Kidney International, Vol. 55 (1999), pp. 2062–2069 TECHNICAL NOTE Polymerase chain reaction detection of Puumala virus RNA in formaldehyde-fixed biopsy material ANDREAS HEISKE,BA ¨ RBEL ANHEIER,JU ¨ RGEN PILASKI,HANS-DIETER KLENK,HERMANN-JOSEF GRO ¨ NE, and HEINZ FELDMANN Institut fu ¨ r Virologie, Philipps-Universita ¨t, Marburg; Medizinisches Institut fu ¨ r Umwelthygiene, Du ¨ sseldorf; and Experimentelle Pathologie, Deutsches Krebsforschungsinstitut, Heidelberg, Germany Polymerase chain reaction detection of Puumala virus RNA pulmonary syndrome” (HPS), became a serious public in formaldehyde-fixed biopsy material. health problem in North and South America. Several Background. Infections with hantaviruses, mainly Clethrio- new hantaviruses have been isolated or at least identified nomys-derived Puumala viruses, are known causes of acute and characterized by molecular methods from different renal failure [hemorrhagic fever with renal syndrome (HFRS)] in western Europe. Laboratory diagnosis is primarily based on rodent species worldwide [1, 2]. Their ubiquity and po- serology. At the time of clinical symptoms, viral RNA can tential for causing severe human diseases make hantavi- hardly be detected in the blood or urine, indicating that poly- ruses prototypes of emerging/re-emerging human patho- merase chain reaction (PCR) is of little diagnostic value for gens [3]. these infections. Biopsy material is usually formaldehyde-fixed and, thus, regarded as poor quality for PCR applications. The Hantaviruses are rodent-borne, negative-stranded aim of this study was to establish a technique to retrieve such RNA viruses that belong to the hantavirus genus of the material for laboratory diagnostic. Bunyaviridae family. Their genome consists of three seg- Methods. Formaldehyde-fixed, paraffin-embedded kidney ments, with the L (large) encoding the viral polymerase biopsies of 14 patients with renal failure either clinically sus- protein, the M (middle) encoding a glycoprotein precur- pected for HFRS (7 cases) or caused by unknown (2 cases) or known other causes (drugs, sarcoidosis; 5 cases) were histologi- sor, and the S (small) encoding the nucleocapsid protein cally investigated. An established S segment-specific PCR assay (N) [4]. All hantavirus-associated illnesses caused by was applied to RNA isolated from the biopsies, and amplifica- Hantaan (HTN), Dobrava/Belgrade (DOB/BEL), Seoul tion products were verified by direct sequence determination. (SEO), and Puumala (PUU) viruses share various de- Results. Investigations revealed a typical histopathological appearance for hantavirus infections in all seven suspected grees of fever and renal involvement, with or without HFRS cases and one case of unknown cause. With five of the hemorrhagic manifestations, and are referred to collec- suspected HFRS cases, hantavirus-specific RNA was detected. tively as HFRS [5, 6]. The pronounced pulmonary Sequence comparison revealed a close relationship to corre- involvement associated with the human pathogenic sponding nucleoproteins of known Puumala viruses. Conclusion. The established technique provides a simple “New World” hantaviruses is a hallmark of HPS [7, 8]. and powerful tool that expands the diagnostic possibilities, A routine diagnosis for hantavirus infections is based especially for otherwise unidentified or retrospective cases. It on serological methods, such as IgM m-capture and IgG further allows insight into the molecular epidemiology of HFRS- enzyme-linked immunosorbent assays (ELISAs) [9–14]. causing agents. Polymerase chain reaction (PCR) was thought to have little diagnostic value for HFRS, because at the time of The “hemorrhagic fever with renal syndrome” (HFRS), clinical symptoms it is quite difficult to demonstrate the a clinical syndrome associated with hantavirus infections, presence of hantaviruses in clinical samples, such as urine has been endemic for decades in parts of Asia and Eu- or blood [1]. Appropriate samples for PCR detection rope. Recently, a previously unknown disease, “hantavirus are fresh or frozen tissues and, to some extent, urine and peripheral blood mononuclear cells collected early in the course of the disease [11, 15–18]. Unfortunately, Key words: hantavirus infection, HFRS, acute renal failure, polymerase chain reaction, kidney biopsy. biopsy materials usually get formaldehyde fixed. Until now, this material has been shown to be of limited quality Received for publication August 17, 1998 for PCR applications, because formaldehyde fixation and in revised form December 14, 1998 Accepted for publication December 14, 1998 seems to lead to RNA degradation [19] and inhibition of polymerase activity [20]. In addition, the yield of isolated 1999 by the International Society of Nephrology 2062
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