Detection of toxigenic Clostridioides [Clostridium] difficile : Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates

Legaria, María C.; Rollet, Raquel; Martino, Ana Di; Castello, Liliana; Barberis, Claudia; Rossetti, María A.; Guardati, María C.; Canigia, Liliana Fernández; Carloni, G.; Litterio, Mirta; Rocchi, Marta; Anchart, E.; Trejo, Fernando Miguel; Minnaard, Jessica; Klajn, Diana; Predari, Silvia C.

doi:10.1016/j.ram.2017.01.002

Cited by 6 publications

(5 citation statements)

References 54 publications

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“…Although this sample was included as a false-positive result for the ELISA and GDH tests, it is more likely to be a failed growth of the isolate in the medium used in the TC protocol [9,14]. The performance of the GDH detection seen in the two commercial tests corroborates previous studies that reported the usefulness of this method as a screening test for the diagnosis of CDI [2,[15][16][17][18]. Interestingly, the similarities between the results obtained by these lateral flow tests demonstrate a very high concordance between the tests, with a K value of 0.975 (95% CI 0.926-1.000) [13].…”

supporting

confidence: 80%

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

et al. 2020

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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. Keywords Pseudomembranous colitis. Nosocomial diarrhea Clostridioides (Clostridium) difficile is responsible for most cases of antibiotic-associated diarrhea worldwide [1]. Despite the severity of the disease, the best laboratory diagnostic approach to diagnose C. difficile infection (CDI) is a subject of ongoing debate [2]. The diagnosis of CDI is frequently based on the clinical history and the detection of A/B toxins, and/or toxigenic isolates, by a combination of laborious methods [3]. In this context, the use of rapid tests for the detection of C. difficile glutamate dehydrogenase (GDH) is increasing as a screening method due to its low cost, high sensitivity, and fast results [4, 5]. However, if this method is not capable of differentiating non-toxigenic from toxigenic C. difficile strains, a stool sample positive for GDH is usually subjected to a second test involving a toxin-detecting assay, such as enzyme immunoassays (ELISA) and lateral flow tests, or a DNA-based test, commonly nucleic acid amplification-based tests [3]. Although the use of multiple tests is highly recommended to diagnose CDI [3], the cost of these algorithms commonly exceeds the affordability in developing countries [5]. In Brazil, despite the low sensitivity of ELISAs for the detection of A/B toxins, this method is still largely used as a single test in several hospitals [6, 7]. The use of a low sensitivity test can jeopardize the efficacy of control measures if infected individuals are kept without the necessary isolation and are not properly treated [6, 8]. Thus, to improve CDI diagnosis, this study aimed to evaluate two commercial assays for CDI diagnosis through detection of the GDH component and A/B toxins in hospitalized adults with diarrhea in a university hospital in Brazil.

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confidence: 80%

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

et al. 2020

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“…Genomic DNA was extracted from pure colonies of C. difficile isolates using the boiling method as described previously (Legaria et al 2018 ; Abbasi Montazeri et al 2020 ). Also, DNA was extracted directly from stool specimens using the QIAamp DNA Stool Minikit kit (QIAGEN, Hiden, Germany), according to the manufacturer's recommendations.…”

Section: Methodsmentioning

confidence: 99%

“…Also, DNA was extracted directly from stool specimens using the QIAamp DNA Stool Minikit kit (QIAGEN, Hiden, Germany), according to the manufacturer's recommendations. The concentration and purity of extracted DNA were measured using a NanoDrop spectrophotometry (Thermo Scientific, Waltham, MA, USA) at 260 nm (Legaria et al 2018 ; Abbasi Montazeri et al 2020 ).…”

