Evaluation of latex agglutination and microtube coagulase tests for detection of Staphylococcus aureus

DIAGNMICROBIOLINFECTDIS 1984;2:287-291

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Evaluation of Latex Agglutination and Microtube Coagulase Tests for Detection of Staphylococcus aureus Majid Pourshadi and John Klaas

In a blind study, a latex agglutination test (Serostat Staphylococcus, Scott Laboratories) and a microtube coagulase test (Staphase, API) were evaluated for their ability to detect Staphylococcus aureus. Of 289 isolates of catalase-positive, gram-positive cocci, 122 were identified as S. aureus based on positive reactions in at least three of the following tests: tube coagulase, slide coagulase, DNase production, or anaerobic fermentation of mannitol. The latex agglutination test gave positive reactions for all S. aureus isolates and 10 (8%) non-S, aureus isolates. The slide coagulase test was positive for 121 S. aureus isolates and three (2%) non-S, aureus isolates. The microtube coagulase test detected 53, 90, and 98% of the S. aureus isolates after 2, 4, and 24 hr, respectively. In contrast, the conventional tube coagulase test detected 97% of the S. aureus isolates after 2 hr, and 98% after 4 and 24 hr. Two isolates of S. aureus gave negative tube coagulase reactions at 37°C, but positive reactions at room temperature after 24 hr. The combination of tube and slide coagulase tests provided the most reliable results. The slide and tube coagulase tests gave more reliable results than the latex agglutination and microtube coagulase tests, respectively.

The p r o d u c t i o n of coagulase is generally accepted as the most significant criterion for differentiation of S t a p h y l o c o c c u s aureus from other species of staphylococci (Baird-Parker et al., 1976; Kloos and Smith, 1980). Recently, a latex slide agglutination test and a microtube coagulase test have been p r o p o s e d for the identification of S. aureus. The former test, w h i c h was d e v e l o p e d by Essers and Radebold (1980), uses plasma-coated latex particles that can detect both protein A and c l u m p i n g factor. Bacteria that p r o d u c e c l u m p i n g factor will react with fibrinogen in the plasma, whereas bacteria containing protein A will react w i t h i m m u n o g l o b n l i n G (IgG) (Essers and Radebold, 1980; Myrick and Ellner, 1982). This test is c o m m e r c i a l l y available as the Serostat Staphylococcus test (Scott Laboratories, Fiskeville, RI). The microtube coagulase test, c o m m e r c i a l l y marketed as the Staphase test (Analytab Products Inc., Plainview, NY), is essentially a tube coagulase test packaged in the API microtube format. The p u r p o s e of this study was to determine the ability of the Serostat Staphylococcus test and the Staphase test to identify isolates of S. aureus.

From the Medical Laboratory Science Program, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA. Address reprint requests to: Majid Pourshadi, Medical Laboratory Science Program, College of Pharmacy and Allied Health Professions, Northeastern University, Boston, MA 02115. Received September 12, 1983; revised and accepted January 30, 1984.

© 1984 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017

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M. Pourshadi and John Klaas

MATERIALS AND METHODS

Organisms A total of 289 catalase-positive, gram-positive cocci were isolated from human clinical specimens submitted for routine bacteriologic examination. Prior to testing, all isolates were coded to prevent observer bias during testing, subcultured on Trypticase Soy Agar plates containing 5% sheep blood (Scott), and incubated aerobically for 24 hr at 37°C. Biochemical tests of each isolate were performed in duplicate, using Staphylococcus epidermidis (ATCC 12228) and S. aureus (ATCC 25923) as control organisms. Isolates were considered to be S. aureus if they were positive in at least three of the following standard tests: tube coagulase, slide coagulase, anaerobic fermentation of mannitol, or deoxyribonuclease production.

Tube Coagulase Test One or two isolated colonies of each organism were transferred with a sterile wooden applicator stick into a test tube containing 0.4 ml of rabbit plasma with EDTA (BBL Microbiology Systems, Cockeysville, MD). The tubes were incubated at 37°C and examined after 1, 2, 4, and 24 hr. Any degree of clotting was considered a positive reaction (Kloos and Smith, 1980; Sonnenworth, 1980).

Slide Coagulase Test A heavy suspension of each organism was prepared in sterile distilled water. One drop of this suspension was mixed with one drop of EDTA-rabbit plasma (BBL) on a glass slide. The slide was gently rotated for 10-20 sec and examined against a black background for any degree of clumping (Cadness-Graves et al., 1943; Kloos and Smith, 1980; Sonnenworth, 1980).

Anaerobic Fermentation of Mannitol This test was performed as described by Mossel (1962), except that the inoculated tubes were overlaid with 3-4 ml of mineral oil to ensure anaerobic conditions. Development of any distinct yellow color after incubation for 48 hr at 37°C was considered a positive reaction.

