title: Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: a useful adjunct to conventional tissue-based diagnostic methods.
abstract: 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).
  
  
  METHODS
  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.affiliation: Infectious Disease Pathology Activity, Centers for Disease Control and Prevention, 1600 Clifton Road, N.E., Mailstop G32, Atlanta, GA 30333, United States. JBhatnagar@cdc.gov
authors.forename: Julu
authors.initials: J
authors.lastname: Bhatnagar
authors.forename: Jeannette
authors.initials: J
authors.lastname: Guarner
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Paddock
authors.forename: Wun-Ju
authors.initials: WJ
authors.lastname: Shieh
authors.forename: Robert S
authors.initials: RS
authors.lastname: Lanciotti
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Marfin
authors.forename: Grant L
authors.initials: GL
authors.lastname: Campbell
authors.forename: Sherif R
authors.initials: SR
authors.lastname: Zaki
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D005557:Formaldehyde; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012699:Serology / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014901:West Nile Fever / Q000821:virology*; D014902:West Nile virus / Q000302:isolation & purification*
chemical_list: D005557:Formaldehyde