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Comparison of TechLab Clostridium difficile Tox-A enzyme immunoassay and bartels prima system Toxin-A EIA
DIAGN MICROBIOLINFECTDIS 1994;20:1-5
1
BACTERIOLOGY
Comparison of TechLab Clostridium difficile Tox-A Enzyme Immunoassay and Bartels Prima System Toxin-A EIA Kevin R. Forward, Maurice T. Dalton, Elizabeth Kerr, Nadeen Paisley, and Geraldine Cooper
We evaluated the Bartels Clostridium difficile toxin A test and the TechLab Tox-A test to detect C. difficile toxin A in stool. The results were compared with C. difficile cytotoxicity assays. Of the 463 specimens tested 82 (17.7%) tested positive by cytotoxicity assay. The sensitivity, specificity, and positive and negative predictive values of the TechLab EIA were 86.6%, 93.7%, 74.7%, and 97.0%, respectively. For the Bar-
tels Prima EIA, sensitivity, specificity, and positive and negative predictive values were 95.1%, 95.5%, 82.1%, and 98.9%, respectively. The differences in sensitivity were statistically significant. Indeterminate results requiring repeat testing were more common with the TechLab EIA than with the Bartels Prima EIA. Of the two kits, the Bartels EIA is preferable, primarily because of its increased sensitivity.
INTRODUCTION
and Shah, 1993; Struelens et al., 1991). Earlier diagnosis of CDAC may also reduce the cost of other microbiologic examinations. To more rapidly identify patients with CDAC, a number of investigators have developed methods designed for the same-day detection of either C. difficile itself or one or both of the C. difficile toxins. Several enzyme immunoassays (EIA) detecting one or both of the C. difficile toxins are commercially available. The Premier C. difficile toxin A (Premier, Meridian Diagnostics Inc., Cincinnati, OH, USA), VIDAS C. difficile toxin A (BioM6rieux Vitek Systems, Hazelwood, MO, USA), Cytoclone A + B EIA (Cambridge Biotech Corp., Worcester, MA, USA), and C. diff-CUBE test (Difco Laboratories, Detroit, MI, USA) are among those recently evaluated (Shanholtzer et al., 1992; K n a p p et al., 1993; Kurzynski et al., 1992; Whittier et al., 1993; Doern et al., 1992; DiPersio et al., 1991; Barbut et al., 1993). The availability of two new EIA to detect C. difficile cytotoxin A led us to evaluate the Bartels C. difficile toxin-A EIA (Baxter Diagnostics Inc., Deerfield, IL, USA) and the TechLab C. difficile Tox-A EIA (TechLab, Blacksburg, VA, USA) and to compare results with conventional C. difficile tissue culture cytotoxicity testing.
Clostridium difficile is an important cause of community- and hospital-acquired diarrheal disease. Although the incidence of pseudomembranous colitis may have decreased in recent years, the incidence of C. difficile-assodated diarrhea (CDAC) appears to be increasing (Lyerly et al., 1988). A large proportion of cases of C. difficile associated diarrhea appear to be nosocomially acquired (Malamou-Lodas et al., 1983; McFarland et al., 1989). Rapid testing of stool specimens may allow for earlier discontinuation of contributing antibiotics, treatment with vancomycin or metronidazole, and the institution of necessary infection control measures (Brazier, 1993; Bartlett, 1990 and 1992; Gerding and Brazier, 1993; Fekety
From the Department of Microbiology,Victoria General Hospital, and Department of Microbiologyand Immunology, Dalhousie University, Halifax, Nova Scotia, Canada. Address reprint requests to Dr. K.R. Forward, FRCPC, Department of Microbiology,Victoria General Hospital, 1278Tower Road, Halifax, Nova Scotia B3H 2Y9, Canada. Received 17 February 1994; revised and accepted 8 June 1994. © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/94/$7.00
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MATERIALS A N D M E T H O D S Clinical Specimens We e x a m i n e d 463 diarrheal stool specimens from patients hospitalized at the Victoria General Hospital in Halifax, N o v a Scotia, b e t w e e n February 1992 and January 1993. Diarrhea was defined as liquid or s e m i f o r m e d stools that a s s u m e d the shape of the specimen container. Duplicate or r e p e a t e d specim e n s were not included in the analysis. Testing was p e r f o r m e d b y different technologists w h o were unaware of the results of the other studies. Stool specimens for EIA testing were refrigerated u p o n receipt, and if testing could not be carried out on the same day, frozen at - 2 0 ° C .
