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01.02.2013 | Clinical and Epidemiological Study

Validation of a multiplex reverse transcriptase PCR ELISA for the detection of 19 respiratory tract pathogens

verfasst von: W. Puppe, J. Weigl, B. Gröndahl, M. Knuf, S. Rockahr, P. von Bismarck, G. Aron, H. G. M. Niesters, A. D. M. E. Osterhaus, H.-J. Schmitt

Erschienen in: Infection | Ausgabe 1/2013

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Abstract

Introduction

Since acute respiratory tract infections inflict a high burden of disease in children worldwide, a multiplex reverse transcription polymerase chain reaction combined with a microwell hybridization assay (m-RT-PCR–ELISA) to detect 19 different respiratory pathogens was developed and validated.

Methods

A total of 430 respiratory specimens were retrospectively tested in parallel by both the advanced 19-valent m-RT-PCR–ELISA as well as by culture or individual RT-PCR assays used in clinical routine.

Results

The mean (median) sensitivity of the m-RT-PCR–ELISA in the retrospective test was 93.3% (95.1%; range 83.3–100 %), and the mean (median) specificity was 99.8 and 100 % (range 98.6–100 %), respectively. The mean positive predictive value was 99.3 % (range 93.4–100 %) and the mean negative predictive value was 95.3 % (range 98.4–100 %). Feasibility and clinical value of the 19-valent method was prospectively shown on 16,231 incoming clinical specimens from patients between 0 and 16 years of age with acute respiratory tract infections admitted to pediatric hospitals or private practices from October 2003 to June 2010 in three regions in Germany (Kiel, Mainz, Freiburg; Freiburg to June 2007 only). At least one microorganism was detected in 10,765 of 16,231 (66.3 %) clinical specimens: 5,044 RV, 1,999 RSV, 1,286 AV, 944 EV, 737 seasonal IVA, 173 pandemic IVA H1N1-2009, 899 MPV, 518 CV, 383 PIV3, 268 PIV1, 259 Mpn, 205 IVB, 164 PIV2, 144 PIV4, 103 Bp, 29 Cpn and 29 Bpp, while reovirus and Lpn were not present in these specimens from a pediatric population. More than one organism could be detected in 13.4 % of the specimens.

Conclusions

The m-RT-PCR–ELISA evaluated here improves the spectrum for diagnosing respiratory infections and is a feasible instrument for individual diagnostic and epidemiological studies.

Leowski J. Mortality from acute respiratory infections in children under 5 years of age: global estimates. World Health Stat Q. 1986;39:138–44.PubMed

Gröndahl B, Puppe W, Hoppe A, Kühne I, Weigl JA, Schmitt HJ. Rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-PCR: feasibility study. J Clin Microbiol. 1999;37:1–7.PubMed

Puppe W, Weigl JA, Aron G, Gröndahl B, Schmitt HJ, Niesters HG, Groen J. Evaluation of a multiplex reverse transcriptase PCR ELISA for the detection of nine respiratory tract pathogens. J Clin Virol. 2004;30:165–74.PubMedCrossRef

Brunstein JD, Cline CL, McKinney S, Thomas E. Evidence from multiplex molecular assays for complex multipathogen interactions in acute respiratory infections. J Clin Microbiol. 2008;46:97–102.PubMedCrossRef

Gruteke P, Glas AS, Dierdorp M, Vreede WB, Pilon JW, Bruisten SM. Practical implementation of a multiplex PCR for acute respiratory tract infections in children. J Clin Microbiol. 2004;42:5596–603.PubMedCrossRef

Kehl SC, Henrickson KJ, Hua W, Fan J. Evaluation of the Hexaplex assay for detection of respiratory viruses in children. J Clin Microbiol. 2001;39:1696–701.PubMedCrossRef

Létant SE, Ortiz JI, Bentley Tammero LF, Birch JM, Derlet RW, Cohen S, Manning D, McBride MT. Multiplexed reverse transcriptase PCR assay for identification of viral respiratory pathogens at the point of care. J Clin Microbiol. 2007;45:3498–505.PubMedCrossRef

Nolte FS, Marshall DJ, Rasberry C, Schievelbein S, Banks GG, Storch GA, Arens MQ, Buller RS, Prudent JR. MultiCode-PLx system for multiplexed detection of seventeen respiratory viruses. J Clin Microbiol. 2007;45:2779–86.PubMedCrossRef

Syrmis MW, Whiley DM, Thomas M, Mackay IM, Williamson J, Siebert DJ, Nissen MD, Sloots TP. A sensitive, specific, and cost-effective multiplex reverse transcriptase-PCR assay for the detection of seven common respiratory viruses in respiratory samples. J Mol Diagn. 2004;6:125–31.PubMedCrossRef

10.

