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.

In recent years, nosocomial infections caused by Clostridium difficile (C. difficile) have risen, becoming a leading cause of hospital-acquired diarrhea. The global prevalence of C. difficile infection (CDI) varies across regions and populations. The diagnosis relies primarily on laboratory testing, including toxin, glutamate dehydrogenase, and nucleic acid amplification tests. Treatment strategies for CDI include antimicrobial therapy (e.g., metronidazole, vancomycin, and fidamycin), fecal transplantation, and immunotherapy (e.g., belotozumab), depending on the patient's specificity and severity. This paper reviews recent research on CDI's epidemiological characteristics, risk factors, diagnosis, treatment, and prevention, aiming to support hospitals and public health initiatives in implementing effective detection, prevention, and treatment strategies.

The diagnosis, pharmacologic management, and surgical options for Clostridioides difficile infection (CDI) are rapidly evolving, which presents a challenge for the busy surgeon to remain up to date on the latest clinical guidelines. This review provides an evidence-based practical guide for CDI management tailored to the needs of surgeons and surgical intensivists. Historically, the diagnosis of CDI relied on slow cell culture cytotoxicity neutralization assays, but now, the rapidly resulting nucleic acid amplification tests and enzyme immunoassays have become mainstream. In terms of antibiotic therapy, metronidazole and oral vancomycin were the main "workhorse" antibiotics in the early 2000s, but large randomized controlled trials have now demonstrated that fidaxomicin produces superior results. Regarding surgical intervention, total abdominal colectomy was once the only procedure of choice; however, diverting loop ileostomy with colonic lavage is emerging as a viable alternative. Finally, novel adjuncts such as fecal microbiota transplantation and targeted therapy against toxin B (bezlotoxumab) are playing an increasingly important role in the management of CDI.

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.

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.

Multiplex polymerase chain reaction (PCR) panels for stool testing may be used to diagnose Clostridioides difficile, which can circumvent more appropriate targeted C. difficile testing, resulting in treatment of incidentally detected colonization. We sought to reduce C. difficile diagnosis via a gastrointestinal pathogen panel (GIPP).

Quasi-experimental, pre/post, retrospective cohort study from January 1, 2022, to January 31, 2024.

Mayo Clinic Arizona-a single academic medical center and associated clinics.

Adult patients receiving C. difficile testing and/or treatment.

Preferred C. difficile testing consisted of glutamate dehydrogenase and toxin antigen immunoassay, followed by toxin gene testing for discrepant results. The GIPP contained 22 targets during the baseline period with C. difficile removed during the postintervention period. Surveys were provided to provider and nursing groups, separately, to identify C. difficile ordering practices and knowledge gaps.

At baseline, from January 1, 2022, to January 31, 2023, 2,772 GIPPs were completed for 2,307 unique patients (∼7 per day), primarily for outpatients (1,805 of 2,772, 65%). The most common positive target was C. difficile (517 of 1,018, 51%), which resulted in treatment for C. difficile infection in 94.9% (337 of 355) of cases. Following GIPP C. difficile target removal, GIPP orders decreased from 3.23 to 2.7 per 1,000 patient visits (P < .001). Prescribing of C. difficile treatments decreased in the postintervention period in inpatient and outpatient settings. There were no cases of delayed C. difficile diagnosis during the postintervention period.

Removing C. difficile from the GIPP resulted in effective diagnostic and antimicrobial stewardship without resulting in delayed diagnoses.

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.

Infectious diseases in older adults present a significant challenge to the healthcare system, marked by increased morbidity, mortality, and rising costs of care. Age-related changes (ARCs) in the immune system, including immunosenescence and inflammaging, contribute to heightened susceptibility to severe infections and reduced vaccine responsiveness. Additionally, alterations in the normal microbial flora due to aging and factors such as antibiotic therapy predispose older individuals to infections and age-related diseases. Changes in body composition also affect the pharmacokinetics and pharmacodynamics of drugs, complicating the management of antibiotics and leading to potential overdoses, adverse drug reactions, or underdoses that foster antibiotic resistance. The inappropriate use of antibiotics has exacerbated the emergence of multidrug-resistant pathogens, posing a critical global concern. This narrative review provides an overview of immunosenescence and inflammaging and focuses on three major infectious diseases affecting older adults: bacterial pneumonia, urinary tract infections, and Clostridium difficile infections. Through this exploration, we aim to highlight the need for targeted approaches in managing infectious diseases in the aging population.

