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Original Articles

Additional molecular testing of saliva specimens improves the detection of respiratory viruses

, , , , , , , , , & show all
Pages 1-7 | Received 17 Jan 2017, Accepted 09 Apr 2017, Published online: 15 Jan 2019

Abstract

Emerging infectious diseases in humans are often caused by respiratory viruses such as pandemic or avian influenza viruses and novel coronaviruses. Microbiological testing for respiratory viruses is important for patient management, infection control and epidemiological studies. Nasopharyngeal specimens are frequently tested, but their sensitivity is suboptimal. This study evaluated the incremental benefit of testing respiratory viruses in expectorated saliva using molecular assays. A total of 258 hospitalized adult patients with suspected respiratory infections were included. Their expectorated saliva was collected without the use of any special devices. In the first cohort of 159 patients whose nasopharyngeal aspirates (NPAs) tested positive for respiratory viruses during routine testing, the viral load was measured using quantitative reverse transcription PCR. Seventeen percent of the patients (27/159) had higher viral loads in the saliva than in the NPA. The second cohort consisted of 99 patients whose NPAs tested negative for respiratory viruses using a direct immunofluorescence assay. Their NPA and saliva specimens were additionally tested using multiplex PCR. In these patients, the concordance rate by multiplex PCR between NPA and saliva was 83.8%. Multiplex PCR detected viruses in saliva samples from 16 patients, of which nine (56.3%) had at least one virus that was not detected in the NPA. Decisions on antiviral or isolation precautions would be affected by salivary testing in six patients. Although NPAs have high viral loads and remain the specimen of choice for most patients with respiratory virus infections, supplementary molecular testing of saliva can improve the clinical management of these patients.

Emerging Microbes & Infections (2017) 6, e49 doi:10.1038/emi.2017.35; published online 7 June 2017

Introduction

Viruses play important roles in respiratory tract infections.Citation1, Citation2, Citation3, Citation4 Recently, several novel respiratory viruses have emerged, including the 2009 pandemic influenza virus A(H1N1)pdm09,Citation5 the avian influenza viruses A(H7N9) and A(H5N6),Citation6, Citation7, Citation8 and the Middle East Respiratory Syndrome (MERS) coronavirus.Citation9 The novel avian influenza viruses and the MERS coronavirus are associated with high mortality rates of over 30%.

Prompt and accurate detection of respiratory viruses is important for guiding antiviral treatment.Citation10 For influenza virus infection, earlier administration of neuraminidase inhibitors is associated with faster resolution of symptoms in randomized clinical trials and improved survival in retrospective studies.Citation11, Citation12 Hyperimmune intravenous immunoglobulin confers survival benefit for severe influenza only if administered within 5 days after symptom onset.Citation13 Antibiotic usage can be reduced if a respiratory virus is detected without evidence of bacterial infection.Citation14 Early detection is also important for infection control and public health measures.

Nasopharyngeal aspirate (NPA), nasopharyngeal swabs (NPS) (including flocked swabs), and nasal or throat swabs/washes are the recommended upper respiratory tract specimen types for diagnostic testing of respiratory viruses.Citation15, Citation16 Nasopharyngeal specimens are usually considered to have the highest detection rate for respiratory viruses. Nasopharyngeal specimens are often used as the only specimen type in routine clinical practice and in many surveillance studies for the detection of respiratory viruses.Citation17 However, in studies that have tested multiple specimen types, nasopharyngeal specimens have been found to be negative in some patients with respiratory virus infections.Citation18, Citation19, Citation20, Citation21, Citation22 Sputum or other lower respiratory tract specimens may contain a higher viral load for some patients, which will improve the detection of viruses. Jeong et al showed that nasopharyngeal swabs were negative in 25% of adult patients with sputum positive for respiratory viruses.Citation19 However, many patients do not have sputum production or cannot expectorate good quality sputum. Additionally, the collection of tracheal or bronchial specimens involves invasive procedures that are associated with significant discomfort and risk to the patient and pose a risk to healthcare workers.Citation23, Citation24

