Molecular Detection and Typing of Dengue Viruses from Archived Tissues of Fatal Cases by RT-PCR and Sequencing: Diagnostic and Epidemiologic Implications

Julu Bhatnagar Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Dianna M. Blau Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Wun-Ju Shieh Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Christopher D. Paddock Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Clifton Drew Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Lindy Liu Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Tara Jones Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Mitesh Patel Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Sherif R. Zaki Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia

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Diagnosis of dengue virus (DENV) infection in fatal cases is challenging because of the frequent unavailability of blood or fresh tissues. For formalin-fixed, paraffin-embedded (FFPE) tissues immunohistochemistry (IHC) can be used; however, it may not be as sensitive and serotyping is not possible. The application of reverse transcription-polymerase chain reaction (RT-PCR) for the detection of DENV in FFPE tissues has been very limited. We evaluated FFPE autopsy tissues of 122 patients with suspected DENV infection by flavivirus and DENV RT-PCR, sequencing, and DENV IHC. The DENV was detected in 61 (50%) cases by RT-PCR or IHC. The RT-PCR and sequencing detected DENV in 60 (49%) cases (DENV-1 in 16, DENV-2 in 27, DENV-3 in 8, and DENV-4 in 6 cases). No serotype could be identified in three cases. The IHC detected DENV antigens in 50 (40%) cases. The RT-PCR using FFPE tissue improves detection of DENV in fatal cases and provides sequence information useful for typing and epidemiologic studies.

Author Notes

*Address correspondence to Julu Bhatnagar, Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Bldg.18/SB-105, MS-G32, 1600 Clifton Road NE, Atlanta, GA 30333. E-mail: JBhatnagar@cdc.gov

Authors’ addresses: Julu Bhatnagar, Dianna M. Blau, Wun-Ju Shieh, Christopher D. Paddock, Clifton Drew, Lindy Liu, Tara Jones, Mitesh Patel, and Sherif R. Zaki, Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, GA, E-mails: JBhatnagar@cdc.gov, DBlau@cdc.gov, WShieh@cdc.gov, CPaddock@cdc.gov, CDrew1@cdc.gov, LLiu1@cdc.gov, TJones5@cdc.gov, MPatel1@cdc.gov, and SZaki@cdc.gov.

Reprint requests: Julu Bhatnagar, Infectious Diseases Pathology Branch, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Bldg.18/SB-105, MS-G32, Atlanta, GA, Tel: 404-639-2826, Fax: 404-639-3043, E-mail: JBhatnagar@cdc.gov.

  • 1.

    Gubler DJ, 1998. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev 11: 480–496.

  • 2.

    Gubler DJ, 1988. Dengue. Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Volume 2. Boca Raton, FL: CRC Press, 223–260.

  • 3.

    Centers for Disease Control and Prevention, 2010. Dengue Clinical Guidance. Available at: http://www.cdc.gov/dengue/clinicalLab/clinical.html. Accessed April 31, 2011.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4.

    Halstead SB, 1988. Pathogenesis of dengue: challenges to molecular biology. Science 239: 476–481.

  • 5.

    Gubler DJ, 1998. The global pandemic of dengue/dengue hemorrhagic fever: current status and prospects for the future. Ann Acad Med Singapore 27: 227–234.

  • 6.

    World Health Organization, 2009. Dengue and Dengue Hemorrhagic Fever. Fact sheet no. 117. Available at: http://www.who.int/mediacentre/factsheets/fs117/en/index.html. Accessed March 31, 2011.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Wilder-Smith A, Gubler DJ, 2008. Geographic expansion of dengue: the impact of international travel. Med Clin North Am 92: 1377–1390 x.

  • 8.

    World Health Organization, 2009. Dengue: Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva, Switzerland: World Health Organization.

  • 9.

    Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A, 1984. Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue viruses. Am J Trop Med Hyg 33: 158–165.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV, 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30: 545–551.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 11.

    Makino Y, Tadano M, Saito M, Maneekarn N, Sittisombut N, Sirisanthana V, Poneprasert B, Fukunaga T, 1994. Studies on serological cross-reaction in sequential flavivirus infections. Microbiol Immunol 38: 951–955.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 12.

    Chaterji S, Allen JC Jr, Chow A, Leo YS, Ooi EE, 2011. Evaluation of the NS1 rapid test and the WHO dengue classification schemes for use as bedside diagnosis of acute dengue fever in adults. Am J Trop Med Hyg 84: 224–228.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 13.

    Bessoff K, Phoutrides E, Delorey M, Acosta LN, Hunsperger E, 2010. Utility of a commercial nonstructural protein 1 antigen capture kit as a dengue virus diagnostic tool. Clin Vaccine Immunol 17: 949–953.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14.

