Abstract
Aim
This hospital network‐based retrospective observational study aimed to describe the prevalence and seasonality of paediatric and adult viral respiratory pathogens and their rates of co‐infections, following the introduction of a rapid multiplex molecular diagnostic assay.
Methods
All nasopharyngeal samples tested in patients presenting to Monash Health, Melbourne, Australia, from August 2009 to July 2015 by means of multiplex tandem polymerase chain reaction using the Respiratory Pathogen 12Plex kit (AusDiagnostics) were included in the analysis.
Results
There were 28 729 patient samples analysed after duplicate samples were excluded. Positive results were twice as likely in paediatrics, 7573/11 491 (65.9%), compared to adults, 5410/17 238 (31.4%). Co‐infection was more frequent in paediatrics, 1642/7573 (21.7% of positives), compared to adults 299/5410 (5.5%). Adenovirus had a high prevalence as a co‐infection, 639/990 (64.5%), in paediatrics. Testing frequency increased by 179% in the paediatric group and by 949% for adults over the 6 years of observation.
Conclusions
This study demonstrated a significant difference in the positive detection rate of pathogens and co‐infections between the population groups. Adenovirus had a surprisingly high prevalence as a co‐infection, especially in paediatric patients. Over the study period, rapid uptake of the test was observed, especially in adults. This raises concerns about how we can ensure that testing remains rational and is able to be provided in a cost‐effective manner in the future.
Keywords: co‐infection, paediatric, polymerase chain reaction, respiratory, viral
What is already known on this topic
Respiratory tract infections are the most common cause for hospital admission in young children.
Viral pathogens can account for up to 50% of community‐acquired pneumonia in children.
Rapid molecular diagnostics allow for the easy detection of viral pathogens, including multiple pathogens.
What this paper adds
There is a high prevalence of adenovirus in co‐infections in paediatric population.
Although low overall, there is also a high prevalence of adenovirus in co‐infection amongst adults.
The uptake rate of rapid molecular testing over 6 years since its introduction has been small in paediatrics compared to in the adult cohort.
Respiratory tract infections (RTI) are the most common cause of hospitalisation in young children.1, 2 Viral pathogens can account for greater than 50% of community‐acquired pneumonia (CAP) in children,2, 3 with viral aetiologies being attributed to up to 30% of adults admitted with CAP,4 a condition with an overall mortality of 3%.5 This is significant given that over 15–20% of severe CAP cases in adults have been attributed to influenza alone.5, 6
The increased availability of rapid molecular diagnostics for multiple respiratory viruses has allowed for the easier identification of viral pathogens.7 The use of amplification during these diagnostic assays enables the detection of even very low levels of virus, providing a high sensitivity.8, 9 There has been a resultant rapid uptake of testing in recent years, largely replacing less sensitive and/or more labour‐intensive culture and antigen detection methodologies. Given the emergence of improved therapies for viral respiratory disease, identification of the relative contribution of viruses to RTI presentations may aid in both clinical management and informing the health system of the prevalence of these pathogens.
We aimed to describe the frequency of viral respiratory pathogen detection in both paediatric and adult populations at our health network following the implementation of a respiratory multiplex polymerase chain reaction (PCR) assay. Analysis was conducted to describe the prevalence of co‐infections as the improved sensitivity and increased use of multiplex analysis has led to the increased detection of more than one potential pathogen in a single sample.10, 11
Methods
Setting
The study was conducted at Monash Health, Melbourne, Australia, a 2100 inpatient bed hospital regional network, incorporating Monash Children's Hospital and three emergency departments. This encompasses a 2312 km2 catchment region in the southeast of Melbourne. Monash Health services a greater community of 1.3 million residents, covering approximately 24% of Victoria's population, with secondary and tertiary hospitals for both children and adults within the network. All patients who were tested using respiratory multiplex PCRs were included: paediatric was defined as younger than 18 years of age, and adult was defined as 18 years and over.
Data collection
All respiratory multiplex PCR results were retrospectively extracted from the Monash Pathology laboratory information system spanning a 6‐year period from August 2009 to July 2015. Only nasopharyngeal samples tested for all 10 respiratory pathogens on the multiplex panel were included in the study.