Tissue Culture Cytotoxicity Assay Cytotoxicity studies were p e r f o r m e d in h u m a n foreskin fibroblast cells p r e p a r e d in screw-capped culture tubes. Initial screening was p e r f o r m e d with a 1:45 dilution of stool filtrate. A negative control was included with each run. Toxin assays were considered positive if the cytopathic effect was neutralized by C. sordelii antitoxin (Wellmark Diagnostics Ltd., Guelph, Ontario, Canada).
Enzyme Immunoassays The TechLab Tox-A EIA test was p e r f o r m e d according to m a n u f a c t u r e r ' s instructions. In brief, the specimen was first emulsified and vortexed in manufacturer-supplied diluent. The stool specimen was a d d e d to microtiter containing immobilized affinitypurified polyclonal a n t i b o d y against C. difficile toxin A in the p r e s e n c e of a monoclonal a n t i b o d y against toxin A c o n j u g a t e d to h o r s e r a d i s h p e r o x i d a s e . Plates were incubated, w a s h e d , and dried, and substrate was a d d e d to each well. After 15 min incubation at r o o m t e m p e r a t u r e a color intensifier was added. Plates w e r e read o n an EIA reader, at an optical density of 450 n m within 10 min of adding the intensifier. Results w e r e interpreted as per manufacturer's i n s t r u c t i o n s - - t h a t is, a reading of >0.2 was c o n s i d e r e d to be positive; a reading of <0.1 was c o n s i d e r e d to be negative; a sample giving a reading b e t w e e n 0.101 and 0.2 was c o n s i d e r e d to be indeterminate. O n e positive and one negative control well w e r e i n c l u d e d in e a c h test r u n as r e c o m m e n d e d by the manufacturer. Bartels Prima Clostridium difficile toxin-A EIA was p e r f o r m e d in strict a d h e r e n c e to the manufacturer's instructions. Stools w e r e emulsified in the treatment b u f f e r s u p p l i e d w i t h the kit a n d v o r t e x e d . The emulsified specimen and negative and positive controis were a d d e d to microtiter stripwells coated with
K.R. Forward et al.
m o u s e i m m u n o g l o b u l i n G to C. difficile toxin A. Plates were incubated, washed, and dried. Rabbit i m m u n o g l o b u l i n to C. difficile toxin A and peroxidase-labeled goat anti-rabbit antibodies were a d d e d to the well in a coincubation step. Plates were incubated at 35°C for 30 min, w a s h e d , and dried, and the e n z y m e substrate a d d e d for a further 15 min incubation at r o o m temperature. A stop solution (1 M p h o s p h o r i c acid) was a d d e d and the plates were read on a microplate EIA reader with an absorbance of 450 nm. The test was considered positive if the result was >0.15 more than the m e a n negative control. An indeterminate range was defined as being b e t w e e n the m e a n negative value plus 0.1 and the m e a n negative value plus 0.15. Three negative controls and one positive control were r u n for each plate as per the manufacturer's directions. Specim e n s falling within the i n d e t e r m i n a t e range of either EIA were retested.
Statistical Analysis M c N e m a r ' s test was u s e d to detect differences bet w e e n the results of the two EIAs. Repeatedly indeterminate results were considered negative for the p u r p o s e s of analysis. Results were analyzed against cytotoxicity assay results.
RESULTS We examined 463 diarrheal stools samples. Eightytwo (17.7%) tested positive b y cytotoxicity assay (Table 1). Seventy were positive by all three assays. One stool was positive by cytotoxicity assay and by the TechLab EIA, but not the Bartels Prima EIA. Eight stools were positive b y tissue culture cytotoxicity assay and the Bartels assay but negative by the TechLab Tox-A assay. Three stools w e r e positive by the cytotoxicity assay and negative by b o t h EIA. By all tests, 349 specimens were negative. W h e n tissue culture cytotoxicity assay was u s e d as the " g o l d
TABLE 1 C o m p a r i s o n of ToxLab Tox-A and Bartels Prima E n z y m e I m m u n o a s s a y s and Cytotoxicity Assay n
TechLab Tox A
Bartels Prima
Cytotoxicity Assay
349 70 15 9 8 8 3
+ + + -
+ + + + -
+ + +
1
+
-
+
TechLab Tox-A vs Bartels Prima Clostridium difficile Toxin Assays
TABLE 2
3
Comparison of TechLab Tox-A and Bartels Prima Enzyme Immunoassays
Standard
EIA Kit
True Positive
False Positive
True Negative
False Negative
Cytotoxicity positive (n = 82)
TechLab Bartels
71 78
24 17
357 364
11 4
TABLE 3
Sensitivities, Specificities, and Predictive Values of Clostridium
difficile Toxin-A Enzyme Immunoassays Standard Cytotoxicity Assay
EIA Kit
Sensitivity
Specificity
PPV
NPV
Cytotoxicity positive (n = 82)
TechLab Bartels
86.6 95.1a
93.7 95.5b
74.7 82.1
97.0 98.9
