Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay.
Wastewater
/ virology
Influenza A virus
/ genetics
Humans
Influenza B virus
/ genetics
Multiplex Polymerase Chain Reaction
/ methods
Sensitivity and Specificity
Respiratory Syncytial Viruses
/ genetics
Reproducibility of Results
Influenza, Human
/ diagnosis
Respiratory Syncytial Virus, Human
/ genetics
Real-Time Polymerase Chain Reaction
/ methods
SARS-CoV-2
/ genetics
Influenza A
Influenza B
RT-ddPCR
Respiratory Syncytial Virus
Wastewater
Journal
Human genomics
ISSN: 1479-7364
Titre abrégé: Hum Genomics
Pays: England
ID NLM: 101202210
Informations de publication
Date de publication:
20 May 2024
20 May 2024
Historique:
received:
29
12
2023
accepted:
01
05
2024
medline:
21
5
2024
pubmed:
21
5
2024
entrez:
20
5
2024
Statut:
epublish
Résumé
After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.
Sections du résumé
BACKGROUND
BACKGROUND
After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M).
RESULTS
RESULTS
The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen's correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance.
CONCLUSIONS
CONCLUSIONS
Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.
Identifiants
pubmed: 38769549
doi: 10.1186/s40246-024-00614-8
pii: 10.1186/s40246-024-00614-8
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
48Informations de copyright
© 2024. The Author(s).
Références
Chow EJ, Uyeki TM, Chu HY. The effects of the COVID-19 pandemic on community respiratory virus activity. Nat Rev Microbiol. 2022;21:195–210.
pubmed: 36253478
pmcid: 9574826
World Health Organization (WHO). https://www.who.int/ . Accessed 10 Nov 2023.
Centers for Disease Control and Prevention. https://www.cdc.gov/ . Accessed 10 Nov 2023.
Ferrani S, Prazuck T, Béchet S, Lesne F, Cohen R, Levy C. Diagnostic accuracy of a rapid antigen triple test (SARS-CoV-2, respiratory syncytial virus, and influenza) using anterior nasal swabs versus multiplex RT-PCR in children in an emergency department. Infect Dis Now. 2023;53:104769.
doi: 10.1016/j.idnow.2023.104769
pubmed: 37572988
Bayart JL, Gillot C, Dogné JM, Roussel G, Verbelen V, Favresse J, et al. Clinical performance evaluation of the Fluorecare® SARS-CoV-2 & influenza A/B & RSV rapid antigen combo test in symptomatic individuals. J Clin Virol. 2023;6:96.
Papagiannis D, Perlepe G, Tendolouri T, Karakitsiou P, Damagka G, Kalaitzi A, et al. Proportion of respiratory syncytial virus, SARS-CoV-2, influenza A/B, and adenovirus cases via rapid tests in the community during winter 2023—a cross sectional study. Diseases. 2023;11:122.
doi: 10.3390/diseases11030122
pubmed: 37754318
pmcid: 10529898
Dinç HÖ, Karabulut N, Alaçam S, Uysal HK, Daşdemir FO, Önel M, et al. Evaluation of the diagnostic performance of a SARS-CoV-2 and influenza A/B combo rapid antigen test in respiratory samples. Diagnostics. 2023;13:63.
doi: 10.3390/diagnostics13050972
Merckx J, Wali R, Schiller I, Caya C, Gore GC, Chartrand C, et al. Diagnostic accuracy of novel and traditional rapid tests for influenza infection compared with reverse transcriptase polymerase chain reaction. Ann Intern Med. 2017;167:395–409.
doi: 10.7326/M17-0848
Alotaibi BS, Tantry BA, Bandy A, Ahmad R, Khursheed SQ, Ahmad A, et al. Simultaneous detection of influenza A/B, respiratory syncytial virus, and SARS-CoV-2 in nasopharyngeal swabs by one-tube multiplex reverse transcription polymerase chain reaction. Trop Med Infect Dis. 2023;8:693.
Sahajpal NS, Mondal AK, Ananth S, Njau A, Jones K, Ahluwalia P, et al. Clinical validation of a multiplex PCR-based detection assay using saliva or nasopharyngeal samples for SARS-Cov-2, influenza A and B. Sci Rep. 2022;12:1–7.
doi: 10.1038/s41598-022-07152-0
Hayes EK, Gouthro MT, Leblanc JJ, Gagnon GA. Simultaneous detection of SARS-CoV-2, in fl uenza A, respiratory syncytial virus, and measles in wastewater by multiplex RT-qPCR. Sci Total Environ. 2023;889:164261.
doi: 10.1016/j.scitotenv.2023.164261
pubmed: 37201817
Toribio-avedillo D, Gómez-gómez C, Sala-comorera L, Rodríguez-rubio L. Monitoring influenza and respiratory syncytial virus in wastewater. Beyond COVID-19. 2023;5:693.
