Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: a useful adjunct to conventional tissue-based diagnostic methods.
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.
Bhatnagar
Julu
J
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
Guarner
Jeannette
J
Paddock
Christopher D
CD
Shieh
Wun-Ju
WJ
Lanciotti
Robert S
RS
Marfin
Anthony A
AA
Campbell
Grant L
GL
Zaki
Sherif R
SR
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*
D005557:Formaldehyde