Results of a prospective, 18-month clinical evaluation of culture, cytotoxin testing, and culturette brand (CDT) latex testing in the diagnosis of Clostridium difficile-associated diarrhea

doi:10.1016/0732-8893(88)90045-4

Diagnostic Microbiology and Infectious Disease

Volume 10, Issue 2, June 1988, Pages 85-91

https://doi.org/10.1016/0732-8893(88)90045-4 Get rights and content

Abstract

An 18-mo evaluation of culture, cytotoxin, and latex testing for $Clostridium$ $difficile$ was performed between July 1, 1985, and December 31, 1986, on 1,536 specimens from 1,406 patients during evaluation of diarrhea. All cases with at least one test positive were investigated for clinical status. There were 144 $Clostridium$ $difficile$ -associated diarrhea (CAD) patients; 139 (97%) were positive by culture, 96 (67%) by cytotoxin, and 98 (68%) by latex testing. In the 1,262 non-CAD patients with diarrheal stool, 89 (7.1%) were positive by culture, 18 (1.4%) by cytotoxin, and 68 (5.4%) by the latex test. No CAD patient was positive by cytotoxin testing only, and two were positive by latex testing only. The culture and cytotoxin positivity were similar to our previous reports of 90–97% and 70–73%, respectively. Latex sensitivity (68%) was comparable to that of cytotoxin testing in this large group of patients (p 0.5). Overall, in the 1,262 patients without clinical evidence of $Clostridium$ $difficile$ disease, positive tests by latex testing (5.4%) were intermediate between those of culture (7.1%, p < 0.1) and cytotoxin (1.4%, p < 0.001).

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CCCNA is technically demanding, takes 2 to 4 days to turn around a negative result, and is generally only performed by large clinical laboratories with the capacity to maintain cell cultures. Compared with identification of toxin-producing C difficile using anaerobic culture (termed toxigenic culture; discussed in the section on microbiology), CCCNA was observed to be 67% to 78% sensitive,140,141 but the extent to which either CCCNA or toxigenic culture could be considered a reference standard was never established. Both CCCNA and toxigenic culture identify patients with colonization and are therefore susceptible to false positives for the end point of CDI.
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As there are nontoxigenic C difficile strains, it is necessary to confirm that cultured isolates produce toxin in vitro. As with CCTA, there are also variations in the exact methodologies used for CC, with differences in the culture conditions and the way C difficile is identified, how and whether alcohol shock is performed, and how in vitro toxin production is detected.31–36 CC may take up to 5 days to produce a result.
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Strains of C. difficile that do not produce toxins are common and considered nonpathogenic, which is why culture methods alone are not sufficient for diagnosis of CDI. Both older published results32 and the conclusions of newer studies2,5 suggest that the CCCN test lacks adequate sensitivity for detection of toxin-producing strains, partially because of the degradation of the toxin over time.43 Eastwood et al2 compared the results of EIA, CCCN, and toxigenic culture for detection of C. difficile from a series of stool samples.
The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. Rather, two-step algorithms that use glutamate dehydrogenase (GDH) assays to screen for C. difficile in stool specimens, followed by either direct cytotoxin testing or culture to identify toxin-producing C. difficile isolates, were recommended in one guideline and either GDH algorithms or NAATs were recommended in another guideline. Unfortunately, neither culture nor direct cytotoxin testing is widely available. In addition, this two-step approach requires 48 to 92 hours to complete, which may delay the initiation of therapy and critical infection control measures. Recent studies also show the sensitivity of several GDH assays to be <90%. This review considers the role of NAATs for diagnosing CDI and explores their potential advantages over two-step algorithms, including shorter time to results, while providing comparable, if not superior, accuracy.
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Some authors, however, cite culture with CCTA as being a more sensitive assay.6,9–12 However, C. difficile may be cultured in the stool of hospitalised patients without evidence of diarrhoea and that toxgenic culture may be a poorer predictor of CDI than CCTA directly on stool.13–16 We found that agreement between these two tests is not exact (93%) and that toxigenic culture detected fewer positives than the CCTA.
Commonly used immunoassays have limitations as stand-alone tests for the diagnosis of Clostridium difficile infection (CDI). In particular, the specificity of these assays means that these tests generate a relatively large number of false-positive results. We introduced a two-stage regimen for CDI as routine. Unformed stool samples received in our laboratory were initially tested with a Meridian Premier enzyme immunoassay (EIA) and positive samples were retested with reference testing methods (toxigenic culture and cell cytotoxicity assay). Clinicians received diagnostically useful information on the day that the sample arrived in the laboratory, with definitive negative and provisional positive results made available. We reviewed the first 3643 unformed stool specimens of which 158/3643 (4.3%) were provisionally positive by EIA. Of the 158 samples that were EIA positive, 119 were confirmed as being positive by at least one of the reference methods, giving a positive predictive value in this population of 75% (95% confidence interval: 67.6–81.7%). Comparison of the optical density values of the EIA lying between true and false-positive results suggests that the introduction of a second cut-off value would improve diagnostics. A test with two cut-offs would give the following results: ‘positive’, ‘negative’ and ‘indeterminate result, please perform confirmatory test’. This algorithm was a simple and cost-effective method to immediately improve diagnostics, but there is an urgent need for further research in laboratory diagnosis for CDI.
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Patient stool is filtered and incubated with human fibroblast cells with and without C difficile antitoxin for up to 48 hours, and if the antitoxin-free well shows a cytopathic effect and the antitoxin well does not, the presence of C difficile toxin in stool is confirmed (see Fig. 2). Historically, CCCNA was 67% to 78% sensitive when compared with TC as the gold standard,67,68 but over time such assays came to be regarded as a clinical gold standard in their own right to which more rapid assays were compared. More recent studies, however, confirm the imperfect sensitivity of CCCNA itself compared with TC.69,70

