Comparative study of nasopharyngeal aspirate and nasal swab specimens for diagnosis of acute viral respiratory infection

Paired nasopharyngeal aspirate (NPA) and nasal swab (NS) samples from 475 children hospitalized for acute respiratory infection were studied for the detection of influenza virus, parainfluenza virus, respiratory syncytial virus, and adenovirus by immunofluorescence test, viral culture, and multiplex PCR assay. The overall sensitivity of viral detection with NPA specimens was higher than that obtained with NS specimens.     

Nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses

To determine the usefulness of nasal swabs as a simple method for detection of respiratory viruses, we compared nasal swabs and nasopharyngeal aspirates obtained at the same time from the opposite nostrils of 230 children with upper respiratory infection. The sensitivity of nasal swabs was comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses except respiratory syncytial virus.     

Rapid diagnosis of respiratory syncytial virus infection by using Pernasal swabs.

The sensitivity and specificity of direct immunofluorescence microscopy performed on Pernasal swab specimens and compared with those of nasopharyngeal aspirates were 93 and 99%, respectively. Posterior nasopharyngeal swabs applied immediately to microscope slides allow a rapid and simple screening procedure for the diagnosis of acute respiratory syncytial virus infections.     

Differential detection of rhinoviruses and enteroviruses RNA sequences associated with classical immunofluorescence assay detection of respiratory virus antigens in nasopharyngeal swabs from infants with bronchiolitis

To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT-PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. Respiratory syncytial viruses (A&B) were detectable in 45 of 84 (53.6%) nasopharyngeal aspirates from infants with bronchiolitis, whereas coronaviruses, influenza viruses, and parainfluenza viruses were not detectable in the same samples. Adenoviruses were detectable by PCR in 11 of 84 (13.1%) nasopharyngeal swabs. By using a picornavirus RT-PCR assay followed by a differential molecular hybridisation, rhinovirus and enterovirus RNA sequences were detected in 16 of 84 (19%) and in 10 of 84 (11.9%) of the nasopharyngeal swabs tested. Positive human rhinovirus or enterovirus RT-PCR assay, however, was the only evidence of respiratory infection in 8 of 84 (9.5%) and in 7 of 84 (8.33%) of the studied patients. Respiratory syncytial viruses, human rhinoviruses, adenoviruses, and enteroviruses occur in dual infections detected in 18 of 84 (21.4%) respiratory samples tested. The median duration of stay in hospital was not significantly different between the patients demonstrating a single viral infection and those with a dual viral infection (6.22 +/- 2.07 vs. 5. 04 +/- 0.95 days; P > 0.05). In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants.     

Swabbing for respiratory viral infections in older patients: a comparison of rayon and nylon flocked swabs

The purpose of this study was to compare the sampling efficacy of rayon swabs and nylon flocked swabs, and of oropharyngeal and nasopharyngeal specimens for the detection of respiratory viruses in elderly patients. Samples were obtained from patients 60 years of age or above who were newly admitted to Sorlandet Hospital Arendal, Norway. The patients were interviewed for current symptoms of a respiratory tract infection. Using rayon swabs and nylon flocked swabs, comparable sets of mucosal samples were harvested from the nasopharynx and the oropharynx. The samples were analysed using real-time polymerase chain reaction (PCR) methods. A total of 223 patients (mean age 74.9 years, standard deviation [SD] 9.0 years) were swabbed and a virus was recovered from 11% of the symptomatic patients. Regardless of the sampling site, a calculated 4.8 times higher viral load (95% confidence interval [CI] 1.3–17, p = 0.017) was obtained using the nylon flocked swabs as compared to the rayon swabs. Also, regardless of the type of swab, a calculated 19 times higher viral load was found in the samples from the nasopharynx as compared to the oropharynx (95% CI 5.4–67.4, p < 0.001). When swabbing for respiratory viruses in elderly patients, nasopharyngeal rather than oropharyngeal samples should be obtained. Nylon flocked swabs appear to be more efficient than rayon swabs.     

