G.C. Test; Pharmacia Diagnostics, Piscataway, N.J.), and meningococci were confirmed with grouping sera (Wellcome. Diagnostics, Dartford, England).
Vol. 24, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1986, p. 141-142
0095-1137/86/070141-02$02.00/0 Copyright © 1986, American Society for Microbiology
Preliminary Evaluation of a Rapid Colorimetric Method for Identification of Pathogenic Neisseria MARION E. HOSMER,' MENASHI A. COHENFORD,2 AND PAUL D. ELLNER1* Clinical Microbiology Service, The Presbyterian Hospital, New York, New York 10032,' and Oncology Laboratories, Inc., West Warwick, Rhode Island 028932 Received 27 January 1986/Accepted 20 March 1986
A rapid colorimetric method for the identification of pathogenic Neisseria (Identicult-Neisseria; Scott Laboratories, Inc.) based on P-galactosidase, y-glutamylaminopeptidase, and y-prolylaminopeptidase is described. Ail 82 clinical isolates of Neisseria gonorrhoeae, 9 clinical isolates of N. meningitidis, and 5 clinical isolates of N. lactamica were correctly determined to the species level, as were 4 isolates of Branhamella catarrhalis. Reactions were prompt and easily interpreted. The system should be extremely useful in clinical laboratories. appear on the y-prolylaminopeptidase spot. A red color on the -y-prolylaminopeptidase spot only identifies the organism as N. gonorrhoeae. If no color appears at any of the spots, the organism is presumptively identified as Branhamella catarrhalis. One hundred organisms, all of which were fresh clinical isolates from patients at The Presbyterian Hospital, were tested with the new systein. The identification was confirmed by fermentation of glucose, maltose, or sucrose on Columbia agar slants (Scott) and 3-galactosidase activity (o-nitrophenyl-,-D-galactosidase tablets; Key Scientific Products Co., Inc., Los Angeles, Calif.). Additionally, gonococci were confirmed by coagglutination (Phadebact, G.C. Test; Pharmacia Diagnostics, Piscataway, N.J.), and meningococci were confirmed with grouping sera (Wellcome Diagnostics, Dartford, England). The isolates included 82 N. gonorrhoeae from genital cultures, 9 N. meningitidis from blood or cerebrospinal fluid, 5 N. lactamica from respiratory specimens, and 4 Branhamella catarrhalis from middle ear exudates. All of the organisms were correctly identified by the Identicult-Neisseria strip test. Color appeared within 15 s after the addition of developer solution and was easily interpreted. It is important that the test be performed only on colonies from a selective medium because N. sicca, N. flavescens, and N. subflava produce y-glutamylaminopeptidase and -y-prolylaminopeptidase. These organisms would be misidentified as N. meningitidis, but they do not grow on modified Thayer-Martin or Martin-Lewis medium. The Identicult-Neisseria test is an analogous product to one recently described for the identification of group A streptococci and enterococci (2). Enzymatic methods have a number of distinct advantages over other techniques. Colorimetric spot tests on paper are less subjective than coagglutination tests and are faster than other enzymatic tests. The paper strips are easy to handle and stable at 2 to 8°C, and the cost per test is competitive with that of other tests. The Identicult-Neisseria test promises to provide a rapid and reliable approach to the identification of pathogenic Neisseria spp. in the clinical laboratory.
Commonly used procedures for the identification of Neisseria spp. in the laboratory are based on carbohydrate fermentation patterns. These methods rely on bacterial growth and require incubation periods of 3 to 24 h. The results of such tests may sometimes be misleading, or the organism may fail to grow. Nongrowth carbohydrate degradation tests may be used in which results are available within 1 to 4 h. These tests usually require a heavy inoculum, and unreliable results may occur with colonies from plates containing antimicrobial agents (4). D'Amato et al. (1) determined the enzymatic profiles of Neisseria gonorrhoeae, N. meningitidis, and related species using chromogenic substrates as a means of rapidly identifying these organisms. Yajko et al. (7) was able to rapidly confirm the identification of N. gonorrhoeae using chromogenic substrates of ,-galactosidase, -y-glutamylaminopeptidasé, and y-prolylaminopeptidase and agglutination by wheat germ and soybean lectins. Welborn et al. (6) described a commercial system (Gonochek LI; Du Pont Diagnostic Systems, Wilmington, Del.) for the rapid identification of pathogenic Neisseria spp. that combined several chromogenic substrates in a single tube. This system compared favorably with coagglutination and nongrowth carbohydrate degradation systems (3, 5). A new product for the rapid identification of pathogenic Neisseria spp. (Identicult-Neisseria; Scott Laboratories, Inc.) has recently become available. This system employs a filter paper strip with three spots containing synthetic chromogenic substrates for ,B-galactosidase, -y-glutamylaminopeptidase, and y-prolylaminopeptidase. To perform the test, 5 to 10 oxidase-positive colonies from a selective medium such as Martin-Lewis or NYC medium were picked with a swab or applicator stick and applied to each of the three substrate spots. Three drops of buffer were added to each spôt, and the strip was incubated at 35°C for 15 min. A blue-green color appearing on the P-galactosidase spot indicates that the isolate is N. lactamica. If no color is present on this spot, three drops of developer solution supplied by the manufacturer were added to the other two substrate spots. The appearance of a deep blue color on the -y-glutamylaminopeptidase spot is evidence that the organism is N. meningitidis, regardless of a red color that might *
LITERATURE CITED 1. D'Amato, R. F., L. A. Eriquei, K. M. Tomfohrde, and E. Singerman. 1978. Rapid identification of Neisseria gonorrhoeae and Neisseria meningitidis by using enzymatic profiles. J. Clin.
Corresponding author.
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Microbiol. 7:77-81. 2. Ellner, P. D., D. A. Williams, M. E. Hosmer, and M. A. Cohenford. 1985. Preliminary evaluation of a rapid colorimetric method for the presumptive identification of group A streptococci and enterococci. J. Clin. Microbiol. 22:880-881. 3. Janda, W. M., M. G. Ulanday, M. Bohnhoff, and L. LeBeau. 1985. Evaluation of the RIM-N, Gonocheck II, and Phadebact systems for the identification of pathogenic Neisseria spp. and Branhamella catarrhalis. J. Clin. Microbiol. 21:734-737. 4. Kellogg, D. S. Jr., K. K. Holmes, and G. A. Hill. 1976. Cumitech 4, Laboratory diagnosis of gonorrhea. Coordinating ed., S.
J. CLIN. MICROBIOL.
Marcus and J. C. Sherris. American Society for Microbiology, Washington, D.C. 5. Philip, A., and G. C. Garton. 1985. Comparative evaluation of five commercial systems for the rapid identification of pathogenic Neisseria species. J. Clin. Microbiol. 22:101-104. 6. Welborn, P. P., C. T. Uyeda, and N. Ellison-Birang. 1984. Evaluation of Gonochek II as a rapid identification system for pathogenic Neisseria species. J. Clin. Microbiol. 20:680-683. 7. Yajko, D. M., A. Chu, and W. K. Hadley. 1984. Rapid confirmatory identification of Neisseria gonorrhoeae with lectins and chromogenic substrates. J. Clin. Microbiol. 19:380-382.
