Toxigenic Culture Research Articles

Comparison of the STANDARD M10 C. difficile, Xpert C. difficile, and BD MAX Cdiff assays as confirmatory tests in a two-step algorithm for diagnosing Clostridioides difficile infection.

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.

Association Between the Duration of Diarrhea and the Length of Hospitalization Among Clostridioides difficile Patients in Northern Nigeria

The United States Centers for Disease Control and Prevention (CDC) has categorized Clostridioides difficile infection (CDI) as a significant concern in extended-care facilities, hospitals, and outpatient clinics. However, little is known about CDI in low- and middle-income countries. This study determined CDI prevalence and impact in outpatient adults presenting with diarrhea in Nigeria. Toxigenic culture and PCR were used to detect and validate C. difficile. Prior antibiotic use, medical history, and demographic data were also obtained. Descriptive and inferential statistics were used for data analysis. The patient demographics were 35.48% (22/62) for the 18–24 years age group and 32.26% (20/62) for both the 25–30 years age group and the 31+ years group, with an average age of 29.7 years. Forty-eight percent of the patients (30/62) tested positive for CDI, and the prevalence increased with age. Most patients (86.67%, 52/60) reported moderate/severe cases of diarrhea and 67.7% had no knowledge of antibiotics. The results showed that 62.30% of the cases were hospitalized with the duration of diarrhea being significantly associated (r = 0.98, p ˂ 0.001) with the length of hospitalization. These results suggest that C. difficile is common among diarrhea patients in this population and that Nigerian hospitals’ infection prevention and control measures must include this pathogen.

Comparative evaluation of the STANDARD M10 and Xpert C. difficile assays for detection of toxigenic Clostridioides difficile in stool specimens.

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.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridioides difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC), should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays or single-molecule array assays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has been advocated by international guidelines (IDSA/SHEA and ESCMID) in order to optimize diagnostic accuracy. As a result, a survey performed in 2018-2019 in Europe revealed that most of all hospital sites reported using more than one test to diagnose CDI. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal microbiota biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Analysis of Clostridioides difficile Infection in Children with Diarrhea in Two Hospitals in Southern Brazil.

Clostridioides difficile infection (CDI) has been increasingly observed in children, but there is a lack of epidemiological and molecular data on CDI in Latin America. This prospective cohort study aimed to investigate the role of CDI in children with diarrhea. It included 105 children with antimicrobial-associated diarrhea (AAD) and analyzed the molecular characteristics of strains isolated from two hospitals in southern Brazil between 2017 and 2020. Fecal samples from the participants were tested for glutamate dehydrogenase (GDH) and A/B toxins using a rapid enzyme immunoassay. GDH-positive samples underwent automated real-time polymerase chain reaction and toxigenic culture. Toxigenic C. difficile isolates were selected for whole genome sequencing. Out of the 105 patients, 14 (13.3%) met the criteria for CDI. Children with a history of previous CDI and the presence of mucus in their stool were more likely to have CDI. Metronidazole was the most used treatment (71.4%), and three patients (23.1%) experienced CDI recurrence (rCDI). Although the number of sequenced isolates was limited, a wide diversity of sequence types (ST) was observed. In addition to toxin genes (tcdA, tcdB, cdtA, and cdtB), the isolates also exhibited virulence factors involved in adhesion (cwp66, groEL, slpA, fbpA/fbp68) and immune evasion (rmlA, rmlB, rmlC, gnd, rfbA-1), along with multiple resistance factors (gyrA mutation, norA, ermB, dfrF, and vanG). These findings highlight the prevalence and recurrence of CDI among hospitalized children. Longitudinal studies are needed to better understand the characteristics of CDI-associated diarrhea and its impact on the healthcare system in this population.

