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Frequency of Sample Submission for
Optimal Utilization of the Cell Culture
Cytotoxicity Assay for Detection of
Clostridium difficile Toxin
Anita P. Borek, Deborah Z. Aird and Karen C. Carroll
J. Clin. Microbiol. 2005, 43(6):2994. DOI:
10.1128/JCM.43.6.2994-2995.2005.
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JOURNAL OF CLINICAL MICROBIOLOGY, June 2005, p. 2994–2995
0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.6.2994–2995.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 6
Frequency of Sample Submission for Optimal Utilization of the Cell
Culture Cytotoxicity Assay for Detection of Clostridium
difficile Toxin
Anita P. Borek,* Deborah Z. Aird, and Karen C. Carroll
Division of Microbiology, Department of Pathology, The Johns Hopkins Medical Institutions, Meyer B1-193,
600 N. Wolfe St., Baltimore, Maryland 21287
Received 17 November 2004/Returned for modification 20 January 2005/Accepted 16 February 2005
Clostridium difficile is a leading cause of diarrhea in patients
who have received antimicrobials and chemotherapeutic treatments. In the era of shrinking laboratory resources, the timely,
accurate diagnosis of C. difficile continues to be a challenge. A
variety of testing options are available. These include anaerobic culture on selective media, direct toxin testing for either
toxin A alone or both toxins A and B, detection of C. difficile
antigen with subsequent toxin testing of positive samples, and
finally nucleic acid amplification techniques. Each of these
methods has advantages and disadvantages (1–4, 7, 9).
Direct toxin testing is the most frequently used approach.
Cell culture systems that are sensitive to the effects of toxin
followed by neutralization of cytopathic effect have been the
gold standard. Analytical sensitivity of the cell culture cytotoxicity assay (CCCA) is as low as 10 pg of toxin (4). Such testing
requires the availability of cell culture lab facilities and skilled
personnel. In addition, this system is labor-intensive and may
require up to 72 h for a final result. For these reasons, many
labs have abandoned CCCA in favor of enzyme immunoassays
(EIAs). In general, EIAs are less sensitive than CCCA (2–4, 7).
A previous publication from this tertiary-care, urban medical center reported the value in testing multiple samples from
patients when an EIA is used (5). In the attempt to overcome
perceived suboptimal recovery, the laboratory received two,
three, or more samples per week. After a series of problems
related to consecutive use of two commercial toxin A/B EIAs
in our laboratory, a decision was made to return to testing with
an in-house CCCA previously reported to be more sensitive
than an EIA method (5). The purpose of this study was to
determine the utility of multiple sample submissions when a
CCCA is used.
CCCA was performed using a modified version of an inhouse procedure (5, 6). In brief, stool specimens were diluted
1:5 in sterile phosphate-buffered saline. Specimens were mixed
by vortexing and were centrifuged at 4,000 rpm at 4°C for 30
min. Supernatants were filter sterilized through an 0.45-␮m
membrane filter (Millipore, Billerica, MA). Dilutions of the
stool filtrates and positive control toxin (1:2 and 1:10) were
prepared with sterile phosphate-buffered saline and antitoxin.
Twenty microliters of each dilution of control toxin, control
toxin plus antitoxin, stool filtrate, and stool filtrate plus antitoxin was added to duplicate wells of microtiter plates containing confluent human foreskin fibroblast cells (Diagnostic Hybrids, Inc., Athens, OH). Thus, the final dilutions tested were
1:60 and 1:300. The plates were incubated at 37°C in 5% CO2
and were read for cytopathic effect at 24 and 48 h. Dilutions of
toxin control reagent mixed with buffer were observed for the
expected cell rounding in the 1:60 and 1:300 dilutions. Dilutions of toxin control reagent mixed with antitoxin were observed for neutralization in the 1:60 and 1:300 dilutions. In
mixtures containing dilutions of fecal filtrate plus buffer, ⱖ50%
cell rounding indicated the presence of cytotoxic activity. If this
was neutralized in dilutions of fecal filtrate plus antitoxin, the
presence of C. difficile toxin was confirmed. All consecutive
stool samples with sufficient volume were accepted for testing
7 days per week.
All CCCA results over a 3-month period were reviewed for
multiple requests within 7 days. All data were entered into
EXCEL and analyzed using STATA version 7.0.
There were 2,940 samples tested from 670 patients. Overall,
there were 219 positives from 141 different patients for a sample positivity rate of 7.4%. In 1,101 instances a second sample
from the same patient was submitted within 7 days. Of those
1,101 instances, two consecutive samples were negative for
1,063 specimens and in 38 instances both were positive. The
first two samples were 100% concordant (Table 1). Two hundred forty-seven patients were tested using a third sample
during the same week. For 245 of these 247 patients, results of
the third sample matched the results of the initial two samples.