Non-invasive sample collection for respiratory virus testing by multiplex PCR

Background

Identifying respiratory pathogens within populations is difficult because invasive sample collection, such as with nasopharyngeal aspirate (NPA), is generally required. PCR technology could allow for non-invasive sampling methods.

Objective

Evaluate the utility of non-invasive sample collection using anterior nare swabs and facial tissues for respiratory virus detection by multiplex PCR.

Study design

Children aged 1 month–17 years evaluated in a pediatric emergency department for respiratory symptoms had a swab, facial tissue, and NPA sample collected. All samples were tested for respiratory viruses by multiplex PCR. Viral detection rates were calculated for each collection method. Sensitivity and specificity of swabs and facial tissues were calculated using NPA as the gold standard.

Results

285 samples from 95 children were evaluated (92 swab-NPA pairs, 91 facial tissue-NPA pairs). 91% of NPA, 82% of swab, and 77% of tissue samples were positive for ≥ 1 virus. Respiratory syncytial virus (RSV) and human rhinovirus (HRV) were most common. Overall, swabs were positive for 74% of virus infections, and facial tissues were positive for 58%. Sensitivity ranged from 17 to 94% for swabs and 33 to 84% for tissues. Sensitivity was highest for RSV (94% swabs and 84% tissues). Specificity was ≥95% for all viruses except HRV for both collection methods.

Conclusions

Sensitivity of anterior nare swabs and facial tissues in the detection of respiratory viruses by multiplex PCR varied by virus type. Given its simplicity and specificity, non-invasive sampling for PCR testing may be useful for conducting epidemiologic or surveillance studies in settings where invasive testing is impractical or not feasible.     

Simultaneous detection of human bocavirus and adenovirus by multiplex real-time PCR in a Belgian paediatric population

Since the discovery of human bocavirus (hBoV), the virus has been detected worldwide in respiratory tract samples from young children by various polymerase chain reaction (PCR) assays and real-time PCRs (Q-PCR). Until now, no data have been reported on the presence of hBoV in Belgium and the detection of hBoV in a multiplex Q-PCR setting has not been described. The aim of this study was to develop a fast and reliable multiplex Q-PCR for the simultaneous detection of hBoV DNA and adenovirus (AdV) DNA. During the winter of 2004–2005, 445 nasopharyngeal aspirates (NPAs) were analysed from 404 Belgian children up to 5 years old with acute respiratory tract infections (ARTIs). (Co)infections with hBoV, AdV, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza A virus were investigated. A viral agent was detected in 61% (n = 272/445) of the NPAs. Multiplex Q-PCR found a prevalence of 11% (n = 51/445) hBoV and 13% (n = 58/445) AdV. Coinfections were more frequently found with AdV (62%; n = 36/58) than with hBoV (49%; n = 25/51). Follow-up samples were available from 22 patients with ARTIs. In three patients, hBoV DNA persisted for one month. Multiplex Q-PCR may help in closing the diagnostic gap by addressing a broader range of potential respiratory pathogens.     

Detection of respiratory viruses by PCR assay of nasopharyngeal swabs stored in skim milk-tryptone-glucose-glycerol transport medium

We analyzed 129 paired nasopharyngeal aspirates (stored in viral transport medium [VTM]) and nasopharyngeal swabs (stored in skim milk-tryptone-glucose-glycerol [STGG] bacterial transport and storage medium) using PCRs to detect adenoviruses, influenza virus A or B, and respiratory syncytial virus (RSV). Overall, swabs stored in STGG medium without antimicrobials were found to be an acceptable alternative to aspirates stored in antimicrobial-containing VTM, with PCR agreement of 90.2% (kappa of 0.8).     

Practical recommendations for the detection of pediatric respiratory syncytial virus infections.

