Detection of Clostridium difficile among diarrheic children using cultural and polymerase chain reaction technique.

INTRODUCTION: Clostridium difficile is the most common cause of antibiotic-associated diarrhea and colitis. Several methods are available for the detection of C. difficile in stool samples. This study aimed to use glutamate dehydrogenase (GDH), toxin detection, culture and polymerase chain reaction (PCR) techniques for the diagnosis of this pathogen. METHODOLOGY: A total of 300 stool samples were collected from children with hospital acquired diarrhea (HA-D), community acquired diarrhea (CA-D), and hospitalized non-diarrheic children as control with ages ranging from 6 months to 6 years (mean 3.7 ± 1.7). Each stool sample was divided into two parts; one part was tested for the enzyme GDH, toxin A and B and then cultured on selective media; and the other part for direct DNA extraction. RESULTS: From a total of 300 stool samples, 9 (3.0%) were positive for C. difficile by the PCR technique, 7 (7%) samples of which were from HA-D cases and 2 (2.0%) from CA-D cases; the control group samples were negative. The enzyme GDH was detected in 12 (12%) samples and toxins A and B in 8 (8%) samples from HA-D cases compared to 5 (5%) and 2 (2%), respectively from CA-D cases. Both GDH and the toxins were negative in control samples. Only 19 (19.0%) samples from HA-D cases gave suspected growth and all of these were negative by PCR. CONCLUSIONS: Based on the results of this study, we conclude that the PCR technique is the only reliable method for the diagnosis of this pathogen.

Laboratory diagnosis of Clostridioides difficile infection in symptomatic patients: what can we do better?

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.

Prevalence and Seasonality of Clostridiodes difficile over 12 Years at a Tertiary Hospital in Brazil.

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.

Influence of dietary carbohydrate profile on the dairy cow rumen meta-proteome.

Diet starch and fiber contents influence the rumen microbial profile and its fermentation products, yet no information exists about the effects of these dietary carbohydrate fractions on the metabolic activity of these microbes. The objective of this experiment was to evaluate the effects of dietary carbohydrate profile changes on the rumen meta-proteome profile. Eight cannulated Holstein cows were assigned to the study as part of a 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement including four 28-d periods. Cows received 1 of 4 dietary treatments on a dry matter (DM) basis. Diets included different concentrations of rumen fermentable starch (RFS) and physically effective undigested NDF (peuNDF240) content in the diet: (1) low peuNDF240, low RFS (LNLS); (2) high peuNDF240, low RFS (HNLS); (3) low peuNDF240, high RFS (LNHS); and (4) high peuNDF240, high RFS (HNHS). Rumen fluid samples were collected from each cow on the last 2 d of each period at 3 time points (0600, 1000, and 1400 h). The microbial protein fraction was isolated, isobarically labeled, and analyzed using liquid chromatography combined with tandem mass spectrometry techniques. Product ion spectra were searched using the SEQUEST search on Proteome Discoverer 2.4 (Thermo Scientific) against 71 curated microbe-specific databases. Data were analyzed using PROC MIXED procedure in SAS 9.4 (SAS Institute Inc.). A total of 138 proteins were characterized across 26 of the searched microbial species. In total, 46 proteins were affected by treatments across 17 of the searched microbial species. Of these 46 proteins, 28 were affected by RFS content across 13 microbial species, with 20 proteins having higher abundance with higher dietary RFS and 8 proteins having higher abundance with lower dietary RFS. The majority of these proteins have roles in energetics, carbon metabolism, and protein synthesis. Examples include pyruvate, phosphate dikinase (Ruminococcus albus SY3), 30S ribosomal protein S11 (Clostridium aminophilum), and methyl-coenzyme M reductase subunit α (Methanobrevibacter ruminantium strain 35063), which had higher abundances with higher dietary RFS. Conversely, glutamate dehydrogenase (Butyrivibrio fibrisolvens) and 50S ribosomal protein L5 (Pseudobutyrivibrio ruminis) and L15 (Ruminococcus bromii) had lower abundances with higher dietary RFS content. Among the remaining 18 proteins unaffected by RFS content alone, 5 proteins were affected by peuNDF240 content, and 13 were affected by peuNDF240 × RFS interactions. Our results suggest that the RFS content of the diet may have a greater influence on rumen microbial protein abundances than dietary peuNDF240 content or peuNDF240 × RFS interactions. This research highlights that dietary carbohydrate profile changes can influence rumen microbial protein abundances. Further research is needed to fully characterize the effects of diet on the rumen meta-proteome and manipulate the various roles of rumen microbes. This will aid in designing the strategies to maximize the efficiency of nutrient use in the rumen.

