Published online August 25, 2008
PEDIATRICS Vol. 122 No. 3 September 2008, pp. e615-e620 (doi:10.1542/peds.2008-0691)
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ARTICLE

Comparing Nose-Throat Swabs and Nasopharyngeal Aspirates Collected From Children With Symptoms for Respiratory Virus Identification Using Real-Time Polymerase Chain Reaction

Stephen B. Lambert, MBBSa,b, David M. Whiley, PhDa,b, Nicholas T. O'Neill, BNursa, Emily C. Andrews, BNursa, Fiona M. Canavan, BNursa, Cheryl Bletchly, PhDc, David J. Siebert, MBBSc, Theo P. Sloots, PhDa,b,c,d and Michael D. Nissen, MBBSa,b,c,d

a Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Royal Children's Hospital and Health Service District, Herston, Queensland, Australia
b Clinical Medical Virology Centre
d Department of Paediatrics and Child Health, University of Queensland, Brisbane, Queensland, Australia
c Microbiology Division, Clinical and Statewide Services Division, Pathology Queensland, Royal Brisbane Hospital Campus, Herston, Queensland, Australia


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
OBJECTIVES. The objective of this study was to calculate sensitivity values for the detection of major respiratory viruses of childhood by using combined nose-throat swabs and nasopharyngeal aspirates.

METHODS. Children who had symptoms and presented to a pediatric teaching hospital and had a diagnostic respiratory specimen collected were enrolled, and paired nose-throat swab and nasopharyngeal aspirate specimens were collected. Parents were asked to collect the nose-throat swab specimen in the first instance but could defer to a health care worker if unwilling. Nose-throat swab collectors were asked to rate perceived quality of collection. All nasopharyngeal aspirates were collected by a health care worker by using a standard protocol. Real-time polymerase chain reaction for 8 respiratory viruses was performed in our hospital's diagnostic laboratory.

RESULTS. Paired nose-throat swab/nasopharyngeal aspirate specimens were collected during 303 illnesses, with at least 1 respiratory virus identified in 186 (61%). For the major pathogens of childhood, influenza A virus and respiratory syncytial virus, collection by using the nose-throat swab had a sensitivity of 91.9% and 93.1%, respectively. A health care worker collected 219 (72%) of the nose-throat swab specimens; concordance with the nasopharyngeal aspirate was not related to health care worker collection or perceived quality of collection.

CONCLUSIONS. Nose-throat swab specimens, in combination with sensitive molecular testing, are a less invasive diagnostic respiratory specimen with adequate sensitivity for use in the clinic and hospital outpatient settings and large-scale community studies through parent collection. For children who present to a hospital in which an avian or pandemic strain of influenza virus is reasonably part of the differential diagnosis, nasopharyngeal aspirates or a similar collection technique (eg, nasal washes) should continue to be used.

Key Words: respiratory viruses • real-time PCR • diagnostic methods • children

Abbreviations: NTS—nose-throat swab • NPA—nasopharyngeal aspirate • ARI—acute respiratory infection • PCR—polymerase chain reaction • RSV—respiratory syncytial virus • PIV—parainfluenza virus • CI—confidence interval • NAAT—nucleic acid–amplification test

In a number of areas, the use of more sensitive molecular diagnostics allows for the collection of a less invasive clinical specimen while increasing sensitivity of diagnosis for viruses and other fastidious organisms. Furthermore, there remains a relative absence of recent community epidemiology and transmission data by using molecular methods for the identification of respiratory viruses in the literature. This lack is highlighted by the need to use historical data about influenza transmission, collected without the benefit of sensitive molecular methods, to populate a mathematical model for containment interventions in the event of an influenza pandemic.1