Saliva can be easily provided by patients without any invasive procedures. However, saliva is rarely used for the detection of respiratory viruses, because it is generally considered to have inferior sensitivity compared with other respiratory tract specimens. MUC5B, salivary gp-340, histatins, and human neutrophil defensins in saliva have been shown to neutralize influenza A virus.Citation25 Moreover, saliva is not a suitable specimen type for direct immunofluorescence assay (DFA) because it does not contain a sufficient amount of infected respiratory epithelial cells. Recently, there has been renewed interest in using saliva for the detection of respiratory viruses with molecular assays.Citation18, Citation26, Citation27, Citation28, Citation29 Some of these studies showed that the detection rate of respiratory viruses was actually higher for saliva than for nasopharyngeal specimens.Citation18 However, these studies were conducted either in childrenCitation28 or among adult patients with mild symptoms who did not require hospitalization.Citation18, Citation27 Some studies only evaluated the detection of influenza virus.Citation26, Citation29 Furthermore, many of these studies used special collection devices that are not usually available in most hospitals. The utility of saliva for the detection of respiratory viruses among hospitalized adult patients has not been comprehensively studied.

The current study sought to evaluate the benefit of testing expectorated saliva in addition to NPA among adult patients hospitalized for a suspected respiratory tract infection. The aim of the first part of the study was to assess the difference in the viral load between NPA and saliva using quantitative PCR with reverse transcription (RT-PCR). The aim of the second part of the study was to evaluate the incremental benefit of testing saliva in addition to NPA using a commercially available molecular assay. As the current study was designed to enable the easy application of our saliva specimen collection method to any clinical setting, we asked the study participants to provide saliva specimens by simple expectoration into a sterile bottle without using any special collection devices.

Materials and Methods

Study design and participants

This study was a 1-year prospective study conducted between 1 March 2015, and 29 February 2016, in Queen Mary Hospital, which is a teaching hospital in Hong Kong with 1600 beds. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Written informed consent was obtained from all study participants. Patients were eligible for recruitment if they were hospitalized adult patients aged 18 years or above with a suspected respiratory tract infection and had NPA samples obtained for respiratory virus testing. Saliva specimens were collected from the enrolled patients when the result of the routine clinical testing for respiratory viruses in the clinical microbiology laboratory of Queen Mary Hospital was known. Therefore, a patient’s saliva specimen was always collected after the NPA specimen. Patients were excluded from the study if they were discharged from the hospital before enrollment, were unable to provide a saliva specimen, or refused or were unable to provide written informed consent.

The first part of the study consisted of patients whose NPA samples tested positive for respiratory viruses by DFA or the influenza A virus M gene by real-time RT-PCR during routine respiratory virus testing in our clinical microbiology laboratory (). The viral load was determined in both the NPA and the saliva samples using quantitative RT-PCR (qRT-PCR). During the first phase (1 March to 15 June 2015), only patients who tested positive for influenza A virus were included. During the second phase (16 June 2015 to 29 February 2016), patients who tested positive for any respiratory viruses were included.

The second part of the study consisted of patients whose NPA specimens either tested negative for respiratory viruses by DFA or had insufficient nasopharyngeal columnar epithelial cells (NPCs) in the NPA sample for DFA during routine clinical testing (). Insufficient NPCs was defined as <20 NPCs in the entire well. Multiplex PCR was used to test for respiratory viruses in both the NPA and the saliva samples.

Figure 1 Study design. nasopharyngeal aspirate, NPA; quantitative PCR with reverse transcription, qRT-PCR. aRoutine clinical testing was performed using antigen detection by the DFA, which included the influenza A and B viruses, parainfluenza virus types 1–3, respiratory syncytial virus, human metapneumovirus and adenovirus. From 1 March to 8 April 2015 (during the peak influenza A virus season), monoplex real-time RT-PCR for the influenza A M gene was performed for patients admitted to the general medical ward. bPatients whose NPA specimens either tested negative for respiratory viruses by DFA or had insufficient NPCs for DFA during routine clinical testing. Insufficient NPCs is defined as <20 NPCs in the entire well.