In our private clinic-hospital setting, respiratory syncytial virus (RSV) was isolated from infants more frequently and sooner from nasal washes (84%; 4.2 days) than from throat swabs (45%; 5.5 days) or nasopharyngeal swabs (39%; 5.7 days). Immunofluorescence of nasal wash cells identified 72% of the infants with virus isolations from nasal washes in less than one day. We therefore recommend the combination of isolation and immunofluorescence on nasal wash specimens for optimal detection of RSV-infected infants. Immunofluorescence of respiratory tract cells was also useful for monitoring the presence of RSV antigen in intubation secretions during ribavirin antiviral therapy. RSV infectivity was maintained in phosphate-buffered saline at room temperature for 6 h. Transport and inoculation of specimens in less than 6 h yielded RSV isolates from 50% of sampled infants during the two RSV seasons examined. For optimal RSV isolation, we recommend inoculation of HEp-2 tubes less than or equal to 4 days old. Replacing medium after 3 days as compared with 7 days did not increase recovery of RSV and provided little practical reduction in time to detection of cytopathology.     

Association of respiratory picornaviruses with acute bronchiolitis in French infants.

BACKGROUND: Human rhinoviruses and enteroviruses (Picornaviridae) are suspected to be major viral etiological causes of bronchiolitis in infants. OBJECTIVES: In the present study, we assessed the potential role of the respiratory picornaviruses as causative agents of bronchiolitis in French infants. STUDY DESIGN: From September 2001 to June 2002, we prospectively selected 192 infants < or =36 months of age and hospitalized for acute bronchiolitis. The detection of common respiratory viruses (respiratory syncytial virus, influenza virus A and B, parainfluenza virus 1, 2, 3 and adenovirus) was performed using classical immunofluorescence antigen and cell-culture detection assays on nasopharyngeal aspirates whereas the detection of human metapneumovirus (HMPV) was performed by a real-time RT-PCR assay. The presence of rhinovirus and/or enterovirus was assessed in respiratory samples by a picornavirus RT-PCR detection assay followed by a differential Southern blotting procedure. RESULTS: A potential causative virus was detected in 72.5% of the 192 study infants. RSV (30%), rhinovirus (21%), enterovirus (9%), influenza virus A (6%) and human metapneumovirus (4%) were the most frequent causative agents detected. Rhinoviruses or enteroviruses were detected as the only evidence of respiratory viral tract infection in 57 (30%) of 192 infants, whereas rhinovirus or enterovirus occurred in mixed viral infection detected in 25 (13%) of 192 study cases (30% versus 13%, p<10(-3)). CONCLUSIONS: Our data suggest that respiratory picornaviruses are one of the leading etiological causes of bronchiolitis in French infants. These findings highlight the need to implement a rapid picornavirus RT-PCR detection assay for the clinical diagnosis of respiratory infections in pediatric patients with bronchiolitis.     

Comparison of four different sampling methods for detecting pharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae in children.

Samples from 96 children with acute respiratory infection were obtained simultaneously with nasal, nasopharyngeal, and oropharyngeal swabs and by nasopharyngeal aspiration and were cultured on chocolate and blood agar plates. The rates of isolation of Streptococcus pneumoniae and Haemophilus influenzae detected by the four sampling methods were compared. Nasopharyngeal aspirates were optimal for the detection of both S. pneumoniae (isolation rate, 33%) and H. influenzae (isolation rate, 31%). When a nasopharyngeal aspirate is not available, such as for healthy children or children with no obtainable secretions, the nasopharyngeal swab seems optimal for the detection of both S. pneumoniae and H. influenzae among children younger than 13 months of age. Among older children, similarly, the nasopharyngeal swab seems optimal for the detection of S. pneumoniae; however, for H. influenzae, the oropharyngeal swab seems optimal.     

Evaluation and clinical validation of an alcohol-based transport medium for preservation and inactivation of respiratory viruses