The Molecular Epidemiology of Clostridioides difficile Infection in Central India: A Prospective Observational Cohort Study

This prospective observational cohort study aimed to establish and compare baseline rates of Clostridioides difficile infection (CDI) in community and hospitalized patients in Nagpur and rural Melghat Maharashtra, including adults aged ≥18 years with a diagnosis of diarrhoea as defined as 3 or more loose stools in a 24 h period. All diarrhoeal samples were tested for CDI using the C. diff Quik Chek Complete enzyme immunoassay. C. difficile-positive stool samples were characterised by toxigenic culture, antimicrobial susceptibility testing and PCR ribotyping. C. difficile testing was performed on 1683 patients with acute diarrhoea. A total of 54 patients (3.21%; 95% CI: 2.42–4.17) tested positive for both the GDH antigen and free toxin. The risk factors for CDI included the presence of co-morbidities, antibiotic usage, and immunosuppression. The detected PCR ribotypes included 053-16, 017, 313, 001, 107, and 216. Our findings show that toxigenic C. difficile is an important but neglected aetiologic agent of infective diarrhoea in Central India. These results underscore the need to enhance the awareness and testing of patients with diarrhoea in India regarding the presence of toxigenic C. difficile, particularly in high-risk individuals with multiple co-morbidities, immunosuppression, and recent or ongoing antibiotic exposure or hospitalization.

CLINICAL OUTCOME AND SEVERITY OF CLOSTRIDIOIDES (CLOSTRIDIUM) DIFFICILE INFECTION AT A TERTIARY REFERRAL HOSPITAL IN BRAZIL.

•The outcomes of CDI were evaluated in 65 patients with CDI in a Brazilian tertiary hospital. •Lack of clinical improvement after treatment and the severity score (ATLAS) increased the risk of death. •The use of multiple antimicrobial agents was associated with longer hospital stays. •Patients with high Charlson comorbidity index (>7) were more likely to recur. Background - Clostridioides difficile infection (CDI) is a potentially severe disease that can present with refractoriness, recurrence, and evolution to death. In Brazil, the epidemiology of CDI seems to differ from that of the United States and most European countries, with only one ribotype (RT) 027-related case and a high prevalence of RT106. Objective - The aim of this study was to evaluate the outcomes of CDI and its possible association with ribotypes at a university hospital in Brazil. Methods - A total of 65 patients with CDI were included and stool samples were submitted to A/B toxin detection and toxigenic culture, and toxigenic isolates (n=44) were also PCR ribotyped. Results - Patients' median age was 59 (20-87) years and there were 16 (24.6%) deaths. The median Charlson comorbidity index (CCI) was 4 (0-15) and 16.9% of the patients had CCI ≥8. The ATLAS score and non-improvement of diarrhea were related to higher mortality. A longer length of hospitalization was related to the enteral nutrition and use of multiple antibiotics. The period between CDI diagnosis and hospital discharge was longer in those who received new antibiotics after diagnosis, multiple antibiotics, and required intensive care treatment. Recurrence was associated with CCI >7. Twenty ribotypes were identified and RT106 was the most frequently detected strain (43.2%). No relationship was observed between the ribotypes and outcomes. CDI was present in patients with more comorbidities. Conclusion - Risk factors for higher mortality, longer hospital stay and recurrence were identified. A diversity of ribotypes was observed and C. difficile strains were not related to the outcomes.

VE303, a Defined Bacterial Consortium, for Prevention of Recurrent Clostridioides difficile Infection

