Roman Wölfel

Publications of Roman Wölfel

• Phylogenetic analysis of Puumala virus subtype Bavaria, characterization and diagnostic use of its recombinant nucleocapsid protein.

  Authors: Marc Mertens, Eveline Kindler, Petra Emmerich, Jutta Esser, Christiane Wagner-Wiening, Roman Wölfel, Rasa Petraityte-Burneikiene, Jonas Schmidt-Chanasit, Aurelija Zvirbliene, Martin H Groschup, Gerhard Dobler, Martin Pfeffer, Gerald Heckel, Rainer G Ulrich, Sandra S Essbauer

  Virus genes. 05/2011; 43(2):177-91.

  Puumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak inPuumala virus (PUUV) is the predominant hantavirus species in Germany causing large numbers of mild to moderate cases of haemorrhagic fever with renal syndrome (HFRS). During an outbreak in South-East Germany in 2004 a novel PUUV subtype designated Bavaria was identified as the causative agent of HFRS in humans [1]. Here we present a molecular characterization of this PUUV strain by investigating novel partial and almost entire nucleocapsid (N) protein-encoding small (S-) segment sequences and partial medium (M-) segment sequences from bank voles (Myodes glareolus) trapped in Lower Bavaria during 2004 and 2005. Phylogenetic analyses confirmed their classification as subtype Bavaria, which is further subdivided into four geographical clusters. The entire N protein, harbouring an amino-terminal hexahistidine tag, of the Bavarian strain was produced in yeast Saccharomyces cerevisiae and showed a slightly different reactivity with N-specific monoclonal antibodies, compared to the yeast-expressed N protein of the PUUV strain Vranica/Hällnäs. Endpoint titration of human sera from different parts of Germany and from Finland revealed only very slight differences in the diagnostic value of the different recombinant proteins. Based on the novel N antigen indirect and monoclonal antibody capture IgG-ELISAs were established. By using serum panels from Germany and Finland their validation demonstrated a high sensitivity and specificity. In summary, our investigations demonstrated the Bavarian PUUV strain to be genetically divergent from other PUUV strains and the potential of its N protein for diagnostic applications.

• Typhus and other rickettsioses: emerging infections in Germany.

  Authors: Gerhard Dobler, Roman Wölfel

  Deutsches Ärzteblatt international. 06/2009; 106(20):348-54.

  BACKGROUND: Rickettsioses are diseases caused by rickettsiae, obligate intracellular bacteria that are transmitted by arthropods to humans. They cause various types of spotted fever and typhus.BACKGROUND: Rickettsioses are diseases caused by rickettsiae, obligate intracellular bacteria that are transmitted by arthropods to humans. They cause various types of spotted fever and typhus. METHODS: A review of the literature is presented along with the authors' own findings. RESULTS: Six indigenous species of rickettsiae have been found in Germany to date, five of which have been described as human pathogens in other countries. Rickettsia slovaca causes tick-borne lymphadenitis (TIBOLA). Rickettsia helvetica is a known pathogen of nonspecific fever; its role in endocarditis is still under investigation. Rickettsia felis causes so-called flea-borne spotted fever. Rickettsia monacensis and Rickettsia massiliae were recently shown to cause the classical form of tick-borne spotted fever. The sixth indigenous species in Germany, Rickettsia sp. RpA4, has not yet been associated with any human disease. The most important rickettsioses imported to Germany by travelers are African tick bite fever and Mediterranean spotted fever. CONCLUSIONS: Modern molecular biological techniques have enabled the detection of a number of rickettsial species in Germany. The medical importance of these illnesses in Germany remains to be determined. In travel medicine, imported rickettsioses play a role that should not be underestimated.

• Low-Density Macroarray for Rapid Detection and Identification of Crimean-Congo Hemorrhagic Fever Virus.

