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Rapid detection of Clostridium difficile in stool using the VIDASRC. difficile Toxin A II assay
Diagnostic Microbiology and Infectious Disease 45 (2003) 117–121
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Rapid detection of Clostridium difficile in stool using the VIDASR C. difficile Toxin A II assay S.M. Lipsona* , G. Tortoraa, A. Temponea, D.P. Fedorkob, E.D. Spitzera a
Clinical Microbiology Laboratory, Department of Laboratories, SUNY at Stony Brook, Stony Brook, NY, USA b Infectious Diseases Laboratory, NIH, Bethesda, MD, USA Received 29 August 2002; accepted 23 October 2002
A rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is important in patient management and in the administration of appropriate therapeutic modalities. The VIDASR C. difficile Toxin A II (CDA 2) assay (bioMerieux, Inc., Hazelwood, MO) was compared with the cell culture cytotoxicity assay (CCA) for the rapid detection of C. difficile in stool from patients in whom C. difficile infection was suspected. Thirty-eight consecutively collected CCA-positive stool specimens, and 33 CCA-negative stool specimens were tested by the CDA 2 assay. Where appropriate, discordant specimens were repeated and/or tested by isolation utilizing cycloserine-cefoxitinfructose agar (CCFA). Among 12 discordant stool specimens, 7 were VIDASR-/cytotoxicity⫹, 2 were VIDASR equivocal (E)/cytotoxicity⫹, 2 were VIDASR E/cytotoxicity⫺, and 1 was VIDASR⫹/cytotoxicity⫺. One VIDASR E/cytotoxicity⫹ lacked sufficient stool to be repeated. From the single VIDASR⫹/cytotoxicity⫺ specimen, C. sordelli was isolated. Specimens that were equivocal by VIDASR, were omitted from incorporation into this study’s test parameters. The sensitivity, specificity, positive and negative predictive values for the CDA 2 assay were 80.6, 96.8, 96.7, and 81.1%, respectively. The specimens which yielded false negative VIDASR results had low levels of toxin based on endpoint titrations using the cytotoxicity assay. Although the CDA 2 assay displayed a reduced sensitivity compared with the CCA, the automated assay is rapid (results promulgated within 2 h), with computer generated readings obviating visual interpretations. Recognition of the CDA 2 assay’s limitations is important to addressing this test’s clinical utility. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Clostridium difficile is a Gram-positive, spore forming, anaerobic bacillus. This nosocomially transmitted microorganism is responsible for virtually all cases of pseudomembranous colitis and up to 20% of the cases of antibioticassociated diarrhea with colitis. 300,000 to 3,000,000 cases of diarrhea and colitis, due to C. difficile infections, occur annually in the United States (Kelly et al., 1994; Mylonakis et al., 2001). If left untreated, the infection can be fatal. The administration of antibiotics (e.g., cephalosporin group, ampicillin, amoxicillin, clindamycin) for other infections may upset the ecology of the intestinal microbiota, thereby allowing for the growth of toxigenic strains of C. difficile. The disease caused by C. difficile is due to two toxins produced by this microorganism. Toxin A, an enterotoxin, is considered to cause most of the symptoms associated with * Corresponding author. Virology Consultants, Inc., 2364 East 74th Street, Brooklyn, NY 11234, USA. Tel.: ⫹1-718-209-3662; fax: ⫹1-718209-3662. E-mail address: [email protected] (S.M. Lipson).
C. difficile disease. Toxin B is referred to as a cytotoxin, due to its potent cytotoxic (rounding) activity in cell culture. The genes for both toxins are located within a 19 kb “pathogenicity locus” and are coordinately expressed (Johnson et al., 2001). Both toxins A and B are produced during the disease process, and are considered to work synergistically. Strains that harbor a deletion in the toxin A gene and only produce toxin B are uncommon, but cause severe disease (Mani and Dupuy, 2001). The cytotoxicity (cytotoxin) assay alone, or in combination with toxigenic culture, is usually considered the “gold standard” for the diagnosis of C. difficile-associated diarrhea (Alfa et al., 2002; Bartlett, 2002; Fedorko et al., 1999). Performance of toxigenic culture however, is tedious and requires several days to complete. The cytotoxicity assay accordingly, is used in many laboratories for the identification of C. difficile toxin in stool. Notwithstanding, the cytotoxicity assay in itself lacks rapidity, and requires the maintenance of living cells; Interpretation of the cytopathic effect can be subjective. To address these issues, manufacturers have developed numerous enzyme immunoassay
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(EIA) and latex agglutination (LA) assays for the detection of C. difficile toxin A and/or B in stool. These assays have been reported to have sensitivities ranging from 62 to 90% (Barbut et al.,1993; Fedorko et al., 1999; Vargas et al., 1997; Jacobs et al., 1996; Patel et al., 2001). The VIDASR C. difficile Toxin A II (CDA 2) enzyme-linked fluorescent immunoassay is one of the few C. difficile assays that can be performed on an automated instrument, and is currently used in many laboratories (Anonymous, 2002). The purpose of this study, was to evaluate the VIDASR system, using the mini-VIDASR instrument in comparison with the cytotoxicity assay, for the detection of C. difficile in stool specimens.
