Research Article
01 July 2007

Increased Detection of Respiratory Syncytial Virus, Influenza Viruses, Parainfluenza Viruses, and Adenoviruses with Real-Time PCR in Samples from Patients with Respiratory Symptoms

ABSTRACT

Respiratory samples (n = 267) from hospitalized patients with respiratory symptoms were tested by real-time PCR, viral culture, and direct immunofluorescence for respiratory syncytial virus, influenza virus, parainfluenza viruses, and adenoviruses. Compared with conventional diagnostic tests, real-time PCR increased the diagnostic yields for these viruses from 24% to 43% and from 3.5% to 36% for children and adults, respectively.
Determining etiological diagnoses for patients admitted to the hospital with respiratory symptoms remains a clinical and laboratory challenge. Infections with respiratory syncytial virus (RSV), influenza viruses (IVs), parainfluenza viruses (PIVs), and adenoviruses (AdVs) are traditionally diagnosed using viral culture and direct immunofluorescence (DIF) tests. However, over the last 2 decades, nucleic acid amplification techniques, particularly real-time PCR, have become available as diagnostic tools. The aim of the present study was to determine the diagnostic yields of real-time PCR for RSV, IV, PIVs and AdVs in clinical practice, compared with those of traditional tests.
For this purpose, respiratory specimens from hospitalized pediatric and adult patients with respiratory symptoms were collected during one respiratory season (December 2004 through May 2005). Follow-up samples (taken within 4 weeks after a first sample) were excluded. Samples were divided into three aliquots. One aliquot of the samples from children was used for DIF assays to detect RSVs A and B, IVs A and B, PIVs 1 to 3, and AdV using Imagen kits (DaKo, Glostrup, Denmark) according to the manufacturer's protocol. DIF was not performed for samples from adults, because it has been shown to have a very low sensitivity in an adult population (2). One aliquot of the samples was used for immediate viral culture of RSV, IVs, PIVs 1 to 3, and AdV on LLC-MK2, RD, R-HELA, and HEp-2C cells. Cultures were examined every other day for the development of a cytopathologic effect for 14 days, and positive cultures were confirmed by DIF with monoclonal antibodies specific for RSV A or B; IV A or B; PIV 1, 2, or 3; or AdV (DaKo, Glostrup, Denmark). Finally, an aliquot of the samples was used to extract viral nucleic acids using the MagNA Pure LC total nucleic acid isolation kit (Roche Diagnostics, Basel, Switzerland) as described previously (6). Subsequently, cDNA was synthesized using MultiScribe reverse transcriptase and random hexamers (both from Applied Biosystems, Foster City, CA). Detection of viruses was performed using parallel real-time PCR assays for RSVs A and B (8) and IVs A and B as previously described (7, 10). In addition, in-house real-time PCR methods were developed for the detection of PIVs 1 to 4 and AdVs using Primer Express (Applied Biosystems). Conserved target regions were identified using BLAST (www.ncbi.nlm.nih.gov/blast). Sequences of the primers and probes used are summarized in Table 1. Real-time PCR procedures were performed as described previously (6).
A total of 267 samples from patients with respiratory symptoms were analyzed. Of these samples, 181 were from children (152 nasopharyngeal aspirate, 25 sputum, and 4 bronchoalveolar lavage specimens) and 86 from adults (77 nasal and/or throat swab, 6 bronchoalveolar lavage, and 3 sputum specimens). Twelve children and four adults contributed two samples.
For children, viral culture identified viruses in 25 (14%) samples, whereas DIF detected viruses in 35 (19%) samples. Thirty-eight (21%) samples were inadequate for analysis by DIF because of the absence of intact cells. The combination of viral culture and DIF detected viruses in 43 (24%) samples. A total of 78 (43%) samples were positive by real-time PCR; in these samples, 89 viruses were identified. Mixed viral infections were detected by real-time PCR only (seven [3.9%] double infections and two [1.1%] triple infections).
In adults, viral culture identified respiratory viruses in only three (3.5%) samples. Respiratory viruses were detected in 31 (36%) samples by real-time PCR; no mixed viral infections were detected.
The diagnostic yields of conventional methods compared to that of real-time PCR as the reference method are presented in Table 2. The sensitivities of conventional methods (culture and DIF) in diagnosing the pediatric population were 0.46 (confidence interval [CI], 0.38 to 0.53) for RSVs, 0.44 (CI, 0.37 to 0.52) for IVs, 0.63 (CI, 0.55 to 0.70) for PIVs, and 0.24 (CI, 0.17 to 0.30) for AdVs. The sensitivities of conventional methods (viral culture) for detection in adults appeared to be much lower (Table 2) and were 0.00 (CI, 0.00 to 0.00) for RSVs, 0.11 (CI, 0.04 to 0.18) for IVs, 0.00 (CI, 0.00 to 0.00) for PIVs, and 1.00 (CI, 1.00 to 1.00) for AdVs. The specificities of conventional methods for the different respiratory viruses were 0.98 to 1.00 for children and 1.00 for adults.