EIA, enzymeimmunoassay;NPV, negativepredictivevalue; and PPV, positivepredictive value. ~TheBartelsEIAwas significantlymore sensitivethan the TechLabEIA (P < .05, odds ratio 0.125, 85% confidencelimits 0.016 and 0.999, McNemar's test). bDifferencesin the specificityof both EIA was not significant(P K 0.2, McNemar's test). standard," there were 71 true positive tests using TechLab EIA with a sensitivity of 86.6% and 24 false positives with a specificity of 93.7% (Tables 2 and 3). The positive and negative predictive values of the TechLab EIA were 74.7% and 97%, respectively. The Bartels Prima EIA had 78 true positives, with a sensitivity of 95.1%, and 17 false positives with a specificity of 95%. The positive and negative predictive values of the Bartels EIA were 82.1% and 98.9%, respectively. The differences in the sensitivity were statistically significant (P = 0.039; odds ratio 0.125; 95% confidence limits 0.016-0.999). Initial testing with the TechLab EIA resulted in 16 indeterminate results. Ten were negative on repeat testing; cytotoxicity testing was negative in nine. One was positive on repeat, and in this case the cytotoxicity test was negative. Five were indeterminate when repeated; in these five the cytotoxicity assay was negative. The Bartels EIA gave two indeterminate results. One was cytotoxicity assay negative, and the EIA was negative on repeat. In the second case, the cytotoxicity assay was positive and the EIA was indeterminate on repeat. DISCUSSION Rapid and accurate diagnosis of CDAC continues to be a goal of diagnostic microbiology laboratories. Cytotoxicity assays require expertise in cell culture methodology. They are labor intensive and expensive, and results are not usually available for 48-72 h. C. difficile cultures may require several days, and results are nonspecific because many patients are
colonized with nontoxigenic strains. Endoscopy is costly, uncomfortable, and primarily valuable in patients with pseudomembranous colitis. Because of this, physicians are usually reluctant to perform this examination, particularly in patients with mild or moderate symptoms. The latex agglutination assays that detect C. difficile antigens are also nonspecific, and results of a positive test need be confirmed with an assay for cytotoxin. Newly developed EIA for C. difficile toxins offer the promise of rapid and accurate testing for CDAC. It is important that these methods be evaluated in a setting that closely approximates conditions in a busy diagnostic laboratory. We examined specimens from 463 patients; 82 had C. difficile cytotoxin in submitted specimens. These proportions are consistent with studies by Riley et al. (1983) and Aronsson et al. (1985), who reported that 15% to 20% of diarrheal stools from hospitalized patients were positive for C. difficile. The performance of both EIA compared favorably with evaluations of other C. difficile toxin A EIA (Barbut et al., 1993; Stanholtzer et al., 1992; Knapp et al., 1993; Kurzynski et al., 1992; Doern et al., 1992; DiPersio et al., 1991). The Bartels EIA sensitivity was the better of the two and was similar to those observed by Whittier et al. (1993). In their study, they compared both the Bartels and TechLab EIAs with Premier and VIDAS. For the Bartels EIA, they found a sensitivity of 94% compared with our finding of 95.1%; they determined the specificity to be 92%, compared with our finding of 95.5%. The same authors determined the sensitivity of the TechLab EIA to be 93%, compared with our finding of 86.6%; they found the specificity to be
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92%, compared with our finding of 93.7%. We observed high sensitivities for both EIAs despite our freezing a large proportion of specimens before processing, which we did to conserve resources. Duration of freezing did not affect ToxLab EIA results. The mean time frozen was not significantly different for false-negative tests (13.4 days) than for true positives (10.4 days) or true negatives (9.8 days). To determine whether freezing specimens adversely affected the results of the EIA, we compared the mean time that true-positive, true-negative, false-positive, and false-negative samples had been frozen. None of the differences were statistically different. Because the C. difficile tissue culture cytotoxicity assay may not be 100% sensitive, it is possible that some patients, particularly those with both EIA tests positive and negative cytotoxicity assay had CDAD, i.e., that the cytotoxicity assay was falsely negative (Lashner et al., 1986). We attempted to use previously published clinical criteria to access the probability of disease in these patients, but these were too rigorous for a retrospective examination of medical records. Many patients did not have stool numbers counted or recorded on the chart, and antibiotics in the 8 weeks before the development of diarrhea may not have been accurately recorded. Because there are a very large number of causes of diarrhea, and a large number of medications may produce diarrhea, it was not difficult to identify in hospitalized patients other "possible" but not probable explanations for their diarrhea. At our hospital, few physicians stop antibiotics or initiate therapy for CDAD unless the tissue culture cytotoxicity assay is positive. As a result, it was difficult to identify patients who improved with the discontinuation of antibiotics or with the initiation of specific therapy.
