Abstract
Objectives
The performance of the new Respiratory Pathogen panel (fluorescent probe melting curve, FPMC) for the qualitative detection of 12 organisms (chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus, rhinovirus, etc.) was assessed.
Methods
Prospectively collected nasopharyngeal swab (NPS) and sputum specimens (n = 635) were detected by using the FPMC panel, with the Sanger sequencing method as the comparative method.
Results
The overall percent concordance between the FPMC analysis method and the Sanger sequencing method was 100% and 99.66% for NPS and sputum specimens, respectively. The FPMC testified an overall positive percent concordance of 100% for both NPS and sputum specimens. The FPMC analysis method also testified an overall negative percent concordance of 100% and 99.38% for NPS and sputum specimens, respectively.
Conclusions
The FPMC analysis method is a stable and accurate assay for rapid, comprehensive detecting for respiratory pathogens.
Introduction
Respiratory tract infection (RTI) is one of the most important causes for extensive morbidity and mortality among patients worldwide [1]. Different pathogens could induce similar symptoms and signs of RTI, which is mainly characterized by upper respiratory infections such as rhinitis, pharyngitis, laryngitis, tonsillitis, etc. [2]. And some patients with RTI show severe symptoms of lower respiratory infections including tracheitis, bronchitis and pneumonia [3]. It has been demonstrated that most acute respiratory tract infections are induced by viruses including respiratory syncytial virus, adenovirus, influenza A and B viruses, parainfluenza virus, and so on [4]. Respiratory virus infection is one of the most common diseases for people in all age groups [5]. Notably, treatment method, curative effect and disease course vary between patients with RTI induced by different pathogens [6]. Therefore, accurate and timely etiological analysis is not only essential for diagnosis of RTI, but also the basis for reasonable selection of appropriate therapeutic regimen [7, 8]. And it is also urgent to develop new methods of rapid detection of respiratory viruses.
Single-tube multiplex fluorescent probe melting curve (FPMC) technology (four channel detection: Fam, Vic, Rox, Cy5) and fusion curve analysis employed in the new detection method is evaluated in this study. Detection using this new method covers a wide range of 12 kinds of pathogen nucleic acids, including chlamydia pneumoniae, mycoplasma pneumoniae, adenovirus, influenza A virus, influenza B virus, parainfluenza virus (types 1, 2, 3 and 4), rhinovirus, respiratory syncytial virus, Boca virus, metapneumovirus, coronavirus (229E, hku1, nl63 and OC43), or novel coronavirus. Specifically, three channels (Fam, Vic and Rox) were employed to detect the target pathogen, and pathogens in a sample are identified according to the cycle threshold (CT) values in each channel during the process of PCR amplification and the corresponding change rate of peak height within a range of the melting temperatures of the targeted pathogens. Additionally, Cy5 channel was used for detecting the endogenous internal standard in order to monitor the quality of samples and the accuracy of experimental processes.
To have insight into the novel diagnostic assay for clinical application, we here provide important evidence comparing the diagnostic accuracy of the FPMC analysis method and Sanger sequencing method for the detection of RTI.
Methods
Study population
This study included 635 patients of all ages and both genders showing with signs and/or symptoms of respiratory tract infection such as cough, nasal congestion, runny nose, sore throat, loss of smell or taste, dyspnea, lung related diseases, etc. These patients were all from the second affiliated hospital of ** of multiple pathogens with strong sensitivity and high specificity by using the technology of hybridization or polymerase chain reaction. Moreover, on the basis of ensuring the sensitivity and specificity of analysis, it could perform detection by micro-sample handling and make operation procedures more easily.
Our results demonstrate that the overall performance of the FPMC analysis method has an overall percent agreement (true-positive and true-negative results) of > 99% for all available targets tested compared with the sequencing method. Discrepancy between the FPMC analysis method and the sequencing method may be due to three main factors. Firstly, the sensitivity of the sequencing method may be low, which will lead to the negative results for those weakly positive samples with CT value being near the cut off of the FPMC analysis method. Secondly, primers of the sequencing method may not be able to cover all sub-types of organisms, and thus some organisms in a sample could not be detected using the sequencing method. Finally, FPMC analysis method is a new assay based on PCR reaction. So there are occasionally false-positive results due to the PCR contamination during the process of experiments. In this study, the performance characteristics of the new FPMC analysis method were evaluated by assessing agreement with the results of the sequencing method, a generally accepted standard method.
However, there are still some limitations in our study. Firstly, this study is lack of another molecular-based method for discordant sample adjudications. Comparisons of this FPMC analysis method with another multiplex panel would provide useful information about discordant results with the sequencing method. But this is beyond the designs of our current study. Secondly, the pathogen spectrum of the FPMC analysis method does not include all pathogens. Therefore, combination of the FPMC analysis method and other molecular methods detecting bacteria could help to improve ability in diagnostic testing of respiratory pathogens. Finally, the lack of detection of influenza A virus and Covid-2019 in this study limits the data on the performance for these targets. In our subsequent research, relevant samples will be collected to elucidate the diagnostic efficacy of the new assay kit for influenza A virus and Covid-19. Overall, the FPMC analysis method is a rapid, accurate, and easy-to-use assay for detection of organisms in clinical specimens from the respiratory tract in clinical laboratories.
Data availability
No datasets were generated or analysed during the current study.
References
Ferkol T, Schraufnagel D. The global burden of respiratory disease. Annals Am Thorac Soc. 2014;11(3):404–6.
Shahan B, Barstow C, Mahowald M. Respiratory conditions: Upper Respiratory Tract infections. FP Essentials. 2019;486:11–8.
Niederman MS, Torres A. Respiratory infections. Eur Respiratory Review: Official J Eur Respiratory Soc 2022, 31(166).