2. Materials and methods 2.1. Stool specimens Thirty-eight consecutive cytotoxicity-positive stool specimens and 32 randomly selected negative stool specimens that were submitted at the same time as the positive specimens, were included in this study. Most of the specimens were tested by the CDA 2 assay within 24-h after completion of the cytotoxicity assay. CDA 2 discordant specimens were stored for a maximum of 3 to 5 days at 4°C, for repeated testing by the CDA 2 and the CCA. Several specimens were stored at ⫺20°C for subsequent resolution testing. Preliminary testing showed that the storage of stool at 4°C for several days or 1 freeze/thaw cycle, had no effect on specimen potency following inoculation into fibroblast culture. Stool specimens were obtained from patients for whom a C. difficile cytotoxicity test had been ordered as part of their routine care. All specimens were watery or of loose consistency. Most of the specimens were obtained from hospitalized adults at the SUNY University Hospital at Stony Brook. A few specimens were obtained from adults and children seen in outpatient clinics. 2.2. Cytotoxicity (cytotoxin) assay (CCA) The CCA was performed according to manufacturer’s instructions (Anonymous, 1999). Briefly, stool specimens were diluted 1:2 in phosphate-buffered saline, centrifuged, followed by filtration. Equal parts of stool filtrate and working strength antitoxin (C. difficile Toxin/Antitoxin Kit, Tech Lab, Blacksburg, VA) and sterile PBS were incubated for a period of 30 min., followed by inoculation into human lung fibroblast tube cultures (MRC-5; BioWhittaker, Walkersville, MD). After 18-24 and again after 48 h, tube cultures were microscopically visualized for characteristic cytotoxic activity (cell rounding; Fig. 1). Stool filtrates that caused cell rounding in at least 50% of the monolayer that was neutralized by C. difficile antitoxin, were considered “positive for C. difficile toxin.” Monolayers suspected of dis-
playing a C. difficile-induced cytotoxicity, were examined again after 72 h. Positive (toxin control reagent) and negative (PBS) controls were performed in parallel with each run. The Techlab C. difficile Toxin/Antitoxin kit is intended for the laboratory confirmation of C. difficile toxins A and B. 2.3. Semi-quantitation of C. difficile toxin Toxin was extracted from six CCA⫹/VIDAS⫺ specimens as described above. Each filtrate was serially diluted in PBS from 10⫺1 to 10⫺6, and inoculated in triplicate into MRC-5 tube cultures. Tube cultures were monitored for the development of cytotoxicity for a maximum period of 72-h (see Fig. 1). The cytotoxic dose-fifty TD50) was calculated according to the standard end point assay procedure (Lipson, 1992). 2.4. Toxigenic culture The toxigenic culture assay was performed as described previously (Fedorko et al., 1999). Briefly, spores for C. difficile were selected for by alcohol treatment. The alcoholtreated stool specimens were inoculated onto CDC anaerobe blood agar (Remel, Lexena, KS) and anaerobically reduced cycloserine-cefoxitin-fructose agar (CCFA) (Anaerobe Systems, San Jose, CA). Colonies on CCFA suspected of being C. difficile, were identified using the PRO Disc (Carr Scarborough Microbiologicals, Decatur, GA) as previously described (Fedorko and Williams, 1997). Toxin production of the C. sordelli isolate was performed using the Bartels cytotoxin assay and the Wampole C. difficile toxin A ⫹ B ELISA. 2.5. VIDASR C. difficile Toxin A II (CDA 2) Testing by the CDA 2 was performed according to the manufacturer’s specifications (Anonymous, 2001). Briefly, sample diluent was added to standard, positive, negative, and mixed stool specimens, followed by vortexing and high speed centrifugation and use of the supernatants in the mini-VIDAS CDA 2 assay system. The processed standard, control and sample supernatants were inoculated into sample wells of the Toxin A II Reagent Strips. Reagent Strips and the Solid Phase Receptacles (SPRs) were loaded into appropriate instrument section positions. Upon entry of assay code CDA 2, all further steps were executed automatically by the instrument. The CDA 2 assay detects C. difficile enterotoxin A. The CDA 2 assay was performed using coded specimens. At the completion of this assay, the specimen codes were broken. Retesting of stool specimens by the CDA 2 assay, the cytotoxicity assay, and toxigenic culture, was performed on coded specimens as well.