K.R. Forward et al.
We routinely use a 1:45 dilution of stool in performing our cell culture cytotoxicity assay. Although the optimal screening dilution has not been determined, several other investigators (Doern et al., 1992; Shanholtzer et al., 1992; DiPersio et al., 1992) have used a 1:40 dilution when conducting comparative studies. Mattia et al. (1993) suggested that 1:90 is a more appropriate cutoff. Had we initially screened at a higher dilution, the increased sensitivity of the cytotoxicity assay may have resulted in either fewer "false positive" EIA; conversely, a lower screening dilution may have resulted in more "false positive" EIA. Further studies are necessary to determine the optimal dilution for this test if it is to continue to be the gold standard against which newer methods are compared. Staff have found the TechLab EIA to be somewhat easier to perform because it required fewer steps and fewer controls. The four controls required for the Bartels EIA meant that the per-test cost would be quite substantial if low numbers of tests were performed. The manufacturer does suggest that when stat or low-volume testing is performed, the number of negative controls may be reduced to one or two. In summary, the performance of Bartels EIA was superior to that of the TechLab EIA. In our hospital, the negative predictive values of the Bartels EIA would probably make cell culture cytotoxicity assay testing of EIA-negative specimens unnecessary. Similarly, its high positive predictive value would probably make confirmation of positive Bartels EIA unnecessary. Volumes and financial factors permitting, we find the Bartels assay to be an acceptable substitute for conventional C. difficile cytotoxicity assay testing.
REFERENCES Aronsson B, Mollby R, Nord CE (1985) Antimicrobial agents and Clostridium difficile in acute enteric disease: epidemiological data from Sweden, 1980-1982. J Infect Dis 151:476-481. Barbut F, Kajzer C, Planas N, Petit J-C (1993) Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea. J Clin Microbiol 31:963-967. Bartlett JG (1990) Clostridium difficile: clinical considerations. Ref Infect Dis 12(Suppl 2):$243-$251. Bartlett JG (1992) Antibiotic-associated diarrhea. Clin Infect Dis 15:573-581. Brazier JS (1993) Role of the laboratory in investigations of Clostridium difficile diarrhea. Clin Infect Dis 16(Suppl 4):$228-$233. DiPersio JR, Varga FJ, Conwell DL, Kraft JA, Kozak JK, Willis DH (1991) Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease. J Clin Microbiol 29:2724-2730.
Doern GV, Coughlin RT, Wu L (1992) Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody immunoassay for toxin A only and two cytotoxicity assays. J Clin Microbiol 30:2042-2046. Fekety R, Shah AB (1993) Diagnosis and treatment of Clostridium difficile colitis. JAMA 269:71-75. Gerding DN, Brazier JS (1993) Optimal methods for identifying Clostridium difficile infections. Clin Infect Dis 16(Suppl 4):$439-$442. Knapp CC, Sandin RL, Hall GS, Ludwig D, Rutherford I, Washington JA (1993) Comparison of VIDAS Clostridium difficile toxin-A assay and Premier C. difficile toxin-A assay to cytotoxin-B tissue culture assay for the detection of toxins of C. difficile. Diagn Microbiol Infect Dis 17:7-12. Kurzynski TA, Kimball JL, Schultz DA, Shell RF (1992) Evaluation of C. diff.-CUBE test for detection of Clostridium difficile-associated diarrhea. Diagn Microbiol Infect Dis 15:493M98.
TechLab Tox-A vs Bartels Prima Clostridium difficile Toxin Assays
Lashner BA, Todorczuk J, Sahm DF, Hanauer SB (1986) Clostridium difficile culture-positive toxin-negative diarrhea. Am J Gastroenterol 81:940-943. Lyerly DM, Krivan HC, Wilkins TD (1988) Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1:1-18. Malamou-Lodas H, O'Farrell S, Nash JQ, Tabaqchali S (1983) Isolation of Clostridium difficile from patients and the environment of hospital wards. J Clin Pathol 36:8892. Mattia AR, Doern GV, Clark J, Holden J, Wu L, Ferraro MJ (1993) Comparison of four methods in the diagnosis of Clostridium difficile disease. Eur J Clin Microbiol Infect Dis 12:882-886. McFarland LV, Mulligan ME, Kwok RYY, Stamm WE
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(1989) Nosocomial acquisition of Clostridium difficile infection. N Engl J Med 320:204-210. Riley TV, Bowman RA, Carroll SM (1983) Diarrhoea associated with Clostridium difficile in a hospital population. Med J Aust 1:166-169. Struelens MJ, Maas A, Nonhoff C, Deplano A, Rost F, Serruys E, Delmee M (1991) Control of nosocomial transmission of Clostridium difficile based on sporadic case surveillance. Am ] Med 91(Suppl 3B):138S-144S. Whittier S, Shapiro DS, Kelly WF, Waldren TP, Wait KJ, McMillon LT, Gilligan PH (1993) Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases. J Clin Microbiol 31:2861-2865.