The effect of rationally defined nonpathogenic, nontoxigenic, commensal strains of Clostridia on prevention of Clostridioides difficile infection (CDI) is unknown. To determine the efficacy of VE303, a defined bacterial consortium of 8 strains of commensal Clostridia, in adults at high risk for CDI recurrence. The primary objective was to determine the recommended VE303 dosing for a phase 3 trial. Phase 2, randomized, double-blind, placebo-controlled, dose-ranging study conducted from February 2019 to September 2021 at 27 sites in the US and Canada. The study included 79 participants aged 18 years or older who were diagnosed with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence (defined as aged ≥75 years or ≥65 years with ≥1 risk factors: creatinine clearance <60 mL/min/1.73 m2, proton pump inhibitor use, remote [>6 months earlier] CDI history). Participants were randomly assigned to high-dose VE303 (8.0 × 109 colony-forming units [CFUs]) (n = 30), low-dose VE303 (1.6 × 109 CFUs) (n = 27), or placebo capsules (n = 22) orally once daily for 14 days. The primary efficacy end point was the proportion of participants with CDI recurrence at 8 weeks using a combined clinical and laboratory definition. The primary efficacy end point was analyzed in 3 prespecified analyses, using successively broader definitions for an on-study CDI recurrence: (1) diarrhea consistent with CDI plus a toxin-positive stool sample; (2) diarrhea consistent with CDI plus a toxin-positive, polymerase chain reaction-positive, or toxigenic culture-positive stool sample; and (3) diarrhea consistent with CDI plus laboratory confirmation or (in the absence of a stool sample) treatment with a CDI-targeted antibiotic. Baseline characteristics were similar across the high-dose VE303 (n = 29; 1 additional participant excluded from efficacy analysis), low-dose VE303 (n = 27), and placebo (n = 22) groups. The participants' median age was 63.5 years (range, 24-96); 70.5% were female; and 1.3% were Asian, 1.3% Black, 2.6% Hispanic, and 96.2% White. CDI recurrence rates through week 8 (using the efficacy analysis 3 definition) were 13.8% (4/29) for high-dose VE303, 37.0% (10/27) for low-dose VE303, and 45.5% (10/22) for placebo (P = .006, high-dose VE303 vs placebo). Among adults with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence, high-dose VE303 prevented recurrent CDI compared with placebo. A larger, phase 3 study is needed to confirm these findings. ClinicalTrials.gov Identifier: NCT03788434.

Pediatric diarrhea patients living in urban areas have a higher incidence of Clostridioides difficile infection.

Clostridioides difficile infection (CDI) is a major cause of antibiotic-associated diarrhea and an unappreciated contributor to child mortality in low- and middle-income countries where the diagnosis may be difficult. There is little information about the prevalence of CDI among infants, children, and adolescents in Africa. Using a cross-sectional design, seventy-six samples were collected from pediatric patients presenting with diarrhea, including infants (≤ 2 years old), children (2-12 years), and adolescents (13 ≤17 years) from three hospitals between January and December 2019. Demographic data, medical history, and prior antibiotic use were recorded. Toxigenic culture and PCR were used to detect and validate the presence of C. difficile in the samples. Data obtained were analyzed using descriptive and inferential statistics. A total of 29 (38.7%), 39 (52.0%) and 7 (9.3%) samples were from infants, children, and adolescents, respectively. The average age of the patients was 4.4 years. Of these samples, 31 (41%) were positive for C. difficile by culture and were verified by PCR amplification of C. difficile-specific genes (tcdA and tcdB). The most positive cases were children (53.3%) and infants (40.0%) with the majority of them residing in urban areas. Forty-nine (66.2%) of the patients had no known antibiotics exposure, whereas 29.0% and 29.7% reported the use of over-the-counter antibiotics at 14 and 90 days, prior to the hospital visit, respectively. CDI is relatively common among children with diarrhea in Northern Nigeria. Therefore, for effective management and treatment, more attention should be given to testing for C. difficile as one of the causative agents of diarrhea.

A rapid multiplex real-time PCR detection of toxigenic Clostridioides difficile directly from fecal samples

This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/μL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China.

