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      A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

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          Abstract

          Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene ( tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples ( tcdB +, n =  111; tcdB −, n  = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours.

          In a further study ( tcdB +, n =  47; tcdB −, n  = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples ( tcdB +, n =  25; tcdB −, n  = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

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          Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents.

          Michelle Riggs, Ajay K Sethi, Trina F Zabarsky … (2007)
          Asymptomatic fecal carriage of Clostridium difficile is common in patients staying in health care facilities, but the importance of asymptomatic carriers with regard to disease transmission is unclear. We prospectively examined the prevalence of asymptomatic carriage of epidemic North American pulsed-field gel electrophoresis type 1 and nonepidemic toxigenic C. difficile strains among long-term care patients in the context of an outbreak of C. difficile-associated disease and evaluated the frequency of skin and environmental contamination. Molecular typing was performed by pulsed-field gel electrophoresis. Logistic regression was used to assess factors associated with asymptomatic carriage, and a sensitive and specific prediction rule was developed to identify high-risk patients. Thirty-five (51%) of 68 asymptomatic patients were carriers of toxigenic C. difficile, and 13 (37%) of these patients carried epidemic strains. Compared with noncarriers, asymptomatic carriers had higher percentages of skin (61% vs. 19%; P=.001) and environmental contamination (59% vs. 24%; P=.004). Eighty-seven percent of isolates found in skin samples and 58% of isolates found in environmental samples were identical to concurrent isolates found in stool samples. Spores on the skin of asymptomatic patients were easily transferred to investigators' hands. Previous C. difficile-associated disease (P<.001) and previous antibiotic use (P=.017) were associated with asymptomatic carriage, and the combination of these 2 variables was predictive of asymptomatic carriage (sensitivity, 77%; specificity, 58%; positive predictive value, 66%; negative predictive value, 70%). Our findings suggest that asymptomatic carriers of epidemic and nonepidemic C. difficile strains have the potential to contribute significantly to disease transmission in long-term care facilities. Clinical factors, such as previous C. difficile-associated disease and recent antibiotic use, may be predictive of asymptomatic carriage.
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            Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection

            Timothy Planche, Kerrie Davies, Pietro G Coen … (2013)
            Summary Background Diagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection. Methods In this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12 420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative. Findings Clinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12–2·31). Multistage algorithms performed better than did standalone assays. Interpretation We noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection. Funding Department of Health and Health Protection Agency, UK.
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              Narrative review: the new epidemic of Clostridium difficile-associated enteric disease.

              John Bartlett (2006)
              Antibiotic-associated diarrhea and colitis were well established soon after antibiotics became available. Early work implicated Staphylococcus aureus, but in 1978 Clostridium difficile became the established pathogen in the vast majority of cases. In the first 5 years (1978 through 1983), the most common cause was clindamycin, the standard diagnostic test was the cytotoxin assay, and standard management was to withdraw the implicated antibiotic and treat with oral vancomycin. Most patients responded well, but 25% relapsed when vancomycin was withdrawn. During the next 20 years (1983 through 2003), the most commonly implicated antibiotics were the cephalosporins, which reflected the rates of use; the enzyme immunoassay replaced the cytotoxin assay because of speed of results and technical ease of performance; and metronidazole replaced vancomycin as standard treatment, and principles of containment hospitals became infection control and antibiotic control. During the recent past (2003 to 2006), C. difficile has been more frequent, more severe, more refractory to standard therapy, and more likely to relapse. This pattern is widly distributed in the United States, Canada, and Europe and is now attributed to a new strain of C. difficile designated BI, NAP1, or ribotype 027 (which are synonymous terms). This strain appears more virulent, possibly because of production of large amounts of toxins, and fluoroquinolones are now major inducing agents along with cephalosporins, which presumably reflects newly acquired in vitro resistance and escalating rates of use. The recent experience does not change principles of management of the individual patient, but it does serve to emphasize the need for better diagnostics, early recognition, improved methods to manage severe disease and relapsing disease, and greater attention to infection control and antibiotic restraint.
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                Author and article information

                Contributors
                Yung-Fu Chang: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2014
                Publication date (Electronic): 8 January 2014
                Volume: 9
                Issue: 1
                Electronic Location Identifier: e83808
                Affiliations
                [1 ]Lumora Ltd, Ely, Cambridgeshire, United Kingdom
                [2 ]Public Health England, Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge, Cambridgeshire, United Kingdom
                Cornell University, United States of America
                Author notes

                Competing Interests: LCT and OAG are inventors of Bioluminescent Assay in Real-Time (BART). LCT co-founded Lumora Ltd, a company that has full rights for the use of the BART patent. Patent name: Method for Determining the Amount of Template Nucleic Acid Present in a Sample and number WO2006010948. A further pending patent is ongoing. CJM, CRP and LCT are the inventors of heat elution sample preparation technology. CJM, CRP, JES, MJW, OAG and LCT declare financial interests in Lumora Ltd. The following authors were all employees at Lumora Ltd: CJM, CRP, JES, MJW, OAG and LCT, whose company funded this study. There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: CJM CRP NJP MDC SAP OAG NMB LCT. Performed the experiments: CJM CRP NJP MDC LCT AS JES MJW. Analyzed the data: CJM CRP NJP MDC JES LCT. Contributed reagents/materials/analysis tools: CJM CRP JES MJW SAP OAG. Wrote the paper: CJM NJP MDC CRP LCT.

                Article
                Publisher ID: PONE-D-13-19393
                DOI: 10.1371/journal.pone.0083808
                PMC ID: 3885522
                PubMed ID: 24416173
                SO-VID: 9b12e37d-94d9-4b2f-9387-787d3693ca16
                Copyright statement: Copyright @ 2014
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 10 May 2013
                Date accepted : 8 November 2013
                Page count
                Pages: 10
                Funding
                The authors have no support or funding to report.
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Biochemistry
                Subject: Nucleic Acids
                Subject: DNA
                Subject: Biotechnology
                Subject: Applied Microbiology
                Subject: Microbiology
                Subject: Bacterial Pathogens
                Subject: Gram Positive
                Subject: Medical Microbiology
                Subject: Microbial Pathogens
                Subject: Medicine
                Subject: Diagnostic Medicine
                Subject: Test Evaluation
                Subject: Gastroenterology and Hepatology
                Subject: Infectious Diseases
                Subject: Bacterial Diseases
                Subject: Clostridium Difficile
                Subject: Public Health

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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