The clinical and public health importance of influenza and other respiratory viruses has accelerated the development of highly sensitive molecular diagnostics, but data are limited regarding preanalytical stages of diagnostic testing. We evaluated CyMol, an alcohol-based transport medium, for its ability to maintain specimen integrity for up to 21 days of storage at various temperatures; for its ability to inactivate virus; and for its compatibility with antigen- or nucleic acid-based diagnostics for respiratory viruses in clinical samples. In mock-infected samples, both universal transport medium (UTM-RT) and CyMol maintained equivalent viral quantities for at least 14 days at room temperature or colder, whereas a dry swab collection maintained viral quantities only if refrigerated or frozen. CyMol inactivated influenza virus within 5 min of sample immersion. UTM-RT- and CyMol-collected nasal swab specimens from 73 symptomatic students attending a campus health clinic were positive for a respiratory virus in 56.2% of subjects by multiplex PCR testing, including influenza A and B viruses, rhinovirus/enteroviruses, coronaviruses, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and adenovirus. Detection by PCR was equivalent in UTM-RT- and CyMol-collected specimens and in self- and staff-collected swabs. Direct fluorescent antibody (DFA) testing was substantially less sensitive (23.3%) than multiplex PCR, and DFA testing from UTM-RT-collected swabs was more sensitive than that from CyMol-collected swabs. These data indicate that an alcohol-based transport medium such as CyMol preserves respiratory virus integrity, rapidly inactivates viruses, and is compatible with PCR-based respiratory diagnostics.     

Comparison of Midturbinate Flocked-Swab Specimens with Nasopharyngeal Aspirates for Detection of Respiratory Viruses in Children by the Direct Fluorescent Antibody Technique

Paired nasopharyngeal aspirate (NPA) and midturbinate flocked-swab specimens from 153 children with respiratory symptoms were examined by the direct fluorescent antibody (DFA) technique. Seventy-four infants (49%) had a viral infection documented by DFA. The flocked-swab specimens had 93% sensitivity and 96.7% agreement with the NPA specimens, with a kappa coefficient of 93.4% (95% confidence interval, 0.877, 0.991).The direct fluorescent antibody (DFA) technique revolutionized the rapid detection of respiratory viruses. Since its inception in 1968, it has been one of the mainstays in clinical virology laboratories throughout the world (4). The ability of DFA to detect respiratory viruses depends on many things, but it all begins with good specimen collection. The nasopharyngeal aspirate (NPA) has been considered the best specimen to detect respiratory viruses in infants (4). However, it is difficult to collect because it requires special equipment, such as a catheter, trap, and vacuum source, and specialized training. A traditional nasopharyngeal swab is the next best specimen, especially in older children or adults, because it utilizes common supplies; however, the collection end of the swab, comprised of wound Dacron fibers, has limited absorbent capacity to trap virus-infected exfoliated epithelial cells. A nylon nasopharyngeal flocked swab with enhanced absorptive properties introduced in 2006 compared favorably to the NPA for the detection of respiratory viruses by DFA (2). Recently, a midturbinate flocked swab developed by Smieja, et al. (7), and marketed by Copan, Inc., has offered a more intuitive approach for the collection of nasopharyngeal specimens (1). It has compared favorably to the NPA and the flocked nasopharyngeal swab in the diagnosis of respiratory viruses by culture, antigen detection, and PCR, none of which require intact exfoliated epithelial cells for visualization; there is no published experience of midturbinate flocked swabs with DFA in children (1, 5, 6). The midturbinate flocked swab differs from the nasopharyngeal swab. It has a sampling depth indication gauge and also has a larger absorptive capacity than the smaller nasopharyngeal swab.The present study was designed to compare the efficacy of the midturbinate flocked swab with the NPA in the detection of respiratory viruses by DFA.The study was conducted from 5 January 2010 through 11 March 2010. All children 2 years of age or less admitted to the infant''s floor of the hospital with respiratory symptoms were enrolled. The study was reviewed by the Children and Youth Institutional Review Board, who waived the need for a formal review because the study was deemed an evaluation comparing a new specimen collection device to the standard nasopharyngeal aspirate; parents were allowed to opt out of the use of the new specimen device. A nasopharyngeal aspirate specimen was collected through one nostril. A second specimen was collected through the other nostril with a midturbinate FLOQ swab (Copan Diagnostics, Inc., Murrieta, CA) designed for children 2 years of age or less; the swab was inserted up to the collar on the shaft. Both specimens were placed in 3 ml of Copan UTM transport medium, transported to the virus laboratory, and processed within 6 h. The suspension was centrifuged, and the cellular pellet washed. The cells were then spotted to glass slides. The cells were stained for DFA using a D3 Ultra respiratory screening identification kit (Diagnostic Hybrids, Inc. [DHI], Athens, OH). The kit screened for respiratory syncytial virus (RSV), influenza viruses (IFV) A and B, parainfluenza viruses (PFV) 1, 2, and 3, and adenovirus (AdV). An additional stain for human metapneumovirus (hMPV) (DHI) was included. The DFA readers were not blinded to the specimen source. The degree of DFA agreement between specimens collected by NPA and midturbinate flocked swabs was calculated with Cohen''s kappa coefficient of agreement.One hundred fifty-three infants entered the study. Paired specimens were collected from every infant. Respiratory viruses were identified in 74 (48.6%). Respiratory syncytial virus was most frequent, found in 47 patients (30%), with hMPV in 25 (16.3%), PFV in 1 (0.7%), AdV in 1 (0.7%), and IFV in none (0.0%). The 2009 H1N1 influenza A virus had last been identified in the laboratory in November 2009, more than 1 month before the start of the study. DFA of NPA specimens identified all the viruses. DFA of the flocked-swab specimens failed to detect 4 RSV and 1 hMPV isolate that had been detected in the NPA specimens. The negative DFA test results on flocked-swab specimens agreed with the negative DFA test results on NPA specimens. Overall, the positive and negative DFA test results on flocked-swab specimens had 96.7% agreement with the DFA test results on NPA specimens, with a Kappa coefficient of 93.4% (95% confidence interval [CI], 0.877, 0.991; P < 0.00001). The sensitivity of the flocked swab was 93.2% (95% CI, 0.849, 0.978).The midturbinate flocked swab proved to be comparable to the NPA for the detection of common respiratory viruses, such as RSV and hMPV, in a DFA test in the present study. The absence of IFV and the low numbers of AdV and PFV isolates in specimens prevented an assessment of the swab''s utility in detecting these viruses; however, earlier studies with nasopharyngeal flocked swabs suggested that the midturbinate swab would give similar results (3). In an earlier study, the sensitivity of the NPA in detecting either IFV or RSV was greater than the sensitivity of flocked nasopharyngeal swabs, although the difference was not statistically significant; the differences may be attributed to the greater number of respiratory epithelial cells available for examination in NPA specimens (2). The advantage of the midturbinate collection over nasopharyngeal collection resides in the relative ease of collection and the resultant patient cooperation, especially among the very young; however, the observations made in the present study may not extend beyond the pediatric population.     