Since the viral load in a sample can be relatively assessed by determining the cycle threshold (CT) value, we compared the values for the samples that were culture/DIF positive with values for the samples that were culture/DIF negative. High CT values are representative of a low viral load, while a low CT value reflects a high viral load. For the pediatric population, the mean CT value of samples positive for RSV by real-time PCR but in which RSV was not detected by conventional tests was 3.9 cycles higher (CI, 0.36 to 7.4) than that of positive samples subjected to detection by conventional tests (P = 0.03). For AdV, the CT value was 13 cycles higher (CI, 5.4 to 22; P = 0.003). For IV, the trend was the same (5.7 cycles higher [CI, −0.25 to 12]; P = 0.06). For PIVs, no difference was observed. For adults, this analysis could not be performed due to the small number of positive results by viral culture.
In the present study, we showed that real-time PCR for RSV, IV, PIV, and AdV increased the diagnostic yields for these viruses substantially in clinical practice, in comparison with those of conventional diagnostic tests. However, some limitations of our study deserve further discussion. First, there is no gold standard for the detection of respiratory viruses to which both conventional tests and real-time PCR can be compared. This is a problem encountered in all studies evaluating real-time PCR (3, 4). Second, we did not use blind antibody fluorescence staining for viral culture and therefore may have missed positive cultures without cytopathogenic effects.
The findings of our study are in accordance with those of previous reports. van Kraaij et al. identified RSV, IV, and PIV as present in 2% of 52 episodes of respiratory tract infection in adults after stem cell transplantation, whereas real-time PCR was positive for 12%, 4%, and 6% of the samples, respectively (9). Templeton et al. (5) identified RSV, IV, and/or PIV in 19% of 358 respiratory samples by viral culture, compared with 24% of samples by real-time PCR (5). In the present study, we found an association between CT values and conventional tests being positive. This was in agreement with the work of Bredius et al., who found CT values of 27 to 42 for samples positive only by real-time PCR versus CT values of 18 to 22 for culture-positive samples (1).
Apart from being more sensitive than conventional methods, real-time PCR is also able to detect microorganisms that are difficult to culture, microorganisms for which no (commercial) DIF assay is available, or microorganisms that cannot be cultured at all. At our center, real-time PCR is routinely performed for rhinoviruses, coronaviruses, human metapneumovirus, and Mycoplasma pneumoniae. These agents were found, respectively, in 47, 23, 13, and 5 pediatric samples and in 16, 6, 1, and 1 adult samples (unpublished data), increasing the detection rate further to 69% and 57% for children and adults, respectively.
In conclusion, real-time PCR considerably increases the diagnostic yields for respiratory viruses from patients admitted with respiratory symptoms within a clinically relevant time frame. This allows clinicians to initiate optimal patient management and to initiate adequate (future) use of antiviral therapy and optimal infection control.
TABLE 1.
TABLE 1. Primers and probes used for real-time PCR detection
Virus(es)Target geneForward primer(s) (5′-3′)Reverse primer(s) (5′-3′)Probe(s)aReference
RSV ANucleocapsidAGA TCA ACT TCT GTC ATC CAG CAATTC TGC ACA TCA TAA TTA GGA GTA TCA ATFAM-CAC CAT CCA ACG GAG CAC AGG AGA T-TAMRA8
RSV BNucleocapsidAAG ATG CAA ATC ATA AAT TCA CAG GATGA TAT CCA GCA TCT TTA AGT ATC TTT ATA GTGFAM-TTC CCT TCC TAA CCT GGA CAT AGC ATA TAA CAT ACC T-TAMRA8
IV AMatrixAAG ACC AAT CCT GTC ACC TCT GACAA AGC GTC TAC GCT GCA GTC CFAM-TTT GTG TTC ACG CTC ACC GT-TAMRA10
IV BHemagglutininAAA TAC GGT GGA TTA AAC AAA AGC AACCA GCA ATA GCT CCG AAG AAAFAM-CAC CCA TAT TGG GCA ATT TCC TAT GGC-TAMRA7
PIV 1Hemagglutinin-neuraminidaseTGA TTT AAA CCC GGT AAT TTC TCA TCCT TGT TCC TGC AGC TAT TAC AGAFAM-ACG ACA ACA GGA AAT C-MGB 
PIV 2Hemagglutinin-neuraminidaseAGG ACT ATG AAA ACC ATT TAC CTA AGT GAAAG CAA GTC TCA GTT CAG CTA GAT CAFAM-ATC AAT CGC AAA AGC TGT TCA GTC ACT GCT ATA C-TAMRA 
PIV 3Hemagglutinin-neuraminidaseTGA TGA AAG ATC AGA TTA TGC ATA TCCCG GGA CAC CCA GTT GTGFAM-TGG ACC AGG GAT ATA CTA CAA AGG CAA AAT AAT ATT TCT C-TAMRA 
PIV 4NucleocapsidCAA AYG ATC CAC AGC AAA GAT TCATG TGG CCT GTA AGG AAA GCAFAM-GTA TCA TCA TCT GCC AAA TCG GCA ATT AAA CA-TAMRA 
AVsHexonTTT GAG GTG GAY CCM ATG GAAGA ASG GSG TRC GCA GGT AFAM-ACC ACG TCG AAA ACT TCG AA-MGB 
  TTT GAG GTY GAY CCC ATG GAAGA ASG GTG TRC GCA GAT AFAM-ACC ACG TCG AAA ACT TCA AA-MGB 
    FAM-ACA CCG CGG CGT CA-MGB 
a
FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binding.
TABLE 2.
TABLE 2. Comparison of the results of conventional diagnostic tests and real-time PCR
Patient populationConventional test(s)Result of conventional testNo. of specimens with indicated PCR result for:       
   RSVs IVs PIVs AdVs 
   ++++
ChildrenCulture + DIF+211815043
  2513410162317313161
AdultsCulture+00200010
  10761668284085