    Tricou V, Vu HT, Quynh NV, Nguyen CV, Tran HT, Farrar J, Wills B, Simmons CP, 2010. Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viremia and antibody responses. BMC Infect Dis 10: 142.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Ramirez AH, Moros Z, Comach G, Zambrano J, Bravo L, Pinto B, Vielma S, Cardier J, Liprandi F, 2009. Evaluation of dengue NS1 antigen detection tests with acute sera from patients infected with dengue virus in Venezuela. Diagn Microbiol Infect Dis 65: 247–253.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 16.

    Lima Mda R, Nogueira RM, Schatzmayr HG, de Filippis AM, Limonta D, dos Santos FB, 2011. A new approach to dengue fatal cases diagnosis: NS1 antigen capture in tissues. PLoS Negl Trop Dis 5: e1147: .

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 17.

    de Araujo JM, Schatzmayr HG, de Filippis AM, dos Santos FB, Cardoso MA, Britto C, Coelho JM, Nogueira RM, 2009. A retrospective survey of dengue virus infection in fatal cases from an epidemic in Brazil. J Virol Methods 155: 34–38.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 18.

    Rosen LL, Drouet MM, Deubel VV, 1999. Detection of dengue virus RNA by reverse transcription-polymerase chain reaction in the liver and lymphoid organs but not in the brain in fatal human infection. Am J Trop Med Hyg 61: 720–724.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 19.

    Johnson BW, Russell BJ, Lanciotti RS, 2005. Serotype-specific detection of dengue viruses in a fourplex real-time reverse transcriptase PCR assay. J Clin Microbiol 43: 4977–4983.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 20.

    Sariol CA, Pelegrino JL, Martinez A, Arteaga E, Kouri G, Guzman MG, 1999. Detection and genetic relationship of dengue virus sequences in seventeen-year-old paraffin-embedded samples from Cuba. Am J Trop Med Hyg 61: 994–1000.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 21.

    Zaki SR, Peters CJ, 1997. Viral hemorrhagic fevers. Connor DH, Chandler FW, Schawartz DA, Manz HJ, Lack EE, eds. Diagnostic Pathology of Infectious Diseases. Stamford, CT: Appleton and Lange, 347–364.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 22.

    de Macedo FC, Nicol AF, Cooper LD, Yearsley MM, Pires AR, Nuovo GJ, 2006. Histologic, viral, and molecular correlates of dengue fever infection of the liver using highly sensitive immunohistochemistry. Diagn Mol Pathol 15: 223–228.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 23.

    Miagostovich MP, Ramos RG, Nicol AF, Nogueira RM, Cuzzi-Maya T, Oliveira AV, Marchevsky RS, Mesquita RP, Schatzmayr HG, 1997. Retrospective study on dengue fatal cases. Clin Neuropathol 16: 204–208.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 24.

    Bhoopat L, Bhamarapravati N, Attasiri C, Yoksarn S, Chaiwun B, Khunamornpong S, Sirisanthana V, 1996. Immunohistochemical characterization of a new monoclonal antibody reactive with dengue virus-infected cells in frozen tissue using immunoperoxidase technique. Asian Pac J Allergy Immunol 14: 107–113.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 25.

    Rico-Hesse R, Harrison LM, Salas RA, Tovar D, Nisalak A, Ramos C, Boshell J, de Mesa MT, Nogueira RM, da Rosa AT, 1997. Origins of dengue type 2 viruses associated with increased pathogenicity in the Americas. Virology 230: 244–251.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 26.

    Messer WB, Gubler DJ, Harris E, Sivananthan K, de Silva AM, 2003. Emergence and global spread of a dengue serotype 3, subtype III virus. Emerg Infect Dis 9: 800–809.

  • 27.

    Aquino VH, Anatriello E, Goncalves PF, DA Silva EV, Vasconcelos PF, Vieira DS, Batista WC, Bobadilla ML, Vazquez C, Moran M, Figueiredo LT, 2006. Molecular epidemiology of dengue type 3 virus in Brazil and Paraguay, 2002–2004. Am J Trop Med Hyg 75: 710–715.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 28.

    Guarner J, Shieh WJ, Hunter S, Paddock CD, Morken T, Campbell GL, Marfin AA, Zaki SR, 2004. Clinicopathologic study and laboratory diagnosis of 23 cases with West Nile virus encephalomyelitis. Hum Pathol 35: 983–990.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 29.

    Chao DY, Davis BS, Chang GJ, 2007. Development of multiplex real-time reverse transcriptase PCR assays for detecting eight medically important flaviviruses in mosquitoes. J Clin Microbiol 45: 584–589.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 30.

    Shu PY Chang SF, Kuo YC, Yueh YY, Chien LJ, Sue CL, Lin TH, Huang JH, 2003. Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus. J Clin Microbiol 41: 2408–2416.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 31.

    Schmittgen TD, Zakrajsek BA, 2000. Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46: 69–81.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 32.

