Clostridioides difficile infection (CDI) frequently occurs concurrently in patients with inflammatory bowel disease (IBD), and differential diagnosis from IBD flares is critical. However, clinical management of C. difficile in IBD patients with polymerase chain reaction toxin-positive (tPCR+)/enzyme immunoassay toxin-negative (tEIA-) results has not yet been investigated.

We aimed to assess the clinical significance of C. difficile tPCR+/tEIA- in patients with IBD and the impact of antibiotic treatment on IBD outcomes.

This single-center, retrospective cohort study included patients with IBD with CDI test results between January 01, 2018, and August 01, 2022. First, the clinical outcomes of IBD, such as medication escalation, hospitalization, and surgery, were compared between patients with IBD with tPCR-/tEIA- and those with tPCR+/tEIA- using Cox regression and propensity score matching. Next, the clinical outcomes of IBD were assessed based on whether antibiotic treatment for CDI was administered to both groups.

Among 412 patients with IBD with PCR test, 71 (17.2%) showed tPCR+/tEIA- results. The tPCR+/tEIA- group showed no statistically significant difference in IBD outcomes compared to the tPCR-/tEIA- group. The antibiotic-treated tPCR+/tEIA- group showed a higher risk of drug escalation and admission than the tPCR-/tEIA- group, while the antibiotic-untreated tPCR+/tEIA- group did not. After drug escalation during the follow-up, the treated tPCR+/tEIA- group showed IBD outcomes similar to those of the tPCR-/tEIA- group.

In patients with IBD with indeterminate CDI, the need for antibiotics should be thoroughly assessed and proper management of underlying IBD such as drug escalation may lead to favorable outcomes.

To review stool diagnostic tests in acute and chronic diarrhea.

Narrative review of published literature.

In acute diarrhea, stool tests are indicated when there is strong pretest probability of infectious etiology or Clostridioides difficile infection suggested by > 3 unformed bowel movements per 24 hours, symptoms lasting >7 days, and circumstances that are suggestive of infection. Several commercially available rapid tests for bacterial, viral, or protozoal infections may be offered in addition to traditional methods (e.g. culture, microscopy) and provide a result within 6 hours. For C . difficile infections, a highly sensitive test such as glutamate dehydrogenase test is required; however, this does not distinguish infection from carrier state. That differentiation requires specialized nucleic acid amplification test (for toxin B) or enzyme immunoassays for toxin A or B, which are unfortunately not generally offered by microbiology laboratories. Chronic diarrhea may result from inflammatory, fatty, osmotic, or secretory causes; the commonest cause is diarrhea-predominant irritable bowel syndrome/functional diarrhea. Current recommendations in societal guidelines or clinical practice updates regarding stool tests in diarrhea-predominant irritable bowel syndrome/functional diarrhea in the absence of alarm symptoms include testing for Giardia, calprotectin, fecal immunochemical test, and bile acid diarrhea. Comprehensive stool biochemical analyses (osmolality, pH, electrolytes) differentiate osmotic from secretory diarrhea and identify laxative abuse. Specific stool diagnostic tests for bile acid diarrhea and exocrine pancreatic insufficiency can lead to specific diagnosis and treatments. Surrogate markers associated with high fecal output and rapid transit in chronic diarrhea are stool form and colonic transit.

Fecal testing is still very relevant in the practice of gastroenterology and deserves introduction of advanced microbiological and biochemical tests.

This article provides an overview of the advancements in the application of fecal microbiota transplantation (FMT) in treating diseases related to intestinal dysbiosis. FMT involves the transfer of healthy donor fecal microbiota into the patient's body, aiming to restore the balance of intestinal microbiota and thereby treat a variety of intestinal diseases such as recurrent Clostridioides difficile infection (rCDI), inflammatory bowel disease (IBD), constipation, short bowel syndrome (SBS), and irritable bowel syndrome (IBS). While FMT has shown high efficacy in the treatment of rCDI, further research is needed for its application in other chronic conditions. This article elaborates on the application of FMT in intestinal diseases and the mechanisms of intestinal dysbiosis, as well as discusses key factors influencing the effectiveness of FMT, including donor selection, recipient characteristics, treatment protocols, and methods for assessing microbiota. Additionally, it emphasizes the key to successful FMT. Future research should focus on optimizing the FMT process to ensure long-term safety and explore the potential application of FMT in a broader range of medical conditions.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers have become an integral part of all modern clinical microbiology laboratories. They serve as the key tool for pathogen identification and antibiotic resistance determination. However, certain limiting conditions must be fulfilled. The pathogen cannot be tested directly from the sample and requires the cultivation of a pure colony, which means that the standard protocol takes additional time, workforce, and consumables. The testing protocol is also more complicated when it comes to anaerobes. In our work, we focused on the functional detection of Clostridioides difficile, an important nosocomial human pathogen that is responsible for diarrhea and can lead to life-threatening colitis, as a model diagnostic problem. The virulence of C. difficile is mainly caused by two toxins, Toxin A and Toxin B. Established diagnostic methods, including nucleic acid amplification testing methods and immunoassays, detect the presence of the microorganism or the presence and concentration of the toxins, with limited ability to gauge infection severity based on the actual biochemical activity of the toxins and thus their potency to cause harm. This work presents proof-of-concept assays that indirectly determine the toxin activity in the human stool, a very complex matrix sample, using the natural RhoA protein as substrate. The RhoA protein substrate was recombinantly prepared with biotin tag modification, which allows its attachment to the NeutrAvidin MALDI chips. In the assay, the RhoA substrate anchored on the MALDI chip undergoes enzymatic glycosylation when exposed to the Toxin B in the stool sample, and the reaction product is then detected by MALDI-TOF mass spectrometry directly from the MALDI chip. The entire assay, from sampling to final mass spectrometry detection, was performed in situ, on the NeutrAvidin MALDI chip, which was prepared by unique surface modification technology also described in this work. The assay was successfully tested for the detection of Toxin B in a cohort of patient samples as well as in cell culture of C. difficile.