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Fig. 1. Characteristic MRC-5 monolayer degeneration observed in the cytotoxicity assay. a, Uninoculated (control) monolayer; b, 24-h post inoculation of C. difficile-containing toxic stool specimen (100⫻).
2.6. Statistics and resolution of discordant specimens Sensitivity (Sen), specificity (Spec), positive (PPV) and negative predictive values (NPV) were calculated according to standard procedures (Lipson and Zelinsky-Papez, 1989). Discordant specimens were repeated by the CCA and the CDA 2 assays. CCA positive specimens were considered true positives. CCA negative and VIDASR negative specimens (using the CDA 2 assay) were considered true negatives. A single VIDASR positive but CCA negative specimen was resolved by toxigenic culture testing. Toxigenic culture was used only in the resolution of the VIDASR positive/CCA negative specimen.
3. Results During the study period, 450 stool specimens were tested by CCA. Thirty-eight consecutive positive and 33 concurrent negative specimens were tested by both CCA and VIDASR. Fifty-nine of 71 (83%) stool specimens were in accord by both assays.
Among 12 discordant specimens (7 VIDASR⫺/CCA⫹, 2 VIDASR equivocal (E)/CCA⫹, 2 VIDASR E/CCA⫺, 1 VIDASR⫹/CCA⫺), 8 specimens were resolved by repeated and/or additional testing (Table 1). The four specimens that gave equivocal VIDASR results, including one specimen which lacked sufficient stool for repeated testing by both CCA and VIDASR, were not included in the calculations of test performance. Parenthetically, retesting of the four available stool specimens in question by the CDA 2 assay failed to yield a definitive C. difficile laboratory diagnosis. Repeated testing of 3 by CCA, similarly resulted in nonchanged results. Seven VIDASR⫺/CCA⫹ positive specimens were deemed VIDASR false negatives. C. sordelli was isolated from the single VIDASR⫹/CCA⫺ specimen. This result was deemed a VIDASR false positive reaction. Cytotoxicity testing and the testing of the C. sordelli isolate by C. difficile toxin A ⫹ B ELISA, were negative. After resolution testing, the CDA 2 assay revealed sensitivity, specificity, positive, and negative predictive values of 80.6, 96.8, 96.7, and 81.1%, respectively (Table 2). Among 5 VIDASR⫺/CCA⫹ discordants, the VIDASR false negative specimens displayed cytotoxicity end point titers of ⱕ10 (Table 1).
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Table 1 Resolution of discordant specimens Specimen No.a
CCA
CDA 2
TD50
Interpretation
1 2 3 4 5 6 7 8 9 10
⫹b ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺c ⫹ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺
nd ⬍10 ⬍10 ⬍10 ⬍10 n 10 n 1 ⫻ 103 n
FNe FN FN FN FN FN FN FPf Pos. controlg Neg. controlh
a CCA and CDA 2 testing were repeated on refrigerated or frozen stool specimens among negative, positive and equivocal (CDA 2) signals; Assay signals remained unchanged. Four CDA 2 specimens displaying equivocal signals (TV readings: ⱖ0.40 to ⬍1.0) were not incorporated into this study’s test parameters (data not shown). b ⫹, positive assay/signal. c ⫺, negative assay/signal. d n, not done. e FN, false negative; CDA 2 test value (TV) threshold readings ⫽ ⬍0.40. f FP, VIDAS false positive signal (TV threshold reading ⫽ 1.23); Isolation of C. sordelli. g TV reading ⫽ 2.6. h TV reading ⫽ 0.03.
