First Report on Leptospira Species Isolated from Patients in Slovenia.

Leptospirosis is an important worldwide zoonosis, and it has also been reported in Slovenia. The cultivation of Leptospira from human material is difficult. Despite that, we successfully isolated 12 human Leptospira strains isolated from patients between 2002 and 2020 and used various methods for the phenotypic and genotypic characterization of the strains, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) using our own MALDI-TOF data library, melting temperature analysis of the amplified lfb1 gene, determination of Leptospira serogroups using rabbit immune sera, NotI-RFLP of the whole Leptospira genome, multilocus sequence typing (MLST) of seven housekeeping genes, and whole-genome sequencing (WGS)-based typing. We confirmed the presence of four pathogenic Leptospira species (L. kirschneri, L. interrogans, L. borgpetersenii, and L. santarosai) and three serogroups: Grippotyphosa, Icterohaemorrhagiae, and Sejroe. MALDI-TOF identified three of seven isolates at the species level and four isolates at the genus level. Serovars of 8 of the 10 strains were determined using NotI-RFLP. MLST showed that the clinical isolates belonged to sequence types ST17, ST110, and ST155. WGS confirmed the analysis of Leptospira strains using conventional methods. In addition, WGS provided better taxonomic resolution for isolate DDA 10944/10.

Evaluation of real-time PCR targeting the lipL32 gene for diagnosis of Leptospira infection.

BACKGROUND: Different diagnostic methods have been used for the laboratory confirmation of leptospirosis. Molecular diagnostic techniques are not only faster and more sensitive than culture analysis, but can also detect a Leptospira infection before the appearance of antibodies. The aim of the present study was to analyze and compare two different PCR approaches applied to blood and urine specimens obtained from patients with clinical manifestations that were suggestive of leptospirosis. Furthermore, the results of these different PCR approaches were compared with the results of culture and serology analyses. RESULTS: A total of 400 samples (234 blood or 58.5% and 166 urine of 41.5%) from 310 Slovenian patients with clinical manifestations suggestive of leptospirosis were tested using conventional PCR assays targeting the rrs gene and RT-PCR targeting the lipL32 gene. Additionally, culture, serology and sequence analysis were performed for the majority of these samples. The PCR and RT-PCR results were concordant in 376 out of 400 of these samples (94.0%). Conventional PCR was positive for 27 out of 400 samples (6.8%) and RT-PCR was positive for 47 out of 400 samples (11.8%). Culture and microscopic agglutination tests supported these diagnoses. CONCLUSIONS: A comparison of the two PCR methods indicated that the RT-PCR targeting of the lipL32 gene was faster, more sensitive and more specific for the determination of Leptospira DNA in these clinical samples.

Antimicrobial susceptibility of Haemophilus parasuis isolates from Germany by use of a proposed standard method for harmonized testing.

Haemophilus parasuis-related infections, especially among weaners are responsible for major economic losses on pig farms. A method for broth microdilution susceptibility testing of this fastidious organism has recently been developed, but the suitability of this method needs to be validated in a large collection of current field isolates. Using the proposed method, this study tested 123 H. parasuis isolates from different geographic regions in Germany (including five isolates from the Netherlands and Belgium) against a panel of 24 antimicrobial agents and antimicrobial combinations. The isolates were collected between 2013 and 2016. As there are no H. parasuis specific breakpoints available, the tested isolates could not be classified as susceptible, intermediate or resistant. Bi- or multi-modal distributions of minimum inhibitory concentration (MIC) values were observed for some antimicrobial agents (e.g. aminoglycosides, ß-lactams, fluoroquinolones and tetracyclines), indicative of non-wild type populations of H. parasuis. Susceptibility testing revealed broad distributions of MIC values for various antimicrobials (e.g. neomycin, streptomycin, tetracycline, tiamulin, tilmicosin, trimethoprim/sulfamethoxazole and tulathromycin). The lowest MIC90 (i.e. the concentration at which 90% of isolates were inhibited) was obtained for cefotaxime (≤0.015 µg/ml), and the highest MIC90 (512 µg/ml) was obtained for streptomycin. This study tested a large set of current field isolates and included the most common serovars (serovars 4 and 5). The results point to the suitability of the broth microdilution susceptibility testing method proposed previously for determining H. parasuis MIC values. In addition, the study provides a reliable overview of the susceptibility status of H. parasuis at present in Germany.