The diagnostics of Clostridioides difficile infection is usually based on the identification of the bacterial pathogen and/or on the detection of the Toxins A and B. Due to the variance in Toxins A and B activity across species, the toxin concentration determined by standard methods does not necessarily correlate with the severity of the disease. Assays that would target toxins' enzymatic activity are not routinely used because the requirements are unsuitable for clinical laboratories. In this study, we demonstrate a new approach that determines the presence and potency of Toxin B indirectly by determining its enzymatic activity rather than its concentration. This is performed by detecting mass difference due to glycosylation of RhoA substrate by Toxin B, which is then determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented proof-of-concept assay thus offers the possibility to quickly determine the activity of C. difficile toxins directly in the stool samples without pathogen cultivation.

Tuberculosis (TB) continues to be a major public health challenge in low- and middle-income countries (LMICs), where high burdens of coinfections exacerbate the disease's impact. In 2023, an estimated 8.2 million people were newly diagnosed with tuberculosis worldwide, reflecting an increase from 7.5 million in 2022 and 7.1 million in 2019. In LMICs, limited access to healthcare, inadequate nutrition, and poor living conditions contribute to higher coinfection rates among TB patients, leading to delayed diagnosis and treatment, which in turn exacerbates disease severity and facilitates transmission. This narrative review synthesizes the epidemiology, clinical implications, diagnostic challenges, and management strategies related to TB coinfections with viral pathogens including HIV, SARS-CoV-2, and influenza, bacteria such as Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa, fungi such as Aspergillus and Candida species, and parasites. This review highlights that overlapping symptoms, immune system compromise, and socioeconomic barriers in LMICs lead to delayed diagnoses and suboptimal treatment outcomes, while also addressing the challenges of managing drug interactions particularly in HIV-TB coinfections and underscoring the need for integrated diagnostic approaches, improved treatment regimens, and strengthened healthcare systems, thereby consolidating current evidence to inform future research priorities and policy interventions aimed at reducing the overall burden of TB and its coinfections in resource-limited settings.

Clostridioides difficile infection (CDI) is one of the most common causes of diarrhea in healthcare facilities, imposing a significant burden on health-related budgets and resources worldwide. We aimed to study the demographic features, laboratory findings, and outcomes of CDI in Qatar.

A retrospective study involving adult patients with a confirmed diagnosis of CDI was conducted.

Of the 595 patients, 308 (51.8%) were men with a mean age of 58 ± 19.9 years. The median duration of symptoms was 2 days, with an interquartile range values of 2-4 days. The most common symptoms reported were diarrhea (90.6%) and abdominal pain (41.5%). A total of 426 (71.6%) and 422 (70.9%) patients had a history of exposure to antibiotics and proton pump inhibitors, respectively, while 461 (77.7%) patients had a history of contact with a healthcare facility in the prior 3 months. Respiratory tract infections (33.9%) and urinary tract infections (22.4%) were the most common indications for antibiotic use in the study population. Increased C-reactive protein levels were the most frequently observed laboratory findings. Sepsis was identified as the most common complication (10.4 %). Reinfection was observed in 75 (12.6%) patients. Vancomycin monotherapy was the most commonly prescribed treatment.

In Qatar, CDI remains a significant health concern, primarily affecting elderly men, especially those who have had hospital admissions or used proton pump inhibitors or antibiotics. Preventive measures and increased knowledge of contact precautions and hand hygiene, particularly among healthcare workers, will help to reduce transmission. Raising physician awareness regarding the prudent use of antibiotics and antibiotic stewardship will serve as an adjunct to reduce the incidence of CDIs.

A review of the diagnostic and therapeutic management algorithm of the pathogen Clostridioides difficile for daily practice is presented. Its diagnosis, in any unformed stool sample sent to the laboratory, is based on a two-step algorithm, with demonstration of the pathogen by means of its enzyme glutamate dehydrogenase by immunoassay and subsequent PCR (polymerase chain reaction) of its toxin. The mainstay of step therapy, reserved for symptomatic patients, is fidaxomicin, over vancomycin. Metronidazole is not an adequate treatment. Emerging therapies, such as faecal microbiota transplantation or the antibody bezlotoxumab, are gaining importance in patients with risk factors or relapses. Surgery is indicated in patients with worse prognosis and complications. Prevention is essential, based on vigilance and contact precautions, in addition to the elimination of spores from the environment.

German surveillance data from 2022 reported a prevalence of nosocomial infections among hospitalized patients of 5,2%. Clostridioides-difficile-infections (CDI) are the most frequent cause of nosocomial diarrhea. They are usually caused by antibiotic exposure and the subsequent changes in the gut microbiota. Clinical manifestation ranges from asymptomatic colonization over moderate diarrhea to severe pseudomembranous colitis. According to the current German Gastrointestinal Infection Guidelines, fidaxomicin is the preferred treatment option for CDI, especially in patients at high risk of recurrence or those already suffering from recurrence. Vancomycin can also be used as an alternative for initial CDI treatment. Fecal microbiota transplantation is considered a treatment approach for patients with multiple recurrences.