Laboratory testing
Nasopharyngeal samples were collected as aspirates or swabs by using mini‐tipped flocked swabs containing a universal transport medium (Interpath, Melbourne, Australia). Total nucleic acid extraction was performed using the NucliSens easyMAG platform according to the instructions of the manufacturer (bioMerieux, Marcy l'Etoile, France), eluting 200 μL of sample into 50 μL of elution buffer, and then tested using multiplexed tandem PCR (MT‐PCR) and a liquid‐handling robotics system as previously described12 (AusDiagnostics, Melbourne, Australia) using the Respiratory Pathogen 12Plex kit, which detects: influenza A (including H1N1 2009 influenza A), influenza B, respiratory syncytial virus (RSV), picornavirus (human rhinoviruses and human enteroviruses), parainfluenza 1, parainfluenza 2, parainfluenza 3, adenovirus (human types 1, 2, 3, 4, 5, 6, 7 and 8), human metapneumovirus and the bacteria Bordetella pertussis.
Definitions
The following inclusion and exclusion criteria were applied to the dataset to account for duplicate samples:
Any patient with a positive result and a repeat sample within 14 days that demonstrated the same result had the later results excluded for the main analysis.
If the repeat sample demonstrated the same viral pathogen as well as a new viral pathogen, then the repeat result was disregarded; however, the new positive result was included as a co‐infection.
If the repeat sample within 14 days demonstrated a different pathogen from the initial sample, then both results were included as individual cases.
If multiple tests were performed within the same day, then a single result was included, and all others excluded; if both a positive and a negative result were detected from the same patient on the same day, then the positive sample was included.
For definition purposes, a positive sample designated the beginning of the infection period. Beyond a 14‐day period, any repeat test was considered a new sample regardless of result. Single infection was defined as the detection of only one virus from a sample, and co‐infection was defined as the detection of two or more pathogens from the single sample.
Data analysis and statistics
Descriptive analysis was performed using Microsoft Excel 2013 (Microsoft, Redmond, CA, USA). A two‐sample test of proportions was performed using Stata 13 (StataCorp, College Station, TX, USA).
The number and percentage for each pathogen detected, and its co‐infection, were determined. Detection rates for each pathogen and rates of co‐infection were determined. A comparison between the paediatric and adult data for each pathogen was made, including pathogens more commonly isolated in each group.
Ethical approval
We state that the protocol for this quality assurance project was approved by the Monash Health Ethics Committee 11274Q.
Informed consent
As this study was a retrospective extraction of non‐identifiable data from the Monash Health pathology system, informed consent was not obtained from individual patients. The study was conducted with the Monash Health Human Research Ethics Committee approval, as mentioned above.
Results
A total of 28 729 patient samples were analysed following the removal of duplicate samples. Positive results (45.2%) were detected in 7573 paediatric samples (65.9%) and 5410 (31.4%) adult samples (Fig. 1). The most common pathogen detected was picornavirus, with the next most common pathogen being RSV in paediatrics and influenza A in adults (Table 1).
Figure 1.
Analysed respiratory multiplex polymerase chain reaction (PCR) samples obtained from Monash Health, Melbourne, Australia from August 2009 to July 2015.
Table 1.
Individual respiratory pathogen results for all samples, adult and paediatric data, at Monash Health, Melbourne, Australia, from August 2009 to July 2015
| Respiratory pathogen | Paediatric, n (%) | Adult, n (%) |
|---|---|---|
| Influenza A | 467 (4.1) | 1214 (7.0) |
| Influenza B | 263 (2.3) | 430 (2.5) |
| Respiratory syncytial virus | 2038 (17.7) | 664 (3.9) |
| Picornavirus | 4069 (35.4) | 2203 (12.8) |
| Parainfluenza 1 | 134 (1.2) | 57 (0.3) |
| Parainfluenza 2 | 85 (0.7) | 52 (0.3) |
| Parainfluenza 3 | 555 (4.8) | 294 (1.7) |
| Adenovirus | 990 (8.6) | 207 (1.2) |
| Human metapneumovirus | 513 (4.5) | 464 (2.7) |
| Bordetella pertussis | 437 (3.8) | 139 (0.8) |
| Any respiratory pathogen | 7573 (65.9) | 5410 (31.4) |
The seasonality of each pathogen is displayed for each group in Figure 2. Paediatrics showed a consistently high peak for RSV around June, while influenza A was the most prevalent in adults and was seen to peak around September. There was no seasonality noted for the picornavirus amongst either paediatrics or adults.
Figure 2.
Seasonality of detected respiratory pathogen: (a) Paediatric, (b) adult and (c) paediatric and adult picornavirus. (
), Influenza A; (
), influenza B; (
), respiratory syncytial virus; (
), parainfluenza 1; (
), parainfluenza 2; (
), parainfluenza 3; (
), adenovirus; (
), human metapneumovirus; (
), Bordetella pertussis. c: (
), Paediatric picornavirus; (
), adult picornavirus.
