Potential for Misinterpretation in the Laboratory Diagnosis of Clostridioides difficile Infections

<b>Background/Objective</b>. Toxin-producing strains of <i>Clostridioides difficile</i> (<i>C. diff</i>) are the most commonly identified cause of healthcare-associated infection in the elderly. Risk factors include advanced age, hospitalization, prior or concomitant systemic antibacterial therapy, chemotherapy, and gastrointestinal surgery. Patients with unspecified and new-onset diarrhea with ≥3 unformed stools in 24 h are the target population for <i>C. diff</i> infection (CDI) testing. To present data on the risks of laboratory misdiagnosis in managing CDI. <b>Materials</b>. In two general hospitals, we examined 116 clinical stool specimens from hospitalized patients with acute diarrhea suspected of nosocomial or antibiotic-associated diarrhea (AAD) due to <i>C. diff.</i> Enzyme immunoassay (EIA) tests for the detection of <i>C. diff</i> toxins A (cdtA) and B (cdtB) in stool, automated CLIA assay for the detection of <i>C. diff</i> GDH antigen and qualitative determination of cdtA and B in human feces and anaerobic stool culture were applied for CDI laboratory diagnosis. MALDI-TOF (Bruker) was used to identify the presumptive anaerobic bacterial colonies. The following methods were used as confirmatory diagnostics: the LAMP method for the detection of Salmonella spp. and simultaneous detection of <i>C. jejuni</i> and <i>C. coli</i>, an <i>E. coli</i> Typing RT-PCR detection kit (ETEC, EHEC, STEC, EPEC, and EIEC), API 20E and aerobic stool culture methods. <b>Results</b>. A total of 40 toxigenic strains of <i>C. diff</i> were isolated from all 116 tested diarrheal stool samples, of which 38/40 produced toxin B and 2/40 strains were positive for both cdtA and cdtB. Of the stool samples positive for cdtA (6/50) and/or cdtB (44/50) by EIA, 33 were negative for <i>C. diff</i> culture but positive for the following diarrheal agents: <i>Salmonella enterica</i> subsp. <i>arizonae</i> (1/33, LAMP, culture, API 20E); <i>C. jejuni</i> (2/33, LAMP, culture, MALDI TOF); ETEC O142 (1/33), STEC O145 and O138 (2/33, <i>E. coli</i> RT-PCR detection kit, culture); <i>C. perfringens</i> (2/33, anaerobic culture, MALDI TOF); hypermycotic enterotoxigenic <i>K. pneumonia</i> (2/33) and enterotoxigenic <i>P. mirabilis</i> (2/33, culture; PCR encoding LT-toxin). Two of the sixty-six cdtB-positive samples (2/66) showed a similar misdiagnosis when analyzed using the CLIA method. However, the PCR analysis showed that they were cdtB-negative. In contrast, the LAMP method identified a positive result for <i>C. jejuni</i> in one sample, and another was STEC positive (stx1+/stx2+) by RT-PCR. We found an additional discrepancy in the CDI test results: EPEC O86 (RT-PCR eae+) was isolated from a fecal sample positive for GHA enzyme (CLIA) and negative for cdtA and cdtB (CLIA and PCR). However, the culture of <i>C. diff</i> was negative. These findings support the hypothesis that certain human bacterial pathogens that produce enterotoxins other than <i>C. diff</i>, as well as intestinal commensal microorganisms, including Klebsiella sp. and Proteus sp., contribute to false-positive EIA card tests for <i>C. diff</i> toxins A and B, which are the most widely used laboratory tests for CDI. <b>Conclusions</b>. CDI presents a significant challenge to clinical practice in terms of laboratory diagnostic management. It is recommended that toxin-only EIA tests should not be used as the sole diagnostic tool for CDI but should be limited to detecting toxins A and B. Accurate diagnosis of CDI requires a combination of laboratory diagnostic methods on which proper infection management depends.