Templeton KE, Scheltinga SA, Beersma MF, Kroes AC, Claas EC. 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. 2004;42:1564–9.PubMedCrossRef

11.

Wasem S, Weichert S, Walther S, Weigl JA, Puppe W, Ihorst G, Schmitt HJ, Forster J. Lower respiratory tract disease in children: constant pathogens—constant management?! Klin Padiatr. 2008;220:291–5.PubMedCrossRef

12.

Weigl JA, Puppe W, Schmitt HJ. Incidence of respiratory syncytial virus-positive hospitalizations in Germany. Eur J Clin Microbiol Infect Dis. 2001;20:452–9.PubMed

13.

Weigl JA, Puppe W, Schmitt HJ. The incidence of influenza-associated hospitalizations in children in Germany. Epidemiol Infect. 2002;129:525–33.PubMedCrossRef

14.

Weigl JA, Puppe W, Schmitt HJ. Can respiratory syncytial virus etiology be diagnosed clinically? A hospital-based case–control study in children under two years of age. Eur J Epidemiol. 2003;18:431–9.PubMedCrossRef

15.

Weigl JA, Puppe W, Schmitt HJ. Variables explaining the duration of hospitalization in children under two years of age admitted with acute airway infections: does respiratory syncytial virus have a direct impact? Klin Padiatr. 2004;216:7–15.PubMedCrossRef

16.

Weigl JA, Puppe W, Gröndahl B, Schmitt HJ. Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse-transcriptase polymerase chain reaction. Eur J Clin Microbiol Infect Dis. 2000;19:336–43.PubMedCrossRef

17.

Weigl JA, Puppe W, Rockahr S, Schmitt HJ. Burden of disease in hospitalized RSV-positive children in Germany. Klin Padiatr. 2002;214:334–42.PubMedCrossRef

18.

Weigl JA, Puppe W, Belke O, Neusüss J, Bagci F, Schmitt HJ. The descriptive epidemiology of severe lower respiratory tract infections in children in Kiel, Germany. Klin Padiatr. 2005;217:259–67.PubMedCrossRef

19.

Weigl JA, Puppe W, Belke O, Neusüss J, Bagci F, Schmitt HJ. Population-based incidence of severe pneumonia in children in Kiel, Germany. Klin Padiatr. 2005;217:211–9.PubMedCrossRef

20.

Weigl JA, Puppe W, Meyer CU, Berner R, Forster J, Schmitt HJ, Zepp F. Ten years’ experience with year-round active surveillance of up to 19 respiratory pathogens in children. Eur J Pediatr. 2007;166:957–66.PubMedCrossRef

21.

Weigl JA, Puppe W, Meyer CU, Berner R, Forster J, Schmitt HJ, Zepp F. PID-ARI.net—a pediatric infectious diseases network on acute respiratory infections and the added value of a multilevel research network. Klin Padiatr. 2008;220:281–6.PubMedCrossRef

22.

du Prel JB, Puppe W, Gröndahl B, Knuf M, Weigl JA, Schaaff F, Schmitt HJ. Are meteorological parameters associated with acute respiratory tract infections? Clin Infect Dis. 2009;49:861–8.PubMedCrossRef

23.

Gröndahl B, Puppe W, Weigl J, Schmitt HJ. Comparison of the BD Directigen Flu A+B Kit and the Abbott TestPack RSV with a multiplex RT-PCR ELISA for rapid detection of influenza viruses and respiratory syncytial virus. Clin Microbiol Infect. 2005;11:848–50.PubMedCrossRef

24.

Huck B, Scharf G, Neumann-Häfelin D, Puppe W, Weigl J, Falcone V. Novel human metapneumovirus sublineage. Emerg Infect Dis. 2006;12:147–50.PubMedCrossRef

25.

Schützle H, Weigl J, Puppe W, Forster J, Berner R. Diagnostic performance of a rapid antigen test for RSV in comparison with a 19-valent multiplex RT-PCR ELISA in children with acute respiratory tract infections. Eur J Pediatr. 2008;167:745–9.PubMedCrossRef

26.

Toschke AM, Arenz S, von Kries R, Puppe W, Weigl JA, Höhle M, Heininger U. No temporal association between influenza outbreaks and invasive pneumococcal infections. Arch Dis Child. 2008;93:218–20.PubMedCrossRef

27.

Li Z, Jansen DL, Finn TM, Halperin SA, Kasina A, O’Connor SP, Aoyama T, Manclark CR, Brennan MJ. Identification of Bordetella pertussis infection by shared-primer PCR. J Clin Microbiol. 1994;32:783–9.PubMed

28.