The laboratory diagnosis of Clostridioides difficile infection (CDI) is controversial. Nucleic acid amplification tests (NAAT) and toxin enzyme immunoassays (EIA) are most widely used, often in combination. However, the interpretation of a positive NAAT and negative toxin immunoassay (NAAT+/EIA-) is uncertain. PubMed and EMBASE were searched for studies reporting clinical outcomes in NAAT+/EIA- versus NAAT+/EIA+ patients. Forty-six studies comprising 33,959 patients were included in this meta-analysis. All-cause mortality (RR 0.96, 95% CI 0.80-1.15), attributable mortality (RR 0.61, 95% CI 0.20-1.91), fulminant CDI (RR 0.83, 95% CI 0.57-1.20), radiographic evidence of CDI (RR 0.87, 95% CI 0.65-1.16), total CDI complications (RR 0.95, 95% CI 0.59-1.53), colectomies (RR 0.78, 95% CI 0.34-1.79), and ICU admission (RR 1.04, 95% CI 0.84-1.30) did not significantly differ between NAAT+/EIA- and NAAT+/EIA+ patients. However, rates of recurrent (RR 0.62, 95% CI 0.50-0.77) or severe (RR 0.74, 95% CI 0.63-0.88) CDI were significantly lower in NAAT+/EIA- patients than in NAAT+/EIA+ patients. The pooled prevalence of NAAT+/EIA- patients who were treated with antibiotics for CDI was 73.4% (pooled proportion 0.72, 95% CI 0.52-0.88). NAAT+/EIA- patients have lower rates of recurrence and are at reduced risk for severe CDI compared with NAAT+/EIA+ patients but have a risk of CDI-related complications and mortality comparable to that of NAAT+/EIA+ patients. Toxin results cannot rule in or rule out CDI, and the decision whether to treat symptomatic NAAT+/EIA- patients for CDI should be based on clinical presentation and not on the toxin result.IMPORTANCEClostridioides difficile infection (CDI) is a common cause of healthcare-associated infections and the leading cause of antibiotic-associated diarrhea. However, the laboratory diagnosis of CDI, primarily done by nucleic acid amplification test (NAAT) and enzyme immunoassay (EIA), is controversial, especially in patients who test positive by NAAT but negative by EIA. In this systematic review, we compared the clinical outcomes of NAAT+/EIA- versus NAAT+/EIA+ patients and found that the two groups have similar risk of mortality and CDI-related complications. However, NAAT+/EIA- patients had significantly lower rates of recurrence and severe CDI than NAAT+/EIA+ patients, and most NAAT+/EIA- patients received CDI therapy. Toxin testing can help to predict the likelihood of CDI recurrence or severe infection, but the toxin result should not be a determining factor in the administration of CDI therapy. The decision on whether to treat NAAT+/EIA- patients should be based on clinical assessment.

Current guidelines recommend a two-step algorithm rather than relying solely on a single test for diagnosing Clostridioides difficile infection. This algorithm starts with enzyme immunoassay (EIA) for detecting glutamate dehydrogenase (GDH) and toxins A/B, followed by nucleic acid amplification test (NAAT) for GDH-positive but toxin-negative cases. This study compared the performance of three commercial NAATs: the STANDARD M10 C. difficile, Xpert C. difficile, and BD MAX Cdiff assays, utilized as confirmatory testing of the two-step algorithm. Two hundred archived stool specimens, previously tested GDH-positive but toxin-negative by EIA, were analyzed in parallel with these NAATs and toxigenic culture, which served as the reference standard. Sensitivity, specificity, positive predictive value, and negative predictive value were 89.1%, 92.6%, 94.6%, and 85.2%, respectively, for the M10 assay; 95.8%, 86.4%, 91.2%, and 93.3%, respectively, for the Xpert assay; and 89.8%, 91.4%, 93.8%, and 86.0%, respectively, for the BD MAX assay. The rates of invalid results were 1.0%, 0.5%, and 1.0% for the M10, Xpert, and BD MAX assays, respectively. In conclusion, the M10 assay is a reliable diagnostic tool, performing comparably to the Xpert and BD MAX assays when used as confirmatory testing in the two-step algorithm.IMPORTANCEWhile numerous studies have assessed nucleic acid amplification tests (NAATs) as stand-alone tests for diagnosing Clostridioides difficile infection, limited research has compared their performance as confirmatory tests in a two-step algorithm. This study evaluated the performance of three commercial NAATs (M10, Xpert, and BD MAX assays) using 200 archived stool specimens initially tested as glutamate dehydrogenase (GDH)-positive but toxin-negative by GDH/toxin A/B enzyme immunoassay, the first step in the two-step algorithm. All three assays demonstrated high sensitivity (89.1% to 95.8%) and specificity (86.4% to 92.6%), with low rates of invalid results (≤1%). Our findings suggest that the M10 assay performs comparably to the Xpert and BD MAX assays when used as confirmatory testing in the two-step algorithm. Offering similar performance and turnaround time to these widely used assays at a slightly lower cost, the M10 assay serves as a practical alternative in this setting.