4. Discussion The CCA is an excellent test for the laboratory identification of C. difficile-associated diarrhea. However, the assay mandates a 24 to 48 hour (or longer) incubation period, and the test may be subject to interpretive differences between some laboratory technologists. The CDA 2 assay is rapid (e.g., ca., 90 min to complete), and results are analyzed by the instrument’s computer. Results are printed for each sample. The CDA 2 assay however, is less sensitive than CCA (viz., 80.6% vs.100%, respectively). Failure of the VIDASR CDA 2 assay to detect C. difficile enterotoxin A may be ascribed to low levels of toxin A in the 7 stool specimens in question. Determination of cyto-
Table 2 Determination of VIDASR test parametersa
toxic dose-fifty (TD50) endpoints supports this contention. Infection with toxin A negative/toxin B positive (toxA⫺/ toxB⫹) strains of C. difficile have been reported (Moncrief et al., 2000). These strains would be missed by an EIA that detects toxin A, only. It should be pointed out however, that toxA⫺/toxB⫹ species are rarely encountered in the routine clinical setting (Halstead et al., 2002). The sole CDA 2 false positive signal was due to the presence of C. sordelli. C. sordelli is recognized to cross react with the rabbit antitoxin A component in the CDA 2 assay (Toma et al., 1999). A failure to resolve VIDASR equivocal results by repeated testing of originally submitted specimens, remains problematic. Collection of additional specimens among those patients in question, may result in interpretatable data permitting a laboratory diagnosis of C. difficile-associated diarrhea. The CDA 2 assay has an advantage of a rapid turnaround time. The early submission and then screening of stool specimens using the CDA 2 assay would alert the clinician within hours that patients were infected. Recognition by the medical staff of the assay’s false negative rate would be required to initiate upon request, testing of the same specimen by the CCA to attain a definitive laboratory diagnosis. Because of the relatively low prevalence of C. difficile infection (ca., 10%) in our hospital setting, CCA testing of all VIDASR-negative specimens would be impractical. In summary, the bioMerieux mini-VIDASR CDA 2 assay may be completed within 2 h of specimen receipt. However, the assay is less sensitive and slightly less specific than CCA. The CDA 2 assay eliminates interpretive differences which might occur between laboratory personnel. Some clinicians may accept the CDA 2 assay’s reduced sensitivity rate (compared with the CCA) as an initial screening procedure. Negative specimens or selected patients may be retested upon physician request by the 24 – 48 h, but more sensitive CCA.
Acknowledgments The authors appreciate H. P. Lipson, for proofreading the manuscript.
References
R
VIDAS
Positive C C
Negative
Equivocal
Positive
29b
7
2
Negative
1
30
2
A a The VIDASR CDA 2 system was tested in parallel with the cytotoxicity assay. b Sen: 29/36 ⫽ 80.6%; Spec: 30/31 ⫽ 96.8%; PPV: 29/30 ⫽ 96.7; NPV: 30/37 ⫽ 81.1%.
Alfa, M. J., Swan, B., VanDekerhove, B., Pang, P., & Harding, G. K. (2002). The diagnosis of Clostridium difficile-associated diarrhea: Comparison to triage C. difficile panel, EIA for Tox A/B and cytotoxin assays. Diagn Microbiol Infect Dis, 43, 257–263. Anonymous. (1999). Package Insert RMS #93-045-00 (rev. 08/11/1999). Clostridium difficile Toxin/Antitoxin Kit. A Toxin/Antitoxin Kit for the Detection of C. difficile toxin in Clinical Specimens. TechLab, Blacksburg, VA. Anonymous. (2001). Package Insert No. 018820-7 (rev. 01/01). VIDASR C. difficile Toxin a II (CDAR 2). bioMerieux, Inc., Hazelwood, MA. Anonymous. (2002). Participant Summary Report: Bacteriology D-A. College of American Pathologists. Northfield, IL.
S.M. Lipson et al. / Diagnostic Microbiology and Infectious Disease 45 (2003) 117–121 Barbut, F., Kajzer, C., Planas, N., & Petit, J. C. (1993). Comparison of three enzyme immunoassays, a cytotoxicity and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea. J Clin Microbiol, 31, 963–967. Bartlett, J. G. (2002). Antibiotic-associated diarrhea. N Engl J Med, 346, 334 –339. Fedorko, D. P., & Williams, E. C. (1997). Use of cycloserine-cefoxitinfructose agar and L-proline-aminopeptidase (PRO disc) in the rapid identification of Clostridium difficile. J Clin Microbiol, 35, 1258 –1259. Fedorko, D. P., Engler, H. D., O’Shaughnessy, E. M., Williams, E. C., Reichelderfer, C. J., & Smith, W. I. Jr. (1999). Evaluation of two rapid assays for detection of Clostridium difficile toxin A in stool specimens. J Clin Microbiol, 37, 3044 –3047. Halstead, D. C., Abid, J., Gomez, N., & Gordon, M. (2002). Multicenter comparison of five assays for detection of Clostridium difficile. 102nd Gen. Mtg., Am. Soc. Microbiol. C-183, p. 132. Jacobs, J., Rudensky, B., Dresner, J., Berman, A., Sonnenblick, M., van Dijk, Y., & Yinnon, A. M. (1996). Comparison of four laboratory tests for diagnosis of Clostridium difficule-associated diarrhea. Eur J Clin Microbiol Infect Dis, 15, 561–566. Johnson, S. S., Kent, A., O’Leary, K. J., Merrigan, M. M., Sambol, S. P., Peterson, L. R., & Gerding, D. N. (2001). Fatal pseudomembranous colitis associated with a variant Clostridium difficile strain not detected by toxin A immunoassay. Ann Intern Med, 135, 434 – 438. Kelly, C. P., Pothoulakis, C., & LaMont, J. T. (1994). Clostridium difficile colitis. N Engl J Med, 330, 257–262.