We previously conducted a community-based cohort study to examine the epidemiology and costs of common respiratory viruses in preschool-aged children.2–4 The specimen type used in that study was a parent-collected combined nose-throat swab (NTS). It was beyond the scope of that study to include a head-to-head comparison of the NTS with nasopharyngeal aspirates (NPAs) for virus detection. With this study, we sought to fill that gap and determine the clinical utility of a less invasive specimen by comparing sensitivity values for common viruses by using NTS specimens and NPAs collected from children who had acute respiratory illnesses (ARIs) and presented to a pediatric teaching hospital.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
We sought to enroll any child who presented to our hospital who had an ARI and for whom an NPA was judged to be clinically indicated and collect a combined NTS at the same time. NTS specimens were collected before NPAs, with the throat swab collected first followed by an anterior nares swab from a single nostril. No instruction was provided regarding the use of different nares for the NPA and NTS collection. Both the nose and throat swabs (Dryswab woodstick shaft in labeled tube [Medical Wire & Equipment, Corsham, Wiltshire, England]) were pooled in a single tube of 1.8 mL of viral transport medium (bioMérieux, Baulkham Hills, New South Wales, Australia). We asked parents to collect the NTS in the first instance, but when they were unwilling or unable to do so but were happy for the extra specimen to be collected, a clinical staff member did this. NTS collectors reported the perceived quality of their collection technique as very good, good, or poor. These categories were assessed entirely subjectively by collectors without additional instruction. We collected basic demographic and illness information at the time of collection. All NPAs were collected by clinical staff in accordance with a standard protocol. All specimens were transported and processed along with other routine diagnostic material.

Since 2004, the onsite pathology service for our hospital (Molecular Diagnostic Unit, Clinical and Statewide Services Division, Pathology Queensland, Herston, Queensland, Australia) has routinely conducted real-time polymerase chain reaction (PCR) testing on nasopharyngeal aspirates and other respiratory specimens for 8 respiratory viruses: influenza A, influenza B, respiratory syncytial virus (RSV), parainfluenza virus I (PIV I), PIV II, PIV III, adenovirus, and human metapneumovirus. The assays for influenza B, PIV I, and PIV II were developed in our laboratory and have been validated for use in a routine diagnostic setting (unpublished data). Testing was performed using the Qiagen One-Step RT-PCR Kit (Qiagen, Doncaster, Victoria, Australia) in 4 PCRs that comprised 3 multiplex reactions and 1 monoplex reaction (Table 1). Amplification and detection were performed on an ABI7500 instrument (Applied Biosystems, Scoresby, Victoria, Australia). As well as a positive control for each of the targeted viruses, each test run included 3 no-target controls that consisted of reaction mixture and 5 µL of PCR-grade water. Test runs were assumed to be contaminated and deemed invalid when any of the 3 no-target controls produced positive amplification curves. Our laboratory does not routinely perform viral culture or antigen detection for respiratory viruses, and these tests were not done in parallel with molecular testing for this study.


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  		TABLE 1 Details of PCR assays used to identify respiratory viruses

 
The specificity of real-time PCR for any virus by using either an NPA or an NTS specimen is likely to be 100% or very close to this value.5 Given this, similar to previous studies,6 we used a consensus standard to assess sensitivity of each testing method: we considered a specimen positive by either method for any virus to represent a true-positive. This approach means that the specificity of either specimen type for any virus will be, by definition, 100%. Using this standard, we calculated sensitivity with 95% confidence intervals (CIs) using Stata 9.2 for Windows (Stata Corp, College Station, TX). Ethics committee approval for the study was granted by the Royal Children's Hospital and Health Services District Ethics Committee, and informed consent was obtained from a parent or guardian before study participation.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Between July 2006 and August 2007, we collected 303 sets of paired NPA/NTS specimens, with most collected during our winter/spring seasons. These specimens were collected from 295 children: 6 children provided 2 paired specimens, and 1 child provided 3 paired specimens. Study participants were more likely to be male (189; 62%), and most were from younger age groups: 136 (45%) <12 months; 130 (43%) 12 months to <5 years; 27 (9%) 5 years to <10 years; and 7 (2%) ≥10 years. The median age of study children was 1.1 years (range: 5 days to 17.5 years). Out-of-home child care or school was reported for 141 (37%) children. The emergency department was the site of collection for 284 (94%) of the paired specimens. Hospital admission occurred for 124 (41%) presentations, with a mean length of stay of 3.9 days (median: 3 days; range: 1–19 days).

The median delay between paired specimen collection and testing was 16 hours. PCR results for 270 (89%) paired specimens were concordant, with the same result in the NPA and NTS specimens (Table 2). By either collection method, there was at least 1 virus detected in 186 ARIs (61%): 163 with 1 virus and 23 with 2 viruses. This left 117 (39%) ARIs with no virus identified in either specimen.