Data collection

Patient data concerning demographics, underlying diseases, clinical findings and radiological changes were recorded in a predesigned database. The Charlson comorbidity score was calculated.Citation30 Pneumonia was defined by radiological evidence of new or increased pulmonary infiltrate in a chest radiograph and at least one of the following symptoms (cough with or without sputum production, dyspnea, tachypnea, or pleuritic chest pain) plus one auscultatory finding or one sign of infection (core body temperature >38 °C, shivers, leukocyte count >10 000 cells/μL or <4000 cells/μL) as described previously.Citation31

Routine respiratory virus testing in the clinical microbiology laboratory

NPA samples were collected in viral transport medium (VTM) as described previously.Citation32 Routine testing for respiratory viruses was performed using antigen detection by DFA (D3 Ultra 8 DFA Respiratory Virus Screening and Identification Kit, Diagnostic Hybrids, Inc., Quidel, San Diego, CA, USA), which included influenza A virus, and influenza B virus, parainfluenza viruses 1–3, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and adenovirus. From 1 March to 8 April 2015 (during the peak influenza A virus season in Hong Kong), real-time RT-PCR for the influenza A virus M gene was performed for patients admitted to the general medical ward as described previously.Citation33 During this period, DFA was not performed if the NPA samples tested positive for influenza A virus using RT-PCR.

Saliva specimen collection

Patients were instructed to expectorate saliva into a sterile container and 2 mL of VTM was added to the container in the microbiology laboratory. The volume of saliva ranged between ~0.5 and 1 mL.

Quantitative RT-PCR for respiratory viruses

Total nucleic acid (TNA) extraction was performed using the easyMAG instrument (bioMerieux, Boxtel, Netherlands) as described previously.Citation34, Citation35 NPA or saliva specimens in VTM (250 μL) were mixed with lysing buffer. After extraction, the nucleic acids were recovered using 55 μL of elution buffer.

qRT-PCR for influenza A virus detection was carried out using Superscript III Platinum One-Step RT-PCR reagents (Invitrogen, Carlsbad, CA, USA) as described previously with modifications.Citation13, Citation33, Citation34 The reagent mixture (25 μL) contained the 1 × Reaction Mix, Superscript III RT/Platinum Taq Mix, ROX reagent, 0.8 μM of the forward and reverse primers, 0.2 μM of the probe and 5 μL of TNA as the template. The thermal cycling conditions were 30 min at 50 °C for reverse transcription, 2 min at 95 °C for RT inactivation/initial denaturation, and 50 cycles of 15 s at 95 °C and 30 s at 55 °C. All reactions were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

The qRT-PCR for the detection of influenza B virus, parainfluenza viruses 1–3, hMPV and respiratory syncytial virus was carried out using AgPath-ID One-Step RT-PCR reagents (Applied Biosystems). The reagent mixture (25 μL) contained 1 × RT-PCR Buffer, 1 × RT-PCR Enzyme Mix, 0.4 μM of the forward and reverse primers, 0.12 μM of the probe and 5 μL of TNA as the template. The thermal cycling conditions were 10 min at 45 °C for reverse transcription, 10 min at 95 °C for RT inactivation/initial denaturation, and 50 cycles of 15 s at 95 °C and 45 s at 55 °C. All reactions were performed using the LightCycler 96 Real-Time PCR System (Roche, Basel, Switzerland). The primer and probe sequences are shown in Table .

Table 1 Primers and probes used in the quantitative reverse transcription PCR

Multiplex PCR panel for respiratory viruses

The multiplex PCR for respiratory viruses was performed using the NxTAG Respiratory Pathogen Panel (IVD) (Luminex, Austin, TX, USA) according to the manufacturer’s instructions. Respiratory viruses detected by this assay included influenza A virus, influenza B virus, RSV A and B, enterovirus/rhinovirus (EV/RV), parainfluenza viruses 1–4, hMPV, adenovirus, coronaviruses HKU1, NL63, 229E and OC43, and human bocavirus. Extracted TNA was added to pre-plated Lyophilized Bead Reagents. The reaction was amplified via RT-PCR, and the reaction product underwent bead hybridization within the sealed reaction well. The hybridized and tagged beads were sorted and read on the MAGPIX instrument, and the signals were analyzed using the NxTAG Respiratory Pathogen Panel Assay File for the SYNCT Software (Luminex, Austin, TX, USA).