1
BACTERIOLOGY
Comparison of TechLab Clostridium difficile Tox-A Enzyme Immunoassay and Bartels Prima System Toxin-A EIA Kevin R. Forward, Maurice T. Dalton, Elizabeth Kerr, Nadeen Paisley, and Geraldine Cooper
We evaluated the Bartels Clostridium difficile toxin A test and the TechLab Tox-A test to detect C. difficile toxin A in stool. The results were compared with C. difficile cytotoxicity assays. Of the 463 specimens tested 82 (17.7%) tested positive by cytotoxicity assay. The sensitivity, specificity, and positive and negative predictive values of the TechLab EIA were 86.6%, 93.7%, 74.7%, and 97.0%, respectively. For the Bar-
tels Prima EIA, sensitivity, specificity, and positive and negative predictive values were 95.1%, 95.5%, 82.1%, and 98.9%, respectively. The differences in sensitivity were statistically significant. Indeterminate results requiring repeat testing were more common with the TechLab EIA than with the Bartels Prima EIA. Of the two kits, the Bartels EIA is preferable, primarily because of its increased sensitivity.
INTRODUCTION
and Shah, 1993; Struelens et al., 1991). Earlier diagnosis of CDAC may also reduce the cost of other microbiologic examinations. To more rapidly identify patients with CDAC, a number of investigators have developed methods designed for the same-day detection of either C. difficile itself or one or both of the C. difficile toxins. Several enzyme immunoassays (EIA) detecting one or both of the C. difficile toxins are commercially available. The Premier C. difficile toxin A (Premier, Meridian Diagnostics Inc., Cincinnati, OH, USA), VIDAS C. difficile toxin A (BioM6rieux Vitek Systems, Hazelwood, MO, USA), Cytoclone A + B EIA (Cambridge Biotech Corp., Worcester, MA, USA), and C. diff-CUBE test (Difco Laboratories, Detroit, MI, USA) are among those recently evaluated (Shanholtzer et al., 1992; K n a p p et al., 1993; Kurzynski et al., 1992; Whittier et al., 1993; Doern et al., 1992; DiPersio et al., 1991; Barbut et al., 1993). The availability of two new EIA to detect C. difficile cytotoxin A led us to evaluate the Bartels C. difficile toxin-A EIA (Baxter Diagnostics Inc., Deerfield, IL, USA) and the TechLab C. difficile Tox-A EIA (TechLab, Blacksburg, VA, USA) and to compare results with conventional C. difficile tissue culture cytotoxicity testing.
Clostridium difficile is an important cause of community- and hospital-acquired diarrheal disease. Although the incidence of pseudomembranous colitis may have decreased in recent years, the incidence of C. difficile-assodated diarrhea (CDAC) appears to be increasing (Lyerly et al., 1988). A large proportion of cases of C. difficile associated diarrhea appear to be nosocomially acquired (Malamou-Lodas et al., 1983; McFarland et al., 1989). Rapid testing of stool specimens may allow for earlier discontinuation of contributing antibiotics, treatment with vancomycin or metronidazole, and the institution of necessary infection control measures (Brazier, 1993; Bartlett, 1990 and 1992; Gerding and Brazier, 1993; Fekety
From the Department of Microbiology,Victoria General Hospital, and Department of Microbiologyand Immunology, Dalhousie University, Halifax, Nova Scotia, Canada. Address reprint requests to Dr. K.R. Forward, FRCPC, Department of Microbiology,Victoria General Hospital, 1278Tower Road, Halifax, Nova Scotia B3H 2Y9, Canada. Received 17 February 1994; revised and accepted 8 June 1994. © 1994 Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/94/$7.00
2
MATERIALS A N D M E T H O D S Clinical Specimens We e x a m i n e d 463 diarrheal stool specimens from patients hospitalized at the Victoria General Hospital in Halifax, N o v a Scotia, b e t w e e n February 1992 and January 1993. Diarrhea was defined as liquid or s e m i f o r m e d stools that a s s u m e d the shape of the specimen container. Duplicate or r e p e a t e d specim e n s were not included in the analysis. Testing was p e r f o r m e d b y different technologists w h o were unaware of the results of the other studies. Stool specimens for EIA testing were refrigerated u p o n receipt, and if testing could not be carried out on the same day, frozen at - 2 0 ° C .
