Aug 9, 1988 - Marion Laboratories for C. difficile toxin A (CDT latex test) was disputed by ..... produce occasional false-positive latex tests of stool speci- mens.
Vol. 26, No. 11
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1988, p. 2452-2455 0095-1137/88/112452-04$02.00/0 Copyright C 1988, American Society for Microbiology
Evaluation of a Commercial Latex Test for Clostridium difficile for Reactivity with C. difficile and Cross-Reactions with Other Bacteria BRIAN L. MILES, JEAN A. SIDERS, AND STEPHEN D. ALLEN* Department of Pathology, Indiana University School of Medicine, Indianapolis, Indiana 46223 Received 30 June 1988/Accepted 9 August 1988
Seventy-eight species of bacteria (739 isolates) were tested for reactivity with a commercial latex test for Clostridium difficile. All noncytotoxic as well as cytotoxic strains of C. difficile reacted positively. Immunospecific cross-reactions were found only with C. sporogenes, proteolytic C. botulinum, and Peptostreptococcus anaerobius. A rapid latex agglutination immunoassay for the detection of Clostridium difficile toxin A, based on the latex assay described by Banno et al. (3), was marketed in January of 1986 (Marion Laboratories, Inc., Kansas City, Mo.). Subsequently, the specificity of the commercial latex test of Marion Laboratories for C. difficile toxin A (CDT latex test) was disputed by Lyerly and Wilkins (12), and it has been reported that the latex assay cross-reacts with C. sporogenes and some proteolytic strains of C. botulinum (4, 7), C. haemolyticum (C. A. Gaydos, B. E. Laughon, L. M. Mundy, R. G. Bennett, and L. Bobo, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, B216, p. 61), and Bacteroides asaccharolyticus (4). The purpose of this study was to assay broth culture supernatants of cytotoxin-positive and cytotoxin-negative C. difficile and à wide variety of other bacteria to establish further the reactivity of the CDT latex test with C. difficile and to examine the assay for potential cross-reactions with other bacteria. In this study, 739 isolates, representing 78 species of bacteria, were tested by using the CDT latex test. Of these isolates, 63 species were anaerobes and 15 species were facultative anaerobes. Most of the organisms were isolated in the clinical microbiology laboratory at Indiana University Hospital from clinical specimens or from feces during fecal flora studies; others were obtained from the American Type Culture Collection and the Virginia Polytechnic Institute. Prior to inclusion in the study, the anaerobic bacteria were identified by using conventional methods and criteria (1, 6), and facultatively anaerobic species were also identified by using established procedures (9) to ensure purity and correct identifications. All isolates were grown anaerobically in brain heart infusion broth (Carr-Scarborough Microbiologicals, Inc., Atlanta, Ga.) for 48 h at 35°C prior to testing with the CDT latex test. Brain heart infusion broth cultures were centrifuged at 1,500 x g for 10 min to pellet the cells. The test sample consisted of 0.5 ml of the culture supernatant suspended in 0.5 ml of the buffer included in the CDT latex test kit. The latex test was performed according to the manufacturer's procedure. Test cards were rotated on a mechanical rotator for 3 min. A positive result was determined when there was agglutination in the test circle greater than that which occurred in the negative control circle. Positive control tests were performed routinely. All of the C. difficile isolates included in this study and four of the C. sporogenes isolates were assayed for cytotoxic activity. For the cyto*
toxicity assay, brain heart infusion broth culture supernatants as described above were filtered through 0.45-,um (pore size) Millipore membrane filters. The culture supernatant filtrates were tested for C. difficile cytotoxin by using the Toxi-titer microtiter plate system (Bartels Immunodiagnostic Supplies, Bellevue, Wash.). The cytotoxicity assay was performed in accordance with the procedure outlined by the manufacturer. The wells of Toxi-titer plates were examined for neutralizable cytopathic effects by using an inverted microscope after 24 and 48 h of incubation at 35°C. All 139 cytotoxin-positive strains of C. difficile and 35 of 35 cytotoxin-negative strains of C. difficile tested gave a positive reaction in the CDT latex test. One strain of C. botulinum (proteolytic, type E) and all 25 strains of C. sporogenes tested were positive. We tested only three strains of C. botulinum, and the two strains that were negative were nonproteolytic. Although we did not test many strains of C. botulinum, the proteolytic group I strains of C. botulinum are indistinguishable from C. sporogenes except by toxin assays (15). Therefore, we predict that most proteolytic C. botulinum strains should be positive in the latex test. Cytotoxicity assays performed on four strains of C. sporogenes were negative. None of the other Clostridium species tested reacted in the CDT latex test. Of the nonclostridia tested, 10 of 10 strains of Peptostreptococcus anaerobius showed positive CDT latex agglutination reactions. In addition, 13 of 17 strains of Staphylococcus aureus had nonspecific agglutination reactions (i.e., agglutination took place in the negative control circles and in the test circles). Bacterial species tested that did not show agglutination reactions in the CDT latex test are listed in Table 1. This study demonstrated that the CDT latex agglutination test agglutinated all of a reasonably large number of toxigenic as well as nontoxigenic strains of C. difficile. The latex test agglutinated only 3 of the 77 additional species tested. Although none of the five strains of B. asaccharolyticus we tested were positive, others have reported some strains of this species that were CDT latex positive (4, 10). One strain of C. haemolyticum was reported to react in the CDT test (Gaydos et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1987). We have not examined C. haemolyticum for reactivity with this test. Results of D. M. Lyerly and associates indicate that the CDT latex test reacts with a nontoxic 43,000-dalton protein that is distinct from toxin A and that is produced by both nontoxigenic and toxigenic strains of C. difficile (10). In addition, Lyerly et al. found that C. sporogenes and P. anaerobius also produce an antigen containing a subunit of a
Corresponding author. 2452
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VOL. 26, 1988
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TABLE 1. Bacterial strains that did not react with the latex test No. of strains tested
Organism(s) Anaerobic bacteria .................. Actinoinyces israelii Bacteroides asaccharolvyicus .................... B. biv'ius ................................... B. disiens .................. ................. B. distasonis ................................... B. fragilis ................................... B. melaninogenicius .................................
Other pigmented Bacteroides spp
.........
Organism(s)
No. of strains tested
Anaerobic bacteria
Fusobacteriuin gonidiaforinans. F. norferun.... F. nav:if(orne. F. necroporn....
1 S 2 1 9 44 3
9
B. oralis ................................... 1 B. splanchnicuts .................................. 1 B. thetaiotaoanicron ................................. B. uniformis ................................... 4 B. ureolyticus ................................... 2 B. v'ulgatus ................................... 10 Bacteroides spp . 8 ................................. Bifidobacteriumn spp ................................. Capnocytophaga oclracea . .2 14 Clostridiuîn barati . ................................. 3 C. bifermenrans........s............................. C. butyricuin .................................. 8 C. cadaveris .................................. 8 C. closridiiforine..8 C. glyc olicumln .......................... ........ 4 C. innociuurn .................................. 9 C. paraputrificuin .................................. 9 C. perfringens ................................... 21 C. puirificuin .................................. 2 C. ramosum .................................. 6 C. septicim n . ...............................3 C. sordellii . ................................12 C. splienoides . ................................2 C. subtermninale . 2 .................................. C. tertiumn ........................ 6 4 Clostridiumn spp .................................... 9 Eubacteriuin limnsumn . ............................. E. rectale ................................... 1 Eubacteriumn spp .................................. 6
4 4 1 4 F. nucleatum .6 F. russii. 3 F. varinm .13
Fusobacteriuin spp. Lactobacillus jensenii. Lactobacillus spp. 9Peplostreptococcus asaccliarol!ticus .