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.

Laboratory diagnosis of Clostridioides (Clostridium) difficile infection in domestic animals: A short review.

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.

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

OBJECTIVES: Clostridioides 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). METHODS: Glutamate 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. RESULTS: The 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. CONCLUSIONS: The two qPCR methodologies evaluated could offer an adequate tool as part of an algorithm in the laboratory diagnosis of CDI.

Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

INTRODUCTION: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. OBJECTIVE: To define if GDH determination is redundant to that of toxins. METHODS: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. RESULTS: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. CONCLUSIONS: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

INTRODUCCIÓN: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. OBJETIVO: Definir si la determinación de GDH es redundante a la de las toxinas. MÉTODOS: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. RESULTADOS: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. CONCLUSIONES: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Trends in Clostridioides difficile diagnosis before and after a change in testing algorithm.

Clostridioides difficile (Clostridium difficile) (CD) infection remains a challenging diagnosis in hospitalized patients given the myriad of testing procedures and array of alternative causes for diarrhea. We identified 100 consecutive inpatients with positive CD testing in a single tertiary center before and after changing from nucleic acid amplification testing (NAAT) alone to a two-step algorithm involving Glutamate Dehydrogenase enzyme immunoassays (GDHEIA) followed by an enzyme immunoassay for CD toxins (EIA). Detailed clinical information was obtained retrospectively to assess for risk factors, clinical features, and treatment outcomes to correlate test results with clinical cases. We demonstrate that using a 2-step testing algorithm identifies patients with a consistent clinical illness for CD disease significantly more often than nucleic acid amplification testing alone without an increase in cases of severe CD disease. Our data suggest that NAAT alone results in an increase in unnecessary treatment of CD colonization.

¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Clostridioides difficile laboratory diagnostic techniques: a comparative approach of rapid and molecular methods.

Clostridioides difficile infection is a public health problem because of it is easily spread; with harmful consequences, it is essential to reduce hospital costs and prevent its dissemination by having a precise diagnosis. The gold standard for its diagnosis is polymerase chain reaction (PCR); however, the technique is not available for all laboratories due to the high cost. New approaches using non-molecular tests to detect C. difficile and toxin A/B production has been proposed to improve cost benefits. The objective of this study is to compare molecular methods (PCR) and rapid methods (immunochromatographic test and enzymatic immunoassay). A series of tests comprising these diagnostic techniques was performed with 50 patients with a clinical diagnosis for Clostridioides difficile on GeneXpert® devices test; a calculation of the sensitivity was executed, followed by a comparison of the efficiency of all techniques. Greater sensitivity was observed in the PCR-based methods (BD MAX™ and BioFire FilmArray®) and the GDH-based assays (RIDASCREEN® and Alere Techlab®). The proposed algorithm represents minor monetary disadvantages but a significant temporal optimization of 10%. Future studies concerning both positive and negative results could be advantageous because of the possibility of calculating more method concordance indexes, such as the specificity and Kappa index, in addition to being able to indicate a monetary profit if the proposed algorithm was applied due to the nonproceeding PCR cases.

A 2-step algorithm combining glutamate dehydrogenase and nucleic acid amplification tests for the detection of Clostridioides difficile in stool specimens.

The optimized diagnosis algorithm of Clostridioides difficile infection (CDI) is worldwide concerns. The purpose of this study was to assess the toxigenic C. difficile test performance and propose an optimal laboratory workflow for the diagnosis of CDI in mild virulent epidemic areas. Diarrhea samples collected from patients were analyzed by glutamate dehydrogenase (GDH), toxin AB (CDAB), and nucleic acid amplification test (NAAT). We assessed the performance of GDH, the GDH-CDAB algorithm, and the GDH-NAAT algorithm using toxigenic culture (TC) as a reference method. In this study, 186 diarrhea samples were collected. The numbers of TC-positive and TC-negative samples were 39 and 147, respectively. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and kappa of the GDH assay were 100%, 80.3%, 57.4%, 100%, and 0.63; of the GDH-CDAB algorithm were 48.7%, 97.3%, 82.6%, 87.7%, and 0.54; and of the GDH-NAAT algorithm were 74.4%, 100%, 100%, 93.6%, and 0.82, respectively. The GDH-NAAT algorithm has great concordance with TC in detecting toxigenic C. difficile (kappa = 0.82), while the sensitivity of the GDH-CDAB algorithm was too low to meet the demand of CDI diagnosis clinically. GDH-NAAT algorithm is recommended for the detection of toxigenic C. difficile with high specificity, increased sensitivity, and cost-effective.