A diverse and well-functioning gut microbiota normally serves as a protective shield against the invasion of harmful bacteria or the proliferation of opportunistic pathogens. Clostridioides difficile infection (CDI) is predominantly associated with the overuse of antibiotics, resulting in a significant alteration in the gut's microbial balance. Unfortunately, the lack of global standardization does not allow for the identification of a set of biomarkers associated with the onset and progression of this disease. In this study, we examined the composition of the gut microbiota in patients at the time of the initial detection of CDI compared to a control group of CDI-negative individuals, with a focus on identifying potential CDI biomarkers for diagnosis. While no significant differences in the alpha and beta diversity between CDI-negative and CDI-positive individuals were found, we found that certain genera (such as Clostridium XIVa and Clostridium XVIII) showed different abundance patterns in the two groups, indicating potential differences in gut microbiota balance. In conclusion, am enrichment in Clostridium XI and a decrease in Faecalibacterium emerged in the CDI-positive patients and following antibiotic treatment, indicating that changes in the Clostridium/Faecalibacterium ratio may be a promising biomarker that warrants further investigation for CDI diagnosis.

Diagnosis of Clostridioides difficile infection (CDI) remains challenging as it involves in the first instance recognition (clinical awareness) of the patients' symptoms for clinical suspicion of CDI to warrant testing, and secondly, different laboratory tests have been described for CDI. Due to the overwhelming amount of information in the literature on CDI tests and their performance, with separately published guidelines, this review aims to provide a comprehensive but concise summary of the current state of CDI diagnostic testing. Current knowledge and the impact of using different laboratory diagnostic procedures for CDI, including the most recommended approach as a two-step algorithm and the concept of diagnostic stewardship, are being discussed. This review provides an updated overview and valuable take-home messages in the field of CDI laboratory testing and highlights that timely diagnosis is important for the clinical management of CDI and that the recommended testing procedures are increasingly becoming more widely accepted.

To evaluate the risk of recurrent Clostridioides difficile infection (CDI) in solid-organ transplant (SOT) recipients.

Retrospective multicenter study including SOT recipients with a first CDI episode in the year after transplantation (Jan 2017-June 2020). The primary outcome measure was recurrence, defined as a new CDI ≤56 days from the first episode. A competing risk analysis was performed using the sub-distribution hazard model multivariable analysis.

191 SOT recipients were included: 101 (52.9%) were kidney, 66 (34.6%) liver, 11 (5.8%) lung, 8 (4.2%) simultaneous pancreas-kidney, 4 (2.1%) heart and 1 (0.5%) pancreas alone recipients. Treatment for the first CDI were: vancomycin (n = 114,59.7%), vancomycin+metronidazole (n = 39,20.4%), metronidazole (n = 26,13.6%), fidaxomicin (n = 9,4.7%), 3 patients did not receive any therapy. After the first CDI, 17/191 (8.9%) patients died within 56-day mortality without having a recurrence, while 23/191 (12%) patients had a recurrence. Among patients with recurrent CDI, 56-day mortality rate was 30.4% (7/23 patients). On multivariable analysis, severe CDI (sHR4.01, 95% CI 1.77-9.08, p < .001) and metronidazole monotherapy (sHR 3.65, 95% CI 1.64-8.14, p = .001) were factors independently associated with recurrence.

Metronidazole monotherapy is associated with increased risk of recurrent CDI in SOT recipients. Therapeutic strategies aimed to reduce the risk of recurrence should be implemented in this setting.

We evaluated the analytical performance of three commercial molecular assays for rapid detection of Clostridioides difficile toxin B in stool samples. The results were compared with results from the BD MAX™ Cdiff assay. We analyzed forty negative and thirty-two positive stool samples with three rapid assays: Roche cobas® Liat® Cdiff, SD Biosensor STANDARD™ M10 C. difficile and Cepheid Xpert® C. difficile BT. The assays demonstrated a sensitivity of 96.9 %, 96.9 % and 100.0 % and a specificity of 100 %, 97.5 % and 97.5 %, respectively. There is limited data available on the analytical performance of the newly introduced STANDARD™ M10 C. difficile assay. In this study, all three rapid assays demonstrated similarly high analytical performance and can be used for detection of toxigenic C. difficile.

Clostridioides difficile infection (CDI) causes alterations in the intestinal microbiota, frequently associated with changes in the gut metabolism of bile acids and cholesterol. In addition to the impact on microbiome composition and given the metabolic changes occurring during CDI, our work focuses on the importance to know the effects at the local and systemic levels, both during the infection and its treatment, by paying particular attention to plasma lipid metabolism due to its relationship with CDI pathogenesis. Specific changes, characterized by a loss of microbial richness and diversity and related to a reduction in short-chain acid-producing bacteria and an increase in bile salt hydrolase-producing bacteria, were observed in the gut microbiota of CDI patients, especially in those suffering from recurrent CDI (RCDI). However, gut microbiota showed its ability to restore itself after treatment, resembling healthy individuals, in those patients treated by fecal microbiome transfer (FMT), in contrast with those treated with antibiotics, and displaying increased levels of Eubacterium coprostanoligenes, a cholesterol-reducing anaerobe. Interestingly, changes in plasma lipidome revealed a global depletion in circulating lipids in CDI, with the largest impact on cholesteryl esters. CDI patients also showed a specific and consistent decrease in the levels of lipid species containing linoleic acid-an essential fatty acid-which were only partially recovered after antibiotic treatment. Analysis of the plasma lipidome reflects CDI impact on the gut microbiota and its metabolism, evidencing changes in sterol and fatty acid metabolism that are possibly related to specific alterations observed in gut microbial communities of CDI patients.