  Authors: Roman Wölfel, Janusz T Paweska, Nadine Petersen, Antoinette A Grobbelaar, Patricia A Leman, Roger Hewson, Marie-Claude Georges-Courbot, Anna Papa, Volker Heiser, Marcus Panning, Stephan Günther, Christian Drosten

  Journal of clinical microbiology. 03/2009;

  Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis which occurs throughout Africa, Eastern Europe, and Asia and results in an approximately 30% fatality rate. A RT-PCR assayCrimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral zoonosis which occurs throughout Africa, Eastern Europe, and Asia and results in an approximately 30% fatality rate. A RT-PCR assay including a competitive internal control was developed on the basis of the most up-to-date genome information. Biotinylated amplification products were hybridised to DNA macroarrays on the surface of polymer supports and hybridisation events were visualized by incubation with a streptavidin-horse radish peroxidase (HRP) conjugate and the formation of a visible substrate precipitate. Optimal assay conditions were established for detection of as few as 6.3 genome copies per reaction. 18 geographic and historic diverse CCHF virus strains representing all clinical relevant isolates were detected. The feasibility of the assay for clinical diagnosis was validated with acute phase patient samples from South Africa, Iran and Pakistan. The assay provides a specific, sensitive, and rapid method for CCHF virus detection without requiring sophisticated equipments. It has usefulness for clinical diagnosis and surveillance of CCHF infections under limited laboratory conditions in developing countries or in field situations.

• Seroepidemiological study in a Puumala virus outbreak area in South-East Germany.

  Authors: Marc Mertens, Roman Wölfel, Katrin Ullrich, Kumiko Yoshimatsu, Jana Blumhardt, Ina Römer, Jutta Esser, Jonas Schmidt-Chanasit, Martin Groschup, Gerhard Dobler, Sandra Essbauer, Rainer Ulrich

  Medical microbiology and immunology. 02/2009;

  Puumala virus (PUUV) is the cause of the majority of haemorrhagic fever with renal syndrome cases in Germany. In 2004, a nephropathia epidemica outbreak was recorded in Lower Bavaria, South-EastPuumala virus (PUUV) is the cause of the majority of haemorrhagic fever with renal syndrome cases in Germany. In 2004, a nephropathia epidemica outbreak was recorded in Lower Bavaria, South-East Germany. For a seroepidemiological study in this region including the resident population at four locations (n = 178) and soldiers from one location (n = 208) indirect immunoglobulin M (IgM) and immunoglobulin G (IgG) enzyme-linked immunosorbent assays (ELISAs) and immunoblot tests based on a yeast-expressed PUUV nucleocapsid protein were established. The validation using human serum panels originating from Germany revealed a diagnostic sensitivity and specificity of 98/100% for the IgM ELISA, 99/99% for the IgG ELISA, 99/100% for the IgM immunoblot test and 100/96% for the IgG immunoblot test. Using the novel IgG assays as well as a commercial IgG ELISA and an immunofluorescence assay for the resident population an average prevalence of 6.7% (12 of 178) with a range of 0% (0 of 21) to 11.9% (7 of 59) was observed. Positive serological results were equally distributed between males and females with an average age of 63 for males and 52 for females. The seroprevalence in the soldier group was found to be about 1% with one positive male of 203 (age 46 years) and one positive female of five (age 47 years). In conclusion, the PUUV seroprevalence in the residents of the outbreak region in Lower Bavaria was found to be up to fivefold higher than the average hantavirus seroprevalence of the German population.

• [Prevalence of tick-borne encephalitis virus and rickettsiae in ticks of the district Burgenland, Austria.]

  Authors: Gerhard Dobler, Sandra Essbauer, Rahime Terzioglu, Astrid Thomas, Roman Wölfel

  Wiener klinische Wochenschrift. 11/2008; 120 Suppl 4:45-8.