2
1 7 25 P. inagnus .15 P. micros.16 P. prevotii. 3 2 Peptostreptococcus spp. 1 Propiobibacieriuin .17 aces P. avidCen ......................................i.1 2 Propionibacteriumn spp. S 3aphloccocus sacclarovuticus .4 Streptococcus iniermnedius .4 S. mnorbillorun. 1 Veilloella 19 ...............................8.Vei parv lla.. Nonanaerobic bacteria Acinetobacter calcoaceticus subsp. anitratuts .3 Bacillus spp. 3 7 Enterobacter cloacae. Enterococcus spp .16 Escherichlia coli .23 Klebsiella oaxvioa .3 K. pneumnoniae. 9 Morganella mnorganii .1 Proteus mnirabilis .10 1 Providencia stuariii ................ Pseiudoinonas aeruginosa .12 Serratia mnarcescens. 3 Staphllocaaccus epidermnidis .15 S .reptococcus 1 pnenniae..
43,000-dalton cross-reactive protein which is biochemically and immunologically very similar to the protein of C. difficile that reacts in the CDT latex test (10). The 43,000-dalton cross-reactive protein was not detected in B. asaccharolvticus, which apparently must contain a different antigen or involve a different mechanism of agglutination from that for C. difficile, C. sporogenes, and P. anaerob ius (10). The fact that the commercial CDT latex test detects neither toxin A nor toxin B of C. difficile and that it produces false-positive reactions with four to five other species does not mean that the test is not clinically useful. In the clinical evaluation of Peterson et al. (involving 161 fresh fecal specimens) (13), the CDT latex test was superior to cell culture (for the cytotoxin of C. difficile) in diagnosing patients with C. difficile-associated diarrhea, and the sensitivity of the latex test was equivalent to that of culture of the organism. Interestingly, the percentages of false-positive results in fresh fecal specimens from non-C. difficile-associated diarrhea cases were 3% for the latex test, 3% for the tissue culture assay, and 23% for culture of the organism. Kelly et al. reported similar findings (8) in their study of fecal specimens from 626 patients tested by the CDT latex test, cell culture for cytotoxin, and bacteriological culture. The results of the three procedures agreed for 88% of the specimens tested. Kelly et al. reviewed the clinical findings
for 93 patients, 40 of whom were reported to have C.
difficile-associated diarrhea and 53 of whom did not (8). The sensitivities of the cytotoxin assay, latex test, and culture were 70, 78, and 90%, respectively. The specificities of these tests were 98, 92, and 96%, respectively. Kelly et al.
concluded that the accuracy of detecting C. dzfficile-associated diarrhea was improved by using the latex test in combination with either culture or the cytotoxin assay (8). A somewhat less extensive study than that of Kelly et al., involving 201 stool samples, indicated that the sensitivity of the latex test compared with that of a tissue culture assay for cytotoxin was 83%; the specificity was 93% (14). The positive and negative predictive values of the latex test results were 72 and 96%, respectively. Sherman et al. concluded that the CDT latex test may be a useful screening procedure but that a positive latex test result should be confirmed by an assay for toxin (14). In another study of 207 fecal specimens from patients with diarrhea, there was complete agreement for 76% of the specimens for which the latex test, culture for C. difficile, and a cytotoxin assay were all negative. Positive latex test results agreed with positive culture results for 15.6% of the specimens, while positive latex test results agreed with positive cytotoxin assays for 7.3% of the specimens (4). In this study, there was better correlation between a positive
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J. CLIN. MICROBIOL.
