Modern algorуthm of microbiological diagnostics of antibiotic-associated diarrhea and pseudomembranous colitis due to Clostridium difficile

Abstract

Introduction. Today, the problem of C.difficile-associated infection (Cdl-AI) is most often associated with two clinical manifestations - antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC), which are frequent side effects of antibacterial drugs and are manifested from a  mild form  to a severe course of the disease. According to the authors, AAD is registered in 3.2–29.0% of patients, and the risk of developing PMK in operated patients ranges from 14 to 27%. The problem of detection and treatment of Cdl-AI is extremely relevant today due to the increased frequency of AAD and PMK registration in patients with coronavirus disease (COVID-19), who received high doses of antibiotics and cytokine storm blockers during treatment. In modern foreign literature, AAD and PMK are described as "nosocomial colitis".  The category of patients - asymptomatic carriers of C. difficile, which are becoming an epidemiologically dangerous category, maintaining the circulation of the pathogen in hospitals is of particular concern. The relevance of Cdl-AI requires the search for effective methods and measures of laboratory diagnosis of AAD and PMK, aimed at identifying and studying the biological properties of pathogens of these diseases. Materials and methods. To achieve this goal, a literature search was conducted using the English-language text database PubMed using the keywords "C.difficile-associated infection", "antibiotic-associated diarrhea", "pseudomembranous colitis". Results and discussion. The basis of research algorythms in the diagnosis of Cdl-AI is the detection of the enzyme glutamate dehydrogenase (GDG) with the  determination of  the presence of exotoxins or genes encoding GDG, TcdA, TcdB and binary toxin and bacteriological examination of the material to identify pure cultures of the pathogen and study its biological properties. The American and European Societies of Microbiologists recommend a two-step algorythm that includes a screening test for the detection of GDG in feces and the detection of exotoxins (TcdA, TcdB) using serological methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic analysis. If the ELISA or IHA test is positive, a bacteriological examination of the material is performed, followed by a study of the toxigenic properties and sensitivity of the isolated C. difficile strains to ABP. The most optimal is a three-step algorythm for laboratory diagnosis of Cld-AI, which involves the determination of genes encoding GDG, toxins A / B and binary toxin C. difficile. Obtaining a negative PCR result makes it possible to stop the search for the pathogen in the test material, and vice versa: obtaining a positive result (the first stage)  involves a bacteriological examination of samples to isolate pure culture of the pathogen (the second stage) and study its sensitivity to antibiotics (the third stage). To increase the specificity of the PCR method, a two-step PCR protocol has been proposed, with using primers to the 16S region of C. difficile ribosomal RNA, which made it possible to  identify more than 150 ribotypes and 24 C. difficile toxinotypes. As an alternative to PCR ribotyping, the MALDI-TOF method of mass spectrometry (Matrix assisted laser desorption / ionization time-of-flight mass spectrometry) has been proposed to investigate epidemic outbreaks. profiles of bacteria and identify pure cultures of C.difficile. MALDI-TOF mass spectrometry and PCR ribotyping can be used to monitor the spread of the pathogen and to carry out anti-epidemic measures. Conclusions. Under the current conditions of uncontrolled use of antibiotics in health care facilities, the incidence of Cdl-AI has a steady upward trend. Adherence to a certain algorythm for the diagnosis of Cdl-AI is necessary for the timely detection of AAD and PMK and prevent the development of severe forms. Consistent implementation of the stages of diagnosis of AAD and PMK avoids the spread of nosocomial strains of clostridia in a hospital.

Key words: C.difficile-associated infection, antibiotic-associated diarrhea, pseudomembranous colitis, laboratory diagnostics.

DOI: 10.5281/zenodo.6350087