Detection of Clostridioides difficile toxin B gene in clinical stool specimens using rapid diagnostic quenching probe-polymerase chain reaction assay

We tested the accuracy of quenching probe-polymerase chain reaction (QP-PCR) for detecting Clostridioides difficile toxin B gene (tcdB) in stools from inpatients with suspected C. difficile infection and compared the results with other nucleic acid amplification tests (NAATs). Toxigenic culture results were used as reference for comparison. QP-PCR had comparable diagnostic accuracy with other NAATs and prior bead-beating enabled detection of tcdB in specimens judged as negative, without bead-beating. Taken together, the QP-PCR either with or without bead-beating showed sufficient effectiveness for detecting tcdB in stool specimens.

Evaluation of Two Rapid Diagnostic Clostridioides difficile Infection Tests in a Chinese Hospital: A Real-world Analysis

Background: Accurate diagnosis is essential for optimal prevention and treatment of Clostridioides difficile infection (CDI), and various diagnostic methods must be evaluated. Objectives: We aimed to evaluate and compare the performance of VIDAS C. difficile, C. DIFF QUIK CHEK COMPLETE (QCC), and toxigenic culture (TC) tests for diagnosing CDI and further determine the relationships between clinical factors and the toxin status of patients. Methods: Stool samples were randomly selected for VIDAS or QCC testing according to the manufacturer’s instructions between May 2017 and May 2021, and their performance was compared with that of TC. Clinical information was obtained from the hospital’s electronic medical records. Results: Among 10,897 samples tested, 6,435 and 4,462 samples were assigned for VIDAS and QCC tests, respectively. A total of 9.1% (996/10,897) of the samples were positive for TC. The sensitivity, specificity, positive predictive value, and negative predictive value were 36.6%, 98.6%, 72.1%, and 87.6% for VIDAS toxins A and B testing and 31.6%, 98.2%, 64.0%, and 87.8% for QCC toxin testing, respectively. Our results showed that the clinical data of the patients with positive and detectable toxins were not significantly different. Conclusions: The VIDAS and QCC tests provide rapid screening assays for the laboratory diagnosis of CDI. However, a more specific test to detect free toxins is required to confirm the diagnosis for glutamate dehydrogenase (GDH)-positive and toxin-negative samples. The clinical characteristics and outcomes of this cohort were similar, regardless of the results of toxins A and B testing.

Comparison of commercial assays and two-step approach to detect Clostridioides difficile in South Africa.

BackgroundClostridioides difficile is the number one cause of hospital-acquired diarrhoea. Accurate diagnosis of C. difficile is of utmost importance as it guides patient management and infection control practices. Studies evaluating the performance of commercially available nucleic acid amplification tests (NAATs) versus algorithms are lacking in resource-limited settings.ObjectiveThis study assessed the performance of three commercially available tests and a two-step approach for the diagnosis of C. difficile infection using toxigenic culture (TC) as the gold standard.MethodsTwo hundred and twenty-three non-duplicate loose stool samples were submitted to the National Health Laboratory Service Microbiology Laboratory at Tygerberg Hospital, Cape Town, South Africa, from October 2017 to October 2018. The samples were tested in parallel using the C. DIFF QUIK CHEK COMPLETE enzyme immunoassay (EIA) and two NAATs (Xpert C. difficile and BD MAX Cdiff), and the results were compared to TC. The performance of a two-step approach consisting of the C. DIFF QUIK CHEK COMPLETE followed by the Xpert C. difficile was also determined.ResultsOf 223 faecal specimens tested, 37 (16.6%) were TC-positive. The sensitivity and specificity of the C. DIFF QUIK CHEK COMPLETE were 54.1% and 98.9%; Xpert C. difficile, 86.4% and 96.8%; BD MAX Cdiff, 89.2% and 96.8%; and two-step approach, 89.2% and 96.2%.ConclusionThe C. DIFF QUIK CHEK COMPLETE, in a two-step approach with the Xpert C. difficile, performed similarly to the NAATs on their own and offer advantages in terms of cost and workflow in low-resource settings.