NOTES
latex test and culture of C. difficile than between a positive culture and the cytotoxin assay. When compared with results in the cytotoxin assay, four of the latex test results were interpreted to be false-positive and four were falsenegative. The presence of C. sporogenes in one specimen and B. asaccharolyticius in another, both of which reacted in vitro with the latex test, probably explains two of the four tests that were interpreted to be false-positive. Thus, this study provides evidence that cross-reacting organisms may produce occasional false-positive latex tests of stool specimens. In our experience, the percentage of positive latex test results for culture-negative, cytotoxin-negative stools has been low (i.e., 0.7% of 588 fresh stool specimens; S. D. Allen, B. L. Miles, and J. A. Siders, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, C6, p. 324). There is a theoretical but probably remote possibility of finding a false-positive latex test result owing to the presence of a cross-reacting proteolytic strain of C. botilinion in a stool specimen, especially if the laboratory performs CDT latex tests on feces of infants. Although C. botidinum is usually not considered to be part of the indigenous microbiota of the intestinal tract of humans, Arnon reported that C. botulinurn type A was found in the feces of 1 of 395 healthy infants (2). There is also a report of a study by Thompson et al. from Utah (16) in which C. botulinum was isolated from 20 children without neurologic manifestations of botulism. These children ranged from less than 1 month to 11 years of age. To our knowledge, these interesting and significant findings have not as yet been confirmed in other populations of children, or in adults. Several extensive studies reviewed by Finegold and colleagues did not indicate the detection of C. sporogenes (or of C. botulinum) in any of the fecal specimens examined from 141 adult volunteers (5). Likewise, we did not detect C. sporogenes or C. botidlinum in the feces of 30 healthy adult volunteers studied in Indianapolis (S. D. Allen, J. A. Siders, M. D. Cromer, N. B. O'Bryan, C. G. Kindberg, J. W. Suttie, P. M. Allison, N. U. Bang, and G. G. Brier, Abstract, Microecol. Ther. 15:283-284, 1985). P. anaerobizis was encountered in the feces of 1.4% of the 141 subjects studied by Finegold and colleagues (5) and in 6.6% of the 30 volunteers whose feces were studied by us. Whether or not C. sporogenes or P. anaerobius is harbored any more frequently in the stools of individuals who have diarrhea than in the formed stools of healthy volunteers is unknown. Thirteen ofthe 17 S. aureus strains tested in the CDT latex test showed nonspecific agglutination (uninterpretable results). The broth culture supernatants ofthese 13 S. aureus strains were highly reactive with the negative control latex beads, as well as with latex beads that were coated with antibody directed against C. difficile antigen. Nonspecific agglutination results occurred in only about 1% of the stools we have tested using the CDT latex test (unpublished data). Thus, in practice, this kind of uninterpretable reaction does not seem to be a significant problem. However, S. aureus is not uncommon in stool samples of healthy volunteers and possibly could cause nonspecific agglutination reactions in occasional fecal samples. Preliminary evidence (unpublished data) suggests that the CDT latex test does not react with strains of S. aureus that lack protein A. The CDT latex test appears to be relatively specific for an antigen shared by most, if not all, cytotoxin-positive and toxin-negative strains of C. difficile. The chemical and physical characteristics of the latex agglutinating antigen(s) detected in C. difficile, C. sporogenes, and P. anaerobius were
determined recently (10, 11). In another study (B. L. Miles, N. U. Hill, and S. D. Allen, Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, B220, p. 61), we found no correlation between the presence or absence of enterotoxin (toxin A) production by C. difficile, as detected by using a rabbit ileal loop model, and reactivity of C. difficile in the CDT test. Our results tend to confirm the findings of Lyerly and associates (10-12) that the antigen detected by using the CDT test is not toxin A; neither is it toxin B. Therefore, the test is an indicator of the presence of the organisms per se and not toxin. Again, it is noteworthy that only 3 of 77 species tested in our in vitro culture study, other than C. difficile, showed false-positive agglutination reactions in the latex test. As alluded to above, data are lacking relative to the frequencies of C. botulinuin, C. sporogenes, and P. anaerobius in diarrheic fecal specimens received by clinical laboratories for C. difficile analyses. Nonetheless, these organisms are potential causes of false-positive CDT latex test results. These observations probably support arguments for the need to confirm positive stool CDT latex test results with another test (i.e., cytotoxin assay [14] or culture [8] or both), when warranted clinically.