Tissue Culture Cytotoxicity Assay Cytotoxicity studies were p e r f o r m e d in h u m a n foreskin fibroblast cells p r e p a r e d in screw-capped culture tubes. Initial screening was p e r f o r m e d with a 1:45 dilution of stool filtrate. A negative control was included with each run. Toxin assays were considered positive if the cytopathic effect was neutralized by C. sordelii antitoxin (Wellmark Diagnostics Ltd., Guelph, Ontario, Canada).
Enzyme Immunoassays The TechLab Tox-A EIA test was p e r f o r m e d according to m a n u f a c t u r e r ' s instructions. In brief, the specimen was first emulsified and vortexed in manufacturer-supplied diluent. The stool specimen was a d d e d to microtiter containing immobilized affinitypurified polyclonal a n t i b o d y against C. difficile toxin A in the p r e s e n c e of a monoclonal a n t i b o d y against toxin A c o n j u g a t e d to h o r s e r a d i s h p e r o x i d a s e . Plates were incubated, w a s h e d , and dried, and substrate was a d d e d to each well. After 15 min incubation at r o o m t e m p e r a t u r e a color intensifier was added. Plates w e r e read o n an EIA reader, at an optical density of 450 n m within 10 min of adding the intensifier. Results w e r e interpreted as per manufacturer's i n s t r u c t i o n s - - t h a t is, a reading of >0.2 was c o n s i d e r e d to be positive; a reading of <0.1 was c o n s i d e r e d to be negative; a sample giving a reading b e t w e e n 0.101 and 0.2 was c o n s i d e r e d to be indeterminate. O n e positive and one negative control well w e r e i n c l u d e d in e a c h test r u n as r e c o m m e n d e d by the manufacturer. Bartels Prima Clostridium difficile toxin-A EIA was p e r f o r m e d in strict a d h e r e n c e to the manufacturer's instructions. Stools w e r e emulsified in the treatment b u f f e r s u p p l i e d w i t h the kit a n d v o r t e x e d . The emulsified specimen and negative and positive controis were a d d e d to microtiter stripwells coated with
K.R. Forward et al.
m o u s e i m m u n o g l o b u l i n G to C. difficile toxin A. Plates were incubated, washed, and dried. Rabbit i m m u n o g l o b u l i n to C. difficile toxin A and peroxidase-labeled goat anti-rabbit antibodies were a d d e d to the well in a coincubation step. Plates were incubated at 35°C for 30 min, w a s h e d , and dried, and the e n z y m e substrate a d d e d for a further 15 min incubation at r o o m temperature. A stop solution (1 M p h o s p h o r i c acid) was a d d e d and the plates were read on a microplate EIA reader with an absorbance of 450 nm. The test was considered positive if the result was >0.15 more than the m e a n negative control. An indeterminate range was defined as being b e t w e e n the m e a n negative value plus 0.1 and the m e a n negative value plus 0.15. Three negative controls and one positive control were r u n for each plate as per the manufacturer's directions. Specim e n s falling within the i n d e t e r m i n a t e range of either EIA were retested.
Statistical Analysis M c N e m a r ' s test was u s e d to detect differences bet w e e n the results of the two EIAs. Repeatedly indeterminate results were considered negative for the p u r p o s e s of analysis. Results were analyzed against cytotoxicity assay results.