The detection of multiple respiratory pathogens was common as shown in Figure 3. There were 1454 paediatric and 284 adult samples that identified two pathogens; the most common combination for both groups was RSV and picornavirus. Amongst adults, this was closely followed by influenza A and picornavirus (Table 2). There were 179 paediatric samples with three pathogens (10.9%), yielding 35 different pathogen combinations. The most common three‐pathogen combination was RSV, adenovirus and picornavirus. There were nine samples with four pathogens (0.5%) detected, with eight different pathogen combinations. There were only 15 adult samples with three pathogens (5.0%), yielding nine different pathogen combinations. The majority of adenovirus detected in paediatrics was seen in the setting of other pathogens, 639 (64.5%), with adults also having a considerable proportion detected as a co‐infection, 55 (26.6%).
Figure 3.
Comparison of prevalence of respiratory pathogen detection as co‐infection between paediatrics and adults. (), Adults; (), paediatrics. HMPV, human metapneumovirus; RSV, respiratory syncytial virus.
Table 2.
Co‐infections with two respiratory pathogens only in paediatrics and adults presenting to Monash Health, Melbourne, Australia, from August 2009 to July 2015
| Paediatrics co‐infections | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Influenza A | Influenza B, n | RSV, n | Picornavirus, n | Parainfluenza 1, n | Parainfluenza 2, n | Parainfluenza 3, n | Adenovirus, n | HMPV, n | Bordetella pertussis, n | |
| Influenza A | NA | 0 | 21 | 36 | 0 | 0 | 6 | 19 | 11 | 6 |
| Influenza B | NA | 12 | 19 | 0 | 0 | 1 | 7 | 2 | 2 | |
| RSV | NA | 400 | 4 | 1 | 20 | 91 | 22 | 18 | ||
| Picornavirus | NA | 30 | 16 | 108 | 319 | 92 | 95 | |||
| Parainfluenza 1 | NA | 0 | 0 | 3 | 1 | 1 | ||||
| Parainfluenza 2 | NA | 1 | 5 | 1 | 0 | |||||
| Parainfluenza 3 | NA | 39 | 6 | 5 | ||||||
| Adenovirus | NA | 22 | 5 | |||||||
| HMPV | NA | 7 | ||||||||
| B. pertussis | NA | |||||||||
| Adult co‐infections | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Influenza A | Influenza B, n | RSV, n | Picornavirus, n | Parainfluenza 1, n | Parainfluenza 2, n | Parainfluenza 3, n | Adenovirus, n | HMPV, n | B. pertussis, n | |
| Influenza A | NA | 0 | 13 | 45 | 0 | 2 | 2 | 5 | 13 | 8 |
| Influenza B | NA | 9 | 8 | 3 | 1 | 1 | 4 | 4 | 1 | |
| RSV | NA | 46 | 0 | 0 | 1 | 9 | 6 | 5 | ||
| Picornavirus | NA | 1 | 4 | 11 | 25 | 20 | 21 | |||
| Parainfluenza 1 | NA | 0 | 0 | 0 | 0 | 0 | ||||
| Parainfluenza 2 | NA | 0 | 1 | 0 | 1 | |||||
| Parainfluenza 3 | NA | 3 | 3 | 5 | ||||||
| Adenovirus | NA | 1 | 2 | |||||||
| HMPV | NA | 0 | ||||||||
| B. pertussis | NA | |||||||||
Co‐infections of three or more are not included in this data.
HMPV, human metapneumovirus; NA, not applicable; RSV, respiratory syncytial virus.
There was an increase in the number of samples performed over the observation period. In the final year (August 2014–July 2015), there were 2352 paediatric samples and 5988 adult samples analysed, compared with only 1314 paediatric and 631 adult samples in the first year (Table 3).
Table 3.
Comparison of frequency of testing, and rate of positivity, per annum over the 6‐year period from August 2009 to July 2015
| Year | Paediatric, n (%) | Adult, n (%) |
|---|---|---|
| 2009 | 1314 (69.1) | 631 (33.0) |
| 2010 | 1844 (68.7) | 1491 (37.0) |
| 2011 | 1836 (67.8) | 2365 (37.8) |
| 2012 | 1923 (63.1) | 2673 (30.8) |
| 2013 | 2222 (65.8) | 4090 (28.5) |
| 2014 | 2352 (62.7) | 5988 (29.5) |
| Total | 11 491 (65.9) | 17 238 (31.4) |
Each 12‐month period referenced refers to August–July.