Section: Methodsmentioning

confidence: 99%

Loop mediated isothermal amplification of Clostridioides difficile isolates in gastrointestinal patients

et al. 2022

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This study investigated the prevalence of Clostridioides difficile by culture, multiplex polymerase chain reaction (M-PCR), and loop mediated isothermal amplification (LAMP) in patients with suspected C. difficile infections (CDIs). Also, the results of three methods were compared. All stool specimens collected from CDI suspected patients were cultured on selective C. difficile cycloserine-cefoxitin fructose agar and incubated in an anaerobic jar up to 7 days. The bacterial isolates were identified using standard tests. Multiplex-PCR (M-PCR) was performed for detection of tcdA, tcdB, and tpi genes. The LAMP assay was performed to detect the tcdB gene of C. difficile. C. difficile was isolated from 20.0% (n = 10/50) of samples by culture. M-PCR showed that 34.0% (n = 17/50) of the specimens were positive for C. difficile based on the presence of tpi gene. Out of the 17 C. difficile, 13 strains (76.0%) were positive for tcdB gene using M-PCR. However, the LAMP assay showed that 30.0% (15/50) of specimens were positive for the presence of tcdB gene. M-PCR and LAMP methods showed 100.0% sensitivity compared to the culture method. However, the specificity of the LAMP (87.5%) was relatively higher than the M-PCR (82.5%) compared to the culture. Based on the results of this study, the prevalence of toxigenic C. difficile strains was high in suspected CDI patients. So, the differentiation between toxigenic and non-toxigenic strains is necessary. Our data showed that the LAMP assay is a good method for direct detection of toxigenic C. difficile strains from stool specimens.

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“…After isolation in CCFA medium, the presence of C. difficile was confirmed by gram stain, colony morphology, and detection of “horse-barn” odor. 16…”

Section: Methodsmentioning

confidence: 99%

“…After isolation in CCFA medium, the presence of C. difficile was confirmed by gram stain, colony morphology, and detection of "horse-barn" odor. 16 Molecular methods: DNA extraction: Each isolated strain from cultures was transferred with an inoculating loop into a 1.5 mL microcentrifuge tube containing 200 mL of sterile PBS buffer, and bacterial DNA was extracted using the QIAamp kit (Qiagen, Germany), according to the manufacturer's instructions.…”

Section: Toxigenic Culturementioning

confidence: 99%

A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates

Khodaparast

Mobarez

Saberifiroozi

2019

Middle East J Dig Dis

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BACKGROUND Clostridium difficile is the major causative agent of nosocomial antibiotic-associated colitis. The gold standard for C. difficile detection is stool culture followed by cytotoxic assay, although it is laborious and time-consuming. We developed a screening test based on a two-step conventional polymerase chain reaction (PCR) approach to detect gluD, the glutamate dehydrogenase (GDH) enzyme gene, which is a marker for screening of C. difficile. Targeting gluD comparing to the conserved stable genetic element of pathogenicity locus (PaLoc), with an accessory gene of Cdd3, was an effective method for the detection of this pathogen from patients with enterocolitis. METHODS Fresh fecal samples of the patients who were clinically suspicious for antibiotic-associated colitis were collected. Stool specimens were cultured on the cycloserine-cefoxitin fructose agar (CCFA) in an anaerobic condition, following alcohol shock treatment and enrichment in Clostridium difficile Brucella broth (CDBB). On confirmed colonies, PCR was carried out for detection of PaLoc subsidiary gene, Cdd3, and toxicogenic genes, tcdA and tcdB. The gluD that is GDH gene detection was performed by conventional PCR on the extracted DNA from 578 fresh stool samples. RESULTS 57 (9.8%) strains of C. difficile were approved by conventional PCR for gluD and Cdd3 genes, in which 37 (6.4%) colonies had tcdA+/tcdB+ genotype, 2 (0.3%) tcdA+/tcdB-, 4 (0.7%) tcdA-/ tcdB+ and the remaining 14 (2.4%) colonies were tcdA and tcdB negative. CONCLUSION These results demonstrate that targeting gluD by PCR is quite promising for rapid detection of C. difficile from fresh fecal samples. Furthermore, the multiple-gene analysis for tcdA and tcdB assay proved a reliable approach for diagnosing of toxigenic strains among clinical samples.

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Detection of toxigenic Clostridioides [Clostridium] difficile : Usefulness of two commercially available enzyme immunoassays and a PCR assay on stool samples and stool isolates

Cited by 6 publications

References 54 publications

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

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

Loop mediated isothermal amplification of Clostridioides difficile isolates in gastrointestinal patients

A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates

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