Deoxyribonuclease Production DNase production was tested by making a heavy streak of the test organism on DNase test agar containing toluidine blue dye (Scott). The plates were examined for a pinkcolored zone around the colonies after a 48-hr incubation at 37°C.

Latex Slide Agglutination Test Following the directions of the manufacturer, one drop of heavy bacterial suspension was mixed with one drop of the latex reagent (Serostat Staphylococcus test, Scott) on a glass slide. The slide was tilted back and forth over a black background and observed for clumping within 45 sec.

Microtube Coagulase Test The Staphase microtubes, containing lyophilized rabbit plasma with EDTA, were reconstituted with two to three drops of sterile distilled water, and inoculated with

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Detection of S. aureus

one or two isolated colonies according to the directions of the manufacturer. The microtubes were examined for gelation of plasma after 2, 4, and 24 hr of incubation at 37°C.

Data Analysis Sensitivity of a test was expressed as the percentage of S. aureus isolates correctly detected by the test. Specificity of a test was expressed as the percentage of non°S. aureus isolates correctly detected by the test (Galen and Gambino, 1975). Cochran's Q test and a two-tailed McNemar test were used to determine significant differences among methods (Daniel, 1978). A difference was considered significant if P ~< 0.05. RESULTS Of the 289 isolates tested, 122 were identified as S. aureus. As shown in Table 1, the latex agglutination test correctly detected all isolates of S. aureus. However, this test gave false-positive reactions with 10 (6%) non-S, aureus isolates. The API StaphIdent System was used to determine whether these 10 isolates were similar. Five of the isolates were identified as Staphylococcus warneri, two as Staphylococcus xylosus, one as Staphylococcus hominis, and one as S. epidermidis. The one additional isolate was identified as Micrococcus spp. based on its failure to produce acid from glucose u n d e r anaerobic conditions (Kloos and Smith, 1980). The slide coagulase test gave positive reactions for 121 of the 122 isolates of S. aureus and three isolates of non-S, aureus. These three isolates were identified as S. warneri, S. xylosus, and S. epidermidis by the API Staph-Ident System. Eight nonS. aureus isolates agglutinated in water but not in saline, and saline was substituted for water as the s u s p e n d i n g m e d i u m in testing these organisms for c l u m p i n g factor. Thirteen other isolates autoagglutinated in both saline and water, and could not be tested by either the latex agglutination or slide coagulase test. The sensitivity and

TABLE 1. Comparison of Latex Agglutination Test with Standard Methods for Identification of Staphylococcus aureus Method

Latex agglutination Positive Negative Slide coagulase Positive Negative Tube coagulation Positive Negative DNase production Positive

Negative Anaerobic fermentation of mannitol Positive Negative

S. a u r e u s (Total, 122)

Non-S. aureus (Total, 167)

122 0

10 144°

121 1

3 151°

120 2

0 167

118 4

5 162

118 4

20 147

~Thirteen isolates were not tested because of autoagglutination in water or saline.

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TABLE 2. Comparison of Standard Coagulase with API Microtube Coagulase for Detection of Staphylococcus aureus

Method Tube coagulase Microtube coagulase

Cumulative no. of positive reactions at

Total tested

1 hr

2 hr

4 hr

24 hr

122

59 (48%)

118 (97%)

120 (98%)

120 (98%)

122

NT°

65 (53%)

110 (90%)

119 (98%)

QNT= not tested.

specificity of latex agglutination test were calculated to be 100% and 94%, respectively. The slide coagulase test had a sensitivity of 99% and a specificity of 98%. Among the 122 isolates of S. aureus tested with the API microtube coagulase system, 65 (53%) were detected after 2 hr, 110 (90%) after 4 hr, and 119 (98%) after 24 hr of incubation at 37 ° C (Table 2). In contrast, the conventional tube coagulase test detected 118 (97%) of these isolates after 2 hr, and 120 (98%) after 4 and 24 hr of incubation. Two isolates of S. aureus gave negative tube coagulase reactions at 37 ° C, but positive reactions at room temperature after 24 hr of incubation. These two strains gave positive reactions for the latex agglutination, slide coagulase, DNase production, and anaerobic fermentation of mannitol, but negative reactions in the API microtube coagulase test at both 37 ° C and room temperature. The identification of these two isolates as S. aureus was also confirmed by the API Staph-Ident System. DISCUSSION A simple, single test for rapid and reliable identification of S. aureus has been elusive (Boothby et al., 1979). Traditionally, clinical laboratories have relied on the tube coagulase test (Engels et al., 1981). Recently, the latex agglutination test has been suggested as an alternative method for the identification of S. aureus (Doern, 1982; Essers and Radebold, 1980; Myrick and Ellner, 1982). Based on our results, there was no significant difference between the sensitivity of latex agglutination as compared to the sensitivity of either slide or tube coagulase test (p > 0.05). However, the specificity of 94% for the latex agglutination test was significantly less than the specificity of 98% for the slide test or 100% for the tube coagulase test (p < 0.01). Myrick and Ellner (1982) reported a 99% specificity for the latex agglutination test, but this may be an overestimate. They included as S. aureus all isolates that gave positive reactions with the slide coagulase test. In our testing, we found three non-S, oureus isolates which were positive in both the slide coagulase and latex agglutination tests. Had we followed the criterion of Myrick and Ellner (1982) for identification of S. aureus and considered these isolates as S. aureus, our specificity for the latex agglutination test would have increased. This finding suggests that the slide coagulase test, which detects only clumping factor (Kloos and Smith, 1980), should not be used as the sole criterion for identification of S. aureus. All isolates with positive slide coagulase reactions should be confirmed with another test, such as tube coagulase. In an evaluation of the API microtube coagulase test, one study (Goldstein and Roberts, 1982) reported a 99% sensitivity and 100% specificity with coagulase-positive staphylococci with 4 hr of incubation at 370 C. However, only 90% of our S, aureus isolates had positive reactions with this test within 4 hr. When the incubation

