Repeated enzyme immunoassays have limited utility in diagnosing Clostridium difficile
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- Drees, M., Snydman, D.R. & O’Sullivan, C.E. Eur J Clin Microbiol Infect Dis (2008) 27: 397. doi:10.1007/s10096-007-0452-8
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Many clinical laboratories use enzyme immunoassays (EIA) to diagnose Clostridium difficile-associated disease (CDAD). Clinicians frequently order three EIAs to “rule out” CDAD. We performed a retrospective cohort study to determine the clinical utility of repeating EIA testing to diagnose CDAD. We reviewed all EIAs performed by our laboratory during 2005, determined the total number of tests per patient and per testing episode, and calculated the relative negative predictive value (NPV) of one EIA compared to ≥2 EIAs. The laboratory performed 2,938 EIAs, of which 253 (8.6%) tests were positive. Most patients (85%) were diagnosed by the first EIA performed. Of >1,000 testing episodes that included ≥2 EIAs within 7 days, only 15 patients had a positive second or third test after negative initial testing. The relative NPV of the first EIA was 97.4%. These data suggest that using newer generation EIAs, repeated testing is of limited benefit in diagnosing CDAD.
Recently noted trends toward higher incidence and increased severity of Clostridium difficile-associated disease (CDAD)[1, 2] have made rapid and accurate diagnosis imperative. The cellular cytotoxin assay (CCA), currently the accepted gold standard, has reported sensitivities that typically exceed 90% , but sensitivities as low as 67% have been reported . Many clinical laboratories now use enzyme immunoassays (EIA) exclusively. These tests are rapid, less expensive, and technically easy to perform, but have variable sensitivities (<60% to 99%) [4, 5]. Similar to the use of sputum analysis in diagnosing tuberculosis, many clinicians frequently order three EIAs to definitively “rule out” CDAD. However, there has been limited research evaluating the clinical performance of repeated diagnostic testing, particularly with the newer generations of EIAs [6, 7]. We conducted this retrospective cohort study to evaluate the clinical performance of the EIA used in our tertiary care academic medical center and to determine the diagnostic utility of repeating EIA testing for CDAD.
We reviewed all C. difficile EIAs performed by the microbiology laboratory during 2005 at Tufts-New England Medical Center (Boston, MA). The laboratory used the Premier Toxins A&B EIA (Meridian, Bioscience, Inc.), which has published sensitivities of 94.7–96.8% and negative predictive values of 98.9–99.8%, compared to the CCA [8, 9]. The test was performed according to the manufacturer’s directions, without any confirmatory testing. Laboratory policy included rejection of greater than two stool samples sent for C. difficile testing within a 5-day period, but was not strictly enforced. For all patients with positive EIAs, the electronic medical record was reviewed to confirm the clinical diagnosis and retrieve demographic information, comorbidities and prior history of CDAD. Repeat EIAs performed within 7 days of initial testing were considered part of a single testing episode, while EIAs performed after 7 days were considered a new testing episode, assuming that sufficient time had elapsed for new exposure to C. difficile. Because we were most interested in the utility of repeated EIAs rather than in comparing the EIA to a gold standard [from which we could calculate a negative predictive value (NPV)], we calculated a “relative” NPV for the first EIA performed compared to multiple EIAs. We defined true negatives as patients who had at least two EIAs performed and who were never diagnosed with CDAD by laboratory testing.
Frequency of EIA testing in patients with and without positive test results
Patients with any positive EIA
Patients without any positive EIAs
N = 189
N = 1,358
Total number of tests performed per patient*
Testing episodes in patients with positive EIAs†
Testing episodes in patients without any positive EIAs
N = 217
N = 2,010
Number of tests performed within each testing episode
Few studies have examined the common practice of repeating diagnostic testing for C. difficile. Several have found little value in repeating testing using the CCA [10, 11], but the number of EIAs required to definitively rule out CDAD has not been studied using more recently developed assays. A 1995 study  found that repeating EIA testing increased yield by 12%, but a more recent study  found that of 396 patients with negative initial EIA results, only 1 of 78 patients who underwent repeat EIA testing had a positive test result. Our study found multiple testing within 7 days to be extremely common, but the yield of these repeated tests was low, identifying only 15 additional patients from more than 1,000 patients tested at least twice within 7 days.
This study is subject to certain limitations. The clinical suspicion for CDAD when the tests were ordered is unknown. We were unable to detect patients with CDAD but without a positive EIA result who were treated empirically. We assumed that positive EIAs indicated CDAD, although this may have overestimated cases of disease. However, we did review the medical record of all patients with positive EIAs to confirm that CDAD was also clinically diagnosed. Our microbiology laboratory did not routinely perform confirmatory testing, so a gold standard with which to calculate sensitivity, specificity and negative predictive value was not available. Instead, we calculated the relative NPV of a first negative EIA compared to multiple EIAs. While this does not answer the question of EIA sensitivity, it is useful for clinicians whose microbiology laboratories perform EIA testing exclusively. However, the interpretation of this high relative NPV, and the implications of negative initial testing, will vary based on clinical suspicion of CDAD and local disease prevalence and virulence.
Most cases of CDAD in our institution were diagnosed by the first EIA performed. We found the relative NPV of a single EIA, compared to multiple EIAs, to be quite high (97%). For difficult-to-diagnose infectious diseases such as tuberculosis, repeated testing is recommended to increase diagnostic yield . We found that repeating the EIA was of little benefit in diagnosing CDAD, as few of these repeat tests were positive. This suggests either increased sensitivity of newer generation EIAs or that an alternate testing modality in addition to EIA should be used, as has been suggested [4, 13]. Avoidance of unnecessary repeat diagnostic testing for CDAD is desirable for several reasons, including the cost of additional tests as well as costs and adverse events related to empiric antibiotic use and patient isolation while awaiting these additional tests. We believe that our results support the practice of repeating diagnostic testing only when the clinical suspicion for CDAD is relatively high, and then performing no greater than two EIAs within a 7-day period. While further study of this issue is needed, repeating EIA testing for CDAD may be unnecessary for many patients.
Dr. Drees was supported by NRSA grant 5 T32 HS 000060–13. There was no external funding for this study.
The authors would like to thank Linda Perry, Director, Tufts-New England Medical Center Microbiology Laboratory, for her assistance with this study.