RESULTS We examined 463 diarrheal stools samples. Eightytwo (17.7%) tested positive b y cytotoxicity assay (Table 1). Seventy were positive by all three assays. One stool was positive by cytotoxicity assay and by the TechLab EIA, but not the Bartels Prima EIA. Eight stools were positive b y tissue culture cytotoxicity assay and the Bartels assay but negative by the TechLab Tox-A assay. Three stools w e r e positive by the cytotoxicity assay and negative by b o t h EIA. By all tests, 349 specimens were negative. W h e n tissue culture cytotoxicity assay was u s e d as the " g o l d
TABLE 1 C o m p a r i s o n of ToxLab Tox-A and Bartels Prima E n z y m e I m m u n o a s s a y s and Cytotoxicity Assay n
TechLab Tox A
Bartels Prima
Cytotoxicity Assay
349 70 15 9 8 8 3
+ + + -
+ + + + -
+ + +
1
+
-
+
TechLab Tox-A vs Bartels Prima Clostridium difficile Toxin Assays
TABLE 2
3
Comparison of TechLab Tox-A and Bartels Prima Enzyme Immunoassays
Standard
EIA Kit
True Positive
False Positive
True Negative
False Negative
Cytotoxicity positive (n = 82)
TechLab Bartels
71 78
24 17
357 364
11 4
TABLE 3
Sensitivities, Specificities, and Predictive Values of Clostridium
difficile Toxin-A Enzyme Immunoassays Standard Cytotoxicity Assay
EIA Kit
Sensitivity
Specificity
PPV
NPV
Cytotoxicity positive (n = 82)
TechLab Bartels
86.6 95.1a
93.7 95.5b
74.7 82.1
97.0 98.9
EIA, enzymeimmunoassay;NPV, negativepredictivevalue; and PPV, positivepredictive value. ~TheBartelsEIAwas significantlymore sensitivethan the TechLabEIA (P < .05, odds ratio 0.125, 85% confidencelimits 0.016 and 0.999, McNemar's test). bDifferencesin the specificityof both EIA was not significant(P K 0.2, McNemar's test). standard," there were 71 true positive tests using TechLab EIA with a sensitivity of 86.6% and 24 false positives with a specificity of 93.7% (Tables 2 and 3). The positive and negative predictive values of the TechLab EIA were 74.7% and 97%, respectively. The Bartels Prima EIA had 78 true positives, with a sensitivity of 95.1%, and 17 false positives with a specificity of 95%. The positive and negative predictive values of the Bartels EIA were 82.1% and 98.9%, respectively. The differences in the sensitivity were statistically significant (P = 0.039; odds ratio 0.125; 95% confidence limits 0.016-0.999). Initial testing with the TechLab EIA resulted in 16 indeterminate results. Ten were negative on repeat testing; cytotoxicity testing was negative in nine. One was positive on repeat, and in this case the cytotoxicity test was negative. Five were indeterminate when repeated; in these five the cytotoxicity assay was negative. The Bartels EIA gave two indeterminate results. One was cytotoxicity assay negative, and the EIA was negative on repeat. In the second case, the cytotoxicity assay was positive and the EIA was indeterminate on repeat. DISCUSSION Rapid and accurate diagnosis of CDAC continues to be a goal of diagnostic microbiology laboratories. Cytotoxicity assays require expertise in cell culture methodology. They are labor intensive and expensive, and results are not usually available for 48-72 h. C. difficile cultures may require several days, and results are nonspecific because many patients are
colonized with nontoxigenic strains. Endoscopy is costly, uncomfortable, and primarily valuable in patients with pseudomembranous colitis. Because of this, physicians are usually reluctant to perform this examination, particularly in patients with mild or moderate symptoms. The latex agglutination assays that detect C. difficile antigens are also nonspecific, and results of a positive test need be confirmed with an assay for cytotoxin. Newly developed EIA for C. difficile toxins offer the promise of rapid and accurate testing for CDAC. It is important that these methods be evaluated in a setting that closely approximates conditions in a busy diagnostic laboratory. We examined specimens from 463 patients; 82 had C. difficile cytotoxin in submitted specimens. These proportions are consistent with studies by Riley et al. (1983) and Aronsson et al. (1985), who reported that 15% to 20% of diarrheal stools from hospitalized patients were positive for C. difficile. The performance of both EIA compared favorably with evaluations of other C. difficile toxin A EIA (Barbut et al., 1993; Stanholtzer et al., 1992; Knapp et al., 1993; Kurzynski et al., 1992; Doern et al., 1992; DiPersio et al., 1991). The Bartels EIA sensitivity was the better of the two and was similar to those observed by Whittier et al. (1993). In their study, they compared both the Bartels and TechLab EIAs with Premier and VIDAS. For the Bartels EIA, they found a sensitivity of 94% compared with our finding of 95.1%; they determined the specificity to be 92%, compared with our finding of 95.5%. The same authors determined the sensitivity of the TechLab EIA to be 93%, compared with our finding of 86.6%; they found the specificity to be
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92%, compared with our finding of 93.7%. We observed high sensitivities for both EIAs despite our freezing a large proportion of specimens before processing, which we did to conserve resources. Duration of freezing did not affect ToxLab EIA results. The mean time frozen was not significantly different for false-negative tests (13.4 days) than for true positives (10.4 days) or true negatives (9.8 days). To determine whether freezing specimens adversely affected the results of the EIA, we compared the mean time that true-positive, true-negative, false-positive, and false-negative samples had been frozen. None of the differences were statistically different. Because the C. difficile tissue culture cytotoxicity assay may not be 100% sensitive, it is possible that some patients, particularly those with both EIA tests positive and negative cytotoxicity assay had CDAD, i.e., that the cytotoxicity assay was falsely negative (Lashner et al., 1986). We attempted to use previously published clinical criteria to access the probability of disease in these patients, but these were too rigorous for a retrospective examination of medical records. Many patients did not have stool numbers counted or recorded on the chart, and antibiotics in the 8 weeks before the development of diarrhea may not have been accurately recorded. Because there are a very large number of causes of diarrhea, and a large number of medications may produce diarrhea, it was not difficult to identify in hospitalized patients other "possible" but not probable explanations for their diarrhea. At our hospital, few physicians stop antibiotics or initiate therapy for CDAD unless the tissue culture cytotoxicity assay is positive. As a result, it was difficult to identify patients who improved with the discontinuation of antibiotics or with the initiation of specific therapy.