Statistical analysis

The Mann-Whitney U-test and Fisher’s exact test were used for comparisons of continuous and categorical variables, respectively. The Wilcoxon matched-pairs signed rank test was used for the comparison between the NPA and saliva viral loads. Correlations between the viral loads in the NPA and saliva specimens were determined using Spearman’s correlation coefficient. McNemar’s test was used to compare the positivity rates between the NPA and the saliva specimens. Log-transformed data were used for statistical calculations of the viral load. The statistical analysis was performed using SPSS 21.0 (IBM Corp., Armonk, NY, USA) or GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA).

Results

First cohort: comparison of the viral loads between the NPA and the saliva samples

The first cohort consisted of 159 patients with known respiratory virus infection (Table and Supplementary Table S1). The median age was 69 years, with a range from 20 to 98 years. The median Charlson comorbidity score was 1. Forty (25.2%) patients had pneumonia, two patients (1.3%) required admission to the intensive care unit, and two patients (1.3%) died. Most of the saliva specimens were collected <2 days after the collection of the NPA sample (91.8%, 146/159). Among this first cohort of patients with a respiratory virus detected in the NPA sample during routine clinical testing, the qRT-PCR for the same virus was positive in the NPA specimens of all patients and in the saliva specimens of 91.8% (146/159) of the patients (Table ). Among the 99 patients who tested positive for influenza A virus, the NPA specimens of 12 patients were tested by influenza A virus M gene RT-PCR only during the routine clinical service. The saliva sample was positive by qRT-PCR in 11 out of these 12 patients (91.7%). Although the median viral load was significantly greater in the NPA than in the saliva samples (7.23 vs 5.30 log10 copies per mL, P<0.0001), 17.0% (27/159) of the patients had a higher viral load in the saliva than in their NPA sample (). No significant correlation was found between the viral load in the NPA and that in the saliva (P=0.071). Among the patients with influenza A or influenza B virus infection, no significant difference was found in the median salivary viral load between the patients with saliva collected before and those with saliva collected after oseltamivir treatment (5.43 vs 4.90 log10 copies per mL, P=0.476; ). No significant difference in the median viral load was detected in the saliva specimens between patients with pneumonia and those without pneumonia (5.12 vs 5.13 log10 copies per mL, P=0.218; ).

Figure 2 Viral loads in the NPA and the saliva specimens for all patients in the first cohort. The number of patients infected with each of the respiratory viruses is outlined in Table . (A) Comparison of viral loads between the NPA and saliva specimens. (B) Comparison of the saliva viral load of influenza A and influenza B in patients with saliva collected before and after oseltamivir treatment. (C) Comparison of the saliva viral loads in patients with or without pneumonia. Medians, quartiles, and ranges are shown. nasopharyngeal aspirate, NPA.

Table 2 Detection of viruses using quantitative real-time reverse transcription PCR in the first cohort of patients

Second cohort: multiplex PCR testing of the NPA and the saliva samples

In the second part of the study, multiplex PCR using the Luminex NxTAG Respiratory Pathogen Panel was performed on the saliva and NPA specimens of 99 patients whose NPA specimens tested negative by DFA (n=80) or were considered unsuitable for DFA due to insufficient NPCs (n=19) during routine clinical testing (Supplementary Table S1). Twenty-five patients (25.3%) had pneumonia. At least one respiratory virus was detected in 22.2% (22/99) of the patients, with one virus detected in 20.2% (20/99) of the patients, two viruses detected in 1.0% (1/99) of the patients, and three viruses detected in 1.0% (1/99) of the patients. Hence, a total of 25 viruses were detected (Tables and ). At least one respiratory virus was detected in 14.1% (14/99) and 16.2% (16/99) of the NPA and the saliva samples, respectively (Table ; P=0.789). Eighty-three patients (83.8%) showed complete concordance, with the multiplex PCR on the NPA and the saliva samples showing the presence or absence of exactly the same respiratory viruses (κ coefficient of 0.335 (95% confidence interval: 0.081–0.589)).

