JOURNAL OF CLINICAL MICROBIOLOGY, May 1985, p. 734-737 Vol. 21, No. 5 0095-1137/85/050734-04$02.00/0 Copyright (© 1985, American Society for Microbiology Evaluation of the RIM-N, Gonochek II, and Phadebact Systems for the Identification of Pathogenic Neisseria spp. and Branhamella catarrhalis WILLIAM M. JANDA,1,2* MYRNA G. ULANDAY,' MARJORIE BOHNHOFF,3 AND LEON J. LEBEAU' The University of Illinois Hospital,' The University of Chicago Medical Center,3 and The Howard Brown Memorial Clinic,2 Chicago, Illinois 60612 Received 6 November 1984/Accepted 22 January 1985 Methods for identifying Neisseria spp. include conventional and modified carbohydrate degradation procedures, chromogenic enzyme substrate tests, and immunologic coagglutination tests for Neisseria gonorrhoeae. In this study, we evaluated the abilities of the RIM-N carbohydrate degradation system (American MicroScan, Campbell, Calif.), the Gonochek II enzymatic identification system (Du Pont Co., Wilmington, Del.), and the Phadebact Gonococcus coagglutination test (Pharmacia Diagnostics, Piscataway, N.J.) to identify pathogenic Neisseria spp. and Branhamella catarrhalis. Both stock strains and clinical isolates, including 176 N. gonorrhoeae, 173 Neisseria meningitidis, 48 Neisseria lactamica, and 12 B. catarrhalis strains, were tested. The RIM-N identified 98% of the gonococci, 99% of the meningococci, 94% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 1 h. The Gonochek II system identified 99% of the gonococci, 97% of the meningococci, 100% of the N. lactamica strains, and 100% of the B. catarrhalis strains within 30 min. Phadebact coagglutination provided clearly positive results for only 77% of the N. gonorrhoeae strains, producing negative or equivocal results with 23% of the strains. The RIM-N and Gonocheck II tests generally produced clear-cut reactions. An additional advantage of the Gonocheck II system was the small inoculum required for the performance of the test compared with the other systems, thus allowing the identification of N. gonorrhoeae directly from the primary isolation medium. Current methods for identifying Neisseria spp. isolated (Pharmacia Diagnostics, Piscataway, N.J.), a COA proce- from clinical specimens include conventional and modified dure for the identification of N. gonorrhoeae. carbohydrate degradation procedures, enzymatic tests with (This work was presented at the 24th Interscience Confer- chromogenic substrates, and immunologic coagglutination ence on Antimicrobial Agents and Chemotherapy, 8 to 10 (COA) tests for confirmation of Neisseria gonorrhoeae. October 1984, Washington, D.C.) Whereas conventional sugar utilization tests may require prolonged incubation for organism growth and test results, MATERIALS AND METHODS the modified procedures rely on a heavy bacterial inoculum for rapid identification (2, 6, 8). Chromogenic enzyme sub- Table 1 lists the sources of the stock strains and clinical strate tests, in combination with certain modified conven- isolates examined in this evaluation. tional tests, have also proven useful for identification of Clinical isolates. Clinical isolates of N. gonorrhoeae (102 Neisseria spp. and other bacteria, and commercial systems strains), Neisseria meningitidis (101 strains), Neisseria lac- in which this method is used generally provide results in 4 h tamica (2 strains), and B. catarrhalis (3 strains) were ob- (3, 5, 10). In COA test methods, antibody directed against tained from patients at the University of Illinois Hospital and gonococcal antigens is utilized to produce specific agglutina- the University of Chicago Medical Center and from homo- tion reactions, allowing rapid confirmation of N. gonor- sexual men at the Howard Brown Memorial Clinic in Chi- rhoeae (1, 2, 7). Although not currently commercially avail- cago. Specimens from the University of Illinois Hospital and able, specific agglutination of N. gonorrhoeae by lectins has the University of Chicago Medical Center were received on also been proposed as a useful test for confirmatory identi- JEMBEC plates (Ames Co., Division of Miles Laboratories, fication of gonococci (4). The use of inoculum directly from Elkhart, Ind.) containing modified Thayer-Martin (MTM) the primary selective medium is a distinct advantage with medium (Remel, Lenexa, Kans.) or in swab transport sys- many of these test procedures. tems. Swab specimens were inoculated onto MTM medium In the present study, the rapid identification method for and chocolate (CH) agar (BBL Microbiology Systems, Co- Neisseria (RIM-N; American MicroScan, Campbell, Calif.), ckeysville, Md.) in the laboratory. Specimens from the a rapid carbohydrate test system, and the Gonochek II Howard Brown Memorial Clinic were inoculated directly system (GCII; Du Pont