    Lee IK, Liu JW, Yang KD, 2009. Clinical characteristics, risk factors, and outcomes in adults experiencing dengue hemorrhagic fever complicated with acute renal failure. Am J Trop Med Hyg 80: 651–655.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 33.

    Seneviratne SL, Malavige GN, de Silva HJ, 2006. Pathogenesis of liver involvement during dengue viral infections. Trans R Soc Trop Med Hyg 100: 608–614.

  • 34.

    Nimmannitya S, Thisyakorn U, Hemsrichart V, 1987. Dengue hemorrhagic fever with unusual manifestations. Southeast Asian J Trop Med Public Health 18: 398–406.

  • 35.

    Halstead SB, 2007. Dengue. Lancet 370: 1644–1652.

  • 36.

    Bhatnagar J, Guarner J, Paddock CD, Shieh WJ, Lanciotti RS, Marfin AA, Campbell GL, Zaki SR, 2007. Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: a useful adjunct to conventional tissue-based diagnostic methods. J Clin Virol 38: 106–111.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 37.

    Laue T, Emmerich P, Schmitz H, 1999. Detection of dengue virus RNA in patients after primary or secondary dengue infection by using the TaqMan automated amplification system. J Clin Microbiol 37: 2543–2547.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 38.

    Singh K, Lale A, Eong Ooi E, Chiu LL, Chow VT, Tambyah P, Koay ES, 2006. A prospective clinical study on the use of reverse transcription-polymerase chain reaction for the early diagnosis of Dengue fever. J Mol Diagn 8: 613–616, quiz 617–620.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 39.

    Tricou V, Minh NN, Farrar J, Tran HT, Simmons CP, 2011. Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with Dengue. PLoS Negl Trop Dis 5: e1309.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 40.

    Shieh WJ, Blau DM, Denison AM, Deleon-Carnes M, Adem P, Bhatnagar J, Sumner J, Liu L, Patel M, Batten B, Greer P, Jones T, Smith C, Bartlett J, Montague J, White E, Rollin D, Gao R, Seales C, Jost H, Metcalfe M, Goldsmith CS, Humphrey C, Schmitz A, Drew C, Paddock C, Uyeki TM, Zaki SR, 2009. Pandemic influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States. Am J Pathol 177: 166–175.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 41.

    Rico-Hesse R, 2007. Dengue virus evolution and virulence models. Clin Infect Dis 44: 1462–1466.

  • 42.

    Lourenco J, Recker M, 2010. Viral and epidemiological determinants of the invasion dynamics of novel dengue genotypes. PLoS Negl Trop Dis 4: e894.

  • 43.

    Balmaseda AA, Hammond SN, Pérez LL, Tellez YY, Saborío SI, Mercado JC, Cuadra RR, Rocha JJ, Pérez MA, Silva SS, Rocha CC, Harris EE, 2006. Serotype-specific differences in clinical manifestations of dengue. Am J Trop Med Hyg 74: 449–456.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 44.

    Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S, Endy TP, Raengsakulrach B, Rothman AL, Ennis FA, Nisalak A, 2000. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis 181: 2–9.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 45.

    Nisalak AA, Endy TP, Nimmannitya SS, Kalayanarooj SS, Thisayakorn UU, Scott RM, Burke DS, Hoke CH, Innis BL, Vaughn DW, 2003. Serotype-specific dengue virus circulation and dengue disease in Bangkok, Thailand from 1973 to 1999. Am J Trop Med Hyg 68: 191–202.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 46.

    Kumaria R, 2010. Correlation of disease spectrum among four Dengue serotypes: a five years hospital based study from India. Braz J Infect Dis 14: 141–146.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 47.

    Sumarmo, Wulur H, Jahja E, Gubler DJ, Suharyono W, Sorensen K, 1983. Clinical observations on virologically confirmed fatal dengue infections in Jakarta, Indonesia. Bull World Health Organ 61: 693–701.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 48.

    Tomashek KM, Rivera A, Munoz-Jordan JL, Hunsperger E, Santiago L, Padro O, Garcia E, Sun W, 2009. Description of a large island-wide outbreak of dengue in Puerto Rico, 2007. Am J Trop Med Hyg 81: 467–474.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 49.

    Jessie K, Fong MY, Devi S, Lam SK, Wong KT, 2004. Localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization. J Infect Dis 189: 1411–1418.

    • PubMed
    • Search Google Scholar
    • Export Citation
  • 50.

    Hall WC, Crowell TP, Watts DM, Barros VL, Kruger H, Pinheiro F, Peters CJ, 1991. Demonstration of yellow fever and dengue antigens in formalin-fixed paraffin-embedded human liver by immunohistochemical analysis. Am J Trop Med Hyg 45: 408–417.

    • PubMed
    • Search Google Scholar
    • Export Citation
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