Risk Factors in Diagnosing Clostridioides difficile Infections

Background. Toxin-producing strains of Clostridioides difficile are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalisation, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhoea with ≥3 unformed stools in 24 hours are a target population for testing for C. difficile infection (CDI). Objective. To present data on the risk factors in current practices for clinical diagnoses used in managing CDI. Materials and Methods. We studied 116 clinical stool specimens from inpatients with acute diarrhoea suspected of nosocomial or antibiotic-associated diarrhoea (AAD) due to C. difficile from two general hospitals. Enzyme Immunoassay card tests (EIA) for detecting C. difficile toxins A (cdtA) and B (cdtB) from stool, аutomated CLIA assay for the detection of C. diff. GDH antigen and qualitative determination of cdtA and B in human faeces, and anaerobic stool culture were applied for CDI laboratory diagnostic. MALDI-TOF (Bruker) identified the suspected anaerobic bacterial colonies. The following methods were used as differential diagnostics LAMP method for evidence of Salmonella spp. and simultaneous detection of Campylobacter jejuni and C. coli, E. coli Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, EIEC), API 20E, and aerobic stool culture methods. Results. Forty toxigenic C. difficile strains were isolated from all 116 diarrhoeal stool specimens tested, of which 38 (38/40) produced toxin B and two strains (2/40) were positive for both cdtA and cdtB. Of the faecal samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 samples were C. diff culture negative, but positive for the following diarrhoea agents: 1/33 – Salmonella enterica subsp. Arizonae (LAMP, culture, API 20E); 2/33 C. jejuni (LAMP, culture, MALDI TOF); 1/33 – ETEC O142 and 2/33 - STEC O145 and O138 (E. coli Typing RT-PCR detection kit, culture); 2/33 C. perfringens (anaerobic culture, MALDI TOF); 2/33 – hyper-mycotic enterotoxigenic K. pneumonia and 2/33– enterotoxigenic P. mirabilis (culture; PCR LT-toxin-encoding). Two of the 66 cdtB-positive samples (2/66) exhibited a similar misdiagnosis when analysed using the CLIA method. However, the PCR analysis indicated that they were cdtB-negative. In contrast, the LAMP method identified a positive result for C. jejuni in one sample and another one was STEC positive (stx1+/stx2+) by RT-PCR. Furthermore, there is an additional discrepancy in the CDI testing results. EPEC O86 (RT-PCR eae+) was isolated from a faecal sample that was positive for GHA enzyme (CLIA) and negative for cdt A and cdtB (CLIA and PCR assay). However, the C. diff culture was negative. The findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than C. difficile, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EAI card tests for C. difficile A and B toxins, which are the most widely used in the laboratory diagnosis of CDI. In conclusion, C. difficile infection represents a significant challenge to clinical practice, both in terms of management and prevention of recurrence. It is recommended that toxin-only EAI tests should not be used as a stand-alone diagnostic tool for CDI and that their use should be limited to the detection of toxins A and B. Accurate diagnosis of CDI requires a combination of clinical evaluation, diagnostic methods, and management practices.