(H1N1) pandemic: a systematic review and meta-analysis in hospitalized patients. J Infect Dis 2013;207:553-63. [25] Moscona A. Neuraminidase inhibitors for influenza. N Engl J Med 2005;353:1363-73. 14

ACCEPTED MANUSCRIPT [26] Farrar JS, Wittwer CT. Extreme PCR: efficient and specific DNA amplification in 15-60

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seconds. Clin Chem 2015;61:145-53.

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[27] Sundberg SO, Wittwer CT, Howell RM, Huuskonen J, Pryor RJ, Farrar JS,et al.

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Microfluidic genotyping by rapid serial PCR and high-speed melting analysis. Clin Chem 2014;60:1306-13.

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[28] Wheeler EK, Hara CA, Frank J, Deotte J, Hall SB, Benett W,et al. Under-three minute

MA

PCR: probing the limits of fast amplification. Analyst 2011;136:3707–12. [29] Fuchiwaki Y, Nagai H, Saito M, Tamiya E. Ultra-rapid flow-through polymerase chain

TE

D

reaction microfluidics using vapor pressure. Biosens Bioelectron 2011;27:88 –94.

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[30] Maltezos G, Johnston M, Taganov K, Srichantaratsamee C, Gorman J, Baltimore D,et al. Exploring the limits of ultrafast polymerase chain reaction using liquid for thermal

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heat exchange: a proof of principle. Appl Phys Lett 2010;97:264101. [31] Karle M, Miwa J, Czilwik G, Auwärter V, Roth G, Zengerle R,et al. Continuous microfluidic DNA extraction using phase-transfer magnetophoresis. Lab on a Chip 2010;10:3284-90. [32] Berry SM, Alarid ET, Beebe DJ. One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST). Lab on a Chip 2011;11:1747-53. [33] Siegrist J, Gorkin R, Bastien M, Stewart G, Peytavi R, Kido H,et al. Validation of a 15

ACCEPTED MANUSCRIPT centrifugal microfluidic sample lysis and homogenization platform for nucleic acid

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extraction with clinical samples. Lab on a Chip 2010;10:363-71.

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[34] Gan W, Zhuang B, Zhang P, Han J, Li CX, Liu P. A filter paper-based microdevice for

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low-cost, rapid, and automated DNA extraction and amplification from diverse sample types. Lab on a Chip 2014;14:3719-28.

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[35] Van Heirstraeten L, Spang P, Schwind C, Drese KS, Ritzi-Lehnert M, Nieto B,et al.

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Integrated DNA and RNA extraction and purification on an automated microfluidic cassette from bacterial and viral pathogens causing community-acquired lower

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respiratory tract infections. Lab on a Chip 2014;14:1519-26.

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ACCEPTED MANUSCRIPT Figure legends

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Figure 1. Amplification plots by the droplet-RT-PCR for detecting influenza A or B using

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nasopharyngeal swabs and saliva.

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In both nasopharyngeal swabs and saliva, specific amplifications were obtained from patients; whereas no amplifications were from controls. X-axis, PCR cycles and time; Y-axis, arbitrary

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units of fluorescence.

Figure 2. Quantification of the influenza viral gene in nasopharyngeal swabs and saliva.

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Influenza viral gene was quantified by the conventional RT-qPCR using paired

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nasopharyngeal swabs and saliva.

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Figure 3. Detection rate of influenza A after onset of clinical symptoms. The detection rate of influenza A in nasopharyngeal swabs and saliva by the conventional RT-qPCR and droplet RT-PCR assay were calculated from 23 patients available for information on the time from the onset of clinical symptoms. Open bars, nasopharyngeal swabs; solid bars, saliva.

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Figure 1

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ACCEPTED MANUSCRIPT Table 1. Detection of Influenza A or B virus in nasopharyngeal swabs and saliva using conventional RT-qPCR and droplet-RT-PCR

nasopharyngeal swabs (+)

nasopharyngeal swabs (-)

methods

saliva (+)

saliva (-)

saliva (+)

saliva (-)

conventional

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110

28

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droplet-RT-PCR

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RT-qPCR

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PCR-based

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/l of influenza gene. About 50% of saliva was positive within 24 hours after the onset of symptoms.

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