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Lipson, S. M. (1992). The Neutralization Test. In: H. D. Isenberg (Ed), Clinical Microbiology Procedures Handbook (CMPH). Washington, DC: American Society of Microbiology, 8.14.1– 8.14.8. Lipson, S. M., & Zelinsky-Papez, K. (1989). Comparison of four latex agglutination and three enzyme-linked immunosorbent assays for the detection of rotavirus in fecal specimens. Am J Clin Pathol, 92, 637– 642. Mani, N., & Dupuy, B. (2001). Regulation of toxin synthesis in clostridium difficile by an alternative RNA polymerase sigma factor. Proc Natl Acad Sci USA, 98, 5844 –5849. Moncrief, J. S., Zheng, L., Neville, L. M., & Lyerly, D. M. (2000). Genetic characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates by polymerase chain reaction. J Clin Microbiol, 38, 3072–3075. Mylonakis, E., Ryan, E. T., & Calderwood, S. B. (2001). Clostridium difficile-associated diarrhea: a review. Arch Intern Med, 161, 525–533. Patel, J. B., Donahue, A. M., & Nachamkin, I. (2001). Performance of two rapid, single-use immunoassays for the detection of Clostridium difficile toxin A. Diagn Microbiol Infect Dis, 39, 21–24. Toma, C., Nakamura, S., Kamiya, S., Nakasone, N., & Iwanaga, M. (1999). Detection of Clostridium difficile toxin A by reversed passive latex agglutination. Microbiol Immunol, 43, 773–742. Vargas, S. O., Horensky, D., & Onderdonk, A. B. (1997). Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A. J Clin Pathol, 50, 996 –1000.
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Rapid detection of Clostridium difficile in stool using the VIDASR C. difficile Toxin A II assay S.M. Lipsona* , G. Tortoraa, A. Temponea, D.P. Fedorkob, E.D. Spitzera a
Clinical Microbiology Laboratory, Department of Laboratories, SUNY at Stony Brook, Stony Brook, NY, USA b Infectious Diseases Laboratory, NIH, Bethesda, MD, USA Received 29 August 2002; accepted 23 October 2002
A rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is important in patient management and in the administration of appropriate therapeutic modalities. The VIDASR C. difficile Toxin A II (CDA 2) assay (bioMerieux, Inc., Hazelwood, MO) was compared with the cell culture cytotoxicity assay (CCA) for the rapid detection of C. difficile in stool from patients in whom C. difficile infection was suspected. Thirty-eight consecutively collected CCA-positive stool specimens, and 33 CCA-negative stool specimens were tested by the CDA 2 assay. Where appropriate, discordant specimens were repeated and/or tested by isolation utilizing cycloserine-cefoxitinfructose agar (CCFA). Among 12 discordant stool specimens, 7 were VIDASR-/cytotoxicity⫹, 2 were VIDASR equivocal (E)/cytotoxicity⫹, 2 were VIDASR E/cytotoxicity⫺, and 1 was VIDASR⫹/cytotoxicity⫺. One VIDASR E/cytotoxicity⫹ lacked sufficient stool to be repeated. From the single VIDASR⫹/cytotoxicity⫺ specimen, C. sordelli was isolated. Specimens that were equivocal by VIDASR, were omitted from incorporation into this study’s test parameters. The sensitivity, specificity, positive and negative predictive values for the CDA 2 assay were 80.6, 96.8, 96.7, and 81.1%, respectively. The specimens which yielded false negative VIDASR results had low levels of toxin based on endpoint titrations using the cytotoxicity assay. Although the CDA 2 assay displayed a reduced sensitivity compared with the CCA, the automated assay is rapid (results promulgated within 2 h), with computer generated readings obviating visual interpretations. Recognition of the CDA 2 assay’s limitations is important to addressing this test’s clinical utility. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Clostridium difficile is a Gram-positive, spore forming, anaerobic bacillus. This nosocomially transmitted microorganism is responsible for virtually all cases of pseudomembranous colitis and up to 20% of the cases of antibioticassociated diarrhea with colitis. 300,000 to 3,000,000 cases of diarrhea and colitis, due to C. difficile infections, occur annually in the United States (Kelly et al., 1994; Mylonakis et al., 2001). If left untreated, the infection can be fatal. The administration of antibiotics (e.g., cephalosporin group, ampicillin, amoxicillin, clindamycin) for other infections may upset the ecology of the intestinal microbiota, thereby allowing for the growth of toxigenic strains of C. difficile. The disease caused by C. difficile is due to two toxins produced by this microorganism. Toxin A, an enterotoxin, is considered to cause most of the symptoms associated with * Corresponding author. Virology Consultants, Inc., 2364 East 74th Street, Brooklyn, NY 11234, USA. Tel.: ⫹1-718-209-3662; fax: ⫹1-718209-3662. E-mail address: [email protected] (S.M. Lipson).
