OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

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A retrospective serological and genetic study of hantaviruses responsible for hemorrhagic fever with renal syndrome (HFRS) in Greece during the last 17 years is presented. Fifty-one serum samples taken from 30 HFRS cases previously diagnosed by immunofluorescence assay were tested by ELISA for IgG (Hantaan, Dobrava, and Puumala) and IgM antibodies (Hantaan and Puumala). Results were compatible with the majority of infections being related to hantaviruses carried by rodents of the subfamily Murinae. RNA was extracted from 26 selected samples and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers specifically designed for the detection of hanta-viruses associated with murine (MS-N-specific, MM-G1-specific primers) or arvicoline rodents (PPT-N-specific primers). In addition, primers previously designed for the detection of the G2 coding region of the Murine-associated hanta-viruses were also used. Sequencing of the PCR products was then performed, followed by phylogenetic analysis of nucleotide sequence differences. Eleven out of the 26 serum samples tested were found to be positive by PCR with the MS-N primers, whereas four were positive with the MM-G1 primers, and only two with the G2 primers. None of the samples was found positive with the PPT primers. The sequence analysis showed that the virus that was responsible for these 11 HFRS cases was the Dobrava virus, which is endemic throughout the Balkans.

Blood samples were collected from an Albanian and a Greek patient with hemorrhagic fever with renal syndrome and tested by reverse transcriptase-polymerase chain reaction. The genetic detection assay amplified hantavirus-specific DNA fragments from RNA extracted from the blood of the patients; nucleotide sequence analysis revealed that the causative agent of the disease was Dobrava virus. These findings suggest that Dobrava virus (which was originally isolated from the lungs of an Apodenws flavicollis mouse in Slovenia) is endemic throughout the Balkan States and causes overt human disease.

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