Co., Wilmington, Del.), a rapid onto MTM medium. After incubation in a CO2 environment chromogenic substrate system, were evaluated for the iden- at 35°C, colonies of oxidase-positive, gram-negative diplo- tification of pathogenic Neisseria spp. and Branhamella cocci were subcultured onto CH agar. Growth from the first catarrhalis. Both stock strains and fresh clinical isolates or second subculture was used to inoculate all the test were included in this study. In addition, all strains were systems. In one part of the study, 38 urethral and 6 rectal tested for reactivity with the Phadebact Gonococcus test gonococcal isolates were tested on all of the identification systems with growth from the primary MTM medium. On the following day, conventional identification tests and re- peat tests with all of the systems were performed from CH * Corresponding author. agar subcultures. The identities of all of the isolates used in 734 VOL. 21, 1985 IDENTIFICATION OF NEISSERIA SPP. 735 TABLE 1. Sources of clinical isolates and stock strains used in nitroanilide, and a P-naphthyl amino acid derivative) in a the evaluation of the RIM-N, GCII, and COA tests single tube are utilized to detect enzymatic activities asso- Organism Total no. Source (no.) ciated with N. gonorrhoeae, N. meningitidis, and N. lact- amica. After rehydration of the dried substrates in the tube N. gonorrhoeae with 4 drops of phosphate-buffered saline (pH 7.4), 5 to 10 Clinical isolates 102 Anogenital (100) Oropharyngeal (1) medium-to-large colonies of oxidase-positive, gram-negative Systemic (1) diplococci from a pure culture growing on selective medium Stock strains 74 Anogenital (33) or a CH agar subculture are emulsified in the tube with a Systemic (38) wooden applicator stick. The tube is capped with the paired Oropharyngeal (2) stopper and stored at 35°C for 30 min. Specific color reac- Umbilicus (1) tions in the bacterial suspension confirm the isolate as N. meningitidis (hydrolysis of y-glutamyl-p-nitroanilide, yel- N, meningitidis low) or N. lactamica (hydrolysis of 5-bromo-4-chloro-3- Clinical isolates 101 Oropharyngeal (97) indoyl-,3-D-galactopyranoside, blue). If the suspension is Anogenital (3) Systemic (1) colorless at the end of the incubation period, the paired Stock strains 72 Systemic (37) stopper is split apart and the topmost stopper is inserted into Anogenital (31) the tube. The tube is then inverted so that the bacterial Oropharyngeal (2) suspension comes in contact with the diazo dye coupler Pericardial fluid (1) (o-aminoazotoluene diazonium salt [fast garnet]) present on Lymph node biopsy (1) the stopper. The detection of P-naphthylamine released by bacterial prolyliminopeptidase activity (pink) identifies the N. lactamica isolate as N. gonorrhoeae. The absence of a colored product Clinical isolates 2 Oropharyngeal (2) at the completion of the testing and reading steps is a Stock strains 46 Oropharyngeal (45) presumptive identification of B. catarrhalis. Cervix (1) The Phadebact Gonococcus test is a COA test for the B. catarrhalis identification of N. gonorrhoeae (1, 2, 8). This test was Clinical isolates 3 Conjunctival (3) performed from primary MTM or CH agar subcultures by Stock strains 9 Conjunctival (4) the boiled bacterial suspension technique recommended by Tracheal aspirate (3) the manufacturer. The bacterial suspension is allowed to Oropharyngeal (1) react with the antigonococcal antibody COA reagent (test Systemic (1) reagent) and with the control COA reagent (no antibody) on a cardboard slide. Agglutination within 2 min in the test COA reagent and not in the control COA reagent identifies this evaluation were confirmed by colony and Gram stain the organism as N. gonorrhoeae. All isolates in this study morphology, ability to grow on MTM medium, oxidase were tested with both control and test COA reagents, and reactivity, and acid production from appropriate carbohy- agglutination was scored as 2+ or greater (definite aggluti- drates by the rapid technique described previously (6). nation), ± (slight or equivocal agglutination), or - (no Stock strains. All stock strains had been isolated from a agglutination). variety of clinical specimens and were maintained frozen at -86°C. These included 74 N. gonorrhoeae, 72 Neisseria RESULTS meningitidis, 46 Neisseria lactamica, and 9 B. catarrhalis Table 2 summarizes the data for the identification of strains. All N. gonorrhoeae stock strains had been previ- gonococci, meningococci, N. lactamica, and B. catarrhalis ously auxotyped for their nutritional requirements on a modified Catlin medium (9). For testing on the various identification systems, stock strains were subcultured twice on CH agar before inoculation onto the systems. TABLE 2. Identification of Neisseria spp. and