C. difficile disease. Toxin B is referred to as a cytotoxin, due to its potent cytotoxic (rounding) activity in cell culture. The genes for both toxins are located within a 19 kb “pathogenicity locus” and are coordinately expressed (Johnson et al., 2001). Both toxins A and B are produced during the disease process, and are considered to work synergistically. Strains that harbor a deletion in the toxin A gene and only produce toxin B are uncommon, but cause severe disease (Mani and Dupuy, 2001). The cytotoxicity (cytotoxin) assay alone, or in combination with toxigenic culture, is usually considered the “gold standard” for the diagnosis of C. difficile-associated diarrhea (Alfa et al., 2002; Bartlett, 2002; Fedorko et al., 1999). Performance of toxigenic culture however, is tedious and requires several days to complete. The cytotoxicity assay accordingly, is used in many laboratories for the identification of C. difficile toxin in stool. Notwithstanding, the cytotoxicity assay in itself lacks rapidity, and requires the maintenance of living cells; Interpretation of the cytopathic effect can be subjective. To address these issues, manufacturers have developed numerous enzyme immunoassay
0732-8893/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. S0732-8893(02)00520-5
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S.M. Lipson et al. / Diagnostic Microbiology and Infectious Disease 45 (2003) 117–121
(EIA) and latex agglutination (LA) assays for the detection of C. difficile toxin A and/or B in stool. These assays have been reported to have sensitivities ranging from 62 to 90% (Barbut et al.,1993; Fedorko et al., 1999; Vargas et al., 1997; Jacobs et al., 1996; Patel et al., 2001). The VIDASR C. difficile Toxin A II (CDA 2) enzyme-linked fluorescent immunoassay is one of the few C. difficile assays that can be performed on an automated instrument, and is currently used in many laboratories (Anonymous, 2002). The purpose of this study, was to evaluate the VIDASR system, using the mini-VIDASR instrument in comparison with the cytotoxicity assay, for the detection of C. difficile in stool specimens.
2. Materials and methods 2.1. Stool specimens Thirty-eight consecutive cytotoxicity-positive stool specimens and 32 randomly selected negative stool specimens that were submitted at the same time as the positive specimens, were included in this study. Most of the specimens were tested by the CDA 2 assay within 24-h after completion of the cytotoxicity assay. CDA 2 discordant specimens were stored for a maximum of 3 to 5 days at 4°C, for repeated testing by the CDA 2 and the CCA. Several specimens were stored at ⫺20°C for subsequent resolution testing. Preliminary testing showed that the storage of stool at 4°C for several days or 1 freeze/thaw cycle, had no effect on specimen potency following inoculation into fibroblast culture. Stool specimens were obtained from patients for whom a C. difficile cytotoxicity test had been ordered as part of their routine care. All specimens were watery or of loose consistency. Most of the specimens were obtained from hospitalized adults at the SUNY University Hospital at Stony Brook. A few specimens were obtained from adults and children seen in outpatient clinics. 2.2. Cytotoxicity (cytotoxin) assay (CCA) The CCA was performed according to manufacturer’s instructions (Anonymous, 1999). Briefly, stool specimens were diluted 1:2 in phosphate-buffered saline, centrifuged, followed by filtration. Equal parts of stool filtrate and working strength antitoxin (C. difficile Toxin/Antitoxin Kit, Tech Lab, Blacksburg, VA) and sterile PBS were incubated for a period of 30 min., followed by inoculation into human lung fibroblast tube cultures (MRC-5; BioWhittaker, Walkersville, MD). After 18-24 and again after 48 h, tube cultures were microscopically visualized for characteristic cytotoxic activity (cell rounding; Fig. 1). Stool filtrates that caused cell rounding in at least 50% of the monolayer that was neutralized by C. difficile antitoxin, were considered “positive for C. difficile toxin.” Monolayers suspected of dis-