There is increasing evidence about the influence the changes in microbiota and its metabolism has on numerous diseases and infections such as Clostridioides difficile infection (CDI). The knowledge of these changes at local and systemic levels can help us manage this infection to avoid recurrences and apply the best therapies, such as fecal microbiota transfer (FMT). This study shows a better restoration of the gut in FMT-treated patients than in antibiotic-treated patients, resembling healthy controls and showing increased levels of cholesterol-reducing bacteria. Furthermore, it evidences the CDI impact on plasma lipidome. We observed in CDI patients a global depletion in circulating lipids, particularly cholesteryl esters, and a specific decrease in linoleic acid-containing lipids, an essential fatty acid. Our observations could impact CDI management because the lipid content was only partially recovered after treatment, suggesting that continued nutritional support, aiming to restore healthy lipid levels, could be essential for a full recovery.

Detection and identification of microorganisms are the first steps to guide susceptibility testing and enable clinicians to confirm diseases and guide therapy. The faster the pathogen identification is determined, the quicker the appropriate treatment can be started. In the clinical microbiology laboratory, multiple methodologies can be used to identify organisms, such as traditional biochemical testing or more recent methods like MALDI TOF MS and nucleic acid detection/identification assays. Each of these techniques has advantages and limitations, and clinical laboratories need to determine which methodology is best suited to their particular setting in terms of clinical needs, availability of technical expertise and cost. This article presents a concise review of the history, utilization, advantages and limitations of the main methods used for identifying microorganisms in microbiology laboratories.

The nosocomial transmission of toxin-producing Clostridioides difficile is a significant concern in infection control. C. difficile, which resides in human intestines, poses a risk of transmission, especially when patients are in close contact with medical staff.

To investigate the nosocomial transmission of C. difficile in a single center, we analyzed the genetic relationships of the bacteria. This was done using draft whole-genome sequencing (WGS) and examining single nucleotide polymorphisms (SNPs) in core-genome, alongside data regarding the patient's hospital wards and room changes. Our retrospective analysis covered 38 strains, each isolated from a different patient, between April 2014 and January 2015.

We identified 38 strains that were divided into 11 sequence types (STs). ST81 was the most prevalent (n = 11), followed by ST183 (n = 10) and ST17 (n = 7). A cluster of strains that indicated suspected nosocomial transmission (SNT) was identified through SNP analysis. The draft WGS identified five clusters, with 16 of 38 strains belonging to these clusters. There were two clusters for ST81 (ST81-SNT-1 and ST81-SNT-2), two for ST183 (ST183-SNT-1 and ST183-SNT-2), and one for ST17 (ST17-SNT-1). ST183-SNT-1 was the largest SNT cluster, encompassing five patients who were associated with Wards A, B, and K. The most frequent room changer was a patient labeled Pt08, who changed rooms seven times in Ward B. Patients Pt36 and Pt10, who were also in Ward B, had multiple admissions and discharges during the study period.

Additional culture tests and SNP analysis of C. difficile using draft WGS revealed silent transmission within the wards, particularly in cases involving frequent room changes and repeated admissions and discharges. Monitoring C. difficile transmission using WGS-based analysis could serve as a valuable marker in infection control management.

Clostridioides difficile infection (CDI), a leading cause of nosocomial deaths, is a microbiota-mediated disease. As such, the use of broader spectrum antibiotics, such as vancomycin and metronidazole, can prime the gastrointestinal tract to become more prone to CDI recurrences. Fidaxomicin, a narrow-spectrum antibiotic, has been demonstrated to be superior in preventing recurrence and in preserving the intestinal microbiota; however, widespread employment worldwide has been hindered due to high acquisition costs.

To integrate the currently available guidelines on the management of CDI and to shed light on the timeliest employment of fidaxomicin.

An expert panel was gathered to obtain consensus using Delphi methodology on a series of statements regarding the management of CDI and on appropriate antibiotic use.

Consensus was reached on 21 of the 25 statements addressing the management of CDI.

Delphi methodology was used to achieve consensus on the management of CDI, on the identification of patients at risk of recurrences or severe infection, and on the most appropriate use of fidaxomicin, with the final aim of fostering clinical practice application of treatment algorithms proposed by previous guidelines, in absolute synergy. It could be an important tool to promote more appropriate and cost-effective CDI treatments in European settings with limited resources, like Italy.

Clostridioides difficile infection (CDI) caused by toxigenic C. difficile is the leading cause of antimicrobial and healthcare-associated diarrhea. The pathogenicity of C. difficile relies on the synergistic effect of multiple virulence factors, including spores, flagella, type IV pili (T4P), toxins, and biofilm. Spores enable survival and transmission of C. difficile, while adhesion factors such as flagella and T4P allow C. difficile to colonize and persist in the host intestine. Subsequently, C. difficile produces the toxins TcdA and TcdB, causing pseudomembranous colitis and other C. difficile-associated diseases; adhesion factors bind to the extracellular matrix to form biofilm, allowing C. difficile to evade drug and immune system attack and cause recurrent infection. Cyclic diguanylate (c-di-GMP) is a near-ubiquitous second messenger that extensively regulates morphology, the expression of virulence factors, and multiple physiological processes in C. difficile. In this review, we summarize current knowledge of how c-di-GMP differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in C. difficile. We highlight that C. difficile spore formation and expression of toxin and flagella genes are inhibited at high intracellular levels of c-di-GMP, while T4P biosynthesis, cell aggregation, and biofilm formation are induced. Recent studies have enhanced our understanding of the c-di-GMP signaling networks in C. difficile and provided insights for the development of c-di-GMP-dependent strategies against CDI.

