*Article* **Diagnostic Methods of** *Clostridioides difficile* **Infection and** *Clostridioides difficile* **Ribotypes in Studied Sample**

**Elena Novakova <sup>1</sup> , Zuzana Stofkova 1,\*, Vladimira Sadlonova <sup>1</sup> and Lukas Hleba <sup>2</sup>**

	- 94901 Nitra, Slovakia; lukas.hleba@gmail.com

**\*** Correspondence: zuzana.stofkova@gmail.com

**Abstract:** Background: *Clostridioides* (*Clostridium*) *difficile* is the most common nosocomial pathogen and antibiotic-related diarrhea in health-care facilities. Over the last few years, there was an increase in the incidence rate of *C. difficile* infection cases in Slovakia. In this study, the phenotypic (toxigenicity, antimicrobial susceptibility) and genotypic (PCR ribotypes, genes for binary toxins) patterns of *C. difficile* isolates from patients with CDI were analyzed, from July to August 2016, taken from hospitals in the Horne Povazie region of northern Slovakia. The aim of the study was also to identify hypervirulent strains (e.g., the presence of RT027 or RT176). Methods: The retrospective analysis of biological samples suspected of CDI were analyzed by GDH, anaerobic culture, enzyme immunoassay on toxins A/B, multiplex "real-time" PCR and PCR capillary-based electrophoresis ribotyping, and by MALDI TOF MS. Results: *C. difficile* isolates (*n* = 44) were identified by PCR ribotyping, which revealed five different ribotypes (RT001, 011, 017, 081, 176). The presence of hypervirulent RT027 was not identified. The *C. difficile* isolates (RT001, 011, 081, 176) were susceptible to metronidazole and vancomycin. One isolate RT017 had reduced susceptibility to vancomycin. A statistically significant difference between the most prevalent PCR ribotypes, RT001 and RT176, regarding variables such as albumin, CRP, creatinine, the length of hospitalization (*p* = 0.175), and glomerular filtration (*p* = 0.05) was not found. Conclusion: The results of PCR capillary-based electrophoresis ribotyping in the studied samples showed a high prevalence of RT176 and 001.

**Keywords:** *Clostridioides difficile* infection; multi-step algorithm; multiplex "real-time" PCR; PCR capillary-based electrophoresis ribotyping