Acknowledgments

This study was supported by an M.D./Ph.D. grant from the University Medical Center, Utrecht, The Netherlands.

REFERENCES

1.
Bredius, R. G., K. E. Templeton, S. A. Scheltinga, E. C. Claas, A. C. Kroes, and J. M. Vossen.2004. Prospective study of respiratory viral infections in pediatric hemopoietic stem cell transplantation patients. Pediatr. Infect. Dis. J.23:518-522.
2.
Casiano-Colon, A. E., B. B. Hulbert, T. K. Mayer, E. E. Walsh, and A. R. Falsey.2003. Lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults. J. Clin. Virol.28:169-174.
3.
McAdam, A. J.2000. Discrepant analysis: how can we test a test? J. Clin. Microbiol.38:2027-2029.
4.
Murdoch, D. R.2004. Molecular genetic methods in the diagnosis of lower respiratory tract infections. APMIS112:713-727.
5.
Templeton, K. E., S. A. Scheltinga, M. F. Beersma, A. C. Kroes, and E. C. Claas.2004. Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. J. Clin. Microbiol.42:1564-1569.
6.
van de Pol, A. C., T. F. Wolfs, N. J. Jansen, A. M. van Loon, and J. W. Rossen.2006. Diagnostic value of real-time polymerase chain reaction to detect viruses in young children admitted to the paediatric intensive care unit with lower respiratory tract infection. Crit. Care10:R61.
7.
van Elden, L. J., M. Nijhuis, P. Schipper, R. Schuurman, and A. M. van Loon.2001. Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. J. Clin. Microbiol.39:196-200.
8.
van Elden, L. J., A. M. van Loon, A. van der Beek, K. A. Hendriksen, A. I. Hoepelman, M. G. van Kraaij, P. Schipper, and M. Nijhuis.2003. Applicability of a real-time quantitative PCR assay for diagnosis of respiratory syncytial virus infection in immunocompromised adults. J. Clin. Microbiol.41:4378-4381.
9.
van Kraaij, M. G., L. J. van Elden, A. M. van Loon, K. A. Hendriksen, L. Laterveer, A. W. Dekker, and M. Nijhuis.2005. Frequent detection of respiratory viruses in adult recipients of stem cell transplants with the use of real-time polymerase chain reaction, compared with viral culture. Clin. Infect. Dis.40:662-669.
10.
Ward, C. L., M. H. Dempsey, C. J. Ring, R. E. Kempson, L. Zhang, D. Gor, B. W. Snowden, and M. Tisdale.2004. Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. J. Clin. Virol.29:179-188.

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Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 45Number 7July 2007
Pages: 2260 - 2262
PubMed: 17507513

History

Received: 21 April 2007
Revision received: 26 April 2007
Accepted: 7 May 2007
Published online: 1 July 2007

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Authors

Alma C. van de Pol
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Department of Pediatrics, Division of Infectious Disease, Wilhelmina Children's Hospital, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands
Anton M. van Loon
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Tom F. W. Wolfs
Department of Pediatrics, Division of Infectious Disease, Wilhelmina Children's Hospital, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands
Nicolaas J. G. Jansen
Department of Pediatrics, Division of Intensive Care, Wilhelmina Children's Hospital, University Medical Center Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands
Monique Nijhuis
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Els Klein Breteler
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Rob Schuurman
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
John W. A. Rossen [email protected]
Department of Virology, Eijkman-Winkler Center, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Laboratory of Medical Microbiology and Immunology, St. Elisabeth Hospital Tilburg, Hilvarenbeekseweg 60, 5022 GC Tilburg, The Netherlands

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