Zafeiriadou A, Kaltsis L, Kostakis M, Kapes V, Thomaidis NS, Markou A. Wastewater surveillance of the most common circulating respiratory viruses in Athens: the impact of COVID-19 on their seasonality. Sci Total Environ. 2023;900:166136.
doi: 10.1016/j.scitotenv.2023.166136
pubmed: 37567285
Mercier E, Pisharody L, Guy F, Wan S, Hegazy N, D’Aoust PM, et al. Wastewater-based surveillance identifies start to the pediatric respiratory syncytial virus season in two cities in Ontario, Canada. Front Public Heal. 2023;11:1–8.
Ahmed W, Simpson SL, Bertsch PM, Bibby K, Bivins A, Blackall LL, et al. Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance. Sci Total Environ. 2022;5:805.
Tiwari A, Ahmed W, Oikarinen S, Sherchan SP, Heikinheimo A, Jiang G, et al. Application of digital PCR for public health-related water quality monitoring. Sci Total Environ. 2022;837:155663.
doi: 10.1016/j.scitotenv.2022.155663
pubmed: 35523326
Tan C, Fan D, Wang N, Wang F, Wang B, Zhu L, et al. Applications of digital PCR in COVID-19 pandemic. View. 2021;2:1–7.
doi: 10.1002/VIW.20200082
Waseem H, Abid R, Ali J, Oswald CJ, Gilbride KA. Wastewater-based epidemiology of SARS-CoV-2 and other respiratory viruses: bibliometric tracking of the last decade and emerging research directions. Water (Switzerland). 2023;5:15.
Gibson KE, Schwab KJ, Spencer SK, Borchardt MA. Measuring and mitigating inhibition during quantitative real time PCR analysis of viral nucleic acid extracts from large-volume environmental water samples. Water Res. 2012;46:4281–91.
doi: 10.1016/j.watres.2012.04.030
pubmed: 22673345
Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012;113:1014–26.
doi: 10.1111/j.1365-2672.2012.05384.x
pubmed: 22747964
Cao Y, Griffith JF, Dorevitch S, Weisberg SB. Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters. J Appl Microbiol. 2012;113:66–75.
doi: 10.1111/j.1365-2672.2012.05305.x
pubmed: 22497995
Dimitrakopoulos L, Kontou A, Strati A, Galani A, Kostakis M, Kapes V, et al. Evaluation of viral concentration and extraction methods for SARS-CoV-2 recovery from wastewater using droplet digital and quantitative RT-PCR. Case Stud Chem Environ Eng. 2022;6:100224.
doi: 10.1016/j.cscee.2022.100224
pubmed: 37520924
pmcid: 9222221
Galani A, Aalizadeh R, Kostakis M, Markou A, Alygizakis N, Lytras T, et al. SARS-CoV-2 wastewater surveillance data can predict hospitalizations and ICU admissions. Sci Total Environ. 2022;804:150151.
doi: 10.1016/j.scitotenv.2021.150151
pubmed: 34623953
Babler KM, Sharkey ME, Abelson S, Amirali A, Benitez A, Cosculluela GA, et al. Degradation rates influence the ability of composite samples to represent 24-hourly means of SARS-CoV-2 and other microbiological target measures in wastewater. Sci Total Environ. 2023;867:63.
doi: 10.1016/j.scitotenv.2023.161423
Zhan Q, Babler KM, Sharkey ME, Amirali A, Beaver CC, Boone MM, et al. Relationships between SARS-CoV-2 in Wastewater and COVID-19 Clinical Cases and Hospitalizations, with and without Normalization against Indicators of Human Waste. ACS ES T Water. 2022;2:1992–2003.
doi: 10.1021/acsestwater.2c00045
pubmed: 36398131
pmcid: 9664448
Strati A, Lianidou ES, Zavridou M, Paraskevis D, Magiorkinis G, Sapounas S, et al. Development and analytical validation of a one-step five-plex RT-ddPCR assay for the quantification of SARS-CoV-2 transcripts in clinical samples. Anal Chem. 2022;94:12314–22.