Evaluation of nasal and nasopharyngeal swab collection for the detection of Epstein‐Barr virus in nasopharyngeal carcinoma

Epstein‐Barr virus detection using nasopharyngeal swabs has been suggested as a potential screening test that could improve the specificity of current EBV‐based serological assays. However, application requires insertion of the swab deep into the nasopharynx, a procedure not amenable to non‐clinic screening. We reasoned that swabbing the more easily accessible nasal cavity might provide an appealing alternative for NPC detection. Patients > 18 years of age diagnosed with histologically confirmed NPC were recruited from the Otolaryngology Department at the National Taiwan University Hospital. ENT clinicians collected both nasal and nasopharyngeal swabs. EBV DNA and cellular beta‐globulin DNA were quantified using quantitative PCR targeting a highly‐conserved region of the BKRF1 gene. EBV DNA was detectable (non‐zero) in all 34 nasopharyngeal swabs and above the positivity threshold of 1666 EBV copies in 30 (88.2%) patients. EBV DNA was detectable in 50% of 34 nasal swabs and above the positivity threshold in four (11.8%) patients. Average EBV DNA levels were >3‐fold higher (P < 0.001) in nasopharyngeal compared to nasal swabs. Among the 17 NPC patients with detectable EBV DNA in both swab types, we observed correlation (P < 0.01) between EBV DNA measurements. Our data represent the first evaluation of EBV DNA collected from nasal swabs. Given current EBV DNA amplification techniques, nasopharyngeal swabs remain more sensitive than nasal swabs for NPC detection.     