playing a C. difficile-induced cytotoxicity, were examined again after 72 h. Positive (toxin control reagent) and negative (PBS) controls were performed in parallel with each run. The Techlab C. difficile Toxin/Antitoxin kit is intended for the laboratory confirmation of C. difficile toxins A and B. 2.3. Semi-quantitation of C. difficile toxin Toxin was extracted from six CCA⫹/VIDAS⫺ specimens as described above. Each filtrate was serially diluted in PBS from 10⫺1 to 10⫺6, and inoculated in triplicate into MRC-5 tube cultures. Tube cultures were monitored for the development of cytotoxicity for a maximum period of 72-h (see Fig. 1). The cytotoxic dose-fifty TD50) was calculated according to the standard end point assay procedure (Lipson, 1992). 2.4. Toxigenic culture The toxigenic culture assay was performed as described previously (Fedorko et al., 1999). Briefly, spores for C. difficile were selected for by alcohol treatment. The alcoholtreated stool specimens were inoculated onto CDC anaerobe blood agar (Remel, Lexena, KS) and anaerobically reduced cycloserine-cefoxitin-fructose agar (CCFA) (Anaerobe Systems, San Jose, CA). Colonies on CCFA suspected of being C. difficile, were identified using the PRO Disc (Carr Scarborough Microbiologicals, Decatur, GA) as previously described (Fedorko and Williams, 1997). Toxin production of the C. sordelli isolate was performed using the Bartels cytotoxin assay and the Wampole C. difficile toxin A ⫹ B ELISA. 2.5. VIDASR C. difficile Toxin A II (CDA 2) Testing by the CDA 2 was performed according to the manufacturer’s specifications (Anonymous, 2001). Briefly, sample diluent was added to standard, positive, negative, and mixed stool specimens, followed by vortexing and high speed centrifugation and use of the supernatants in the mini-VIDAS CDA 2 assay system. The processed standard, control and sample supernatants were inoculated into sample wells of the Toxin A II Reagent Strips. Reagent Strips and the Solid Phase Receptacles (SPRs) were loaded into appropriate instrument section positions. Upon entry of assay code CDA 2, all further steps were executed automatically by the instrument. The CDA 2 assay detects C. difficile enterotoxin A. The CDA 2 assay was performed using coded specimens. At the completion of this assay, the specimen codes were broken. Retesting of stool specimens by the CDA 2 assay, the cytotoxicity assay, and toxigenic culture, was performed on coded specimens as well.
S.M. Lipson et al. / Diagnostic Microbiology and Infectious Disease 45 (2003) 117–121
119
Fig. 1. Characteristic MRC-5 monolayer degeneration observed in the cytotoxicity assay. a, Uninoculated (control) monolayer; b, 24-h post inoculation of C. difficile-containing toxic stool specimen (100⫻).
2.6. Statistics and resolution of discordant specimens Sensitivity (Sen), specificity (Spec), positive (PPV) and negative predictive values (NPV) were calculated according to standard procedures (Lipson and Zelinsky-Papez, 1989). Discordant specimens were repeated by the CCA and the CDA 2 assays. CCA positive specimens were considered true positives. CCA negative and VIDASR negative specimens (using the CDA 2 assay) were considered true negatives. A single VIDASR positive but CCA negative specimen was resolved by toxigenic culture testing. Toxigenic culture was used only in the resolution of the VIDASR positive/CCA negative specimen.
3. Results During the study period, 450 stool specimens were tested by CCA. Thirty-eight consecutive positive and 33 concurrent negative specimens were tested by both CCA and VIDASR. Fifty-nine of 71 (83%) stool specimens were in accord by both assays.
