Polymerase chain reaction detection of Puumala virus RNA in formaldehyde-fixed biopsy material

Background Infections with hantaviruses, mainly Clethrionomys-derived Puuma la viruses, are known causes of acute renal failure [hemorrhagic fever with renal syndrome (HFRS)] in western Europe. Laboratory diagnosis is primaril y based on serology. At the time of clinical symptoms, viral RNA can hardly be detected in the blood or urine, indicating that polymerase chain reacti on (PCR) is of little diagnostic value for these infections. Biopsy materia l is usually formaldehyde-fixed and, thus, regarded as poor quality for PCR applications. The aim of this study was to establish a technique to retrie ve such material for laboratory diagnostic. Methods. Formaldehyde-fixed, paraffin-embedded kidney biopsies of 14 patien ts with renal failure either clinically suspected for HFRS (7 cases) or cau sed by unknown (2 cases) or known other causes (drugs, sarcoidosis; 5 cases ) were histologically investigated. An established S segment-specific PCR a ssay was applied to RNA isolated from the biopsies, and amplification produ cts 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 un known cause. With five of the suspected HFRS cases, hantavirus-specific RNA was detected. Sequence comparison revealed a close relationship to corresp onding nucleoproteins of known Puumala viruses. Conclusion. The established technique provides a simple and powerful tool t hat expands the diagnostic possibilities, especially for otherwise unidenti fied or retrospective cases. It further allows insight into the molecular e pidemiology of HFRS-causing agents.