The impact of influenza viruses on hospitalizations in infants younger than two years old during epidemics of respiratory syncytial virus infection

In order to evaluate the association of influenza viruses with hospitalizations for acute respiratory infection in infants younger than two years old during epidemics of respiratory syncytial virus infection, we studied 512 nasal washes from this population. The samples were obtained from 1997 to 2000. A total of 337 viruses were isolated: 264 respiratory syncytial viruses, 62 influenza viruses, eight parainfluenza viruses, two adenovirus and one rhinovirus. Hospitalizations for acute respiratory infection were owing to influenza and respiratory syncytial viruses in 18.3% vs. 78.3% of all cases, and 32.5% vs. 65.8%, respectively, in the group of infants between 6 months and 2 years old.     

Comparison of nasopharyngeal aspirate with flocked swab for PCR‐detection of respiratory viruses in children

Fast‐ and high‐throughput molecular workflows require sample matrices to be suitable for automation. Respiratory swabs are better suited for this purpose compared to the more viscous nasopharyngeal aspirates. Samples collected by nasopharyngeal aspiration and nasopharyngeal flocked swab from 81 children were compared for detection and recovery of respiratory viruses. Using real‐time RT‐PCR, no statistically significant differences in virus detection between the two sample types were found, supporting the use of flocked swabs in children aged one month to two years.     

Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for control of MRSA nosocomial transmission. We aimed to evaluate the performance of the GeneXpert real-time PCR system using the Xpert MRSA assay on a collection of 40 representative Belgian MRSA strains and for MRSA screening of geriatric inpatients. Double nasal swabs were used: the first swab for the Xpert MRSA assay and the second for culture onto chromogenic selective medium and enrichment broth. All but 1 of the 40 collection strains were recognized as MRSA by the Xpert MRSA assay. Nares swabs were prospectively collected from 246 inpatients including 25 nasal MRSA carriers. Compared with enriched cultures, the sensitivity, the specificity, and the positive and negative predictive values of the Xpert MRSA assay were 69.2%, 97.7%, 78.3%, and 96.3% respectively. The 7 evaluable false-negative results according to the assay were due to its possible lack of sensitivity (n = 3) and to the occurrence of a Belgian MRSA clone carrying a particular staphylococcal chromosomal cassette mec (SCCmec) type IV variant (n = 4) not targeted by the current Xpert MRSA assay. Because of the evolution of SCCmec in MRSA, new primers should be designed and further studies are warranted to ensure continuous monitoring of this assay.     

Blood MxA protein as a marker for respiratory virus infections in young children

BackgroundType I interferon induced MxA response can differentiate viral from bacterial infections, but MxA responses in rhinovirus or asymptomatic virus infections are not known.ObjectiveTo study MxA protein levels in healthy state and during respiratory virus infection of young children in an observational prospective cohort.Study designBlood samples and nasal swabs were collected from 153 and 77 children with and without symptoms of respiratory infections, respectively. Blood MxA protein levels were measured by an enzyme immunoassay and PCR methods were used for the detection of respiratory viruses in nasal swabs.ResultsRespiratory viruses were detected in 81% of symptomatic children. They had higher blood MxA protein levels (median [interquartile range]) than asymptomatic virus-negative children (695 [345–1370] μg/L vs. 110 [55–170] μg/L; p < 0.001). Within asymptomatic children, no significant difference was observed in MxA responses between virus-positive and virus-negative groups. A cut-off level of 175 μg/L had 92% sensitivity and 77% specificity for a symptomatic respiratory virus infection. Rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, coronavirus, and human metapneumovirus infections were associated with elevated MxA responses. Asymptomatic virus-negative children vaccinated with a live virus vaccine had elevated MxA protein levels (240 [120–540] μg/L), but significantly lower than children with an acute respiratory infection, who had not received vaccinations (740 [350–1425] μg/L; p < 0.001).ConclusionBlood MxA protein levels are increased in young children with symptomatic respiratory virus infections, including rhinovirus infections. MxA is an informative general marker for the most common acute virus infections.