Sains Malaysiana 47(11)(2018): 2821–2829

http://dx.doi.org/10.17576/jsm-2018-4711-25

 

Multiplex Real-Time PCR Detection of Respiratory Viruses in Lower Respiratory Tract Infections in Children

(Multipleks PCR Masa Sebenar untuk Pengesanan Jangkitan Virus Pernafasan dalam Saluran Pernafasan Bahagian Bawah bagi Kanak-Kanak)

 

ZETTI ZAINOL RASHID^1*, PEI CHUEN LEE², UMI KALSOM ALI¹, MUTTAQILLAH NAJIHAN ABDUL SAMAT¹ & SWEE FONG TANG²

 

¹Department of Medical Microbiology & Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Kuala Lumpur, Federal Territory, Malaysia

 

²Department of Paediatrics, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Kuala Lumpur, Federal Territory, Malaysia

 

Diserahkan: 24 Mei 2018/Diterima: 3 Julai 2018

 

ABSTRACT

Acute respiratory infections cause significant morbidity and mortality in children. Several new respiratory viruses have been identified and co-detection of multiple viruses is commonly reported. This is part of a prospective study which aims to detect respiratory viruses by multiplex molecular method and conventional methods. Nasopharyngeal aspirate specimens were taken from hospitalised children aged less than 5 years with lower respiratory tract infections. These were tested using viral culture, immunofluorescence and Seegene Anyplex™ II RV16 real-time polymerase chain reaction. From 102 samples, 69 (67.6%) were positive by PCR, 12 (11.8%) positive by culture method and 13 (12.7%) positive by IF. A single viral pathogen was detected in 48 samples (47.1%), while 21 samples (20.6%) had co-detection of 2 to 4 viral pathogens. Respiratory syncytial virus (RSV) was most common, detected in 17 samples (16.7% of all samples), followed by adenovirus and rhinovirus in 16 (15.7%), respectively. Bocavirus was detected in 15, enterovirus in 15, influenza A in 8 and parainfluenza-4 in 4 samples, with highest occurrences in co-detection (12/15, 10/15, 5/8 and 3/4, respectively). RSV was the least likely detected in co-detection (3/17). In PCR-positive samples, 54/69 (78.3%) were patients aged up to 24 months. Molecular methods detect more viral aetiologies than conventional methods, with simultaneous detection of multiple respiratory viruses. More sensitive, specific and rapid tools to determine aetiological agents could be incorporated into diagnostic algorithms of respiratory tract infections. Interpretations, significance, and applicability in clinical practice could be further explored, particularly for patients up to 2 years old.

 

Keywords: Infections; molecular; multiplex; PCR; respiratory viruses

 

ABSTRAK

Jangkitan pernafasan akut mengakibatkan morbiditi dan mortaliti yang ketara pada kanak-kanak. Beberapa virus pernafasan yang baru telah dikenal pasti dan pengesanan beberapa virus secara serentak biasa dilaporkan. Kajian ini adalah sebahagian daripada kajian prospektif bertujuan untuk mengesan virus pernafasan menggunakan kaedah molekul multipleks dan kaedah konvensional. Spesimen aspirat nasofarinks diambil daripada kanak-kanak berumur kurang daripada 5 tahun yang dimasukkan ke hospital kerana jangkitan saluran pernafasan bahagian bawah. Pengujian adalah menggunakan pengkulturan virus, imunofluoresen dan reaksi rantai polimerase masa sebenar menggunakan Seegene Anyplex ™ II RV16. Daripada 102 sampel, 69 (67.6%) positif PCR, 12 (11.8%) positif dengan kaedah kultur virus dan 13 (12.7%) positif dengan imunofluoresen. Patogen virus tunggal dikesan dalam 48 sampel (47.1%), manakala 21 sampel (20.6%) mempunyai pengesanan bersama 2 hingga 4 patogen virus. Virus pernafasan sinsitium (RSV) paling banyak dikesan, iaitu dalam 17 sampel (16.7% daripada semua sampel), diikuti oleh adenovirus dan rhinovirus dalam 16 (15.7%) masing-masing. Bocavirus dikesan dalam 15, enterovirus dalam 15, influenza A dalam 8 dan parainfluenza-4 dalam 4 sampel dengan kes tertinggi dalam pengesanan bersama (12/15, 10/15, 5/8 dan 3/4 masing-masing). RSV paling sedikit dikesan dalam pengesanan bersama (3/17). Dalam sampel positif PCR, 54/69 (78.3%) adalah pesakit berusia sehingga 24 bulan. Kaedah molekul mengesan lebih banyak virus daripada kaedah konvensional dengan pengesanan serentak pelbagai virus pernafasan. Kaedah yang lebih sensitif, khusus dan cepat untuk menentukan agen penyebab boleh dimasukkan ke dalam algoritma diagnosis jangkitan saluran pernafasan. Tafsiran keputusan ujian dan kegunaannya dalam amalan klinikal boleh diterokai dengan lebih lanjut, terutama untuk pesakit berusia sehingga 2 tahun.