Discussion
We report the results of a hospital network‐based study comparing the detection of viral pathogens in children and adults, presenting from the same background population, using respiratory multiplex PCR assays. Paediatric samples were more than twice as likely to yield a positive result (65.9 vs. 31.4%).
A rapid uptake in the frequency of testing was observed across our study period, especially in the adult population, with an almost 10‐fold increase in tests performed per annum when comparing across the 6 years. In children, a 79% increase was observed. Indications for testing include the ability to change patient management (either directly or for infection control reasons) or assisting communicable disease epidemiology. Respiratory testing is an uncomfortable procedure that incurs expense. The rapid uptake of testing over this period highlights the need for testing behaviours to remain rational and cost‐effective.13 In the setting of prolonged viral shedding long after clinical resolution of disease, a positive result may even disadvantage patients and health‐care providers due to unnecessary periods of respiratory virus isolation in infected inpatients. The ability to perform a test and obtain a positive result alone is insufficient reason to conduct respiratory testing.
The proportion of paediatric PCR‐positive respiratory samples is comparable with other studies with reported rates between 42.7 and 74%.2, 14, 15, 16 In the adult population, there is less available evidence for comparison that is not exclusive to specific sub‐populations or inclusive of paediatric patients. From available data, our proportion of positive samples (31.4%) appears to be comparable.17, 18, 19 Both the annual (17.7%) and seasonal (30.7%; May to August inclusive) detection rates of paediatric RSV infection are similar to other studies, with reported rates of 17.2–67.1%.2, 15, 20, 21, 22 The detection rate of adult influenza A infection (annual 7.0%; seasonal 9.0%) is consistent with other studies.17, 23 RSV had a higher rate amongst the paediatric population compared with adults, whereas influenza A was more likely to be detected amongst the adult population, a previously recognised observation.17
The most commonly identified pathogen in both groups was the picornavirus (48.3% of total positive results, 31.3% paediatrics and 17.0% adults), making it easily the most commonly identified pathogen in both population groups. Across numerous studies, the picornavirus remains the most common respiratory pathogen identified.3, 24, 25 Its presence also contributes to a significant proportion of co‐infections overall and nearly all co‐infections that contain three‐pathogen combinations. This raises the question of a possible role for the picornavirus in respiratory illness presentations in children and adults. This may be as a primary pathogen, a co‐factor for other viral or bacterial infections or as a trigger for non‐infective respiratory presentations such as asthma. It is important to recognise, however, that it is the high detection of the picornavirus amongst healthy individuals has been well described.26 However, recent concomitant blood and respiratory PCR detection of the rhinovirus in children with signs of lower RTI suggests it may play a role in up to one in six children with RTI who test positive for the rhinovirus.27
The incidence of co‐infections in paediatric positive results (21.7%) is marginally higher than described in other studies (11.3–20.6%),2, 22 with previous studies also demonstrating lower co‐infection rates in adults (5.5%).28 The high proportion of adenovirus infections presenting as a co‐infection has also been recently described amongst children with CAP.29 Although not described on this scale previously, the adenovirus is also present in a significant proportion of adult co‐infection.
Recently, there has been a large‐scale prospective study from southern China describing respiratory viral infections in children and adults, usually through PCR techniques.30 There was a different selection of viruses tested: influenza (A, B, C), RSV, adenovirus, human metapneumovirus, parainfluenza virus, human coronavirus and human bocavirus. Their rate of overall positive result (39.2%) was lower. Our identified rate of co‐infection was significantly higher (6.6 vs. 3.4% of all samples and 15.0 vs. 9.6% of positive samples), which may be explained by the differences in viruses tested, population factors or indications for testing.
Our multiplex PCR tested for 10 common respiratory pathogens and therefore introduced several limitations. We were unable to determine the individual contribution of enterovirus or rhinovirus as they were both reported as picornavirus based on the same 5′ target. There are many other respiratory viral pathogens that we did not test for (e.g. coronavirus, bocavirus, influenza C). Inclusion of these may help close the ‘diagnostic gap’ in 34.1% of paediatric and 68.6% of adult tests that were negative. Our study demonstrates relative reproducibility in seasonality in respiratory pathogens amongst those requiring presentation or admission to hospital. In the absence of correlating clinical data, it is not possible to conclude the presence of a respiratory pathogen as causative for clinical disease.
