BACKGROUND: West Nile virus (WNV), a member of genus Flavivirus, causes febrile illness, encephalitis, meningitis, myelitis, and occasional deaths in humans. Although several reverse transcription-polymerase chain reaction (RT-PCR) assays have been developed for detection of WNV in serum, cerebrospinal fluid, and fresh tissues, the usefulness of WNV RT-PCR assays for RNA extracted from formalin-fixed human tissues has not previously been demonstrated. OBJECTIVE: The objective of this study was to evaluate the application of a RT-PCR technique for the detection of WNV in routinely processed, formalin-fixed, paraffin-embedded (FFPE) human tissues, and to compare it with conventional serology and immunohistochemistry (IHC). STUDY DESIGN: We performed two WNV-specific nested RT-PCR assays targeting the viral capsid, premembrane, and envelope genes in FFPE central nervous system tissue samples from 27 patients with fatal WNV encephalitis, as confirmed by serology or IHC, and compared the results. The presence of WNV in RT-PCR-positive samples was confirmed by amplicon sequencing. RESULTS: Twenty (74%) patients were WNV RT-PCR positive while 24 (89%) were seropositive. WNV IHC staining of neurons and neuronal processes was positive in fourteen (52%) patients. The concordance between IHC and serology was 41% (11/27) and between RT-PCR and serology was 63% (17/27). All 11 seropositive/IHC-positive patients and 6 (46%) of 13 seropositive/IHC-negative patients were RT-PCR positive while all 3 seronegatives were positive by both IHC and RT-PCR. CONCLUSIONS: In this study, RT-PCR was significantly more sensitive than IHC in detecting WNV infections and provided specific sequence information about the infecting virus. RT-PCR on FFPE tissues may be a particularly useful diagnostic tool in patients who die relatively soon after disease onset and for whom serology may be negative. Combined use of serology, IHC, and RT-PCR would be expected to have the best overall sensitivity and improve detection of fatal WNV infection.
BACKGROUND:West Nile virus (WNV), a member of genus Flavivirus, causes febrile illness, encephalitis, meningitis, myelitis, and occasional deaths in humans. Although several reverse transcription-polymerase chain reaction (RT-PCR) assays have been developed for detection of WNV in serum, cerebrospinal fluid, and fresh tissues, the usefulness of WNV RT-PCR assays for RNA extracted from formalin-fixed human tissues has not previously been demonstrated. OBJECTIVE: The objective of this study was to evaluate the application of a RT-PCR technique for the detection of WNV in routinely processed, formalin-fixed, paraffin-embedded (FFPE) human tissues, and to compare it with conventional serology and immunohistochemistry (IHC). STUDY DESIGN: We performed two WNV-specific nested RT-PCR assays targeting the viral capsid, premembrane, and envelope genes in FFPE central nervous system tissue samples from 27 patients with fatal WNV encephalitis, as confirmed by serology or IHC, and compared the results. The presence of WNV in RT-PCR-positive samples was confirmed by amplicon sequencing. RESULTS: Twenty (74%) patients were WNV RT-PCR positive while 24 (89%) were seropositive. WNV IHC staining of neurons and neuronal processes was positive in fourteen (52%) patients. The concordance between IHC and serology was 41% (11/27) and between RT-PCR and serology was 63% (17/27). All 11 seropositive/IHC-positive patients and 6 (46%) of 13 seropositive/IHC-negative patients were RT-PCR positive while all 3 seronegatives were positive by both IHC and RT-PCR. CONCLUSIONS: In this study, RT-PCR was significantly more sensitive than IHC in detecting WNV infections and provided specific sequence information about the infecting virus. RT-PCR on FFPE tissues may be a particularly useful diagnostic tool in patients who die relatively soon after disease onset and for whom serology may be negative. Combined use of serology, IHC, and RT-PCR would be expected to have the best overall sensitivity and improve detection of fatal WNV infection.
Authors: Julu Bhatnagar; Dianna M Blau; Wun-Ju Shieh; Christopher D Paddock; Clifton Drew; Lindy Liu; Tara Jones; Mitesh Patel; Sherif R Zaki Journal: Am J Trop Med Hyg Date: 2012-02 Impact factor: 2.345
Authors: Victor Valcour; Aissa Haman; Susannah Cornes; Carson Lawall; Andrew T Parsa; Carol Glaser; Shigeo Yagi; Tarik Tihan; Julu Bhatnagar; Michael Geschwind Journal: Nat Clin Pract Neurol Date: 2008-05-13
Authors: Pennelope K Blakely; Bette K Kleinschmidt-DeMasters; Kenneth L Tyler; David N Irani Journal: J Neuropathol Exp Neurol Date: 2009-10 Impact factor: 3.685
Authors: Vittorio Sambri; Maria R Capobianchi; Francesca Cavrini; Rémi Charrel; Olivier Donoso-Mantke; Camille Escadafal; Leticia Franco; Paolo Gaibani; Ernest A Gould; Matthias Niedrig; Anna Papa; Anna Pierro; Giada Rossini; Andrea Sanchini; Antonio Tenorio; Stefania Varani; Ana Vázquez; Caterina Vocale; Herve Zeller Journal: Viruses Date: 2013-09-25 Impact factor: 5.048
Authors: Julu Bhatnagar; Demi B Rabeneck; Roosecelis B Martines; Sarah Reagan-Steiner; Yokabed Ermias; Lindsey B C Estetter; Tadaki Suzuki; Jana Ritter; M Kelly Keating; Gillian Hale; Joy Gary; Atis Muehlenbachs; Amy Lambert; Robert Lanciotti; Titilope Oduyebo; Dana Meaney-Delman; Fernando Bolaños; Edgar Alberto Parra Saad; Wun-Ju Shieh; Sherif R Zaki Journal: Emerg Infect Dis Date: 2017-03-15 Impact factor: 6.883