Clostridioides difficile (formerly called Clostridium difficile, C. difficile) infection (CDI) is listed as an urgent threat on the 2019 antibiotic resistance threats report in the United States by the Centers for Disease Control and Prevention. Early detection and appropriate disease management appear to be essential. Meanwhile, although the majority of cases are hospital-acquired CDI, community-acquired CDI cases are also on the rise, and this vulnerability is not limited to immunocompromised patients. Gastrointestinal treatments and/or gastrointestinal tract surgeries may be required for patients diagnosed with digestive diseases. Such treatments could suppress or interfere with the patient's immune system and disrupt gut flora homeostasis, creating a suitable microecosystem for C. difficile overgrowth. Currently, stool-based non-invasive screening is the first-line approach to CDI diagnosis, but the accuracy is varied due to different clinical microbiology detection methods; therefore, improving reliability is clearly required. In this review, we briefly summarised the life cycle and toxicity of C. difficile, and we examined existing diagnostic approaches with an emphasis on novel biomarkers such as microRNAs. These biomarkers can be easily detected through non-invasive liquid biopsy and can yield crucial information about ongoing pathological phenomena, particularly in CDI.

Clostridium (reclassified as " Clostridioides ") difficile is an anaerobic, gram-positive bacterium that causes significant disease through elaboration of two potent toxins in patients whose normal gut microbiota has been altered through antimicrobial or chemotherapeutic agents (dysbiosis). The optimum method of laboratory diagnosis is still somewhat controversial. Recent practice guidelines published by professional societies recommend a two-step approach beginning with a test for glutamate dehydrogenase (GDH), followed by a toxin test and/or a nucleic acid test. Alternatively, in institutions where established clinical algorithms guide testing, a nucleic acid test alone is acceptable. Nucleic acid tests are the methods of choice in approximately 50% of laboratories in the United States. These tests are considered as the most sensitive methods for detection of C. difficile in stool and are the least specific. Because of the lower specificity with nucleic acid tests, some clinicians believe that toxin enzyme immunoassays are better predictors of disease, despite their known poor performance in certain patient populations. This review will discuss the advantages and disadvantages of the currently available test methods for the diagnosis of C. difficile with a brief mention of some novel assays that are currently in clinical trials.

Nucleic acid amplification technology (NAAT) has assumed a critical position in disease diagnosis in recent times and contributed significantly to healthcare. Application of these methods has resulted in a more sensitive, accurate and rapid diagnosis of infectious diseases than older traditional methods like culture-based identification. NAAT such as the polymerase chain reaction (PCR) is widely applied but seldom available to resource-limited settings. Isothermal amplification (IA) methods provide a rapid, sensitive, specific, simpler and less expensive procedure for detecting nucleic acid from samples. However, not all of these IA techniques find regular applications in infectious diseases diagnosis. Disease diagnosis and treatment could be improved, and the rapidly increasing problem of antimicrobial resistance reduced, with improvement, adaptation, and application of isothermal amplification methods in clinical settings, especially in developing countries. This review centres on some isothermal techniques that have found documented applications in infectious diseases diagnosis, highlighting their principles, development, strengths, setbacks and imminent potentials for use at points of care.

For the microbiological diagnosis of a Clostridium (C.) difficile infection (CDI), a two-test algorithm consisting of a C. difficile glutamate dehydrogenase (GDH)-immunoassay followed by a toxin-immunoassay in positive cases is widely used. In this study, two chemiluminescent immunoassays (CLIAs), one for GDH and the other for the toxins A and B, have been evaluated systematically using appropriate reference methods. Three-hundred diarrhoeal stool specimens submitted for CDI diagnosis were analysed by the LIAISON CLIAs (DiaSorin). Toxigenic culture (TC) and cell cytotoxicity assay (CCTA) were used as "gold standard" reference methods. In addition, GDH and toxin A and B enzyme immunoassays (EIAs), C. diff Chek-60 and toxin A/B II (TechLab), and the Cepheid Xpert C. difficile polymerase chain reaction (PCR) were performed. C. difficile was grown in 42 (14%), TC was positive in 35 (11.7%) and CCTA in 25 (8.3%) cases. CLIAs were more sensitive but less specific than the respective EIAs. Using culture as reference, the sensitivity of the GDH CLIA was 100%. In comparison to CCTA sensitivity, specificity, positive predictive value and negative predictive value of the two-test algorithm were 88, 99.3, 91.7 and 98.9% by CLIAs and 72, 99.6, 94.7 and 97.5% by EIAs. Discrepant results by CLIAs were more frequent than that by EIAs (9% vs. 6.3%); in those cases, PCR allowed for the accurate detection of toxigenic strains. Due to performance characteristics and testing comfort, CLIAs in combination with PCR represent a favourable option for the rapid laboratory C. difficile diagnostics.

Clostridium difficile is a Gram-positive bacterium commonly found in health care and long-term-care facilities and is the most common cause of antibiotic-associated diarrhea. Rapid detection of this bacterium can assist physicians in implementing contact precautions and appropriate antibiotic therapy in a timely manner. The purpose of this study was to compare the clinical performance of the Quidel Lyra Direct C. difficile assay (Lyra assay) (Quidel, San Diego, CA) to that of a direct cell culture cytotoxicity neutralization assay (CCNA) and enhanced toxigenic culture. This study was performed at three geographically diverse laboratories within the United States using residual stool specimens submitted for routine C. difficile testing. Residual samples were tested using the Lyra assay on three real-time PCR platforms, and results were compared to those for direct CCNA and enhanced toxigenic culture. The test results for all platforms were consistent across all three test sites. The sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500 Fast DX, and ABI QuantStudio DX instruments compared to CCNA were 90.0% and 93.3%, 95.0% and 94.2%, and 93.8% and 95.0%, respectively. Compared to enhanced toxigenic culture, the sensitivity and specificity of the Lyra assay on the SmartCycler II, ABI 7500, and QuantStudio instruments were 82.1% and 96.9%, 89.3% and 98.8%, and 85.7% and 99.0%, respectively. Overall, the Lyra assay is easy to use and versatile and compares well to C. difficile culture methods.