The high sensitivity of molecular diagnostic assays and their ability to detect low levels of virus have led to a high yield for testing, with up to 95% of children presenting with RTI undergoing respiratory viral pathogen detection.9 However, the interpretation of positive results is more complex. It may represent the causative pathogen for the presenting illness, prolonged shedding from a past infection or simply an asymptomatic infection. Since the introduction of this testing, asymptomatic carriage of respiratory pathogens on confirmed testing has been described at between 40 and 68%.9, 31, 32
The high prevalence of the picornavirus in co‐infections has been described previously23; however, as far as we know, this is the first time the high prevalence of the adenovirus has been documented. It does, however, raise the question regarding the clinical significance and burden of disease associated with those presenting with single‐pathogen detection versus co‐infection. With the advent of increasing respiratory viral therapeutics and vaccines in pre‐clinical and clinical development, our ability to understand the potential roles of many of these viruses is likely to deepen, from both clinical trials and post‐licensure data.
Conclusions
The ability of sensitive molecular assays has improved our ability to provide a potential explanation for their symptoms. However, old challenges remain and the high sensitivity of these tests raises new issues. The old challenge of whether a positive test regularly changes the clinical management of the patient tested remains under debate. The ability of the newer assays to detect non‐viable viral RNA or DNA adds to the complexity of interpreting positive results, and may help explain some of the co‐infections. Nevertheless, they continue to improve our ability to understand not only infectious diseases epidemiology but also the complex interaction of potential respiratory pathogens with adult and child hosts.
Conflict of interest: None declared.
References
- 1. Shay D, Holman R, Newman R, Liu L, Stout J, Anderson L. Bronchiolitis‐associated hospitalizations among US children, 1980–1996. JAMA 1999; 282: 1440–6. [DOI] [PubMed] [Google Scholar]
- 2. Bicer S, Giray T, Col D et al Virological and clinical characterizations of respiratory infections in hospitalized children. Ital. J. Pediatr. 2013; 39: 22. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Tsolia MN, Psarras S, Bossios A et al Etiology of community‐acquired pneumonia in hospitalized school‐age children: Evidence for high prevalence of viral infections. Clin. Infect. Dis. 2004; 39: 681–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Sangil A, Calbo E, Robles A et al Aetiology of community‐acquired pneumonia among adults in an H1N1 pandemic year: The role of respiratory viruses. Eur. J. Clin. Microbiol. Infect. Dis. 2012; 31: 2765–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Ishiguro T, Takayanagi N, Yamaguchi S et al Etiology and factors contributing to the severity and mortality of community‐acquired pneumonia. Intern. Med. 2013; 52: 317–24. [DOI] [PubMed] [Google Scholar]
- 6. Mermond S, Berlioz‐Arthaud A, Estivals M, Baumann F, Levenes H, Martin PMV. Aetiology of community‐acquired pneumonia in hospitalized adult patients in New Caledonia. Trop. Med. Int. Health 2010; 15: 1517–24. [DOI] [PubMed] [Google Scholar]
- 7. Munywoki PK, Hamid F, Mutunga M, Welch S, Cane P, Nokes DJ. Improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real‐time PCR using nasopharyngeal flocked swabs. J. Clin. Microbiol. 2011; 49: 3365–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Mahony JB. Detection of respiratory viruses by molecular methods. Clin. Microbiol. Rev. 2008; 21: 716–47. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Jartti T, Söderlund‐Venermo M, Hedman K, Ruuskanen O, Mäkelä MJ. New molecular virus detection methods and their clinical value in lower respiratory tract infections in children. Paediatr. Respir. Rev. 2013; 14: 38–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. de Vries M, Deijs M, Canuti M et al A sensitive assay for virus discovery in respiratory clinical samples. PLoS One 2011; 6: e16118. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Chen Y, Cui D, Zheng S et al Simultaneous detection of influenza A, influenza B, and respiratory syncytial viruses and subtyping of influenza A H3N2 virus and H1N1 (2009) virus by multiplex real‐time PCR. J. Clin. Microbiol. 2011; 49: 1653–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Szewczuk E, Thapa K, Anninos T et al Rapid semi‐automated quantitative multiplex tandem PCR (MT‐PCR) assays for the differential diagnosis of influenza‐like illness. BMC Infect. Dis. 2010; 10: 113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Gill PJ, Richardson SE, Ostrow O, Friedman JN. Testing for respiratory viruses in children: To swab or not to swab. JAMA Pediatr. 2017; 171: 798–804. [DOI] [PubMed] [Google Scholar]