Clostridium difficile infection (CDI) has been increasing due to the effect of recurrent hospitalizations. The use of antibiotics has been shown to alter the gut microbiome and lead to CDIs. The treatment is limited to three major antibiotics; however, the incidence of recurrent CDIs has been increasing and drug resistance is a major concern. This aspect is a growing concern in modern medicine especially in the elderly population, critical care patients, and immunocompromised individuals who are at high risk of developing CDIs. Clostridium difficile can lead to various complications including septic shock and fulminant colitis that could prove to be lethal in these patients. Newer modalities of treatment have been developed including bezlotoxumab, a monoclonal antibody and fecal microbiota transplant. There have been studies showing asymptomatic carriers and drug resistance posing a major threat to the healthcare system. Newer treatment options are being studied to treat and prevent CDIs. This review will provide an insight into the current treatment modalities, prevention and newer modalities of treatment and challenges faced in the treatment of CDIs.

To review the composition, preparation, proposed mechanism of action, safety, efficacy, and current place in therapy of Rebyota (fecal microbiota, live-jslm).

As the first agent in a new class of drugs, live biotherapeutic products (LBPs), fecal microbiota, live-jslm offers another therapeutic approach for the prevention of recurrent Clostridioides difficile infection (rCDI). LBPs are given following antibiotic therapy for C. difficile to reintroduce certain bacteria present in the normal microbiome, as a means to reconstitute the microbiome of infected individuals. This review provides a summary of phase 2 and 3 clinical trials, product information, discussion of data limitations, and recommendations for place in therapy. High efficacy rates compared to placebo with sustained response up to 24 months after administration have been reported. The majority of adverse events identified were mild to moderate without significant safety signals.

Fecal microbiota, live-jslm has consistently been shown in randomized trials to be safe and effective in reducing rCDI. Its approval marks the culmination of decades of work to identify, characterize, and refine the intestinal microbiome to create pharmaceutical products.

Clostridioides difficile infection (CDI) is the main etiologic agent of antibiotic-associated diarrhea. CDI contributes to gut inflammation and can lead to disruption of the intestinal epithelial barrier. Recently, the rate of CDI cases has been increased. Thus, early diagnosis of C. difficile is critical for controlling the infection and guiding efficacious therapy.

A search strategy was set up using the terms C. difficile biomarkers and diagnosis. The found references were classified into two general categories; conventional and advanced methods.

The pathogenicity and biomarkers of C. difficile, and the collection manners for CDI-suspected specimens were briefly explained. Then, the conventional CDI diagnostic methods were subtly compared in terms of duration, level of difficulty, sensitivity, advantages, and disadvantages. Thereafter, an extensive review of the various newly proposed techniques available for CDI detection was conducted including nucleic acid isothermal amplification-based methods, biosensors, and gene/single-molecule microarrays. Also, the detection mechanisms, pros and cons of these methods were highlighted and compared with each other. In addition, approximately complete information on FDA-approved platforms for CDI diagnosis was collected.

To overcome the deficiencies of conventional methods, the potential of advanced methods for C. difficile diagnosis, their direction, perspective, and challenges ahead were discussed.

To describe an outbreak of sequence type (ST)2 Clostridioides difficile infection (CDI) detected by a recently implemented multilocus sequence type (MLST)-based prospective genomic surveillance system using Oxford Nanopore Technologies (ONT) sequencing.

Hemato-oncology ward of a public tertiary referral centre.

From February 2022, we began prospectively sequencing all C. difficile isolated from inpatients at our institution on the ONT MinION device, with the output being an MLST. Bed-movement data are used to construct real-time ST-specific incidence charts based on ward exposures over the preceding three months.

Between February and October 2022, 76 of 118 (64.4%) CDI cases were successfully sequenced. There was wide ST variation across cases and the hospital, with only four different STs being seen in >4 patients. A clear predominance of ST2 CDI cases emerged among patients with exposure to our hemato-oncology ward between May and October 2022, which totalled ten patients. There was no detectable rise in overall CDI incidence for the ward or hospital due to the outbreak. Following a change in cleaning product to an accelerated hydrogen peroxide wipe and several other interventions, no further outbreak-associated ST2 cases were detected. A retrospective phylogenetic analysis using original sequence data showed clustering of the suspected outbreak cases, with the exception of two cases that were retrospectively excluded from the outbreak.