 

Kata kunci: Jangkitan; molekul; multipleks; PCR; virus pernafasan

RUJUKAN

Aberle, J.H., Aberle, S.W., Pracher, E., Hutter, H.P., Kundi, M. & Popow-Kraupp, T. 2005. Single versus dual respiratory virus infections in hospitalized infants: Impact on clinical course of disease and interferon-gamma response. Pediatr. Infect. Dis. J. 24(7): 605-610.

Advani, S., Sengupta, A., Forman, M., Valsamakis, A. & Milstone, A. 2012. Detecting respiratory viruses in asymptomatic children. Pediatr. Infect. Dis. J. 31(12): 1221-1226.

Allander, T., Tammi, M.T., Eriksson, M., Bjerkner, A., Tiveljung- Lindell, A. & Andersson, B. 2005. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc. Natl. Acad. Sci. U.S.A 102: 12891-12896.

Asner, S.A., Rose, W., Petrich, A., Richardson, S. & Tran, D.J. 2014. Is virus coinfection a predictor of severity in children with viral respiratory infections? Clin. Microbiol. Infect. 21(3): 264.e1-264.e6.

Bibby, D.F., McElarney, I., Breuer, J. & Clark, D.A. 2011. Comparative evaluation of the Seegene Seeplex RV15 and real-time PCR for respiratory virus detection. J. Medical Virology 83: 1469-1475.

Brand, H.K., de Groot, R., Galama, J.M., Brouwer, M.L., Teuwen, K., Hermans, P.W., Melchers, W.J. & Warris, A. 2011. Infection with multiple viruses is not associated with increased disease severity in children with bronchiolitis. Pediatr. Pulmonol. 47: 393-400.

Broccolo, F., Falcone, V., Esposito, S. & Toniolo, A. 2015. Human bocaviruses: Possible etiologic role in respiratory infection. J. Clin. Virol. 72: 75-81.

Bruijnesteijn van Coppenraet, L.E.S., Swanink, C.M.A., van Zwet, A.A., Nijhuis, R.H.T., Schirm, J., Wallinga, J.A. & Ruijs, G.J.H.M. 2010. Comparison of two commercial molecular assays for simultaneous detection of respiratory viruses in clinical samples using two automatic electrophoresis detection systems. J. Virological Methods 169: 188-192.

Bryce, J., Boschi-Pinto, C., Shibuya, K. & Black, R.E. 2005. The WHO Child Health Epidemiology Reference Group WHO estimates of the causes of death in children. Lancet 365: 1147-1152.

Cho, C.H., Chulten, B., Lee, C.K., Nam, M.H., Yoon, S.Y., Lim, C.S., Cho, Y. & Kim, Y.K. 2013. Evaluation of a novel real-time RT-PCR using TOCE technology compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses. J. Clin. Virol. 57(4): 338-342.

Couturier, M.R., Barney, T., Alger, G., Hymas, W.C., Stevenson, J.B., Hillyard, D. & Daly, J.A. 2013. Evaluation of the FilmArray® Respiratory Panel for clinical use in a large children›s hospital. J. Clin. Lab. Anal. 27(2): 148-154.

