Polymerase chain reaction detection of Puumala virus RNA in formaldehyde-fixed biopsy material.
BACKGROUND
Infections with hantaviruses, mainly Clethrionomys-derived Puumala viruses, are known causes of acute renal failure [hemorrhagic fever with renal syndrome (HFRS)] in western Europe. Laboratory diagnosis is primarily based on serology. At the time of clinical symptoms, viral RNA can hardly be detected in the blood or urine, indicating that polymerase chain reaction (PCR) is of little diagnostic value for these infections. Biopsy material is usually formaldehyde-fixed and, thus, regarded as poor quality for PCR applications. The aim of this study was to establish a technique to retrieve such material for laboratory diagnostic.


METHODS
Formaldehyde-fixed, paraffin-embedded kidney biopsies of 14 patients with renal failure either clinically suspected for HFRS (7 cases) or caused by unknown (2 cases) or known other causes (drugs, sarcoidosis; 5 cases) were histologically investigated. An established S segment-specific PCR assay was applied to RNA isolated from the biopsies, and amplification products were verified by direct sequence determination.


RESULTS
Investigations revealed a typical histopathological appearance for hantavirus infections in all seven suspected HFRS cases and one case of unknown cause. With five of the suspected HFRS cases, hantavirus-specific RNA was detected. Sequence comparison revealed a close relationship to corresponding nucleoproteins of known Puumala viruses.


CONCLUSIONS
The established technique provides a simple and powerful tool that expands the diagnostic possibilities, especially for otherwise unidentified or retrospective cases. It further allows insight into the molecular epidemiology of HFRS-causing agents.
Heiske
A
A
Institut für Virologie, Philipps-Universität, Marburg, Germany.
Anheier
B
B
Pilaski
J
J
Klenk
H D
HD
Gröne
H J
HJ
Feldmann
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001706:Biopsy; D005404:Fixatives; D005557:Formaldehyde; D006476:Orthohantavirus / Q000235:genetics / Q000302:isolation & purification*; D018778:Hantavirus Infections / Q000150:complications / Q000175:diagnosis* / Q000473:pathology; D006480:Hemorrhagic Fever with Renal Syndrome / Q000473:pathology / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*
D005404:Fixatives; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D005557:Formaldehyde