There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings.

To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile.

One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples.

Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing.

The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and emphasized the value of using CTN assay and culture as parts of an algorithm that ensures accurate diagnosis of toxigenic C difficile.

Clostridium difficile is a formidable nosocomial and community-acquired pathogen, causing clinical presentations ranging from asymptomatic colonization to self-limiting diarrhea to toxic megacolon and fulminant colitis. Since the early 2000s, the incidence of C. difficile disease has increased dramatically, and this is thought to be due to the emergence of new strain types. For many years, the mainstay of C. difficile disease diagnosis was enzyme immunoassays for detection of the C. difficile toxin(s), although it is now generally accepted that these assays lack sensitivity. A number of molecular assays are commercially available for the detection of C. difficile. This review covers the history and biology of C. difficile and provides an in-depth discussion of the laboratory methods used for the diagnosis of C. difficile infection (CDI). In addition, strain typing methods for C. difficile and the evolving epidemiology of colonization and infection with this organism are discussed. Finally, considerations for diagnosing C. difficile disease in special patient populations, such as children, oncology patients, transplant patients, and patients with inflammatory bowel disease, are described. As detection of C. difficile in clinical specimens does not always equate with disease, the diagnosis of C. difficile infection continues to be a challenge for both laboratories and clinicians.

Clostridium difficile is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis, which have significant morbidity and mortality. Accurate and timely diagnosis is critical. Repeat enzyme immunoassay testing for C. difficile toxin has been recommended because of <100% sensitivity. All C. difficile tests between 1 January 2006 and 31 December 2006 were retrospectively analyzed for results and testing patterns. The Wampole C. difficile Tox A/B II enzyme immunoassay kit was used. There were a total of 8,256 tests from 3,112 patients; 49% of tests were repeated. Of the 3,749 initially negative patient tests, 96 were positive upon repeat testing within 10 days of the first test. Of repeat tests, 0.9% repeated on day 0 (same day as the first test), 1.8% on day 1, 3.8% on day 2, 2.6% on day 3, 5.4% on days 4 to 6, and 10.6% on days 7 to 10 were positive. Thirty-eight patients had a positive test within 48 h of an initial negative test, and based on chart review, 18 patients were treated empirically while 16 were treated following the new result. None had evidence of medical complications. Of initially positive patients, 91% were positive upon repeat testing on day 0, 75% on day 1, and 58% on day 2, to a low of 14% on days 7 to 10. Depending on the clinical setting, these data support not repeating C. difficile tests within 2 days of a negative result and limiting repeat testing to >/=1 week of a positive result.

Prompt and precise diagnosis is an important aspect of effective management of Clostridium difficile infection (CDI). CDI causes 15%-25% of all cases of antibiotic-associated diarrhea, the severity of which ranges from mild diarrhea to fulminant pseudomembranous colitis. Several factors, especially advanced age and hospitalization, should be considered in the diagnosis of CDI. In particular, nosocomial diarrhea arising >72 hours after admission among patients receiving antibiotics is highly likely to have resulted from CDI. Testing of stool for the presence of C. difficile toxin confirms the diagnosis of CDI. However, performance of an enzyme immunoassay is the usual method by which CDI is confirmed, but this test appears to be relatively insensitive, compared with the cell cytotoxicity assay and stool culture for toxigenic C. difficile on selective medium. Endoscopy and computed tomography are less sensitive than stool toxin assays but may be useful when immediate results are important or other confounding conditions rank high in the differential diagnosis. Often overlooked aspects of this diagnosis are high white blood cell counts (which are sometimes in the leukemoid range) and hypoalbuminemia.

A two-step algorithm for the detection of Clostridium difficile by the use of C. Diff Quik Chek (TechLab, Blacksburg, VA) and a tissue culture cytotoxicity neutralization assay was found to be more sensitive than the widely used solid-phase enzyme immunoassay (EIA), the Premier toxin A and B EIA (Meridian Bioscience, Cincinnati, OH), and a newly developed, rapid single-test EIA for C. difficile toxins A and B (Tox A/B Quik Chek; TechLab).

Several kinds of laboratory techniques are available to detect Clostridium difficile toxin in fecal samples. Because questions have been raised about the reliability of immunoassays compared to the accepted standard, cytotoxicity assay, we studied three enzyme immunoassays (EIAs) and one rapid EIA, which demonstrated relatively good sensitivities and specificities compared to cytotoxicity assay.

We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were > or = 99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by approximately 75 to 80% and two-step testing was complete in < or = 3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US dollar 143,000 less than if CCNA alone had been performed on all 5,887 specimens.

We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile isolates were tested for the presence of the toxin genes by PCR. If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive. Eighty-seven of 93 and 79 of 93 C. difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively. No test was able to detect toxin in all samples with true-positive results. Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results. Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C. difficile-associated diarrhea, respectively, if used alone.

Clostridium difficile causes approximately 25% of nosocomial antibiotic-associated diarrheas and most cases of pseudomembranous colitis. We evaluated C. DIFF CHEK, a new screening test that detects glutamate dehydrogenase of C. difficile. Our results showed that this test was comparable to PCR in sensitivity and specificity and outperformed bacterial culture.