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Antimicrobial studyResults of a prospective, 18-month clinical evaluation of culture, cytotoxin testing, and culturette brand (CDT) latex testing in the diagnosis of Clostridium difficile-associated diarrhea

Abstract

Gastroenterology

Amer J Infect Control

Gastroenterology

Lancet

Diag Microbiol Infect Dis

Enzyme immunoassay for detection of Clostridium difficile toxins A and B in patients with antibiotic-associated diarrhoea and colitis

Eur J Clin Microbiol

Diagnosis and epidemiology of Clostridium difficile enterocolitis in Sweden

J Antimicrob Chemother

Accuracy detection of Clostridium difficile toxin by direct counterimmunoelectrophoresis of fecal samples from patients with antimicrobial-associated diarrhea and pseudomembranous colitis

Curr Ther Res

Biochemical characterization and biological actions of two toxins (D-1 and D-2) from Clostridium difficile

Rev Infect Dis

Antibiotics associated colitis

Disease A Month

Clinical and laboratory observations in Clostridium difficile colitis

Am J Clin Nutrition

Selective enrichment broth culture for detection of Clostridium difficile and associated cytotoxin

J Clin Microbiol

Significance of stool toxin determination to Clostridium difficile diarrhea

J Diarrheal Dis Res

Cytotoxicity assay in antibiotic associated colitis

J Infect Dis

Antimicrobial agent-associated colitis and diarrhea: Historical background and clinical aspects

Rev Infect Dis

Selective and differential medium for isolation of Clostridium difficile

J Clin Microbiol

Clostridium difficile-associated diarrhea and colitis in adults: A prospective case-controlled epidemiologic study

Arch Intern Med

Growth of Clostridium difficile and production of toxins A and B in complex and defined media

J Med Microbiol

Evaluation of a commercially available latex immonoagglutination test kit for detection of Clostridium difficile D-1 toxin

Microbiol Immunol

Antimicrobial study
Results of a prospective, 18-month clinical evaluation of culture, cytotoxin testing, and culturette brand (CDT) latex testing in the diagnosis of Clostridium difficile-associated diarrhea