Debiaggi, M., Canducci, F., Ceresola, E.R. & Clementi, M. 2012. The role of infections and coinfections with newly identified and emerging respiratory viruses in children. Virol. J. 9: 247.

Gagliardi, T.B., Paula, F.E., Iwamoto, M.A., Proença-Modena, J.L., Santos, A.E., Camara, A.A., Cervi, M.C., Cintra, O.A. & Arruda, E. 2013. Concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus. J. Med. Virol. 85(10): 1852-1859.

García-Arroyo, L., Prim, N., Martí, N., Roig, M.C., Navarro, F. & Rabella, N. 2016. Benefits and drawbacks of molecular techniques for diagnosis of viral respiratory infections. Experience with two multiplex PCR assays. J. Med. Virol. 88(1): 45-50.

Gharabaghi, F., Hawan, A., Drews, S.J. & Richardson, S.E. 2011. Evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children. Clin. Microbiol. Infect. 17: 1900-1906.

Guido, M., Tumolo, M.R., Verri, T., Romano, A., Serio, F., De Giorgi, M., De Donno, A., Bagordo, F. & Zizza, A. 2016. Human bocavirus: Current knowledge and future challenges. World J. Gastroenterol. 22(39): 8684-8697.

Huh, H.J., Park, K.S., Kim, J.Y., Kwon, H.J., Kim, J.W., Ki, C.S. & Lee, N.Y. 2014. Comparison of the Anyplex(TM) II RV16 and Seeplex(®) RV12 ACE assays for the detection of respiratory viruses. Diagn. Microbiol. Infect. Dis. 79(4): 419-421.

Huijskens, E.G., Biesmans, R.C., Buiting, A.G., Obihara, C.C. & Rossen, J.W. 2012. Diagnostic value of respiratory virus detection in symptomatic children using real-time PCR. Virology Journal 9: 276.

Jacobs, S.E., Lamson, D.M., St George, K. & Walsh, T.J. 2013. Human rhinoviruses. Clin. Microbiol. Rev. 26(1): 135-162.

Jartti, T., Lehtinen, P., Vuorinen, T., Koskenvuo, M. & Ruuskanen, O. 2004. Persistence of rhinovirus and enterovirus RNA after acute respiratory illness in children. J. Med. Virol. 72: 695-699.

Jason, T.W.Y., Ramli, N., Yusof, M.A., Vengataraman, N., Hussin, H. & Abdul Latif, Z. 2016. Epidemiological and clinical features of enteroviral acute lower respiratory tract infections in hospitalized paediatric patients in a Malaysian tertiary center. Med. J. Malaysia 71(5): 264-268.

Khor, C.S., I-Ching, S., Hooi, P.S., Quek, K.F. & Chan, Y.F. 2012. Epidemiology and seasonality of respiratory viral infections in hospitalized children in Kuala Lumpur, Malaysia: A retrospective study of 27 years. BMC Pediatrics 12: 32.

Kim, H.K., Oh, S.H., Yun, K.A., Sung, H. & Kim, M.N. 2013. Comparison of Anyplex II RV16 with the xTAG respiratory viral panel and Seeplex RV15 for detection of respiratory viruses. J. Clin. Microbiol. 51(4): 1137-1141.

Ksiazek, T.G., Erdman, D., Goldsmith, C.S., Zaki, S.R., Peret, T., Emery, S., Tong, S., Urbani, C., Comer, J.A., Lim, W., Rollin, P.E., Dowell, S.F., Ling, A.E., Humphrey, C.D., Shieh, W.J., Guarner, J., Paddock, C.D., Rota, P., Fields, B., DeRisi, J., Yang, J.Y., Cox, N., Hughes, J.M., LeDuc, J.W., Bellini, W.J., Anderson, L.J. & SARS Working Group. 2003. A novel coronavirus associated with severe acute respiratory syndrome. N. Engl. J. Med. 348: 1953-1966.