Impact of the introduction of nucleic acid amplification testing on Clostridioides difficile detection and ribotype distribution in Wales.

OBJECTIVES: To determine the impact of the 2018 introduction of nucleic acid amplification tests (NAATs) for C. difficile detection on the laboratory diagnosis of C. difficile infection (CDI), and the distribution of C. difficile ribotypes. METHODS: A retrospective analysis of five years (2015-2019) of C. difficile diagnostic laboratory and PCR ribotyping test results. RESULTS: A total of 255,104 diagnostic results, from 136,353 patients were analysed: 199,794 samples where glutamate dehydrogenase (GDH) was used as the primary screen; and 55,310 where NAATs were employed. An overall decrease in frontline positivity from 2015 to 2019 (10.3% [n = 5017] to 6% [n = 3190] - p < 0.0001) was observed, despite an increase in the number of samples tested (48,778 to 52,839). NAAT positivity was lower than GDH (p < 0.0001) for the two years where it was implemented. The variance was accounted for by increased overall C. difficile isolation and reduced toxin negative strain culture from NAAT positive samples (p < 0.0001). Ribotype distribution (6546 samples) remained stable with decreasing RT27 isolation in each year except 2017 (p < 0.0001). RT78 was associated with toxin A/B EIA positivity (p < 0.0001). CONCLUSIONS: Use of NAAT for the detection of C. difficile, as part of a 2-step algorithm, has not led to an increase in CDI laboratory diagnostic test positivity. In spite of ribotype distribution being comparable for screening in toxin A/B positive samples, there is a significantly greater correlation between NAAT positivity and culture of toxigenic strains compared to GDH.

Simple and Minimally Invasive SID Devices for Native Mass Spectrometry.

We describe a set of simple devices for surface-induced dissociation of proteins and protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few millimeters along the ion path axis) and is easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. Despite the decrease in size and reduction in number of electrodes to 3 (from 10 to 12 in Gen 1 and ∼6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

The impact of diagnostic methods on the diagnosis of Clostridiodes difficile infection.

BACKGROUND: Clostridiodes difficile is a common cause of healthcare-associated diarrhoea. Laboratory testing for C. difficile infection (CDI) remains an area of confusion, as there is not a single accepted reference standard or a single best test. OBJECTIVES: To analyse the impact of different diagnostic methods on reported CDI rates. In addition, CDI incidence rates at Charlotte Maxeke Johannesburg Academic Hospital (CMJAH), Johannesburg, South Africa, were determined. METHODS: Results of stool samples submitted for C. difficile testing at CMJAH from 1 January 2014 to 31 August 2017 were reviewed. From January 2014 to July 2016, samples were tested by polymerase chain reaction (PCR) or toxin immunoassay, and from August 2016 to August 2017, algorithm-based testing (glutamate dehydrogenase and toxin immunoassay followed by PCR) was performed. RESULTS: A total of 4 829 samples were submitted. For the first period, toxin immunoassay and PCR showed a positivity rate of 11.4% and 21.1%, respectively, with an overall positivity rate of 18.7% (95% confidence interval (CI) 15.6 - 21.9). For the second period, the positivity rate was 15.9% (95% CI 11.3 - 17.7). This rate included samples that were GDH-positive and either showed toxin production or had a positive Xpert result. The CDI incidence for the two periods was different, with an incidence rate of 8.8 and 6.1 per 10 000 patient-days for the first and second periods, respectively. CONCLUSIONS: The choice of laboratory testing method has a major impact on the diagnosis of CDI, and therefore on reported rates of CDI. Standardisation of laboratory testing and incidence rate reporting is required in order to obtain robust and reliable data.

Evaluation of glutamate dehydrogenase (GDH) and toxin A/B rapid tests for Clostridioides (prev. Clostridium) difficile diagnosis in a university hospital in Minas Gerais, Brazil.

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.

Immunochromatographic test and ELISA for the detection of glutamate dehydrogenase (GDH) and A/B toxins as an alternative for the diagnosis of Clostridioides (Clostridium) difficile-associated diarrhea in foals and neonatal piglets.