A positive latex test result can be a rapid and useful diagnostic indicator that C. difficile organisms are present in a fecal specimen. On the other hand, a positive latex test by itself does not distinguish between individuals who are simply colonized with the organisms and those patients who have clinical manifestations attributable to C. difficile-associated disease. It should be obvious that the same is true for primary isolation of C. difficile from feces and for detecting the presence of the cytotoxin of C. difficile in feces; namely, that the tests do not measure a host response to the toxins, nor do they measure a host response to other virulence factors of the organisms. Therefore, clinical findings must also be considered by the clinician in interpreting the results of these diagnostic procedures for a given patient. We thank Kelda Harris Walsh, Marchelle Woods, and Amy Henzman for technical assistance.
LITERATURE CITED 1. Allen, S. D. 1985. Clostridiuin, p. 434-444. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 2. Arnon, S. S. 1985. Infant botulism, p. 39-55. In S. P. Borriello (ed.), Clostridia in gastrointestinal disease. CRC Press, Inc., Boca Raton, Fla.
3. Banno, Y., T. Kobayashi, K. Watanabe, K. Veno, and Y. Hozawa. 1984. Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridiîum difficile. Rev. Infect. Dis. 6(Suppl. 1):11-20. 4. Borriello, S. P., F. E. Barclay, P. J. Reed, A. R. Welch, J. D. Brown, and D. W. Burdon. 1987. Analysis of latex agglutination
test for Closiridiumi difficile toxin A (D-1) and differentiation between C. difficile toxins A and B and latex reactive protein. J.
Clin. Pathol. 40:573-580. 5. Finegold, S. M., V. L. Sutter, and B. E. Mathisen. 1983. Normal indigenous flora, p. 3-31. In D. V. Hentges (ed.), Human intestinal microflora in health and disease. Academic Press, Inc., New York. 6. Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.). 1977.
Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg. 7. Kamiya, S., S. Nakamura, K. Yamakawa, and S. Nishida. 1986.
Evaluation of a commercially available latex immunoagglutination test kit for detection of Clostridiiîm difficile D-1 toxin. Microbiol. Immunol. 30:177-181.
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8. Kelly, M. T., S. G. Champagne, C. H. Sherlock, M. A. Noble, H. J. Freeman, and J. A. Smith. 1987. Commercial latex agglutination test for detection of Clostr-idiniiii difficile-associated diarrhea. J. Clin. Microbiol. 25:1244-1247. 9. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., and H. M. Sommers. 1983. Color atlas and textbook of diagnostic microbiology, 2nd ed. J. P. Lippincott Co., Philadelphia. 10. Lyerly, D. M., D. W. Bail, J. Toth, and T. D. Wilkins. 1988. Characterization of cross-reactive proteins detected by Culturette Brand Rapid Latex Test for Clostridii7m difficile. J. Clin. Microbiol. 26:397-400. 11. Lyerly, D. M., H. C. Krivan, and T. D. Wilkins. 1988. Clostridijin difficile: its disease and toxins. Clin. Microbiol. Rev. 1:118. 12. Lyerly, D. M., and T. D. Wilkins. 1986. Commercial latex test
for Closiridinm difficile Microbiol. 23:622-623.
toxin A does not
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detect toxin A. J. Clin.
13. Peterson, L. R., J. J. Holter, C. J. Shanholtzer, C. R. Garrett, and D. N. Gerding. 1986. Detection of Clostridiuin difficile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens. Am. J. Clin. Pathol. 86:208-211. 14. Sherman, M. E., P. C. DaGirolami, G. M. Thorne, J. Kimber, and K. Eichelberger. 1988. Evaluation of a latex agglutination kit for diagnosis of Clostridiuni diJcil/e-associated colitis. Am. J. Clin. Pathol. 89:228-233. 15. Smith, L. D. 1977. Botulism: the organism. its toxins, the disease, p. 15-33. Charles C Thomas, Publisher, Springfield, lll. 16. Thompson, J. A., L. A. Glasgow, J. R. Warpinski, and C. Oison. 1980. Infant botulism: clinical spectrum and epidemiology. Pediatrics 66:936-942.