Table 3 Detection of respiratory viruses in nasopharyngeal aspirate and saliva using NxTAG Respiratory Pathogen Panel in the second cohort of patientsFootnotea

Respiratory viruses were detected in the saliva specimens of 8 (9.4%) of the 85 patients whose NPA specimens tested negative by NxTAG Respiratory Pathogen Panel, including three patients with influenza A virus, two patients with hMPV, two patients with EV/RV and one patient with coronavirus OC43 (Table ). For the 14 patients whose NPA specimens tested positive by the NxTAG Respiratory Pathogen Panel, one patient had an additional virus species detected in her saliva (Patient 4 in Table ). Potential changes in the antiviral treatment or infection control practice would be possible for six of the nine patients with additional viruses detected in their saliva. The three patients with influenza A virus infection could have been given neuraminidase inhibitor, whereas the three patients with hMPV should have been placed under contact precaution.Citation36

Table 4 Respiratory viruses detected using multiplex PCR panel in the second cohort of patientsFootnotea

Table 5 Nine patients with additional respiratory viruses detected only in their saliva for the second cohort of patients

Discussion

Novel molecular assays have been developed for the diagnosis of respiratory virus infections,Citation37, Citation38, Citation39, Citation40, Citation41 which have greatly enhanced the sensitivity of the detection of respiratory viruses. However, very few studies have reported an evaluation of specimen types to improve the detection rate. This study evaluated the use of saliva in addition to NPA for the diagnosis of respiratory viral infections in two parts. In the first part of this study, saliva had a higher viral load than NPA in 17.0% of the patients who tested positive for respiratory viruses by DFA or influenza A virus by RT-PCR in their NPA samples. In the second part of this study, saliva specimens tested positive in eight (9.4%) of the 85 patients whose NPA specimens tested negative by multiplex PCR. Among the 14 patients whose NPA specimens were unsuitable for DFA or tested negative by DFA but positive by multiplex PCR, one patient (7.1%) had an additional respiratory virus detected in her saliva specimen using multiplex PCR. Importantly, a change in antiviral treatment or isolation precaution could have occurred in six out of these 9 patients with additional respiratory viruses detected in their saliva.

In some previous studies, saliva was obtained using a dropperCitation29 or a special sponge on a stick.Citation28 The advantage of using these devices is that the amount of saliva is standardized, and the saliva is less likely to be contaminated by sputum. However, these collection devices are not usually available in a general medical ward setting. Furthermore, the collection of saliva with these devices requires help from healthcare workers. In the present study, we simply asked the patients to expectorate saliva into the standard sterile sputum containers used routinely in our hospital. As no special collection device is required, the use of expectorated saliva can be implemented easily in daily clinical practice. One possible concern is that the expectorated saliva may be mixed with sputum, which may contain a higher viral load than saliva. However, because the aim of using saliva is to increase the detection rate, this perceived limitation is actually favorable.

In real-life clinical practice, testing both NPA and saliva simultaneously is too costly. A more cost-effective approach is to test saliva only if the NPA result is negative. Hence, in the current study, we mimic this situation by collecting saliva only when the NPA result is available. Our previous studies on influenza viruses showed that patients usually had higher viral loads in their NPA upon hospital admission.Citation33, Citation34, Citation42 Therefore, saliva collected later may have a lower viral load and may result in a lower sensitivity of detection.

Although this study was primarily designed to evaluate the incremental benefit of testing saliva in addition to NPA, the results provided some insights into the potential use of testing saliva alone. Among NPA-positive specimens, the detection rate in saliva was 91.8% (146/159) in the first part of the study and 57.1% (8/14) in the second part of the study. Notably, the detection rate of respiratory viruses by multiplex PCR in the second part of the study was slightly higher in saliva (16.2%) than in NPA (14.1%). Kim et al also showed that multiplex PCR of saliva and NPS specimens had similar detection rates.Citation18 Therefore, the use of saliva alone may not be inferior to the use of NPA if only a single type of specimen is tested. Testing saliva over NPA has many advantages. First, collecting saliva rather than NPA avoids patient discomfort.Citation43 Second, the collection of saliva is suitable for patients for whom the collection of nasopharyngeal specimens is contraindicated, such as patients with severe bleeding tendency. Third, patients can provide saliva specimens after simple instruction, whereas the collection of nasopharyngeal specimens must be performed by healthcare personnel. This approach would reduce the delay in specimen collection. Finally, the collection of saliva does not require any special infection control precautions and can be performed in any clinical setting with standard precautions. By contrast, the procedures for the collection of nasopharyngeal specimens are potentially aerosol-generating and therefore pose significant risks to healthcare workers and other patients. Some health authorities have recommended that nasopharyngeal specimens should be collected in a negative pressure isolation room for patients with suspected MERS coronavirus or novel influenza viruses.Citation44

