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Utility of CDC DENV1–4 real time PCR assay and trioplex assay for the diagnosis of dengue in patients with acute febrile illness

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Abstract

Nucleic acid amplification tests (NAATs) have revolutionized reliable detection of dengue virus (DENV) during acute phase of infection. The study evaluated performance of CDC DENV-1–4 real-time assay, trioplex RT-PCR and heminested conventional RT-PCR assay in the diagnosis of DENV. The three NAATs were performed on 107 consecutive samples collected from patients suspected of DENV infection during acute phase of illness. Their performance was compared against composite reference standard, consisting of DENV NS1 antigen ELISA and DENV IgM ELISA. 88/107 study samples were positive by DENV ELISA, either NS1Ag (80), IgM (3) or both (5). The overall sensitivity of CDC DENV-1–4 RT-PCR assay, trioplex RT-PCR assay and conventional multiplex RT-PCR was 68.18%, 54.55% and 38.64%, respectively in diagnosing dengue during acute phase, with an area under the curve of 0.841, 0.773 and 0.693 respectively when compared against composite reference standard. The sensitivity was 82.93%, 73.17% and 51.22%, respectively within three days of illness and 60%, 42.86% and 28.57%, respectively between 4 and 5th day of illness. All the three molecular assays had 100% specificity. Maximum concordance values of 86.9% were recorded among CDC DENV-1–4 rRT-PCR assay and trioplex assay with kappa value of 0.74, suggestive of substantial agreement. CDC DENV-1–4 rRT-PCR assay can be used as a reliable and accurate test for diagnosis of DENV during acute phase of illness.

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References

  1. Guzman MG, Harris E. Dengue. Lancet. 2015;385:453–65.

    Article  PubMed  Google Scholar 

  2. World Health Organization. Dengue and severe dengue key facts [Internet]. World Heal. Organ. 2021. p. 1–13. Available from: https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue.

  3. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature. 2013;496:504–7. https://doi.org/10.1038/nature12060.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. NCVBDC Organogram. National Center for Vector Borne Diseases Control (NCVBDC) [Internet]. 2017 [cited 2022 Aug 13]. p. 0–2. Available from: https://nvbdcp.gov.in/index1.php?lang=1&level=1&sublinkid=5885&lid=3964.

  5. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992;30:545–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. WHO. Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever [Internet]. WHO Reg. Publ. SEARO. 2011. Available from: http://scholar.google.com/scholar?hl=en&btnG=Search&q=intitle:Comprehensive+Guidelines+for+Prevention+and+Control+of+Dengue+and+Dengue+Haemorrhagic+Fever#1.

  7. CDC. CDC DENV-1-4 real-time RT-PCR assay for detection and serotype identification of dengue virus. CDC [Internet]. 2013;1–53. Available from: http://www.cdc.gov/dengue/resources/rt_pcr/CDCPackageInsert.pdf.

  8. CDC. Trioplex real-time RT-PCR assay: instructions of use. Centers Dis Control Prev. 2017;1–29.

  9. Santiago GA, Vázquez J, Courtney S, Matías KY, Andersen LE, Colón C, et al. Performance of the Trioplex real-time RT-PCR assay for detection of Zika, dengue, and chikungunya viruses. Nat Commun. 2018. https://doi.org/10.1038/s41467-018-03772-1.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics. 1977;33:159.

    Article  CAS  PubMed  Google Scholar 

  11. MedCalc Software Ltd. Free statistical calculators [Internet]. 2016 [cited 2022 Aug 13]. p. 20–2. Available from: https://www.medcalc.org/calc/diagnostic_test.php.

  12. Singh MP, Goyal K, Ratho RK. Nonstructural protein NS1: Giving a new structure to dengue diagnosis. J Clin Microbiol. 2010;48:4688.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Hermann LL, Thaisomboonsuk B, Poolpanichupatam Y, Jarman RG, Kalayanarooj S, Nisalak A, et al. Evaluation of a dengue NS1 antigen detection assay sensitivity and specificity for the diagnosis of acute dengue virus infection. PLoS Negl Trop Dis. 2014;8:1–8.