Mandell LA. Etiologies of acute respiratory tract infections. Clin Infect Diseases: Official Publication Infect Dis Soc Am. 2005;41(4):503–6.
Basnayake TL, Morgan LC, Chang AB. The global burden of respiratory infections in indigenous children and adults: a review. Respirology. 2017;22(8):1518–28.
Papadopoulos NG, Megremis S, Kitsioulis NA, Vangelatou O, West P, Xepapadaki P. Promising approaches for the treatment and prevention of viral respiratory illnesses. J Allergy Clin Immunol. 2017;140(4):921–32.
Rogers BB, Shankar P, Jerris RC, Kotzbauer D, Anderson EJ, Watson JR, O’Brien LA, Uwindatwa F, McNamara K, Bost JE. Impact of a rapid respiratory panel test on patient outcomes. Arch Pathol Lab Med. 2015;139(5):636–41.
Lowe CF, Payne M, Puddicombe D, Mah A, Wong D, Kirkwood A, Hull MW, Leung V. Antimicrobial stewardship for hospitalized patients with viral respiratory tract infections. Am J Infect Control. 2017;45(8):872–5.
Walter JM, Wunderink RG. Severe respiratory viral infections: New evidence and changing paradigms. Infect Dis Clin N Am. 2017;31(3):455–74.
Granados A, Peci A, McGeer A, Gubbay JB. Influenza and rhinovirus viral load and disease severity in upper respiratory tract infections. J Clin Virology: Official Publication Pan Am Soc Clin Virol. 2017;86:14–9.
Seppala E, Sillanpaa S, Nurminen N, Huhtala H, Toppari J, Ilonen J, Veijola R, Knip M, Sipila M, Laranne J, et al. Human enterovirus and rhinovirus infections are associated with otitis media in a prospective birth cohort study. J Clin Virology: Official Publication Pan Am Soc Clin Virol. 2016;85:1–6.
Jang YJ, Kwon HJ, Park HW, Lee BJ. Detection of rhinovirus in turbinate epithelial cells of chronic sinusitis. Am J Rhinol. 2006;20(6):634–6.
Corne JM, Marshall C, Smith S, Schreiber J, Sanderson G, Holgate ST, Johnston SL. Frequency, severity, and duration of rhinovirus infections in asthmatic and non-asthmatic individuals: a longitudinal cohort study. Lancet. 2002;359(9309):831–4.
Louie JK, Roy-Burman A, Guardia-Labar L, Boston EJ, Kiang D, Padilla T, Yagi S, Messenger S, Petru AM, Glaser CA, et al. Rhinovirus associated with severe lower respiratory tract infections in children. Pediatr Infect Dis J. 2009;28(4):337–9.
Louie JK, Yagi S, Nelson FA, Kiang D, Glaser CA, Rosenberg J, Cahill CK, Schnurr DP. Rhinovirus outbreak in a long term care facility for elderly persons associated with unusually high mortality. Clin Infect Diseases: Official Publication Infect Dis Soc Am. 2005;41(2):262–5.
Kraft CS, Jacob JT, Sears MH, Burd EM, Caliendo AM, Lyon GM. Severity of human rhinovirus infection in immunocompromised adults is similar to that of 2009 H1N1 influenza. J Clin Microbiol. 2012;50(3):1061–3.
Makela MJ, Puhakka T, Ruuskanen O, Leinonen M, Saikku P, Kimpimaki M, Blomqvist S, Hyypia T, Arstila P. Viruses and bacteria in the etiology of the common cold. J Clin Microbiol. 1998;36(2):539–42.
Arruda E, Pitkaranta A, Witek TJ Jr., Doyle CA, Hayden FG. Frequency and natural history of rhinovirus infections in adults during autumn. J Clin Microbiol. 1997;35(11):2864–8.
Mandell LA. Community-acquired pneumonia: an overview. Postgrad Med. 2015;127(6):607–15.
Cunha BA. The atypical pneumonias: clinical diagnosis and importance. Clin Microbiol Infection: Official Publication Eur Soc Clin Microbiol Infect Dis. 2006;12(Suppl 3):12–24.
Del Silva-Caso V-MJ, Cornejo-Tapia W, Orellana-Peralta A, Verne F, Ugarte E, Aguilar-Luis C, De Lama-Odria MA, Nazario-Fuertes MDC, Esquivel-Vizcarra R. Molecular etiological profile of atypical bacterial pathogens, viruses and coinfections among infants and children with community acquired pneumonia admitted to a national hospital in Lima, Peru. BMC Res Notes. 2017;10(1):688.
Acknowledgements
We thank all the medical technologists and research staff across all the study sites for their important contribution to this work.
Funding
This study was supported by the Science and Technology Planning Project of Shaanxi Province of China support grant (S2023-YF-YBSF-0408).
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception, design, and data collection. Material preparation was performed by Jianhong Zhu, Bo Zhong, Ning Gao, Ke Lei, Zeshi Liu, Chaoliang **ong, **g Lei and Ying Tian. Analysis was performed by Yan Geng, Yiwei Tang, Nan Feng, Weixiao Zhou, Xue Zhang, Dong Chen and **g Li. The first draft of the manuscript was written by Li Xue, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Second Affiliated Hospital, **’an Jiaotong University (NO. 2020-048) according to the Declaration of Helsinki.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
About this article
Cite this article
Xue, L., Zhu, J., Lei, K. et al. Evaluation of the FPMC respiratory panel for detection of respiratory tract pathogens in nasopharyngeal swab and sputum specimens. Virol J 21, 156 (2024). https://doi.org/10.1186/s12985-024-02430-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1186/s12985-024-02430-x