  Large areas of the Central European country Austria are known to be endemic for tick-borne encephalitis virus (TBEV). However, so far only limited data are available for the prevalence, distributionLarge areas of the Central European country Austria are known to be endemic for tick-borne encephalitis virus (TBEV). However, so far only limited data are available for the prevalence, distribution and species composition of rickettsiae of the spotted fever group in Austria. In the present study 306 ticks were collected in summer 2007. Location of sampling was Neudörfl, a known TBEV endemic area in the district of Burgenland in Eastern Austria. 286 ticks belonged to the species Ixodes ricinus and 20 ticks were identified as Dermacentor spp. Detection of TBEV and rickettsial species was done by polymerase chain reaction. We were not able to detect TBEV in any of the ticks examined. However 16 ticks, exclusively of the species Ixodes ricinus showed positive results for rickettsiae. 14 out of 16 rickettsia-positive ticks contains sufficient DNA for a sequence determination. The sequencing of the citrate synthase genes resulted exclusively in the identification of Rickettsia helvetica. The results show that TBEV was not at all or in a low frequency circulating in a known endemic area in summer 2007. However, for the first time the prevalence of 5.7% of Rickettsia helvetica was determined in a tick population in Eastern Austria.

• Diagnostics of tick-borne rickettsioses in Germany: A modern concept for a neglected disease.

  Authors: Roman Wölfel, Sandra Essbauer, Gerhard Dobler

  International journal of medical microbiology : IJMM. 03/2008;

  Tick-borne rickettsioses in humans occur worldwide and are caused by obligate intracellular bacteria belonging to the spotted-fever group (SFG) within the genus Rickettsia (R.). These tick-borneTick-borne rickettsioses in humans occur worldwide and are caused by obligate intracellular bacteria belonging to the spotted-fever group (SFG) within the genus Rickettsia (R.). These tick-borne rickettsioses are among the most underdiagnosed vector-borne diseases in Germany: Due to the variety of unspecific clinical signs, they are not easily recognised. The clinical picture ranges from subclinical to fatal courses, but may be difficult to differentiate from other febrile conditions without specific tests. Even to date, diagnosis is either based on clinical findings, a record of tick exposure, or indirect serological detection of the pathogens. Herein, we briefly discuss modern diagnostic tools for important tick-borne rickettsial infections with emphasis on new molecular diagnostic assays. As one example, we present a novel real-time polymerase chain reaction (PCR) protocol that facilitates genus-specific, rapid, and sensitive detection of rickettsial pathogens. A conserved region of the rickettsial citrate synthase gene (gltA) is amplified and detected by a 5'-nuclease probe in a LightCycler instrument. Sensitivity was consistently high at less than 23 genome copies per reaction. This detection system has been evaluated both as a useful tool in epidemiological investigations in ticks and in human diagnostics. We describe a rational diagnostic approach for the detection of tick-borne human rickettsioses which consists of that real-time PCR, isolation of rickettsiae in cell culture, multi-locus sequence typing, and serology. Its implementation recently led to the first isolation and characterisation of R. africae in Germany from a patient returning from Zimbabwe. In conclusion, tick-borne rickettsioses should be considered in patients presenting with fever, headache, and rash following a tick bite. Further studies are needed to determine the epidemiology and clinical importance of these pathogens in Germany.

• Virus detection and monitoring of viral load in Crimean-Congo hemorrhagic fever virus patients.

  Authors: Roman Wölfel, Janusz T Paweska, Nadine Petersen, Antoinette A Grobbelaar, Patricia A Leman, Roger Hewson, Marie-Claude Georges-Courbot, Anna Papa, Stephan Günther, Christian Drosten

  Emerging infectious diseases. 08/2007; 13(7):1097-100.

  We developed a real-time reverse transcription--PCR that detected 1,164 copies/mL of Crimean-Congo hemorrhagic fever virus per milliliter of serum at 95% probability (probit analysis) and was 100%We developed a real-time reverse transcription--PCR that detected 1,164 copies/mL of Crimean-Congo hemorrhagic fever virus per milliliter of serum at 95% probability (probit analysis) and was 100% concordant with nested PCR on 63 samples from 31 patients with confirmed infection. Infected patients who died appeared to have higher viral loads; low viral loads correlated with IgG detection.

• Evaluation of sampling technique and transport media for the diagnostics of adenoviral eye infections. Adenovirus sampling and transport.

  Authors: Roman Wölfel, Martin Pfeffer, Sandra Essbauer, Sylke Nerkelun, Gerhard Dobler

  Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv für klinische und experimentelle Ophthalmologie. 12/2006; 244(11):1497-504.