Among 12 discordant specimens (7 VIDASR⫺/CCA⫹, 2 VIDASR equivocal (E)/CCA⫹, 2 VIDASR E/CCA⫺, 1 VIDASR⫹/CCA⫺), 8 specimens were resolved by repeated and/or additional testing (Table 1). The four specimens that gave equivocal VIDASR results, including one specimen which lacked sufficient stool for repeated testing by both CCA and VIDASR, were not included in the calculations of test performance. Parenthetically, retesting of the four available stool specimens in question by the CDA 2 assay failed to yield a definitive C. difficile laboratory diagnosis. Repeated testing of 3 by CCA, similarly resulted in nonchanged results. Seven VIDASR⫺/CCA⫹ positive specimens were deemed VIDASR false negatives. C. sordelli was isolated from the single VIDASR⫹/CCA⫺ specimen. This result was deemed a VIDASR false positive reaction. Cytotoxicity testing and the testing of the C. sordelli isolate by C. difficile toxin A ⫹ B ELISA, were negative. After resolution testing, the CDA 2 assay revealed sensitivity, specificity, positive, and negative predictive values of 80.6, 96.8, 96.7, and 81.1%, respectively (Table 2). Among 5 VIDASR⫺/CCA⫹ discordants, the VIDASR false negative specimens displayed cytotoxicity end point titers of ⱕ10 (Table 1).
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Table 1 Resolution of discordant specimens Specimen No.a
CCA
CDA 2
TD50
Interpretation
1 2 3 4 5 6 7 8 9 10
⫹b ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺c ⫹ ⫺
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺
nd ⬍10 ⬍10 ⬍10 ⬍10 n 10 n 1 ⫻ 103 n
FNe FN FN FN FN FN FN FPf Pos. controlg Neg. controlh
a CCA and CDA 2 testing were repeated on refrigerated or frozen stool specimens among negative, positive and equivocal (CDA 2) signals; Assay signals remained unchanged. Four CDA 2 specimens displaying equivocal signals (TV readings: ⱖ0.40 to ⬍1.0) were not incorporated into this study’s test parameters (data not shown). b ⫹, positive assay/signal. c ⫺, negative assay/signal. d n, not done. e FN, false negative; CDA 2 test value (TV) threshold readings ⫽ ⬍0.40. f FP, VIDAS false positive signal (TV threshold reading ⫽ 1.23); Isolation of C. sordelli. g TV reading ⫽ 2.6. h TV reading ⫽ 0.03.
4. Discussion The CCA is an excellent test for the laboratory identification of C. difficile-associated diarrhea. However, the assay mandates a 24 to 48 hour (or longer) incubation period, and the test may be subject to interpretive differences between some laboratory technologists. The CDA 2 assay is rapid (e.g., ca., 90 min to complete), and results are analyzed by the instrument’s computer. Results are printed for each sample. The CDA 2 assay however, is less sensitive than CCA (viz., 80.6% vs.100%, respectively). Failure of the VIDASR CDA 2 assay to detect C. difficile enterotoxin A may be ascribed to low levels of toxin A in the 7 stool specimens in question. Determination of cyto-
Table 2 Determination of VIDASR test parametersa
toxic dose-fifty (TD50) endpoints supports this contention. Infection with toxin A negative/toxin B positive (toxA⫺/ toxB⫹) strains of C. difficile have been reported (Moncrief et al., 2000). These strains would be missed by an EIA that detects toxin A, only. It should be pointed out however, that toxA⫺/toxB⫹ species are rarely encountered in the routine clinical setting (Halstead et al., 2002). The sole CDA 2 false positive signal was due to the presence of C. sordelli. C. sordelli is recognized to cross react with the rabbit antitoxin A component in the CDA 2 assay (Toma et al., 1999). A failure to resolve VIDASR equivocal results by repeated testing of originally submitted specimens, remains problematic. Collection of additional specimens among those patients in question, may result in interpretatable data permitting a laboratory diagnosis of C. difficile-associated diarrhea. The CDA 2 assay has an advantage of a rapid turnaround time. The early submission and then screening of stool specimens using the CDA 2 assay would alert the clinician within hours that patients were infected. Recognition by the medical staff of the assay’s false negative rate would be required to initiate upon request, testing of the same specimen by the CCA to attain a definitive laboratory diagnosis. Because of the relatively low prevalence of C. difficile infection (ca., 10%) in our hospital setting, CCA testing of all VIDASR-negative specimens would be impractical. In summary, the bioMerieux mini-VIDASR CDA 2 assay may be completed within 2 h of specimen receipt. However, the assay is less sensitive and slightly less specific than CCA. The CDA 2 assay eliminates interpretive differences which might occur between laboratory personnel. Some clinicians may accept the CDA 2 assay’s reduced sensitivity rate (compared with the CCA) as an initial screening procedure. Negative specimens or selected patients may be retested upon physician request by the 24 – 48 h, but more sensitive CCA.
Acknowledgments The authors appreciate H. P. Lipson, for proofreading the manuscript.
References
R
VIDAS
Positive C C
Negative
Equivocal
Positive
29b
7
2
Negative
1
30
2
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