Lee, C.Y., Chang, Y.F., Lee, C.L., Wu, M.C., Ho, C.L. & Chang, Y.C. 2015. Molecular viral epidemiology and clinical characterization of acute febrile respiratory infections in hospitalized children in Taiwan. J. Med. Virol. 87(11): 1860-1866.

Lee, J. & Storch, G.A. 2014. Characterization of human coronavirus OC43 and human coronavirus NL63 infections among hospitalized children <5 years of age. Pediatr. Infect. Dis. J. 33(8): 814-20.

Mayer, L.M., Kahlert, C., Rassouli, F., Vernazza, P. & Albrich, W.C. 2017. Impact of viral multiplex real-time PCR on management of respiratory tract infection: A retrospective cohort study. Pneumonia 9: 4. DOI 10.1186/s41479-017- 0028-z.

Nair, H., Nokes, D.J., Gessner, B.D., Dherani, M., Madhi, S.A., Singleton, R.J., O’Brien, K.L., Roca, A., Wright, P.F., Bruce, N., Chandran, A., Theodoratou, E., Sutanto, A., Sedyaningsih, E.R., Ngama, M., Munywoki, P.K., Kartasasmita, C., Simões, E.A., Rudan, I., Weber, M.W. & Campbell, H. 2010. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: A systematic review and meta-analysis. Lancet 375: 1545-1555.

Nair, H., Simões, E.A., Rudan, I., Gessner, B.D., Azziz- Baumgartner, E., Zhang, J.S., Feikin, D.R., Mackenzie, G.A., Moïsi, J.C., Roca, A., Baggett, H.C., Zaman, S.M., Singleton, R.J., Lucero, M.G., Chandran, A., Gentile, A., Cohen, C., Krishnan, A., Bhutta, Z.A., Arguedas, A., Clara, A.W., Andrade, A.L., Ope, M., Ruvinsky, R.O., Hortal, M., McCracken, J.P., Madhi, S.A., Bruce, N., Qazi, S.A., Morris, S.S., El Arifeen, S., Weber, M.W., Scott, J.A., Brooks, W.A., Breiman, R.F., Campbell, H. & Severe Acute Lower Respiratory Infections Working Group. 2013. Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: A systematic analysis. Lancet 381: 1380-1390.

Nathan, A.M., Rani, F., Lee, R.J.Y., Zaki, R., Westerhout, C., Sam, I.C., Lum, L.C.S. & de Bruyne, J. 2014. Clinical risk factors for life-threatening lower respiratory tract infections in children: A retrospective study in an urban city in Malaysia. PLoS ONE 9(10): e111162. doi:10.1371/journal. pone.0111162.

O’Grady, K.F., Grimwood, K., Sloots, T.P., Whiley, D.M., Acworth, J.P., Phillips, N., Goyal, V. & Chang, A.B. 2016. Prevalence, codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom. Clin. Microbiol. Infect. 22(6): 527-534.

Ong, S.B., Lam, K.L. & Lam, S.K. 1982. Viral agents of acute respiratory infections in young children in Kuala Lumpur. Bull. World Health Organ. 60: 137-140.

Popowitch, E.B., O’Neill, S.S. & Miller, M.B. 2013. Comparison of the Biofire FilmArray RP, Genmark eSensor RVP, Luminex xTAG RVPv1, and Luminex xTAG RVP fast multiplex assays for detection of respiratory viruses. J. Clin. Microbiol. 51(5): 1528-1533.

Reddington, K., Tuite, N., Barry, T., O’Grady, J. & Zumla, A. 2013. Advances in multiparametric molecular diagnostics technologies for respiratory tract infections. Curr. Opin. Pulm. Med. 19(3): 298-304.

Rehder, K.J., Wilson, E.A., Zimmerman, K.O., Cunningham, C.K. & Turner, D.A. 2015. Detection of multiple respiratory viruses associated with mortality and severity of illness in children. Pediatr. Crit. Care Med. 16(7): e201-e206.