Japanese Clinical Practice Guidelines for Management of Clostridioides (Clostridium) difficile infection

Toxigenic culture and cytotoxicity assays are standard tests for detecting C. difficile infection (CDI). Others include immunochromatographic strips, a rapid diagnostic test that detects glutamate dehydrogenase (GDH and toxins simultaneously, and the nucleic acid amplification test (NAAT) that detects C. difficile toxin-producing genes. NAAT may not be available in some facilities, so a 2-step method can be adopted where GDH-positive and toxin-negative specimens are selected first, and then toxigenic culture is performed for these specimens.

Performances of the BD MAX™ CDIFF assay for the detection of toxigenic Clostridioides difficile using Cary-Blair preserved samples

Toxigenic Clostridioides difficile infection (CDI) is a high concern because of its increasing prevalence, among both nosocomial infection and community-acquired infection contexts. A total of 1320 prospective stool samples collected on FecalSwab™ (Cary-Blair medium) were screened using BD Max™ CDIFF assay (BDM). Results were compared to the routine algorithm including immunochromatographic testing for glutamate dehydrogenase (GDH) and toxin A/B (tox) using C. DIFF QUIK CHEK COMPLETE® followed by Xpert C. difficile assay in case of GDH+/tox- results. The prevalence of toxigenic C. difficile was estimated to 5.3% (n=70/1320) using the routine algorithm but to 7.4% (n = 98/1320) using BDM. The overall agreement between BDM and the routine algorithm was 97.4%. After resolution of discrepant results between BDM and the algorithm using Xpert C. difficile assay (unless already perform) and toxigenic culture performed by the French National Reference Laboratory, the overall agreement was increased to 99.4%, demonstrating the excellent performances of the test.

Standardisation and validation of an in-house quantitative real-time polymerase chain reaction (qPCR) assay for the diagnosis of Clostridioides difficile infection

ObjectivesClostridioides difficile is an emerging enteric pathogen that causes nosocomial diarrhoea in adults. The excessive cost of commercial molecular tests restricts the access of developing countries to its diagnosis. This study aimed to develop and validate in-house quantitative polymerase chain reaction (qPCR) targeting the C. difficile toxin B gene (tcdB) using two detection methodologies—SYBR Green and hydrolysis probes—for the diagnosis of C. difficile infection (CDI). MethodsGlutamate dehydrogenase (GDH) plus toxigenic culture was the standard reference diagnostic method. The SYBR Green method and hydrolysis probes were used to study 392 samples simultaneously to assess the diagnostic value of these real-time PCR assays in detecting CDI from clinical samples. ResultsThe SYBR Green and hydrolysis probe assays showed 97.9% and 87.5% sensitivity; 99.1% and 100.0% specificity; 94.0% and 100.0% positive predictive value; 99.7% and 98.3% negative predictive value; and 99.0% and 98.5% accuracy, respectively. ConclusionsThe two qPCR methodologies evaluated could offer an adequate tool as part of an algorithm in the laboratory diagnosis of CDI.

Alternative Causes of Infectious Diarrhea in Patients with Negative Tests for Clostridoides Difficile.

Hospitalized patients with diarrhea who have a negative Clostridoides difficile (C. difficile) test are not routinely evaluated for alternative causes of infectious diarrhea. This study assessed for potential infectious causes of diarrhea in hospitalized patients with an order for repeat C. difficile toxin enzyme immunoassay (tEIA) testing after an initial tEIA test was negative. For patients age ≥18 years who had a second C. difficile tEIA test ordered within 96 h after a negative tEIA test, remnant fecal specimens from the first (negative) tEIA test were evaluated using the BioFire FilmArray Gastrointestinal Panel PCR, C. difficile toxigenic culture, and culture on a blood agar plate (BAP) to identify other potential causes of infectious diarrhea. Growth of organisms on the BAP was also used to assess potential disruptions in the gastrointestinal microbiota. Among 84 remnant specimens, toxigenic C. difficile was identified in 9 (11%) by culture or PCR, while potential alternative causes of infectious diarrhea, including norovirus, rotavirus, enteropathogenic Escherichia coli, and Salmonella, were identified in 11 specimens (13%) by PCR. For the majority of patients, no infectious cause of diarrhea was identified, but 84% exhibited disrupted gastrointestinal microbiota, which may contribute to diarrhea. When a hospitalized patient has a negative C. difficile tEIA test but continues to have diarrhea, alternative infectious and noninfectious causes of diarrhea should be considered. If the patient has clinical signs and symptoms suggestive of infection or risk factors for gastrointestinal infection, laboratory testing for other etiologic agents may be appropriate.