Prospective genomic surveillance of C. difficile using ONT sequencing permitted the identification of an outbreak of ST2 CDI that would have otherwise gone undetected.

Multistep laboratory testing is recommended for the diagnosis of Clostridioides difficile infection (CDI). The aim of this study was to present the impact of multistep CDI diagnostic testing in an academic hospital system and evaluate the toxin B gene polymerase chain reaction (PCR) cycle threshold (Ct) values of PCR-positive tests.

In October 2022, our system began reflex testing all PCR-positive stool samples with the C. DIFF QUIK CHEK COMPLETE (Techlab), an enzyme immunoassay-based test with results for the glutamate dehydrogenase antigen (GDH) and C difficile toxin A/B. Hospital-onset (HO) CDI and CDI antibiotic use before and after testing were tracked. Ct values were obtained from the Infectious Diseases Diagnostic Laboratory. Receiver operating curve analysis was used to examine the sensitivity and specificity for identifying GDH+/toxin+ and GDH-/toxin- at various Ct thresholds.

The HO-CDI rate decreased from 0.352 cases per 1000 patient-days to 0.115 cases per 1000 patient-days post-reflex testing (P < .005). Anti-CDI antibiotics use decreased, but the decrease was not commensurate with CDI rates following reflex testing. PCR+/GDH+/toxin+ samples had a lower mean Ct value than PCR+/GDH-/toxin- samples (23.3 vs 33.5, P < .0001). A Ct value of 28.65 could distinguish between those 2 groups. Fifty-four percent of PCR+/GDH+/toxin- samples had a Ct value below that cut-off, suggesting the possibility of CDI with a negative toxin test.

Reflex testing for a laboratory diagnosis of CDI results in rapid, systemwide decreases in the rate of HO-CDI. Additional research is needed to distinguish CDI from C difficile colonization in patients with discordant testing.

Accurately diagnosing Clostridioides difficile infection (CDI) is crucial for effective patient management. A misdiagnosis poses risks to patients, leads to treatment delays, and contributes to infection transmission in healthcare settings. While using polymerase chain reaction (PCR) to amplify the toxin B gene is a sensitive method for detecting toxigenic C. difficile, there is still a risk of false-negative results. These inaccuracies could have significant consequences for diagnosing and treating CDI, emphasizing the need for careful consideration and other diagnostic approaches. This case report highlights a patient with severe CDI who had negative PCR and toxin and a biopsy showing pseudomembranous colitis on further testing due to persistence and worsening of symptoms. In the diagnosis of C. difficile infection, healthcare providers should consider clinical symptoms, although diarrhea, which is a major sign of CDI, can be due to other causes. Even in the presence of negative PCR results, if a patient displays symptoms consistent with C. difficile-associated disease, healthcare providers may still contemplate treatment.

Children with inflammatory bowel disease (IBD) are at increased risk of C. difficile infection (CDI) and experience worse outcomes associated with an infection. In this article, we review recent research on the incidence, diagnosis, complications, and treatment options for CDI in children with IBD.

Children with IBD have an elevated incidence of CDI, but their CDI risk does not associate with established risk factors in adults with IBD. Existing testing methodologies are inadequate at differentiating CDI from C. difficile colonization in children with IBD. Fecal microbiota transplantation offers a durable cure for recurrent CDI. CDI remains a frequent occurrence in children with IBD. Careful clinical monitoring should be used to diagnose CDI and patients with co-occurring IBD and CDI require careful surveillance for worse outcomes. Future research should explore the optimal diagnosis and treatment modalities in this unique patient population.

Clostridioides difficile (C. difficile) infection is still a threat to many healthcare settings worldwide. Clostridioides difficile epidemiology has changed over the last 20 years, largely due to the emergence of hypervirulent and antimicrobial-resistant C. difficile strains. The excessive use of antimicrobials, the absence of optimal antibiotic policies, and suboptimal infection control practices have fueled the development of this pressing health issue. The prudent use of antimicrobials, particularly broad-spectrum agents, and simple infection control measures, such as hand hygiene, can significantly reduce C. difficile infection rates. Moreover, the early detection of these infections and understanding their epidemiological behavior using accurate laboratory methods are the cornerstone to decreasing the incidence of C. difficile infection and preventing further spread. Although there is no consensus on the single best laboratory method for the diagnosis of C. difficile infection, the use of 2 or more techniques can improve diagnostic accuracy, and it is recommended.