  BACKGROUND: Human adenoviruses (HAdV) may cause pharyngoconjunctival fever, follicular conjunctivitis or epidemic keratoconjunctivitis (EKC). Especially, outbreaks of the latter may lead to severeBACKGROUND: Human adenoviruses (HAdV) may cause pharyngoconjunctival fever, follicular conjunctivitis or epidemic keratoconjunctivitis (EKC). Especially, outbreaks of the latter may lead to severe economic losses when preventive measures are implemented too late. Thus, a safe sampling method, proper specimen transport conditions and a fast and sensitive diagnostic technique is mandatory. METHODS: Two commercially available virus transport systems (VTS) were compared with two NaCl-moisturised sampling devices, one of which comprises Dacron-tipped plastic-shafted swabs and the other a cotton-tipped wood-shafted swab, available in most ophthalmologists' offices. Downstream methods for specific detection of HAdV included direct immunofluorescence assay (IFA) of conjunctival swabs, virus isolation by cell culture and quantitative real-time polymerase chain reaction (qPCR). Furthermore, the influence of application of local anaesthetics prior to swabbing on subsequent detection of HAdV was investigated. RESULTS: Application of local anaesthetics had a positive influence on the amount of swabbed cells, thus increasing the chance of obtaining positive results by IFA. Neither isolation of HAdV by cell culture nor by qPCR was negatively influenced by this pretreatment. Surprisingly, both commercially available VTS performed significantly worse than the NaCl-moisturised swabs. This was shown with regard to virus recovery rates in cell culture as well as viral genome copy numbers in the qPCR. CONCLUSIONS: Based on our results, the following recommendations are provided to improve sampling, transport and diagnostic techniques regarding conjunctival swabs for diagnosis of human adenovirus infection: (1) application of local anaesthetics, (2) NaCl-moisturised VTS for shipment of specimens, and (3) detection of HAdV by qPCR. The latter method proved to be superior to virus isolation by cell culture, including subsequent identification by IFA, because it is faster, more sensitive and allows simultaneous handling of a number of samples. Hence, countermeasures to prevent further virus spread in an outbreak situation can be implemented earlier, thus reducing the number of subsequent adenoviral infections.

• Seroprevalence of tick-borne and mosquito-borne arboviruses in European brown hares in Northern and Western Germany.

  Authors: Gerhard Dobler, Roman Wölfel, Heiko Schmüser, Sandra Essbauer, Martin Pfeffer

  International journal of medical microbiology : IJMM. 06/2006; 296 Suppl 40:80-3.

  Tríbec virus (Kemerovo serogroup, genus Orbivirus), Eyach virus (genus Coltivirus), and Tahyna virus (California encephalitis serogroup, genus Bunyavirus) are arthropod-borne viruses known to occurTríbec virus (Kemerovo serogroup, genus Orbivirus), Eyach virus (genus Coltivirus), and Tahyna virus (California encephalitis serogroup, genus Bunyavirus) are arthropod-borne viruses known to occur in Germany. These viruses are also suspected to cause human disease. So far, no information is available on their geographical distribution in Germany and their natural transmission cycles. A total of 166 sera from European brown hares (Lepus europaeus) collected in seven districts of the Federal State of Schleswig-Holstein and in four districts of the Federal State of North Rhine-Westphalia was tested by plaque reduction neutralization test (PRNT) for antibodies against Tríbec virus, Eyach virus, Tahyna virus, and Central European encephalitis virus. One out of 22 sera (4.5%) collected in the district Nord-Friesland in Schleswig-Holstein was found positive (PRNT(90) 1:10) against Tríbec virus. Neither did sera from other regions of Schleswig-Holstein nor from hares from North Rhine-Westphalia react against any of the arboviruses tested. For the first time, antibodies against Tríbec virus could be found in a European brown hare in Germany. The negative serological results for Central European encephalitis virus are in line with the current knowledge of its natural distribution within Germany. The negative serological results for Tahyna virus or Eyach virus argue against an autochthonous circulation of these viruses in the regions tested.