The performance of MALDI-TOF MS combined with analysis platform for identification of toxin-producing Clostridiodes difficile is yet to be known.

Between August 2018 and September 2020, 61 isolates from stool specimens of patients with C. difficile-associated diarrhea were analyzed using the MALDI Biotyper system. A C. difficile toxin-producer detection model was developed using ClinProTools. The model was validated using 28 known strains that differed from the isolates used to develop the model.

The sensitivity and specificity of the Genetic Algorithm (GA) model using isolates grown on Brucella with hemin and vitamin K (BHK) agar plates were 91.7% and 44.4%, respectively. When isolates grown on cycloserine-cefoxitin mannitol agar were analyzed by the model, sensitivity and specificity were 6.3% and 100%, respectively. The GA model using BHK medium showed the highest discriminatory performance in detection of toxin-producing C. difficile. However, a discrepancy in detection of toxin-producing C. difficile was observed in the results generated when the model was being developed and when the model was validated which suggests that incubation conditions may have affected the results.

MALDI-TOF analysis using ClinProTools has a potential to be a cost-effective tool for rapid diagnosis and contribute to antimicrobial stewardship by differentiating toxin-producing C. difficile from non-producers.

The tcdA gene codes for an important toxin produced by Clostridioides difficile (C. difficile), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene.

Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction.

At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X2 = 47, p < 0.01).

LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile, and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA-LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk.

Clostridioides difficile infection (CDI) is a well-known cause of hospital-acquired infectious diarrhea in developed countries, though it has not been a top priority in the healthcare policies of developing countries. In the last decade, several studies have reported a wide range of CDI rates between 1.3% and 96% in developing nations, raising the concern that this could represent a healthcare threat for these nations. This review defines developing countries as those with a human development index (HDI) below 0.8. We aim to report the available literature on CDI epidemiology, diagnostics, management, and prevention in developing countries. We identify limitations for CDI diagnosis and management, such as limited access to CDI tests and unavailable oral vancomycin formulation, and identify opportunities to enhance CDI care, such as increased molecular test capabilities and creative solutions for CDI. We also discuss infection prevention strategies, including antimicrobial stewardship programs and opportunities emerging from the COVID-19 pandemic, which could impact CDI care.

Clostridioides difficile infection (CDI) is a significant cause of mortality, especially in healthcare environments. Reliable biomarkers that can accurately predict mortality in CDI patients are yet to be evaluated. Our study aims to evaluate the accuracy of several inflammatory biomarkers and hemogram-derived ratios in predicting mortality in CDI patients, such as the neutrophil-to-lymphocyte ratio (NLR), the systemic immune-inflammation index (SII), the platelet-to-neutrophil ratio (PNR), the derived neutrophil-to-lymphocyte ratio (dNLR), C-reactive protein (CRP), the platelet-to-lymphocyte ratio (PLR), and procalcitonin (PCT).

NLR showed a sensitivity of 72.5% and a specificity of 58.42% with an area under curve (AUC) = 0.652. SII had a sensitivity of 77.5%, a specificity of 54.74%, and an AUC = 0.64. PNR, neutrophils, dNLR, and lymphocytes had lower AUCs which ranged from 0.595 to 0.616, with varied sensitivity and specificity. CRP, leukocytes, and platelets showed modest predictive values with AUCs below 0.6. PCT had a sensitivity of 100%, a low specificity of 7.41%, and an AUC = 0.528.

We conducted a retrospective analysis of CDI patients from two different hospital settings in Italy and Romania during the COVID-19 pandemic, from 1 January 2020 to 5 May 2023. Statistical analyses included t-tests, Wilcoxon rank-sum tests, χ2 tests, and multivariate logistic regression to identify predictors of mortality. ROC analysis assessed the accuracy of biomarkers and hemogram-derived ratios. A p value < 0.05 was considered significant.

Neutrophils, dNLR, NLR, SII, and PNR are valuable biomarkers for predicting mortality in CDI patients. Understanding these predictors can improve risk stratification and clinical outcomes for CDI patients.

Clostridioides difficile detection in community settings is time-intensive, resulting in delays in diagnosing and quarantining infected individuals. However, with the advent of semi-automated devices and improved algorithms in recent decades, the ability to discern CDI infection from asymptomatic carriage has significantly improved. This, in turn, has led to efficiently regulated monitoring systems, further reducing endemic risk, with recent concerns regarding a possible surge in hospital-acquired Clostridioides difficile infections post-COVID failing to materialize. This review highlights established and emerging technologies used to detect community-acquired Clostridioides difficile in research and clinical settings.

This study compared the performance of two commercial molecular assays, the STANDARD M10 Clostridioides difficile assay (M10) and the Xpert C. difficile assay (Xpert), for detecting toxigenic C. difficile in stool specimens. A total of 487 consecutive stool specimens submitted for routine C. difficile testing between June and November 2023 were included. Following routine testing using C. DIFF QUIK CHEK COMPLETE (QCC), M10 and Xpert were tested in parallel, alongside toxigenic culture (reference standard). Additionally, two-step algorithms, using QCC on the first step and either M10 or Xpert on the second step, were assessed. Both M10 and Xpert demonstrated a sensitivity and negative predictive value (NPV) of 100%. M10 exhibited significantly higher specificity and positive predictive value (PPV; 91.9% and 64.2%, respectively) than Xpert (90.3% and 59.8%, respectively). Both two-step algorithms showed a sensitivity and NPV of 98.4% and 99.8%, respectively. The specificity and PPV of the two-step algorithm using M10 (95.2% and 75.0%, respectively) were slightly higher than those of the one using Xpert (94.8% and 73.2%, respectively), without statistical significance. Receiver operating characteristic curve analysis, assessing the predictive ability of cycle threshold (Ct) values for the detection of free toxin, exhibited an area under the curve of 0.825 for M10 and 0.843 for Xpert. This indicates the utility of Ct values as predictors for the detection of free toxin in both assays. In conclusion, M10 proves to be an effective diagnostic tool with performance comparable to Xpert, whether utilized independently or as part of a two-step algorithm.