The laboratory diagnosis of Clostridioides difficile infection (CDI) is challenging since this bacteria may be detected in healthy people and toxin production detection is not sensitive enough to be used alone. Thus, there is no single test with adequate sensitivity and specificity to be used in laboratory diagnosis. We evaluated the performance of tests used in the diagnosis of CDI in symptomatic patients with risk factors in hospitals in southern Brazil. Enzyme immunoassays (EIA) for glutamate dehydrogenase antigen (GDH) and toxins A/B, real-time polymerase chain reaction (qPCR), GeneXpert system, and a two-step algorithm comprising GDH/TOXIN EIA performed simultaneously followed by GeneXpert for outliers were evaluated. Toxigenic strain in stool culture was considered CDI positive (gold standard). Among 400 samples tested, 54 (13.5%) were positive for CDI and 346 (86.5%) were negative. The diagnosis of the two-step algorithm and qPCR had an excellent performance with an accuracy of 94.5% and 94.2%, respectively. The Youden index showed that GeneXpert as a single test (83.5%) and the two-step algorithm (82.8%) were the most effective assays. Diagnosing CDI and non-CDI diarrhea could be successfully attained by the combination of clinical data with accuracy of laboratory tests.

Arriving at a C. difficile infection (CDI) diagnosis, treating patients and dealing with recurrences is not straightforward, but a comprehensive and well-rounded understanding of what is needed to improve patient care is lacking. This manuscript addresses the paucity of multidisciplinary perspectives that consider clinical practice related and healthcare system-related challenges to optimizing care delivery.

We draw on narrative review, consultations with clinical experts and patient representatives, and a survey of 95 clinical and microbiology experts from the UK, France, Italy, Australia and Canada, adding novel multi-method evidence to the knowledge base.

We examine the patient pathway and variations in clinical practice and identify, synthesize insights on and discuss associated challenges. Examples of key challenges include the need to conduct multiple tests for a conclusive diagnosis, treatment side-effects, the cost of some antibiotics and barriers to access of fecal microbiota transplantation, difficulties in distinguishing recurrence from new infection, workforce capacity constraints to effective monitoring of patients on treatment and of recurrence, and ascertaining whether a patient has been cured. We also identify key opportunities and priorities for improving patient care that target both clinical practice and the wider healthcare system. While there is some variety across surveyed countries' healthcare systems, there is also strong agreement on some priorities. Key improvement actions seen as priorities by at least half of survey respondents in at least three of the five surveyed countries include: developing innovative products for both preventing (Canada, Australia, UK, Italy, and France) and treating (Canada, Australia, and Italy) recurrences; facilitating more multidisciplinary patient care (UK, Australia, and France); updating diagnosis and treatment guidelines (Australia, Canada, and UK); and educating and supporting professionals in primary care (Italy, UK, Canada, and Australia) and those in secondary care who are not CDI experts (Italy, Australia, and France) on identifying symptoms and managing patients. Finally, we discuss key evidence gaps for a future research agenda.

Clostridioides difficile Infection (CDI) has been identified as one of the main causes of nosocomial infection all across the world. Rapid diagnosis of CDI is difficult and poses a significant challenge to physicians worldwide. We undertook a systematic review and meta-analysis to evaluate rapid tests' diagnostic accuracy against toxigenic culture as the reference standard for CDI.

We searched the PubMed/MEDLINE and EMBASE databases for the relevant records. The QUADAS-2 tool was used to assess the quality of the studies. Diagnostic accuracy measures [i.e., sensitivity, specificity, diagnostic odds ratio (DOR), positive likelihood ratios (PLR), negative likelihood ratios (NLR), and the area under the curve (AUC)] were pooled with a random-effects model. All statistical analyses were performed with Meta-DiSc (Version 1.4, Cochrane Colloquium, Barcelona, Spain) and RevMan (version 5.3; The Nordic Cochrane Centre, the Cochrane Collaboration, Copenhagen, Denmark).

We reviewed retrieved records and identified 63 studies that met the inclusion criteria. 26 were about enzyme immunoassay (EIA) (our main index test). The sensitivity of GDH and Tox A/B EIAs were 82% (95% CI: 79-84) and 75% (95% CI: 70-79), respectively. On the other hand, the specificity of GDH EIA was 91% (95% CI: 90-92) and the specificity of Tox A/B EIA was 95% (95% CI: 94-96). Among other index tests, BD Max with 92% has the most sensitivity and cell cytotoxicity neutralization assay (CCNA) has the most specificity (100%).