- 14. Sung RYT, Chan PKS, Tsen T et al Identification of viral and atypical bacterial pathogens in children hospitalized with acute respiratory infections in Hong Kong by multiplex PCR assays. J. Med. Virol. 2009; 81: 153–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Pierangeli A, Gentile M, Di Marco P et al Detection and typing by molecular techniques of respiratory viruses in children hospitalized for acute respiratory infection in Rome, Italy. J. Med. Virol. 2007; 79: 463–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Blaschke AJ, Allison MA, Meyers L et al Non‐invasive sample collection for respiratory virus testing by multiplex PCR. J. Clin. Virol. 2011; 52: 210–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Verani JR, McCracken J, Arvelo W et al Surveillance for hospitalized acute respiratory infection in Guatemala. PLoS One 2013; 8: e83600. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Drieghe S, Ryckaert I, Beuselinck K, Lagrou K, Padalko E. Epidemiology of respiratory viruses in bronchoalveolar lavage samples in a tertiary hospital. J. Clin. Virol. 2014; 59: 208–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Kim JH, Moon BJ, Gong C‐H, Kim NH, Jang YJ. Detection of respiratory viruses in adult patients with perennial allergic rhinitis. Ann. Allergy Asthma Immunol. 2013; 111: 508–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Fuller DG, Davie G, Lamb D, Carlin JB, Curtis N. Analysis of respiratory viral coinfection and cytomegalovirus coisolation in pediatric inpatients. Pediatr. Infect. Dis. J. 2005; 24: 195–200. [DOI] [PubMed] [Google Scholar]
- 21. Hatipoglu N, Somer A, Badur S et al Viral etiology in hospitalized children with acute lower respiratory tract infection. Turk. J. Pediatr. 2011; 53: 508–16. [PubMed] [Google Scholar]
- 22. Khamis FA, Al‐Kobaisi MF, Al‐Areimi WS, Al‐Kindi H, Al‐Zakwani I. Epidemiology of respiratory virus infections among infants and young children admitted to hospital in Oman. J. Med. Virol. 2012; 84: 1323–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Pretorius MA, Madhi SA, Cohen C et al Respiratory viral coinfections identified by a 10‐plex real‐time reverse‐transcription polymerase chain reaction assay in patients hospitalized with severe acute respiratory illness – South Africa, 2009–2010. J. Infect. Dis. 2012; 206 (Suppl. 1): S159–65. [DOI] [PubMed] [Google Scholar]
- 24. Buecher C, Mardy S, Wang W et al Use of a multiplex PCR/RT‐PCR approach to assess the viral causes of influenza‐like illnesses in Cambodia during three consecutive dry seasons. J. Med. Virol. 2010; 82: 1762–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Cho GS, Moon BJ, Lee BJ et al High rates of detection of respiratory viruses in the nasal washes and mucosae of patients with chronic rhinosinusitis. J. Clin. Microbiol. 2013; 51: 979–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Peghin M, Hirsch HH, Len Ó et al Epidemiology and immediate indirect effects of respiratory viruses in lung transplant recipients: A 5‐year prospective study. Am. J. Transplant. 2017; 17: 1304–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Lu X, Schneider E, Jain S et al Rhinovirus viremia in patients hospitalized with community‐acquired pneumonia. J. Infect. Dis. 2017; 216: 1104–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Njouom R, Yekwa EL, Cappy P, Vabret A, Boisier P, Rousset D. Viral etiology of influenza‐like illnesses in Cameroon, January–December 2009. J. Infect. Dis. 2012; 206 (Suppl. 1): S29–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29. Jain S, Williams DJ, Arnold SR et al Community‐acquired pneumonia requiring hospitalization among U.S. children. N. Engl. J. Med. 2015; 372: 835–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Zhang D, He Z, Xu L et al Epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern China. Int. J. Infect. Dis. 2014; 25: 159–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Chonmaitree T, Alvarez‐Fernandez P, Jennings K et al Symptomatic and asymptomatic respiratory viral infections in the first year of life: Association with acute otitis media development. Clin. Infect. Dis. 2015; 60: 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Rhedin S, Lindstrand A, Rotzen‐Ostlund M et al Clinical utility of PCR for common viruses in acute respiratory illness. Pediatrics 2014; 133: e538–45. [DOI] [PubMed] [Google Scholar]