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of microtubes was e x t e n d e d overnight, an additional 8% of our S. aureus isolates were detected. Our data suggest that the API microtube system should be incubated for an a d d i t i o n a l 20 hr if no clot develops after 4 hr. The conventional tube coagulase test, in contrast, detected a significantly larger number of the S. aureus isolates at both 2 and 4 hr (p < 0.01, Table 2). An advantage of the tube coagulase test is that it is not subject to the autoagglutination that p r e v e n t e d our testing of 13 isolates with the latex agglutination and the slide coagulase tests. Some strains of S. aureus may fail to give positive reactions with the tube coagulase test at 370 C (Landau and Kaplan, 1980), as h a p p e n e d with two of our isolates. These isolates can be detected by performing a second test, such as the slide coagulase test, or by incubating the tube coagulase test at room temperature. In summary, the latex agglutination and API microtube coagulase tests d i d not provide as reliable identification for S. aureus as the traditional tube and slide coagulase tests. A combination of the tube and slide coagulase methods provided the most r a p i d and accurate identification of S. aureus. We are grateful to Aida Mitchell and Rose Girouard of the Microbiology Laboratory, Brookline Hospital, and to Louis Maher of the Microbiology Laboratory, New England Deaconess Hospital, Boston, Massachusetts, for providing clinical isolates.

REFERENCES 1. Baird-Parker AC, et al., (1976) Appendix 1. Identification of staphylococci. Subcommittee on the Taxonomy of Staphylococci and Microcci. Int ] Syst Bacterial 26:333. 2. Boothby JC, et al. (1979) Tandem coagulase/thermonuclease agar method for detection of Staphylococcus aureus. Appl Environ Microbial 37:298. 3. Brun Y, et al. (1978) Micromethod for biochemical identification of coagulase-negative staphylococci. J Clin Microbial 8:503. 4. Cadness-Graves B, et al. (1943) Slide-test for coagulase-positive staphylococci. Lancet 1:736. 5. Daniel WW (1978) Applied Nonparametrie Statistics. Hopewell, N.J.: Houghton Mifflin Company, pp. 146-151,241-249. 6. Doern GV (1982) Evaluation of a commercial latex agglutination test for identification of Staphylococcus aureus. J. Clin Microbial 15:416. 7. Engels W, et al. (1981) Rapid and direct staphylocoagulase assay that uses a chromogenic substrate for identification of Staphylococcus aureus. J Clin Microbial 14:496. 8. Essers L, Radebold K (1980) Rapid and reliable identification of Staphylococcus aureus by a latex agglutination test. J Clin Microbial 12:641. 9. Galen RS, Gambino SR (1975) Beyond Normality: The Predictive Value and Efficiency of Medical Diagnoses. New York: John Wiley & Sons Inc., pp. 9-14. 10. Goldstein J, Roberts JW (1982) Microtube coagulase test for detection of coagulase-positive staphylococci. J Clin Microbial 15:848. 11. Kloos WE, Smith PB (1980) Staphylococci, In Manual of Clinical Microbiology, 3rd ed., D.H. Lennette, A. Balows, W.J. Hausler, and J.P. Truant (eds.), Washington, D.C.: American Society for Microbiology, pp. 83-87. 12. Landau W, Kaplan RL (1980) Room temperature coagulase production by Staphylococcus aureus strains. Clin Microbial Newsltr 2:9. 13. Mossel DAA (1962) Attempt in classification of catalase-positive staphylococci and micrococci. J Bacterial 84:1140. 14. Myrick BA, Ellner PD (1982) Evaluation of the latex slide agglutination test for identification of Staphylococcus aureus. J Clin Microbial 15:275. 15. Sonnenwirth AC (1980) Staphylococcus, In Gradwohl's Clinical Laboratory Methods and Diagnosis, 8th ed., A. C. Sonnenwirth, L. Jarett, (eds.), St. Louis: Mosby, pp. 1630-1665.