doi: 10.1021/acs.analchem.2c00868
pubmed: 35960711
Profaizer T, Slev P. A multiplex, droplet digital PCR assay for the detection of T-cell receptor excision circles and kappa-deleting recombination excision circles. Clin Chem. 2020;66:229–38.
doi: 10.1373/clinchem.2019.308171
pubmed: 31672859
Milosevic D, Mills JR, Campion MB, Vidal-Folch N, Voss JS, Halling KC, et al. Applying standard clinical chemistry assay validation to droplet digital PCR quantitative liquid biopsy testing. Clin Chem. 2018;64:1732–42.
doi: 10.1373/clinchem.2018.291278
pubmed: 30237149
Warish Ahmed, Wendy J. M. Smith, Suzanne Metcalfe, Greg Jackson, Phil M. Choi, Mary Morrison, Daniel Field, Pradip Gyawali, Aaron Bivins, Kyle Bibby and SLS. Comparison of RT-qPCR and RT-dPCR Platforms for the Trace Detection of SARS-CoV‑2 RNA in Wastewater.pdf.
Cao Y, Raith MR, Griffith JF. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment. Water Res. 2015;70:337–49.
doi: 10.1016/j.watres.2014.12.008
pubmed: 25543243
Leong NKC, Chu DKW, Chu JTS, Tam YH, Ip DKM, Cowling BJ, et al. A six-plex droplet digital RT-PCR assay for seasonal influenza virus typing, subtyping, and lineage determination. Influenza Other Respi Viruses. 2020;14:720–9.
doi: 10.1111/irv.12769
Nathaniel KCL, Haogao G, Daisy YMN, Lydia DJC, Pavithra K, Samuel SMC, Malik PLLMP. Development of multiplex RT-ddPCR assays for detection of SARS-CoV-2 and other common respiratory virus infections. Science. 2022;5:96.
Lou X, Yan H, Su L, Sun Y, Wang X, Gong L, et al. Detecting the neuraminidase R294K mutation in avian influenza A (H7N9) virus using reverse transcription droplet digital PCR method. Viruses. 2023;6:15.
Wolfe MK, Duong D, Bakker KM, Ammerman M, Mortenson L, Hughes B, et al. Wastewater-based detection of two influenza outbreaks. Environ Sci Technol Lett. 2022;9:687–92.
doi: 10.1021/acs.estlett.2c00350
Wolken M, Sun T, McCall C, Schneider R, Caton K, Hundley C, et al. Wastewater surveillance of SARS-CoV-2 and influenza in preK-12 schools shows school, community, and citywide infections. Water Res. 2022;2023(231):119648.
Markt R, Stillebacher F, Nägele F, Kammerer A, Peer N, Payr M, et al. Expanding the pathogen panel in wastewater epidemiology to influenza and norovirus. Viruses. 2023;15:68.
doi: 10.3390/v15020263
Hughes B, Duong D, White BJ, Wigginton KR, Chan EMG, Wolfe MK, et al. Respiratory syncytial virus (RSV) RNA in wastewater settled solids reflects RSV clinical positivity rates. Environ Sci Technol Lett. 2022;9:173–8.
doi: 10.1021/acs.estlett.1c00963
Roldan-Hernandez L, Boehm AB. Adsorption of respiratory syncytial virus, rhinovirus, SARS-CoV-2, and F+ bacteriophage MS2 RNA onto wastewater solids from raw wastewater. Environ Sci Technol. 2023;57:13346–55.
doi: 10.1021/acs.est.3c03376
pubmed: 37647137
pmcid: 10501194
Boehm AB, Hughes B, Duong D, Chan-Herur V, Buchman A, Wolfe MK, et al. Wastewater concentrations of human influenza, metapneumovirus, parainfluenza, respiratory syncytial virus, rhinovirus, and seasonal coronavirus nucleic-acids during the COVID-19 pandemic: a surveillance study. The Lancet Microbe. 2023;4:e340–8.
doi: 10.1016/S2666-5247(22)00386-X
pubmed: 36965504
pmcid: 10032662
Boehm AB, Wolfe MK, White BJ, Hughes B, Duong D, Bidwell A. More than a tripledemic: influenza A virus, respiratory syncytial virus, SARS-CoV-2, and human metapneumovirus in wastewater during winter 2022–2023. Environ Sci Technol Lett. 2023;10:622–7.
doi: 10.1021/acs.estlett.3c00385
pubmed: 37577361
pmcid: 10413932