    Article  Google Scholar 

  14. Lapphra K, Sangcharaswichai A, Chokephaibulkit K, Tiengrim S, Piriyakarnsakul W, Chakorn T, et al. Evaluation of an NS1 antigen detection for diagnosis of acute dengue infection in patients with acute febrile illness. Diagn Microbiol Infect Dis. 2008;60:387–91.

    Article  PubMed  Google Scholar 

  15. Lai SC, Huang YY, Wey JJ, Tsai MH, Chen YL, Shu PY, et al. Development of novel dengue NS1 multiplex lateral flow immunoassay to differentiate serotypes in serum of acute phase patients and infected mosquitoes. Front Immunol. 2022;13:1–15.

    Article  CAS  Google Scholar 

  16. Poltep K, Nakayama EE, Sasaki T, Kurosu T, Takashima Y, Phadungsombat J, et al. Development of a dengue virus serotype-specific non-structural protein 1 capture immunochromatography method. Sensors. 2021;21:7809.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M. Enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol. 2002;40:376–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, et al. Dengue in the early febrile phase: viremia and antibody responses. J Infect Dis. 1997;176:322–30.

    Article  CAS  PubMed  Google Scholar 

  19. Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis. 2000;181:2–9.

    Article  CAS  PubMed  Google Scholar 

  20. Waggoner JJ, Abeynayake J, Sahoo MK, Gresh L, Tellez Y, Gonzalez K, et al. Comparison of the FDA-approved CDC DENV-1-4 real-time reverse transcription-PCR with a laboratory-developed assay for dengue virus detection and serotyping. J Clin Microbiol. 2013;51:3418–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Carrasquilla MC, Ortiz MI, León C, Rondón S, Kulkarni MA, Talbot B, et al. Entomological characterization of Aedes mosquitoes and arbovirus detection in Ibagué, a Colombian city with co-circulation of Zika, dengue and chikungunya viruses. Parasit Vectors. 2021;14:446.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Balingit JC, Carvajal TM, Saito-Obata M, Gamboa M, Nicolasora AD, Sy AK, et al. Surveillance of dengue virus in individual Aedes aegypti mosquitoes collected concurrently with suspected human cases in Tarlac City, Philippines. Parasites and vectors. BioMed Central. 2020;13:1–13. https://doi.org/10.1186/s13071-020-04470-y.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors gratefully acknowledge the kind support of NIV, Pune, Resource Centre, VRDL for providing the reagent support and the Regional VRDL, Department of Virology, PGIMER, Chandigarh for the technical support.

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Author notes

  1. Subhabrata Sarkar and Ishani Bora have equally contributed as joint first author.

Authors and Affiliations

  1. Department of Virology, Post Graduate Institute of Medical Education and Research, Chandigarh, National Institute of Virology, Pune, India

    Subhabrata Sarkar, Ishani Bora, Kanwalpreet Kaur & Radha Kanta Ratho

  2. Department of Medical Microbiology, Post Graduate Institute of Medical Education and Research, Chandigarh, National Institute of Virology, Pune, India

    Parakriti Gupta & Shveta Shethi

  3. Virus Research and Diagnostic Laboratory, Diagnostic Virology Group, ICMR-NIV, Resource Centre, National Institute of Virology, Pune, Maharashtra, India

    Gajanan Sapkal

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  1. Subhabrata Sarkar
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Contributions

SS: study conceptualization, Implementation of laboratory experimentation, data analysis and writing manuscript. IB: study conceptualization, data analysis, writing manuscript and approving the final manuscript. PG: Data curation, Data retrieval, Writing – review & editing, review of manuscript. GS: Data curation, Data retrieval, and review of manuscript. SS: Data curation, Data retrieval, Writing – review & editing, review of manuscript. KK: Implementation of diagnostic strategy, Implementation of laboratory experimentation and data curation. RKR: Study conceptualization, data analysis, writing manuscript and supervision.

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Correspondence to Radha Kanta Ratho.

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Sarkar, S., Bora, I., Gupta, P. et al. Utility of CDC DENV1–4 real time PCR assay and trioplex assay for the diagnosis of dengue in patients with acute febrile illness. VirusDis. 34, 365–372 (2023). https://doi.org/10.1007/s13337-023-00831-0

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  • DOI: https://doi.org/10.1007/s13337-023-00831-0

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