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  		TABLE 2 NPA and NTS Concordance

 
For most viruses, the sensitivity of NPAs was equal to or higher than that for the NTS specimens (Table 3). The only virus for which this was not the case was influenza B: for 1 ARI of 10 for which influenza B was identified, the NPA was negative and the NTS positive. For the major respiratory viruses of childhood, influenza A and RSV, the sensitivity of NTS specimens was 91.9% and 93.1%, respectively. For adenoviruses, NTS specimens had a markedly lower sensitivity than NPAs: 65.9% compared with 93.2%.


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  		TABLE 3 Sensitivity for the Detection of Different Viruses According to Specimen Type

 
A health care worker collected the NTS specimen in 219 (72%) instances. The remaining 84 NTS specimens were collected by a parent (mother: 66; father: 16), grandmother (1), and interestingly, self by one 10-year-old. The concordance between NPA and NTS specimens for health care workers was 89% and for non–health care workers was 88% (P = .73). We asked the NTS collectors to report their perceived quality of specimen collection. Concordance for NPA and NTS specimens by reported quality of NTS collection was 84% for very good (109 of 129), 92% for good (154 of 167), and 100% (7 of 7) for poor.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
This study provides sensitivity values for NPA and NTS specimens for the identification of respiratory viruses by using a real-time PCR method. The findings demonstrate that for the major viruses of childhood, influenza A and RSV, sensitivity using an NTS specimen was >90%. It confirms previous data that showed that non–health care workers with simple training can collect the minimally invasive NTS specimen2 and that concordance with the more sensitive NPA specimen did not seem to be related to collector perception of quality.4

There are a number of clinical implications of this work. Depending on the setting, the NTS specimen could reasonably replace the NPA as an outpatient procedure for children who are not being admitted to hospital (59% of our study sample). For children who are being admitted to hospital, who may occasionally end up in intensive care, may receive unnecessary antibiotic therapy, or be subject to additional diagnostic, interventions after an initially negative PCR result, we believe an NPA, because of its improved sensitivity, is still the collection method of choice. The data from this study informed pandemic influenza planning at our hospital: with an ~8% likelihood of a false-negative result for children who presented to the hospital and had an influenza A infection, the NTS was not considered a suitable test where an avian (or pandemic) strain virus was reasonably part of the differential diagnosis; however, during a pandemic, home-based self- or parent-collected respiratory specimens that are transported to a central laboratory could provide adequate diagnostic sensitivity and allow for improved infection control and a reduction in community-based transmission by decreasing exposure of infected individuals to others in health care facilities.

Home-collected respiratory specimens, either parents' collecting from children or adults' collecting from themselves or each other, combined with sensitive molecular methods provide the means to collate previously unavailable data about viral infections and their household transmission. Reports from the Seattle Virus Watch project conducted in the second half of last century described home collection of respiratory specimens by a trained parent or other household member, when a study nurse could not arrange a home visit.7 In the articles from this project, there is no additional analysis or discussion about the quality of home specimen collection or success in terms of virus detection. A recent study8 reported in an article from Finland demonstrated the utility of parent-collected and self-collected nose swabs for the identification of rhinoviruses in children who were hospitalized with a positive rhinovirus PCR result and their household contacts. In that study, parent-collected nasal specimens were used to demonstrate the simultaneous presence in households of different rhinovirus strains in individuals with and without symptoms. There is also some evidence to suggest that using parent collection at home is more likely to result in a specimen's being collected, compared with requiring parents to be available for a home visit or to present for a clinic visit with a child with symptoms.9 This study showed that although parent collection and home-visited households identified a similar number of symptomatic periods during the study (47 and 49, respectively), these illness periods in parent collection households were approximately twice as likely to result in specimen collection, although this finding did not reach statistical significance (43% and 24%, respectively; P = .07). Parent-collected specimens may also be more likely to be positive for a virus by virtue of being collected at an earlier point in illness.10 Home-collected specimens were positive for any virus 80% of the time, compared with 67% of clinic-collected specimens (P = .44).9 In our previous community-based study of preschool-aged children, we received a specimen from a previously trained parent in 74% of ARIs identified2 but were able to convince only 28% of parents in this study to collected an NTS specimen. The proximity of a health care worker may have reduced the likelihood of parent collection. It may also be the case that our parent collection results are biased as a result of self-selection, with only confident parent collectors volunteering; however, in this and the previous study,2 we found that self-report of collection quality did not seem to be associated with the likelihood of virus detection. Use of parent collection techniques could fill gaps in knowledge about virus epidemiology and provide more robust and timely parameter estimates for use in event models, such as for an influenza pandemic.

