Implementation of a Clostridioides difficile sentinel surveillance system in Germany: First insights for 2019-2021.

OBJECTIVES: Clostridioides difficile is a major cause of nosocomial diarrhea. Several "hypervirulent" lineages such as ribotype 027 (RT027) and RT078 are of high epidemiological importance, leading to outbreaks and more severe courses of disease. An active surveillance system targeting molecular epidemiology and corresponding antimicrobial resistance has not been established in Germany. METHODS: Since October 2019, University Hospitals throughout Germany collected by two dates every year (1st April and October, respectively) their first ten unselected samples being tested positive for C. difficile. RESULTS: Out of 1026 samples received from 29 sites, 876 toxigenic C. difficile strains could be cultivated. PCR ribotyping of these strains revealed a large strain diversity with RT014 (17.5%) dominating, followed by isolates of the major nosocomial lineage RT001 (7.1%) and the "hypervirulent" lineage RT078 (5.9%). Notably, prevalence of RT027 was low with ∼3.5% at all time points analyzed, while the abundance of RT001 isolates significantly declined from 12.3% to 3.7% during the sampling period (P < 0.001). Antimicrobial resistance against clarithromycin, moxifloxacin, and rifampicin was detected in 18%, 15%, and 4% of the tested isolates, respectively. Highest resistance rates were found among RT027 isolates (83%, 87% and 63% for clarithromycin, moxifloxacin, and rifampicin, respectively). Vancomycin resistance was not detected, and metronidazole resistance was observed only for a single RT027 isolate. CONCLUSIONS: This Germany-wide continuing surveillance effort with a standardized mode of isolate acquisition indicates that isolates of RT027 were only sporadically detected under these strain acquisition conditions, and RT001 seems to become less important in the hospital setting, being replaced by other RTs.

Molecular epidemiology and antimicrobial resistance of Clostridioides difficile in Germany, 2014-2019.

Clostridioides difficile is a Gram positive spore-forming rod and mainly responsible for nosocomial diarrhea in developed nations. Molecular and antimicrobial surveillance is important for monitoring the strain composition including genotypes of high epidemiological importance such as ribotype 027 (RT027) and corresponding resistance patterns. 1535 isolates obtained from samples sent between 2014 and 2019 to the German National Reference Center (NRC) for diagnostic reasons (NRC strain set), and 1143 isolates from a Tertiary Care University Center in Saarland, Germany (non-NRC strain set), were evaluated using antibiotic susceptibility testing and ribotyping. In the NRC strain set, RT027 overtook RT001, the main RT found in the preceding studies, and dominated with 36.2%, followed by RT001 (13.3%), and RT014 (8.5%). Of note, since 2016 a constant decrease of RT027 could be noticed. In the non-NRC strain set a large strain diversity was present with RT014 (18%) and RT001 (8.9%) being most prevalent. In NRC samples, resistance towards metronidazole, vancomycin, moxifloxacin, clarithromycin and rifampicin was 2.7%, 0%, 57.1%, 53.2% and 19.2%, respectively. Metronidazole resistance was almost exclusively found in RT027 isolates. Rifampicin resistance was also observed predominantly in isolates of RT027, constituting an almost four-fold increase, when compared to preceeding studies in this region. In conclusion these data demonstrate that RT027 is a driver for rifampicin and metronidazole resistance, underlining the importance of continuous surveillance efforts.

Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry.

Fascioliasis is a neglected trematode infection caused by Fasciola gigantica and Fasciola hepatica. Routine diagnosis of fascioliasis relies on macroscopic identification of adult worms in liver tissue of slaughtered animals, and microscopic detection of eggs in fecal samples of animals and humans. However, the diagnostic accuracy of morphological techniques and stool microscopy is low. Molecular diagnostics (e.g., polymerase chain reaction (PCR)) are more reliable, but these techniques are not routinely available in clinical microbiology laboratories. Matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a widely-used technique for identification of bacteria and fungi; yet, standardized protocols and databases for parasite detection need to be developed. The purpose of this study was to develop and validate an in-house database for Fasciola species-specific identification. To achieve this goal, the posterior parts of seven adult F. gigantica and one adult F. hepatica were processed and subjected to MALDI-TOF MS to create main spectra profiles (MSPs). Repeatability and reproducibility tests were performed to develop the database. A principal component analysis revealed significant differences between the spectra of F. gigantica and F. hepatica. Subsequently, 78 Fasciola samples were analyzed by MALDI-TOF MS using the previously developed database, out of which 98.7% (n = 74) and 100% (n = 3) were correctly identified as F. gigantica and F. hepatica, respectively. Log score values ranged between 1.73 and 2.23, thus indicating a reliable identification. We conclude that MALDI-TOF MS can provide species-specific identification of medically relevant liver flukes.