Pongthanapisith V, Sukasem C, Premchaiporn K, Srichantaratsamee C, Chantratita W. Clinical performance of three rapid diagnostic tests for influenza virus in nasopharyngeal specimens to detect novel swine-origin influenza viruses. Infection. 2011;39:105–11. Epub 2011 Mar 22. PubMed PMID: 21424855.PubMedCrossRef

29.

Panning M, Eickmann M, Landt O, Monazahian M, Olschläger S, Baumgarte S, Reischl U, Wenzel JJ, Niller HH, Günther S, Hollmann B, Huzly D, Drexler JF, Helmer A, Becker S, Matz B, Eis-Hübinger A, Drosten C. Detection of influenza A(H1N1)v virus by real-time RT-PCR. Euro Surveill. 2009;14:pii=19329.

30.

Engler HD, Selepak ST. Effect of centrifuging shell vials at 3,500 × g on detection of viruses in clinical specimens. J Clin Microbiol. 1994;32:1580–2.PubMed

31.

Rothbarth PH, Groen J, Bohnen AM, de Groot R, Osterhaus AD. Influenza virus serology—a comparative study. J Virol Methods. 1999;78:163–9.PubMedCrossRef

32.

Dawson B, Trapp RG. Probability and related topics for making inferences about data. In: Dawson B, Trapp RG, editors. Basic and clinical biostatistics. 3rd ed. New York: Lange Medical Books/McGraw-Hill; 2001. p. 82–4.

33.

Drosten C, Chiu LL, Panning M, Leong HN, Preiser W, Tam JS, Günther S, Kramme S, Emmerich P, Ng WL, Schmitz H, Koay ES. Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus. J Clin Microbiol. 2004;42:2043–7.PubMedCrossRef

34.

Drosten C, Günther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguière AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Müller S, Rickerts V, Stürmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med. 2003;348:1967–76.PubMedCrossRef

35.

Echevarría JE, Erdman DD, Swierkosz EM, Holloway BP, Anderson LJ. Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR. J Clin Microbiol. 1998;36:1388–91.PubMed

36.

Arden KE, McErlean P, Nissen MD, Sloots TP, Mackay IM. Frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infections. J Med Virol. 2006;78:1232–40.PubMedCrossRef

37.

Renwick N, Schweiger B, Kapoor V, Liu Z, Villari J, Bullmann R, Miething R, Briese T, Lipkin WI. A recently identified rhinovirus genotype is associated with severe respiratory-tract infection in children in Germany. J Infect Dis. 2007;196:1754–60.PubMedCrossRef

38.

Benson R, Tondella ML, Bhatnagar J, Carvalho Mda G, Sampson JS, Talkington DF, Whitney AM, Mothershed E, McGee L, Carlone G, McClee V, Guarner J, Zaki S, Dejsiri S, Cronin K, Han J, Fields BS. Development and evaluation of a novel multiplex PCR technology for molecular differential detection of bacterial respiratory disease pathogens. J Clin Microbiol. 2008;46:2074–7.PubMedCrossRef

39.

Lam WY, Yeung AC, Tang JW, Ip M, Chan EW, Hui M, Chan PK. Rapid multiplex nested PCR for detection of respiratory viruses. J Clin Microbiol. 2007;45:3631–40.PubMedCrossRef

40.

Marshall DJ, Reisdorf E, Harms G, Beaty E, Moser MJ, Lee WM, Gern JE, Nolte FS, Shult P, Prudent JR. Evaluation of a multiplexed PCR assay for detection of respiratory viral pathogens in a public health laboratory setting. J Clin Microbiol. 2007;45:3875–82.PubMedCrossRef

41.

Reijans M, Dingemans G, Klaassen CH, Meis JF, Keijdener J, Mulders B, Eadie K, van Leeuwen W, van Belkum A, Horrevorts AM, Simons G. RespiFinder: a new multiparameter test to differentially identify fifteen respiratory viruses. J Clin Microbiol. 2008;46:1232–40.PubMedCrossRef

42.

Hierholzer JC, Halonen PE, Dahlen PO, Bingham PG, McDonough MM. Detection of adenovirus in clinical specimens by polymerase chain reaction and liquid-phase hybridization quantitated by time-resolved fluorometry. J Clin Microbiol. 1993;31:1886–91.PubMed

43.

Kösters K, Reischl U, Schmetz J, Riffelmann M, Wirsing von König CH. Real-time LightCycler PCR for detection and discrimination of Bordetella pertussis and Bordetella parapertussis. J Clin Microbiol. 2002;40:1719–22.PubMedCrossRef

44.