Background. Target product profiles (TPPs) specify the essential properties tests must have to be able to address an unmet clinical need. Aim. To explore how early economic modeling can help to define TPP specifications based on cost-effectiveness considerations using the example of a new rapid diagnostic for Clostridioides difficile infection (CDI), a contagious health care-associated infection causing potentially fatal diarrhea. Methods. A resource-constrained simulation model was developed to compare a hypothetical test for CDI with current practice (i.e., test with glutamate dehydrogenase enzyme immunoassay first; if positive, test with polymerase chain reaction and cytotoxicity assay) for adult individuals with suspected CDI at the Leeds Teaching Hospital National Health System (NHS) Trust in the United Kingdom. Parameters are taken from UK-based observational data collected between 2018 and 2021, published literature, and expert opinion. A methodological framework was developed 1) to derive minimum diagnostic sensitivity and specificity and maximum price for different test turnaround-time values based on cost-effectiveness considerations from the health care perspective using the National Institute of Health Care Excellence willingness-to-pay threshold of £20,000 per quality-adjusted life-years and 2) to test their robustness using a series of sensitivity analyses. Results. A new rapid test for CDI with a 15-min turnaround time would require a minimum diagnostic sensitivity and specificity both equal to 96% and a maximum price of £44 to maintain cost-effectiveness compared with standard of care. Conclusions. This study provides a framework to inform the essential test properties based on cost-effectiveness considerations and to isolate the most influential model parameters and scenarios via a series of sensitivity analyses. These specifications, in turn, could be used to inform future TPPs for tests.

Target product profiles (TPPs) for new medical tests provide test developers with performance benchmarks and technical requirements for new tests. Early economic evaluation has already been used to identify acceptable ranges for certain performance requirements for new tests. Currently, however, early economic evaluation methods are yet to be used in the context of TPP development, and there is no guidance as to how this could and should be done.A de novo approach was developed to identify the minimum performance requirements and maximum costs for new tests, based on cost-effectiveness considerations, while also isolating most influential parameters. The added value of this framework lies in structuring early economic evaluation methods as a means of informing transparent, evidence-based minimum TPP performance specifications while also accounting as much as possible for the (inevitable) uncertainty surrounding the minimum performance requirements.This study represents the first application of early economic modeling as a means of deriving the minimum performance specifications for a novel point-of-care test for Clostridioides difficile infection as set out in a future TPP.

SUMMARYClostridioides difficile infection (CDI) is one of the major issues in nosocomial infections. This bacterium is constantly evolving and poses complex challenges for clinicians, often encountered in real-life scenarios. In the face of CDI, we are increasingly equipped with new therapeutic strategies, such as monoclonal antibodies and live biotherapeutic products, which need to be thoroughly understood to fully harness their benefits. Moreover, interesting options are currently under study for the future, including bacteriophages, vaccines, and antibiotic inhibitors. Surveillance and prevention strategies continue to play a pivotal role in limiting the spread of the infection. In this review, we aim to provide the reader with a comprehensive overview of epidemiological aspects, predisposing factors, clinical manifestations, diagnostic tools, and current and future prophylactic and therapeutic options for C. difficile infection.

The detection of Clostridioides difficile infections (CDI) relies on testing the stool of patients by toxin antigen detection or PCR methods. Although PCR and antigenic methods have significantly reduced the time to results, delays in stool collection can significantly add to the turnaround time. The use of rectal swabs to detect C. difficile could considerably reduce the time to diagnosis of CDI. We developed a new rapid PCR assay for the detection of C. difficile and evaluated this PCR assay on both stool and rectal swab specimens. We recruited a total of 623 patients suspected of C. difficile infection. Stool samples and rectal swabs were collected from each patient and tested by our PCR assay. Stool samples were also tested by the cell cytotoxicity neutralization assay (CCNA) as a reference. The PCR assay detected C. difficile in 60 stool specimens and 61 rectal swabs for the 64 patients whose stool samples were positive for C. difficile by CCNA. The PCR assay detected an additional 35 and 36 stool and rectal swab specimens positive for C. difficile, respectively, for sensitivity with stools and rectal swabs of 93.8% and 95.3%, specificity of 93.7% and 93.6%, positive predictive values of 63.2% and 62.9%, and negative predictive values of 99.2% and 99.4%. Detection of C. difficile using PCR on stools or rectal swabs yielded reliable and similar results. The use of PCR tests on rectal swabs could reduce turnaround time for CDI detection, thus improving CDI management and control of C. difficile transmission.

Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated diarrhea, resulting in high morbidity, mortality, and economic burden. In clinical laboratories, CDI testing is currently performed on stool samples collected from patients with diarrhea. However, the diagnosis of CDI can be delayed by the time required to collect stool samples. Barriers to sample collection could be overcome by using a rectal swab instead of a stool sample. Our study showed that CDI can be identified rapidly and reliably by a new PCR assay developed in our laboratory on both stool and rectal swab specimens. The use of PCR tests on rectal swabs could reduce the time for the detection of CDI and improve the management of this infection. It should also provide a useful alternative for infection-control practitioners to better control the spread of C. difficile.