Considering the lack of studies evaluating the performance of commercially available methods for diagnosis of Clostridioides (Clostridium) difficile infection (CDI) in animals, the present study aimed to assess an immunochromatographic test for detection of glutamate dehydrogenase (GDH) and A/B toxins of C. difficile, also evaluated by an ELISA kit, in foals and neonatal piglets. Intestinal contents of 47 piglets and feces of 35 foals were tested to GDH antigen and A/B toxins in a lateral flow method (Ecodiagnostica, Brazil). Also, these samples were submitted to A/B toxin detection by an ELISA kit (C. difficile Tox A/B II, Techlab Inc., USA), using the toxigenic culture (TC) as the reference method. The GDH component of the lateral flow test showed sensitivity and negative predictive value (NPV) of 100% and a high specificity in samples of piglets (82.61%) and foals (100%). Detection of A/B toxins using the lateral flow test and the ELISA resulted in a specificity of 100% in samples of both species. On the other hand, the sensibility ranged from 54.2 to 90% for the ELISA and from 12.5 to 60% for the lateral flow test for piglets' and foals' samples, respectively. In conclusion, the present work suggests that the lateral flow test for GDH detection could be a useful method for diagnosing CDI in these species. On the other hand, the low sensitivity of the lateral flow test for A/B toxins might compromise its utility in piglets.

The Evaluation of the Performance of C. Diff Quik Chek Complete and Toxin A + B (Clostridium difficile) DUO Diagnostic Tests Compared with Toxigenic Culture in the Diagnosis of Clostridium difficile Infection.

BACKGROUND: Clostridium difficile is an important cause of nosocomial diarrhea and the best standard laboratory method for the diagnosis of C. difficile infection is controversial. In this study, we aimed to investigate the performance of Toxin A + B (Clostridium difficile) DUO kit which detects C. difficile toxin A and B by the immunochromatographic method and C. Diff Quik Chek Complete (QCC) rapid membrane immunoassay kit which determines the presence of glutamate dehydrogenase (GDH) and C. difficile toxin A and B in stool samples, compared with toxigenic culture in the diagnosis of C. difficile infection. METHODS: One hundred ninety-three stool samples from patients suspected of having C. difficile infection were included in the study. The performances of two commercial tests were compared with toxigenic culture which was accepted as the reference method. RESULTS: The sensitivity and specificity of the GDH component of QCC were 94.4% and 97.7%, the sensitivity and specificity of the toxin component were 92.3% and 100%, respectively. The sensitivity and specificity of Toxin A + B (Clostridium difficile) DUO test were found as 53.8% and 87.8%, respectively. CONCLUSIONS: C. Diff Quik Chek Complete test, which is a rapid test with high sensitivity and specificity, can be used alone for the diagnosis of C. difficile infection while Toxin A + B (Clostridium difficile) DUO test cannot be used for the same purpose due to the low sensitivity and specificity of the test.

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing.

The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at -80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH-/toxin-, and 124 GDH+/toxin- samples, of which 39 were CCNA+ and 85 were CCNA- Clarity had 96.2% negative agreement with GDH-/toxin- samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin-/CCNA- samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin-/CCNA+ samples, 90.0% of GDH+/toxin-/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin-/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

Extremely diversified haplotypes observed among assemblage B population of Giardia intestinalis in Kenya.

In molecular epidemiological studies of Giardia intestinalis, an pathogenic intestinal flagellate, due to the presence of allelic sequence heterogeneity (ASH) on the tetraploid genome, the image of haplotype diversity in the field remains uncertain. Here we employed the nine assemblage B positive stool samples, which had previously reported from Kenyan children, for the clonal sequence analysis of multiple gene loci (glutamate dehydrogenase (GDH), triosephosphate isomerase (TPI), and beta-giardin (BG)). The diversified unique assemblage B haplotypes as GDH (n = 67), TPI (n = 84), and BG (n = 62), and the assemblage A haplotypes as GDH (n = 7), TPI (n = 14), and BG (n = 15), which were hidden in the previous direct-sequence results, were detected. Among the assemblage B haplotypes, Bayesian phylogeny revealed multiple statistically significant clusters (9, 7, and 7 clusters for GDH, TPI, and BG, respectively). A part of the clusters (2 for GDH and 1 for BG), which included >4 haplotypes from an individual sample, indicated the presence of co-transmission with multiple strains sharing a recent ancestor. Locus-dependent discrepancies, such as different compositions of derived samples in clusters and different genotyping results for the assemblages, were also observed and considered to be the traces of both intra- and inter-assemblage genetic recombination respectively. Our clonal sequence analysis for giardial population, which applied firstly in Kenya, could reveal the higher rates of ASH far beyond the levels reported in other areas and address the complex population structure. The clonal analysis is indispensable for the molecular field study of G. intestinalis.