Identification of an optimal Clostridioides (Clostridium) difficile infection (CDI) diagnostic testing strategy in children and adults remains a conundrum.Reference Burnham, Dubberke, Kociolek, Polage and Riley1,Reference Burnham and Carroll2 Although C. difficile toxin enzyme immunoassays (EIAs) are highly specific for CDI, concerns regarding potential suboptimal sensitivity have limited their widespread use.Reference Burnham, Dubberke, Kociolek, Polage and Riley1,Reference Burnham and Carroll2 Recent data in adults suggest that the diagnostic predictive value of toxin EIAs are better than previously reported.Reference Polage, Gyorke and Kennedy3,Reference Planche, Davies and Coen4 These 2 large studies have resulted in revised recommendations for toxin EIA use for CDI evaluation in adults, particularly at institutions without preagreed criteria for C. difficile testing where use of highly sensitive toxin gene polymerase chain reaction (PCR) assays would result in frequent misdiagnosis of CDI in patients with C. difficile carriage.Reference McDonald, Gerding and Johnson5
Compared to adult patients, studies of C. difficile test performance in pediatric patients are relatively limited. A previous pediatric study reported high prevalence (36%) of false-positive toxin EIA results in children.Reference Toltzis, Nerandzic and Saade6 This anomalous finding has not been corroborated in adult or other pediatric populations. In the absence of evidence challenging this finding, concerns about poor specificity in addition to existing concerns about poor sensitivity have precluded recommendations for use of toxin EIAs in pediatric patients.Reference Sandora, Bryant, Cantey, Elward, Yokoe and Bartlett7 Our objective was to assess the frequency of false-positive toxin EIA results using two commercial toxin EIAs in children without C. difficile carriage or infection.
Methods
The clinical microbiology laboratory at Lurie Children’s Hospital performs C. difficile testing on unformed stools from children at least 12 months old. Deidentified clinical stool specimens that tested negative by tcdB PCR (Xpert C. difficile, Cepheid, Sunnyvale, CA) were aliquoted and stored at −80°C for further testing. After a single freeze-thaw cycle, stools underwent 2 different C. difficile lateral-flow EIAs: Quik Chek Complete (QCC; Techlab, Blacksburg, VA), which detects both glutamate dehydrogenase (GDH) and toxins A and B, and ImmunoCard Toxins A&B (Meridian Biosciences, Cincinnati, OH), which detects toxins A and B only. The EIAs were used according to manufacturers’ instructions. Stools also underwent anaerobic stool culture, as previously described.Reference Kociolek, Patel, Shulman and Gerding8 Stool was cultured onto taurocholate-cycloserine-cefoxitin-fructose agar in an anaerobic chamber and incubated at 37°C for 48 hours. Clostridioides difficile colonies, identified based on typical morphology, were then transferred to trypticase soy broth and incubated anaerobically overnight. The broth culture was then centrifuged at 3,000×g for 10 minutes, the supernatant was passed through a 0.45-µm filter, and the filtrate was assessed for presence of GDH and toxin by QCC. Toxigenic strains of C. difficile were identified if culture filtrate was GDH and toxin positive, and nontoxigenic C. difficile strains were identified if culture filtrate was GDH positive and toxin negative.
Results
All 50 stools were confirmed as unformed (Bristol stool scale types 5–7). Of the 50 tcdB PCR-negative stools, 7 (14%; 95% confidence interval [CI], 0.07–0.26) were GDH positive by QCC, but zero of the 50 (0%; 95% CI, 0–0.07) stools tested positive for C. difficile toxins A and B by either QCC or ImmunoCard. C. difficile grew by anaerobic culture from 2 of 50 (4%) stools. Both culture-positive stools were GDH positive, and both C. difficile isolates were GDH positive and toxin negative, confirming that these were nontoxigenic C. difficile strains.
Discussion
In the present study of 50 children with diarrhea and without C. difficile carriage or infection (ie, stools tested negative for toxigenic C. difficile by a commercial tcdB PCR assay and confirmed negative by toxigenic stool culture), none tested positive for C. difficile toxins A or B using 2 different commercial toxin EIAs. The present study challenges the previously reported finding of frequent (36%) false-positive toxin EIA results in pediatric patients.Reference Toltzis, Nerandzic and Saade6
The reasons for these discordant findings are unknown. Although the prior studyReference Toltzis, Nerandzic and Saade6 performed EIA on fresh stools, the present study performed EIA on stools thawed once. Recommendations from the manufacturers of both EIAs used in the present study support reliable assay performance after a single freeze–thaw cycle. Differences in false-positive rates among different EIAs have been described.Reference Burnham and Carroll2 Our study used 2 different lateral-flow commercial EIAs (TechLab Quik Chek Complete and Meridian ImmunoCard Toxins A&B) that were both different than the microwell commercial EIA used in the previous study (Meridian Premier Toxins A&B).Reference Toltzis, Nerandzic and Saade6 There may be differences in false-positive rates among assays, particularly related to use of 2 distinct EIA methodologies between the present and previous studies (ie, lateral flow vs microwell, respectively). However, a comprehensive review of C. difficile diagnostic assays reported similarly infrequent rates of false positivity among these 3 assays.Reference Burnham and Carroll2 False-positive microwell EIA results may occur if the optical density used to define a positive result deviates from the manufacturer’s recommendation,Reference Burnham and Carroll2 but it is unclear whether that occurred in the previous study. Alternatively, the optical density defining a positive toxin result may not yet be appropriately validated in pediatric patients. Although the predominant specific type of toxin EIA used among pediatric hospitals is unknown, toxin EIAs in general, either as stand-alone tests or part of a multistep algorithm, are used at a minority of children’s hospitals.Reference Kociolek and Sandora9,Reference Kociolek, Kutty, Polgreen and Beekman10 Single-specimen assays, such as lateral flow EIAs as used in the present study, may be preferred because they do not require specimen batching to be cost-effective.
In summary, our study in children supports previous studies in adults that reported infrequent false positivity rates for various toxin EIAs.Reference Burnham and Carroll2 Our data challenge a previous pediatric study that anomalously reported that positive toxin EIA results are unreliable.Reference Toltzis, Nerandzic and Saade6 However, further work is required to understand the negative predictive value of toxin EIA results in children prior to recommending toxin EIAs as stand-alone tests for diagnosing CDI in pediatric patients.
Author ORCIDs
Larry K. Kociolek, 0000-0002-8756-3417
Acknowledgments
We thank the Lurie Children’s Clinical Microbiology Laboratory for their assistance with saving clinical stool specimens for this study.
Financial support
L.K.K. is supported by a grant from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (grant no. K23 AI123525).
Potential conflicts of interest
L.K.K. has previously received research supplies from Alere/Techlab but not for the current study. All authors report no other conflicts of interest relevant to this article.