This meta-analysis demonstrated that EIAs could be reliable methods for detecting CDI based on their sensitivity, specificity, time and cost-effectiveness, and simplicity in the procedure. Further work to improve rapid tests would benefit from improvements to the methodology.

Clostridioides difficile (C. difficile) is an important nosocomial infection that is commonly associated with antibiotic use with pseudomembranous colitis being present in only 13% of cases. Disease severity ranges from asymptomatic carriers to severe complicated disease, based on clinical and laboratory findings. There is no single rapid FDA-approved test to diagnose C. difficile infections (CDI) and diagnosis usually requires a multi-step diagnostic approach. C. difficile testing usually begins with the C. difficile toxin and glutamate dehydrogenase antigen screen (GDH). If testing is negative for either, then nucleic acid amplification testing (NAAT) is done to confirm the diagnosis. Endoscopic evaluation may be required in rare instances when there is a high clinical suspicion of disease with negative testing. Here, we present an interesting case of a patient with multiple negative C. difficile toxin and GDH tests. Given the high index of clinical suspicion of CDI, the patient underwent a colonoscopy which revealed diffuse pseudomembranous colitis. The patient was then appropriately treated with oral vancomycin. We aim to shed light on the different testing modalities available to clinicians and the indications for doing a colonoscopy to delineate between false positive testing and active CDI.

Clostridioides difficile is the main pathogen responsible for antibiotic-associated diarrhea in adults. Besides its challenging diagnosis, C. difficile infection (CDI) causes substantial morbidity and mortality. Commercially, there are assays with different targets and performances in sensitivity and specificity. The objectives of this study were to: (1) evaluate the prevalence and seasonal variability of CDI rates at a tertiary hospital in southern Brazil over 12 years and (2) determine the impact of using a two-step algorithm test in the laboratory diagnosis. Between January 2007 and May 2019, fecal samples from 2275 patients were analyzed in a cross-sectional study. Four commercial tests were adopted for the diagnosis of CDI, the immunochromatographic test for toxin A from 2007 to 2010; the enzyme-linked immunosorbent assay method for toxins A and B from 2011 to March 2017; and the rapid enzyme immunoassay (EIA) for GDH and toxins A and B, associated with a Polymerase Chain Reaction (PCR) for the toxin B gene from June 2017 to 2019. The annual prevalence was 8.7% from 2007 to March 2017, increasing between June 2017 and 2019 to 14.7% when the C. diff Quik Chek Complete + GeneXpert C. difficile (two-step algorithm) test was adopted. The number of samples (691) and percentage of CDI cases (10.5%) were higher in winter, but the difference has no statistical significance (P > 0.05). An accurate diagnosis and adequate knowledge of the local seasonality of CDI allow the effective implementation of prevention and control strategies for nosocomial CDI, in addition to effective treatment for patients.

Patients with inflammatory bowel disease (IBD) are at increased risk for Clostridioides difficile infection, although clinically important infections can be difficult to recognise. C. difficile infection does not produce pseudomembranes when it occurs in IBD patients. These individuals may also be colonised by the organism, in which case diarrhoeal symptoms are not necessarily attributed to C. difficile. We performed this study to determine whether any features distinguished C. difficile-associated colitis from an IBD flare.

We reviewed the clinical, endoscopic and biopsy findings from 50 patients with established IBD and worsening diarrhoea, including 22 with concurrent positive C. difficile stool toxin polymerase chain reaction (PCR) assays and 28 with negative C. difficile assay results. We found that C. difficile-infected patients had symptoms and endoscopic findings that were indistinguishable from active IBD. Although most biopsy samples from patients with C. difficile infection showed chronic active colitis indistinguishable from IBD, some displayed neutrophilic infiltrates unaccompanied by plasma cell-rich inflammation involving superficial (41%) and crypt (18%) epithelium as well as neutrophilic infiltrates within lamina propria distant from foci of cryptitis (32%). All three of these features were significantly more common among infected than uninfected patients (4, 0 and 4%; P = 0.002, P = 0.03 and P = 0.02, respectively).

Although colonic biopsies from IBD patients with C. difficile infection usually lack features that aid distinction from colitic flares, some cases show an acute colitis pattern not seen in IBD alone. When identified in biopsies from symptomatic IBD patients, these changes should alert pathologists to the possibility of this clinically important infection.