Sputum is also a non-invasive specimen that has been used for the detection of respiratory viruses, especially in patients with pneumonia.Citation17, Citation19, Citation22 However, many patients with respiratory virus infection do not have sputum production or cannot expectorate good quality sputum. By contrast, saliva specimens can be obtained much more easily than sputum specimens.

DFA is routinely used in our clinical setting for the diagnosis of respiratory virus infections. The advantages of DFA include a relatively low cost, rapid results and simultaneous detection of multiple viral pathogens. However, the sensitivity of DFA is low compared with molecular assays. During the 2009 influenza pandemic, the sensitivity of DFA ranged from 39% to 93%.Citation5 DFA also has a low sensitivity for influenza A H7N9 virus infection.Citation40 Another disadvantage of DFA is that it is not suitable for saliva specimens, because DFA can only detect viral antigens that are present inside infected cells, which are not present in the saliva. Therefore, to avoid bias between different tests, we used a multiplex PCR panel to test both the NPA and the saliva specimens. Although the multiplex PCR panel used in the second cohort (the NxTAG Respiratory Pathogen Panel) is more sensitive than DFA, it has lower sensitivity than real-time RT-PCR assays. The sensitivity of the NxTAG Respiratory Pathogen Panel ranged from 71.4% to 100% compared with real-time PCR or RT-PCR.Citation45 Therefore, some patients with respiratory virus infection may be missed even when NPA and saliva specimens are tested with this multiplex PCR panel.

This study has several limitations. First, our saliva collection method is not suitable for patients who cannot expectorate saliva, such as patients who are unconscious. For these patients, suction aspiration of saliva is required. Second, in the first cohort, we only evaluated viruses included in the routine DFA or influenza A virus RT-PCR. Other important respiratory viruses, such as rhinoviruses and coronaviruses, need to be further evaluated. Third, the number of specimens of each virus species is too small to compare the differences in sensitivity for specific virus species.

Although the viral load in the saliva is lower than the viral load in the NPA for most patients, we have shown that testing both expectorated saliva and NPA can significantly improve the detection of respiratory viruses compared with testing of NPA alone. Saliva should be obtained from patients with a suspected respiratory virus infection but a negative test for respiratory viruses. Furthermore, saliva should be evaluated as the specimen type in a diagnostic testing for novel respiratory viruses. This approach will ultimately lead to improvement in the management of patients and the prevention of community or nosocomial spread of infections.

Supplemental material

Supplementary Table S1

Download MS Word (53.5 KB)

Acknowledgments

We thank Polly KP Pang (Department of Medicine, the University of Hong Kong), Florence KY Mok (Department of Microbiology, the University of Hong Kong) and Patrick Li (Department of Microbiology, the University of Hong Kong) for their assistance with this study. This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster [UW 13-265]. This study was supported by the Respiratory Virus Research Foundation, Hong Kong University Foundation, Providence Foundation Limited in memory of the late Dr Lui Hac Minh and a donation from Larry Chi-Kin Yung.