Roh, K.H., Kim, J., Nam, M.H., Yoon, S., Lee, C.K., Lee, K., Yoo, Y., Kim, M.J. & Cho, Y. 2008. Comparison of the seeplex reverse transcription pcr assay with the r-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses. Annals. of Clin. Lab. Science 38(1): 41-46.

Royston, L. & Tapparel, C. 2016. Rhinoviruses and respiratory enteroviruses: Not as simple as ABC. Viruses 8: 16.

Rudan, I., Tomaskovic, L., Boschi-Pinto, C. & Campbell, H. 2004. Global estimate of the incidence of clinical pneumonia among children under five years of age. Bull. World Health Organ. 82: 895-903.

Salez, N., Nougairede, A., Ninove, L., Zandotti, C., de Lamballerie, X. & Charrel, R.N. 2015. Prospective and retrospective evaluation of the Cepheid Xpert® Flu/RSV XC assay for rapid detection of influenza A, influenza B, and respiratory syncytial virus. Diagn. Microbiol. Infect. Dis. 81(4): 256-258.

Shay, D.K., Holman, R.C., Newman, R.D., Liu, L.L., Stout, J.W. & Anderson, L.J. 1999. Bronchiolitis associated hospitalizations among US children, 1980-1996. JAMA 282: 1440-1446.

van den Hoogen, B.G., de Jong, J.C., Groen, J., Kuiken, T., de Groot, R., Fouchier, R.A. & Osterhaus, A.D. 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 7: 719-724.

van der Hoek, L., Pyrc, K., Jebbink, M.F., Vermeulen-Oost, W., Berkhout, R.J., Wolthers, K.C., Wertheim-van Dillen, P.M., Kaandorp, J., Spaargaren, J. & Berkhout, B. 2004. Identification of a new human coronavirus. Nat. Med. 10: 368-373.

Walker, C.L., Rudan, I., Liu, L., Nair, H., Theodoratou, E., Bhutta, Z.A., O’Brien, K.L., Campbell, H. & Black, R.E. 2013. Global burden of childhood pneumonia and diarrhoea. The Lancet 381: 1405-1416.

Woo, P.C., Lau, S.K., Chu, C.M., Chan, K.H., Tsoi, H.W., Huang, Y., Wong, B.H., Poon, R.W., Cai, J.J., Luk, W.K., Poon, L.L., Wong, S.S., Guan, Y., Peiris, J.S. & Yuen, K.Y. 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79: 884-895.

Yoshida, L.M., Suzuki, M., Nguyen, H.A., Le, M.N., Dinh Vu, T., Yoshino, H., Schmidt, W.P., Nguyen, T.T., Le, H.T., Morimoto, K., Moriuchi, H., Dang, D.A. & Ariyoshi, K. 2013. Respiratory syncytial virus co-infection and paediatric lower respiratory tract infection. Eur. Respir. J. 42: 461-469.

Yun, S.G., Kim, M.Y., Choi, J.M., Lee, C.K., Lim, C.S., Cho, Y. & Suh, I.B. 2017. Comparison of three multiplex PCR assays for detection of respiratory viruses: Anyplex II RV16, AdvanSure RV, and Real-Q RV. J. Clin. Lab. Anal. https:// doi.org/10.1002/jcla.22230.

Zamberi, S., Zulkifli, I. & Ilina, I. 2003. Respiratory viruses detected in hospitalised paediatric patients with respiratory infections. Med. J. Malaysia 58: 681-687.

Zumla, A., Al-Tawfiq, J.A., Enne, V.I., Kidd, M., Drosten, C., Breuer, J., Muller, M.A., Hui, D., Maeurer, M., Bates, M., Mwaba, P., Al-Hakeem, R., Gray, G., Gautret, P., Al-Rabeeah, A.A., Memish, Z.A. & Gant, V. 2014. Rapid point of care diagnostic tests for viral and bacterial respiratory tract infections--needs, advances, and future prospects. Lancet Infect. Dis. 14(11): 1123-1135.

 

*Pengarang untuk surat-menyurat; email: zetti@ppukm.ukm.edu.my