Despite the known importance of Clostridioides (Clostridium) difficile infection (CDI) in animals, there are no published guidelines for the diagnosis of CDI. The performance of the available commercial methods, all standardized for human stool samples, can vary according to the animal species. Thus, the aim of the present study was to review the literature on the detection of C. difficile in pigs, horses, and dogs. The detection of toxins A and B using enzyme immunoassays seems to have low performance in piglet and dog samples, while it shows high sensitivity for the diagnosis of CDI in foals. On the other hand, tests for the detection of glutamate dehydrogenase (GDH) have a high sensitivity towards detection of C. difficile in animal samples, suggesting that it can be an adequate screening method. A few studies have evaluated real-time PCR or nucleic acid amplification tests in animal samples and, so far, these methods have also shown a low performance for the detection of C. difficile in animals. Although the intestinal lesions caused by CDI can vary among animal species, histopathology can be a useful auxiliary tool for postmortem diagnosis in animals.

This study aimed to evaluate an immunochromatographic test used to detect glutamate dehydrogenase (GDH) for the diagnosis of Clostridium difficile infection (CDI) in dogs. Fecal samples of 119 diarrheic dogs were subjected to toxigenic culture as the "gold standard" method and to GDH detection (Ecodiagnostica, Brazil). Samples positive for toxigenic C. difficile strains and those positive in the GDH test were also subjected to A/B toxin detection using an enzyme immunoassay kit (C. difficile Tox A/B II, Techlab Inc., USA). Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were measured for GDH detection and compared with the toxigenic culture results. A total of 19 (15.9%) dogs were positive for toxigenic C. difficile. Of these, 10 (52.6%) dogs were positive for A/B toxins using the enzyme immunoassay kit and 18 (15.2%) were positive in the GDH test, leading to a sensitivity and NPV of 89.4% and 97.9%, respectively. Three animals, two of which were colonized with non-toxigenic strains, were positive for GDH, though not confirmed with CDI, resulting in a high specificity (97%) and PPV (85%). The results suggest that the lateral flow test for GDH detection could be a useful method for diagnosing CDI in dogs, similar to that previously described for humans and other animal species.

For many years, it has been known that Clostridium difficile (CD) is the primary cause of health-care-associated infectious diarrhea, afflicting approximately 1% of hospitalized patients. CD may be simply carried or lead to a mild disease, but in a relevant number of patients, it can cause a very severe, potentially fatal, disease. In this narrative review, the present possibilities of CD infection (CDI) prevention will be discussed. Interventions usually recommended for infection control and prevention can be effective in reducing CDI incidence. However, in order to overcome limitations of these measures and reduce the risk of new CDI episodes, novel strategies have been developed. As most of the cases of CDI follow antibiotic use, attempts to rationalize antibiotic prescriptions have been implemented. Moreover, to reconstitute normal gut microbiota composition and suppress CD colonization in patients given antimicrobial drugs, administration of probiotics has been suggested. Finally, active and passive immunization has been studied. Vaccines containing inactivated CD toxins or components of CD spores have been studied. Passive immunization with monoclonal antibodies against CD toxins or the administration of hyperimmune whey derived from colostrum or breast milk from immunized cows has been tried. However, most advanced methods have significant limitations as they cannot prevent colonization and development of primary CDI. Only the availability of vaccines able to face these problems can allow a resolutive approach to the total burden due to this pathogen.

Clostridioides (Clostridium) difficile is responsible for most cases of nosocomial diarrhea and, despite the high prevalence of the disease worldwide, the best laboratory diagnostic approach to diagnose C. difficile infection (CDI) is a subject of ongoing debate. Although the use of multiple tests is recommended, the cost of these algorithms commonly exceeds the affordability in some countries. Thus, to improve CDI diagnosis in a university hospital in Brazil, this study analyzed two immunochromatographic tests and one enzyme immunoassay (ELISA) to evaluate the detection of glutamate dehydrogenase (GDH) and A/B toxins of C. difficile. Stool samples of 89 adult patients presenting nosocomial diarrhea during hospitalization were included. The toxigenic culture was used as the reference method. GDH detection by both commercial tests showed high sensitivity (100%) and specificity (92.1%). On the other hand, toxin-based methods showed a sensitivity between 19.2 and 57.7%. In conclusion, the results suggest that rapid tests for GDH detection are not only suitable for CDI diagnosis as screening tests but also as a single method.