Nucleic acid–amplification tests (NAATs) may overcome previously documented issues of specimen type and RSV identification.6,11 Both of these studies showed an approximately one-third reduction in sensitivity for RSV detection comparing a less invasive collection method (nasal swabs) with NPAs when an antigen detection technique was used. Despite this, the authors of the Guinea-Bissau article considered that for larger epidemiologic studies, the benefits of reduced costs and the ability to collect from a population-based sample might outweigh this lower sensitivity.6 Improved sensitivity has been seen using less invasive and self-collected specimens in combination with NAATs for sexually transmitted infections.12–14 Our data show that the wide differential in sensitivity for RSV as a result of specimen type seems to be largely overcome by the use of molecular methods.

Adenoviruses were the single virus group responsible for the highest proportion of discordant findings as a result of the decreased sensitivity of virus detection seen using the NTS specimen. Our results do not provide any clarification as to why this discordance is seen. These findings may be attributable to the different nature of adenoviruses (nonenveloped, DNA viruses) compared with the other respiratory viruses that we tested for, all of which are RNA viruses. They may also fit with the concept that the more invasive the respiratory specimen, the more likely a positive PCR result represents adenoviral persistence,15 rather than an acute infection.4 More work is needed to assess properly the predictive value of a positive adenovirus PCR result from a variety of respiratory specimens for detecting acute adenoviral infections, particularly in children.16

Picornaviruses are not part of the routine panel that our hospital's diagnostic laboratory uses for respiratory specimens. There remains some uncertainty about the meaning of a positive rhinovirus PCR result in children with respiratory symptoms.17–19 It is possible that if PCR testing for picornaviruses or rhinoviruses were used, approximately half of the virus-negative ARIs in our study would have been positive.2 Our previous work showed that picornaviruses were identified with a similar frequency in nose-only swabs and combined NTS specimens collected from the same population.4


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study provides comparative sensitivity values of NTS specimens and NPAs for common respiratory viruses. These values confirm that NTS specimens combined with PCR are suitable for diagnostic purposes in clinic and outpatient settings, as well as in community-based studies that are designed to provide current data about the epidemiology and burden of respiratory viruses; however, when a child is unwell enough to require hospital admission or where avian or a pandemic strain of influenza is reasonably suspected on the basis of history, we believe that an NPA is still the test of choice. Just as with sexually transmitted infections, using NAATs for respiratory virus detection seems to overcome the previously observed sensitivity reduction seen when less invasive specimens were combined with conventional, non–amplification-based laboratory tests.


   ACKNOWLEDGMENTS
 
Dr Lambert received a clinical fellowship funded by the Royal Children's Hospital Foundation to conduct this research.

We thank the parents and children who volunteered to participate in this study. The study would not have been possible without the excellent support from clinical staff from the emergency department and Paterson and Robertson wards at the Royal Children's Hospital, Brisbane, and the laboratory staff from the Molecular Diagnostic Unit, Clinical and Statewide Services Division, Pathology, Queensland. Aaron Buckner, Ria Halstead, Lisa Mulhearn, and Jane Yunus from the Queensland Pediatric Infectious Diseases Laboratory provided clinical and administrative support for this study.


   FOOTNOTES
 
Accepted Apr 30, 2008.

Address correspondence to Stephen Lambert, MBBS, Queensland Paediatric Infectious Diseases Laboratory, Royal Children's Hospital, Herston Queensland 4029, Australia. E-mail: sblambert{at}uq.edu.au

The authors have indicated they have no financial relationships relevant to this article to disclose.


What's Known on This Subject

There is little published information about the use of less-invasive respiratory specimens for clinical diagnosis of viruses by using sensitive molecular methods. These methods are also being used in clinical and community-based research without comparative sensitivity data.

 

What This Study Adds

This study provides the first sensitivity values for this less-invasive specimen type in comparison with nasopharyngeal aspirates. These data have direct clinical application and will be useful for influenza pandemic planning and in planning community-based research by using parentally collected specimens.

 


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics




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