Supplementary Information for this article can be found on the Emerging Microbes & Infections website (http://www.nature.com/emi)

References

  • Jain S, Self WH, Wunderink RGet al.Community-acquired pneumonia requiring hospitalization among US adults. N Engl J Med 2015;373: 415–427.
  • Jain S, Williams DJ, Arnold SRet al.Community-acquired pneumonia requiring hospitalization among US children. N Engl J Med 2015;372: 835–845.
  • Prina E, Ranzani OT, Torres A.Community-acquired pneumonia. Lancet 2015;386: 1097–1108.
  • Luyt CE, Brechot N, Chastre J.What role do viruses play in nosocomial pneumonia? Curr Opin Infect Dis 2014;27: 194–199.
  • Cheng VC, To KK, Tse H, Hung IF, Yuen KY.Two years after pandemic influenza A/2009/H1N1: what have we learned? Clin Microbiol Rev 2012;25: 223–263.
  • To KK, Chan JF, Chen H, Li L, Yuen KY.The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities. Lancet Infect Dis 2013;13: 809–821.
  • Chen Y, Liang W, Yang Set al.Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome. Lancet 2013;381: 1916–1925.
  • Pan M, Gao R, Lv Qet al.Human infection with a novel, highly pathogenic avian influenza A (H5N6) virus: virological and clinical findings. J Infect 2016;72: 52–59.
  • Chan JF, Lau SK, To KK, Cheng VC, Woo PC, Yuen KY.Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease. Clin Microbiol Rev 2015;28: 465–522.
  • Rao S, Nyquist AC.Respiratory viruses and their impact in healthcare. Curr Opin Infect Dis 2014;27: 342–347.
  • Dobson J, Whitley RJ, Pocock S, Monto AS.Oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials. Lancet 2015;385: 1729–1737.
  • Muthuri SG, Venkatesan S, Myles PRet al.Effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza A H1N1pdm09 virus infection: a meta-analysis of individual participant data. Lancet Respir Med 2014;2: 395–404.
  • Hung IF, To KK, Lee CKet al.Hyperimmune IV immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe 2009 influenza A(H1N1) infection. Chest 2013;144: 464–473.
  • Gelfer G, Leggett J, Myers J, Wang L, Gilbert DN.The clinical impact of the detection of potential etiologic pathogens of community-acquired pneumonia. Diagn Microbiol Infect Dis 2015;83: 400–406.
  • Baron EJ, Miller JM, Weinstein MPet al.A guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2013 recommendations by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM)(a). Clin Infect Dis 2013;57: e22–e121.
  • World Health Organization.Clinical management of human infection with pandemic (H1N1) 2009: revised guidance. Available at http://www.who.int/csr/resources/publications/swineflu/clinical_management_h1n1.pdf?ua=1 (accessed on 22 December 2015).
  • Ruuskanen O, Lahti E, Jennings LC, Murdoch DR.Viral pneumonia. Lancet 2011;377: 1264–1275.
  • Kim YG, Yun SG, Kim MYet al.Comparison between saliva and nasopharyngeal swab specimens for detection of respiratory viruses by multiplex reverse transcription-PCR. J Clin Microbiol 2016;55: 226–233.
  • 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: 2122–2127.
  • Azadeh N, Sakata KK, Brighton AM, Vikram HR, Grys TE.FilmArray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples. J Clin Microbiol 2015;53: 3784–3787.
  • To KK, Song W, Lau SYet al.Unique reassortant of influenza A(H7N9) virus associated with severe disease emerging in Hong Kong. J Infect 2014;69: 60–68.
  • Yu L, Wang Z, Chen Yet al.Clinical, virological, and histopathological manifestations of fatal human infections by avian influenza A(H7N9) virus. Clin Infect Dis 2013;57: 1449–1457.
  • Ramirez P, Valencia M, Torres A.Bronchoalveolar lavage to diagnose respiratory infections. Semin Respir Crit Care Med 2007;28: 525–533.
  • To KK, Wong SC, Xu Tet al.Use of nasopharyngeal aspirate for diagnosis of pneumocystis pneumonia. J Clin Microbiol 2013;51: 1570–1574.
  • White MR, Helmerhorst EJ, Ligtenberg Aet al.Multiple components contribute to ability of saliva to inhibit influenza viruses. Oral Microbiol Immunol 2009;24: 18–24.