Two hundred thirty-eight patients were negative, and nine
were positive. Of the nine positive samples, two were first-time
positives. Thus, 2 of the 247 patients (0.8%) had a third sample
whose result was positive, compared to the initial two samples,
whose results were negative in the same week. Chart review
revealed that neither patient was treated. One patient had a
* Corresponding author. Mailing address: Johns Hopkins Hospital,
Meyer Building, Room B1-193, 600 N. Wolfe St., Baltimore, MD
21287-7093. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail:
aborek1@jhmi.edu.
2994
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We reviewed the results of repeated sample submissions within a 7-day time frame for Clostridium difficile
toxin testing. A total of 2,940 samples were tested during a 3-month period using a cell culture cytotoxicity
assay (CCCA). The results from all second samples (n ⴝ 1,101) were concordant with the original test result.
In only two cases (0.8%; n ⴝ 247) was a third sample positive when the first two samples were negative. In this
study, submission of multiple samples for CCCA did not increase detection of Clostridium difficile infection.
VOL. 43, 2005
NOTES
TABLE 1. Result concordance on repeated sample submissions
No. of samples
Result
Second sample
Third sample
Positive
Negative
38
1,063
7a
238
Total
1,101
245
a
Two additional samples were first-time positives representing discordant
results. See text for discussion and patient details.
patients submitted for C. difficile toxin testing using CCCA,
36% represented repeated tests. New information was provided in only 0.5 to 0.8% of cases (8). The authors of that study
likewise concluded that repeat CCCA should not be performed
within 7days of the initial test (8).
The practice of sending multiple C. difficile toxin test samples is not warranted when CCCA is used. In our institution,
such behavior likely arose from use by the laboratory of a less
sensitive and less reproducible method for C. difficile toxin
detection and data that supported the value of repeated testing
when an EIA method was used (5). In addition, some of our
clinicians had the mistaken perception that three negative C.
difficile toxin results were required for nursing home placement. Compliance with a policy of rejection of duplicate samples within a 7-day period has been successful with presentation of the above data to medical staff and clarification of state
rules and regulations regarding nursing home placement of C.
difficile-positive patients.
We thank Richard Thompson for his assistance with the cost-effectiveness data analysis.
REFERENCES
1. Bartlett, J. G. 2002. Antibiotic associated diarrhea. N. Engl. J. Med. 346:334–
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2. Fekety, R. 1997. Guidelines for the diagnosis and management of Clostridium
difficile-associated diarrhea and colitis. Am. J. Gastroenterol. 92:739–750.
3. Gerding, D. N., S. Johnson, L. R. Peterson, M. E. Mulligan, and J. Silva. 1995.
Clostridium difficile-associated diarrhea and colitis. Infect. Control Hosp. Epidemiol. 16:459–477.
4. Johnson, S., and G. N. Gerding. 1998. Clostridium difficile-associated diarrhea.
Clin. Infect. Dis. 26:1027–1036.
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Bartlett. 1995. Clostridium difficile colitis: an efficient clinical approach to
diagnosis. Ann. Intern. Med. 123:835–840.
6. Merz, C. S., C. Kramer, M. Forman, L. Gluck, K. Mills, K. Senft, I. Steiman,
N. Wallace, and P. Charache. 1994. Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens. J. Clin. Microbiol. 32:1142–
1147.
7. Mylonakis, E., E. T. Ryan, and S. B. Calderwood. 2001. Clostridium difficileassociated diarrhea. A review. Arch. Intern. Med. 161:525–533.
8. Renshaw, A. A., J. M. Stelling, and M. H. Doolittle. 1996. The lack of value of
repeated Clostridium difficile cytotoxicity assays. Arch. Pathol. Lab. Med.
120:49–51.
9. Stoddard, B., and M. H. Wilcox. 2002. Clostridium difficile. Curr. Opin. Infect.
Dis. 15:513–518.
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gastrointestinal hemorrhage related to a polyp. Sigmoidoscopy, performed to evaluate the bleeding, did not reveal
pseudomembranous colitis or other mucosal abnormalities.
The second patient had diarrhea postoperatively, but there was
no mention of suspicion of antibiotic-associated diarrhea.
Rejection of the third or more sample per week would decrease the annual volume by 1,080 tests. The supply expense
per test is $13.00, resulting in a $14,040 savings. The technologist time per test is 35 min, resulting in a labor saving of 630 h.
Testing one sample per week would decrease the annual volume by 4,404, saving $57,252 in supply expense and 2,569 h of
labor.
During this time, there were 37% repeat samples. As these
data show, when the CCCA is used, there is reasonable certainty of the accuracy of the result from the first sample tested.
Testing a second sample within 7 days did not yield a different
result in any instance. Submission of a third sample resulted in
detection of a first positive result in only two cases (0.8%).
Current practice guidelines recommend submission of additional specimens for C. difficile toxin if a single sample is negative and clinical suspicion is high (2, 3). Few papers, however,
have analyzed the benefit or cost-effectiveness of such recommendations. An initial study from our institution demonstrated
increased sensitivity of C. difficile detection with submission of
a second sample at a time when the lab was using an insensitive
EIA method (5). The results of the current study, using a more
sensitive CCCA method, are similar to those reported by Renshaw et al. (8). In a study of 4,238 specimens from 2,009
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