Evaluation of an immunochromatographic test for the detection of glutamate dehydrogenase for the diagnosis of Clostridioides (Clostridium) difficile infection in dogs.

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.

Improved diagnosis of antibiotic-associated haemorrhagic colitis (AAHC) in faecal specimens by a new qualitative real-time PCR assay detecting relevant toxin genes of Klebsiella oxytoca sensu lato

ObjectiveToxin-producing Klebsiella oxytoca causes antibiotic-associated haemorrhagic colitis (AAHC). The disease-relevant cytotoxins tilivalline and tilimycine produced by certain K. oxytoca isolates are encoded by the non-ribosomal peptide synthetase genes A (npsA) and B (npsB). In this study, the new LightMix® Modular kit for the detection of relevant K. oxytoca sensu lato (s.l.) toxin genes was evaluated. MethodsDNA was extracted on the automated EMAG® platform. Amplification was done on the Light Cycler® 480 II instrument. In total, 130 residual faecal specimens collected from patients with antibiotic-associated diarrhoea were studied to determine the clinical sensitivity and specificity. Toxigenic culture served as reference method. ResultsWith the new kit, the limit of detection was 15 CFU/mL for all targets. For the pehX target specific to K. oxytoca s.l., 65 of 130 clinical specimens were positive, while toxin-specific targets (npsA/npsB) were positive in 47 of 130. The npsA/npsB PCR targets showed a clinical sensitivity of 100% (95%CI 80.5–100%) and a specificity of 73.5% (95%CI 64.3–81.3%) with a positive predictive value of 16.5% (95%CI 12.7–21.2%) and a negative predictive value of 100%. ConclusionCompared with culture, additional clinical specimens positive for K. oxytoca s.l. were detected with real-time PCR. The specificity of the toxin targets appears moderate due to the inferior sensitivity of the culture-based reference method. Since the developed assay is highly sensitive, it may be used as first-line method to improve the diagnosis of AAHC.

Performance of commercial PCR assays to detect toxigenic Clostridioides difficile in the feces of puppies.

Clostridioides difficile is an important enteric pathogen that causes significant morbidity and mortality in humans. With community‐acquired infections on the rise, it is important to identify reservoirs of the pathogen. Companion animals can be asymptomatic carriers of C. difficile and may therefore represent a reservoir, but epidemiological studies of C. difficile within the pet‐owner unit are needed, along with validated methods to detect C. difficile in both people and animals. The goal of this study was to assess the performance of commercial qPCR assays and a multiplex PCR for C. difficile compared to toxigenic culture. These assays were tested on up to 103 fecal samples from puppies, a population in which the prevalence of C. difficile is the highest. The sensitivities, specificities, positive predictive values and negative predictive values were respectively 84.2%, 87.7%, 61.5%, and 95.9% for the Cepheid GeneXpert; 66.7%, 66.7%, 29.6%, and 90.9% for the DiaSorin Simplexa; and 94.4%, 85.0%, 65.4%, and 98.1%, for the multiplex qPCR. The agreement was highest between the GeneXpert and the multiplex PCR (90.1% agreement, with a kappa statistic of 0.77). For diagnostic purposes, the positive predictive values of the assays were low. However, the high sensitivities of the assays could render them useful for epidemiologic purposes.