  • Bilder L, Machtei EE, Shenhar Y, Kra-Oz Z, Basis F.Salivary detection of H1N1 virus: a clinical feasibility investigation. J Dent Res 2011;90: 1136–1139.
  • Goff J, Rowe A, Brownstein JS, Chunara R.Surveillance of acute respiratory infections using community-submitted symptoms and specimens for molecular diagnostic testing. PLoS Curr 2015;7: pii: ecurrents.outbreaks.0371243baa7f3810ba1279e30b96d3b6.
  • Robinson JL, Lee BE, Kothapalli S, Craig WR, Fox JD.Use of throat swab or saliva specimens for detection of respiratory viruses in children. Clin Infect Dis 2008;46: e61–e64.
  • Sueki A, Matsuda K, Yamaguchi Aet al.Evaluation of saliva as diagnostic materials for influenza virus infection by PCR-based assays. Clin Chim Acta 2016;453: 71–74.
  • Charlson ME, Pompei P, Ales KL, MacKenzie CR.A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 1987;40: 373–383.
  • To KK, Lee KC, Wong SSet al.Lipid metabolites as potential diagnostic and prognostic biomarkers for acute community acquired pneumonia. Diagn Microbiol Infect Dis 2016;85: 249–254.
  • Hung IF, Cheng VC, Wu AKet al.Viral loads in clinical specimens and SARS manifestations. Emerg Infect Dis 2004;10: 1550–1557.
  • To KK, Hung IF, Li IWet al.Delayed clearance of viral load and marked cytokine activation in severe cases of pandemic H1N1 2009 influenza virus infection. Clin Infect Dis 2010;50: 850–859.
  • To KK, Chan KH, Li IWet al.Viral load in patients infected with pandemic H1N1 2009 influenza A virus. J Med Virol 2010;82: 1–7.
  • Chan KH, Yam WC, Pang CMet al.Comparison of the NucliSens easyMAG and Qiagen BioRobot 9604 nucleic acid extraction systems for detection of RNA and DNA respiratory viruses in nasopharyngeal aspirate samples. J Clin Microbiol 2008;46: 2195–2199.
  • Siegel JD, Rhinehart E, Jackson M, Chiarello L, Health Care Infection Control Practices Advisory C.2007 Guideline for isolation precautions: preventing transmission of infectious agents in health care settings. Am J Infect Control 2007;35 (10 Suppl 2): S65–164.
  • Kumar S, Henrickson KJ.Update on influenza diagnostics: lessons from the novel H1N1 influenza A pandemic. Clin Microbiol Rev 2012;25: 344–361.
  • Somerville LK, Ratnamohan VM, Dwyer DE, Kok J.Molecular diagnosis of respiratory viruses. Pathology 2015;47: 243–249.
  • Chen JH, Lam HY, Yip CCet al.Clinical evaluation of the new high-throughput Luminex NxTAG respiratory pathogen panel assay for multiplex respiratory pathogen detection. J Clin Microbiol 2016;54: 1820–1825.
  • Chan KH, To KK, Chan JFet al.Assessment of antigen and molecular tests with serial specimens from a patient with influenza A(H7N9) infection. J Clin Microbiol 2014;52: 2272–2274.
  • Chan JF, Choi GK, Tsang AKet al.Development and evaluation of novel real-time reverse transcription-PCR assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses. J Clin Microbiol 2015;53: 2722–2726.
  • Li IW, Hung IF, To KKet al.The natural viral load profile of patients with pandemic 2009 influenza A(H1N1) and the effect of oseltamivir treatment. Chest 2010;137: 759–768.
  • Hansen KB, Westin J, Andersson LM, Lindh M, Widell A, Nilsson AC.Flocked nasal swab versus nasopharyngeal aspirate in adult emergency room patients: similar multiplex PCR respiratory pathogen results and patient discomfort. Infect Dis (Lond) 2016;48: 246–250.
  • Centre for Health Protection. Recommended Personal Protective Equipment (PPE) in hospitals/clinics for suspected or confirmed cases with Middle East Respiratory Syndrome (MERS) under different response levels. Available at http://www.chp.gov.hk/en/guideline/11/35.html (accessed on 17 November 2015).
  • Esposito S, Scala A, Bianchini Set al.Partial comparison of the NxTAG respiratory pathogen panel assay with the Luminex xTAG respiratory panel fast assay V2 and singleplex real-time polymerase chain reaction for detection of respiratory pathogens. Diagn Microbiol Infect Dis 2016;86: 53–57.