K.R. Forward et al.
We routinely use a 1:45 dilution of stool in performing our cell culture cytotoxicity assay. Although the optimal screening dilution has not been determined, several other investigators (Doern et al., 1992; Shanholtzer et al., 1992; DiPersio et al., 1992) have used a 1:40 dilution when conducting comparative studies. Mattia et al. (1993) suggested that 1:90 is a more appropriate cutoff. Had we initially screened at a higher dilution, the increased sensitivity of the cytotoxicity assay may have resulted in either fewer "false positive" EIA; conversely, a lower screening dilution may have resulted in more "false positive" EIA. Further studies are necessary to determine the optimal dilution for this test if it is to continue to be the gold standard against which newer methods are compared. Staff have found the TechLab EIA to be somewhat easier to perform because it required fewer steps and fewer controls. The four controls required for the Bartels EIA meant that the per-test cost would be quite substantial if low numbers of tests were performed. The manufacturer does suggest that when stat or low-volume testing is performed, the number of negative controls may be reduced to one or two. In summary, the performance of Bartels EIA was superior to that of the TechLab EIA. In our hospital, the negative predictive values of the Bartels EIA would probably make cell culture cytotoxicity assay testing of EIA-negative specimens unnecessary. Similarly, its high positive predictive value would probably make confirmation of positive Bartels EIA unnecessary. Volumes and financial factors permitting, we find the Bartels assay to be an acceptable substitute for conventional C. difficile cytotoxicity assay testing.
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Doern GV, Coughlin RT, Wu L (1992) Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody immunoassay for toxin A only and two cytotoxicity assays. J Clin Microbiol 30:2042-2046. Fekety R, Shah AB (1993) Diagnosis and treatment of Clostridium difficile colitis. JAMA 269:71-75. Gerding DN, Brazier JS (1993) Optimal methods for identifying Clostridium difficile infections. Clin Infect Dis 16(Suppl 4):$439-$442. Knapp CC, Sandin RL, Hall GS, Ludwig D, Rutherford I, Washington JA (1993) Comparison of VIDAS Clostridium difficile toxin-A assay and Premier C. difficile toxin-A assay to cytotoxin-B tissue culture assay for the detection of toxins of C. difficile. Diagn Microbiol Infect Dis 17:7-12. Kurzynski TA, Kimball JL, Schultz DA, Shell RF (1992) Evaluation of C. diff.-CUBE test for detection of Clostridium difficile-associated diarrhea. Diagn Microbiol Infect Dis 15:493M98.
TechLab Tox-A vs Bartels Prima Clostridium difficile Toxin Assays
Lashner BA, Todorczuk J, Sahm DF, Hanauer SB (1986) Clostridium difficile culture-positive toxin-negative diarrhea. Am J Gastroenterol 81:940-943. Lyerly DM, Krivan HC, Wilkins TD (1988) Clostridium difficile: its disease and toxins. Clin Microbiol Rev 1:1-18. Malamou-Lodas H, O'Farrell S, Nash JQ, Tabaqchali S (1983) Isolation of Clostridium difficile from patients and the environment of hospital wards. J Clin Pathol 36:8892. Mattia AR, Doern GV, Clark J, Holden J, Wu L, Ferraro MJ (1993) Comparison of four methods in the diagnosis of Clostridium difficile disease. Eur J Clin Microbiol Infect Dis 12:882-886. McFarland LV, Mulligan ME, Kwok RYY, Stamm WE
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(1989) Nosocomial acquisition of Clostridium difficile infection. N Engl J Med 320:204-210. Riley TV, Bowman RA, Carroll SM (1983) Diarrhoea associated with Clostridium difficile in a hospital population. Med J Aust 1:166-169. Struelens MJ, Maas A, Nonhoff C, Deplano A, Rost F, Serruys E, Delmee M (1991) Control of nosocomial transmission of Clostridium difficile based on sporadic case surveillance. Am ] Med 91(Suppl 3B):138S-144S. Whittier S, Shapiro DS, Kelly WF, Waldren TP, Wait KJ, McMillon LT, Gilligan PH (1993) Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases. J Clin Microbiol 31:2861-2865.