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA+B+CDT+, 22 ToxA+B+CDT+ Hv+ and 105 ToxA+B+CDT-) and 50 non-toxigenic (ToxA-B-) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA-B- strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA+B+CDT- strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA+B+CDT+ strains. The classifiers' performances for Hv strain detection were linked to high specificity (≥0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

The prevalence of Clostridioides difficile infection in older and frail population is extremely high and adverse outcomes, including future recurrences and premature mortality, are common. Nonetheless, the clinical risk profile for Clostridioides difficile recurrence in older people is still controversial. We aimed to investigate: 1) the association between Clostridioides difficile recurrence and 6-month mortality; 2) the risk factors for Clostridioides difficile recurrence after hospital discharge.

This is a retrospective study on adults with a first episode of Clostridioides difficile infection admitted to all Internal Medicine and Geriatrics Units of the University Hospital of Ferrara (Italy) between January 2018 and December 2020. For each patient, sociodemographic, clinical and laboratory data were collected through hospital database system. The primary and secondary outcomes were mortality and recurrence within 6 months from the first infectious episode, respectively.

The mean age of the 386 enrolled patients was 77.8 years; 61.7% were females. Twelve percent patients had Clostridioides difficile recurrence and 32.1% patients died during the 6-month follow-up. At Cox analysis, after adjustment for the potential confounders, participants with recurrence reported a twofold risk of death compared to those without recurrence (HR, 95% CI 2.45, 1.59-3.78). Compared to patients treated with metronidazole, those treated with vancomycin showed a lower risk of recurrence (log-rank p < 0.001).

Clostridioides difficile recurrence is associated with a higher risk of mortality and it may itself be a marker of frailty and vulnerability. Vancomycin treatment during the infectious episode was associated with lower recurrence rate, as compared to metronidazole.

Background:Clostridioides difficile infections (CDI) vary in severity from mild diarrhea to life-threatening conditions like pseudomembranous colitis or toxic megacolon, often leading to sepsis and death. The COVID-19 pandemic prompted changes in healthcare practices, potentially affecting CDI incidence, though reported data are inconclusive. We studied factors influencing CDI incidence and outcomes at a university hospital throughout the COVID-19 pandemic years. Methods: We conducted a retrospective study on all adult hospitalized CDI cases from 1 January 2020 to 31 December 2022 in Hospital Universitari de Sant Joan in Reus. We collected demographic information, comorbid conditions, and concurrent infections. Results: While overall CDI and COVID-19 rates decreased in 2022, a notable increase in CDI infections was observed among oncological patients and those undergoing some aggressive treatments, such as colonoscopies or gastroscopies. The prevalence of comorbidities remained unmodified, and there were declines in prior gastrointestinal surgeries and proton pump inhibitor prescriptions. Factors associated with patient fatality or prolonged hospitalization included older age, cancer, chronic kidney disease, higher Charlson and McCabe indices, elevated C-reactive protein, and low albumin concentrations. Conclusions: Our study shows the evolving landscape of CDI during the COVID-19 pandemic and emphasizes the impact of delayed diagnoses and treatments exacerbated by telemedicine adoption. Identified risk factors for CDI-related mortality or prolonged hospital stays underscore the importance of targeted interventions in high-risk populations.

Patients tested for Clostridioides difficile infection (CDI) using a 2-step algorithm with a nucleic acid amplification test (NAAT) followed by toxin assay are not reported to the National Healthcare Safety Network as a laboratory-identified CDI event if they are NAAT positive (+)/toxin negative (-). We compared NAAT+/toxin- and NAAT+/toxin+ patients and identified factors associated with CDI treatment among NAAT+/toxin- patients.

Retrospective observational study.

The study was conducted across 36 laboratories at 5 Emerging Infections Program sites.

We defined a CDI case as a positive test detected by this 2-step algorithm during 2018-2020 in a patient aged ≥1 year with no positive test in the previous 8 weeks.

We used multivariable logistic regression to compare CDI-related complications and recurrence between NAAT+/toxin- and NAAT+/toxin+ cases. We used a mixed-effects logistic model to identify factors associated with treatment in NAAT+/toxin- cases.

Of 1,801 cases, 1,252 were NAAT+/toxin-, and 549 were NAAT+/toxin+. CDI treatment was given to 866 (71.5%) of 1,212 NAAT+/toxin- cases versus 510 (95.9%) of 532 NAAT+/toxin+ cases (P < .0001). NAAT+/toxin- status was protective for recurrence (adjusted odds ratio [aOR], 0.65; 95% CI, 0.55-0.77) but not CDI-related complications (aOR, 1.05; 95% CI, 0.87-1.28). Among NAAT+/toxin- cases, white blood cell count ≥15,000/µL (aOR, 1.87; 95% CI, 1.28-2.74), ≥3 unformed stools for ≥1 day (aOR, 1.90; 95% CI, 1.40-2.59), and diagnosis by a laboratory that provided no or neutral interpretive comments (aOR, 3.23; 95% CI, 2.23-4.68) were predictors of CDI treatment.

Use of this 2-step algorithm likely results in underreporting of some NAAT+/toxin- cases with clinically relevant CDI. Disease severity and laboratory interpretive comments influence treatment decisions for NAAT+/toxin- cases.