Reischl U, Lehn N, Sanden GN, Loeffelholz MJ. Real-time PCR assay targeting IS481 of Bordetella pertussis and molecular basis for detecting Bordetella holmesii. J Clin Microbiol. 2001;39:1963–6.PubMedCrossRef

45.

Gaydos CA, Quinn TC, Eiden JJ. Identification of Chlamydia pneumoniae by DNA amplification of the 16S rRNA gene. J Clin Microbiol. 1992;30:796–800.PubMed

46.

Stephensen CB, Casebolt DB, Gangopadhyay NN. Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay. Virus Res. 1999;60:181–9.PubMedCrossRef

47.

Vabret A, Mouthon F, Mourez T, Gouarin S, Petitjean J, Freymuth F. Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction. J Virol Methods. 2001;97:59–66.PubMedCrossRef

48.

Rotbart HA, Sawyer MH, Fast S, Lewinski C, Murphy N, Keyser EF, Spadoro J, Kao SY, Loeffelholz M. Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay. J Clin Microbiol. 1994;32:2590–2.PubMed

49.

Claas EC, Sprenger MJ, Kleter GE, van Beek R, Quint WG, Masurel N. Type-specific identification of influenza viruses A, B and C by the polymerase chain reaction. J Virol Methods. 1992;39:1–13.PubMedCrossRef

50.

Alexiou-Daniel S, Stylianakis A, Papoutsi A, Zorbas I, Papa A, Lambropoulos AF, Antoniadis A. Application of polymerase chain reaction for detection of Legionella pneumophila in serum samples. Clin Microbiol Infect. 1998;4:144–8.PubMedCrossRef

51.

van Kuppeveld FJ, van der Logt JT, Angulo AF, van Zoest MJ, Quint WG, Niesters HG, Galama JM, Melchers WJ. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1992;58:2606–15.PubMed

52.

Maertzdorf J, Wang CK, Brown JB, Quinto JD, Chu M, de Graaf M, van den Hoogen BG, Spaete R, Osterhaus AD, Fouchier RA. Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages. J Clin Microbiol. 2004;42:981–6.PubMedCrossRef

53.

Fan J, Henrickson KJ. Rapid diagnosis of human parainfluenza virus type 1 infection by quantitative reverse transcription-PCR-enzyme hybridization assay. J Clin Microbiol. 1996;34:1914–7.PubMed

54.

Yuasa T, Bando H, Kawano M, Tsurudome M, Nishio M, Kondo K, Komada H, Ito Y. Sequence analyses of the 3′ genome end and NP gene of human parainfluenza type 2 virus: sequence variation of the gene-starting signal and the conserved 3′ end. Virology. 1990;179:777–84.PubMedCrossRef

55.

Aguilar JC, Pérez-Breña MP, García ML, Cruz N, Erdman DD, Echevarría JE. Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR. J Clin Microbiol. 2000;38:1191–5.PubMed

56.

Andeweg AC, Bestebroer TM, Huybreghs M, Kimman TG, de Jong JC. Improved detection of rhinoviruses in clinical samples by using a newly developed nested reverse transcription-PCR assay. J Clin Microbiol. 1999;37:524–30.PubMed

57.

Pitkäranta A, Arruda E, Malmberg H, Hayden FG. Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-PCR. J Clin Microbiol. 1997;35:1791–3.PubMed

58.

Leary TP, Erker JC, Chalmers ML, Wetzel JD, Desai SM, Mushahwar IK, Dermody TS. Detection of reovirus by reverse transcription-polymerase chain reaction using primers corresponding to conserved regions of the viral L1 genome segment. J Virol Methods. 2002;104:161–5.PubMedCrossRef

59.

Paton AW, Paton JC, Lawrence AJ, Goldwater PN, Harris RJ. Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification. J Clin Microbiol. 1992;30:901–4.PubMed

Titel: Validation of a multiplex reverse transcriptase PCR ELISA for the detection of 19 respiratory tract pathogens
verfasst von: W. Puppe
J. Weigl
B. Gröndahl
M. Knuf
S. Rockahr
P. von Bismarck
G. Aron
H. G. M. Niesters
A. D. M. E. Osterhaus
H.-J. Schmitt
Publikationsdatum: 01.02.2013
Verlag: Springer-Verlag
Erschienen in: Infection / Ausgabe 1/2013
Print ISSN: 0300-8126
Elektronische ISSN: 1439-0973
DOI: https://doi.org/10.1007/s15010-012-0298-6

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Validation of a multiplex reverse transcriptase PCR ELISA for the detection of 19 respiratory tract pathogens

Abstract

Introduction

Methods

Results

Conclusions

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Abstract

Introduction

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Conclusions

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