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Anaerobic Bacteria DEBORAH M JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 457-462 Vol. 29, No. 3 0095-1137/91/030457-06$02.00/0 Copyright ©D 1991, American Society for Microbiology Clinical Evaluation of the RapID-ANA II Panel for Identification of Anaerobic Bacteria DEBORAH M. CELIG1* AND PAUL C. SCHRECKENBERGER" 2 Clinical Microbiology Laboratories, University of Illinois Hospital,' and Department of Pathology,2 University of Illinois at Chicago, Chicago, Illinois 60612 Received 17 October 1990/Accepted 11 December 1990 The accuracy of the RapID-ANA II system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) was evaluated by comparing the results obtained with that system with results obtained by the methods described by the Virginia Polytechnic Institute and State University. Three hundred anaerobic bacteria were tested, including 259 clinical isolates and 41 stock strains of anaerobic microorganisms representing 16 genera and 48 species. When identifications to the genus level only were included, 96% of the anaerobic gram-negative bacilli, 94% of the Clostridium species, 83% of the anaerobic, nonsporeforming, gram-positive bacilli, and 97% of the anaerobic cocci were correctly identified. When correct identifications to the genus and species levels were compared, 86% of 152 anaerobic gram-negative bacilli, 76% of 34 Clostridium species, 81% of 41 anaerobic, nonsporeforming, gram-positive bacilli, and 97% of 73 anaerobic cocci were correctly identified. Eight isolates (3%) produced inadequate identifications in which the correct identification was listed with one or two other possible choices and extra tests were required for separation. A total of 9 isolates (3%) were misidentified by the RapID-ANA II panel. Overall, the system was able to correctly identify 94% of all the isolates to the genus level and 87% of the isolates to the species level in 4 h by using aerobic incubation. Time-consuming biochemical testing under strict anaero- The purpose of this study was to evaluate the accuracy of bic conditions and gas-liquid chromatographic analysis of the RapID-ANA II for the identification of clinically signif- short-chain fatty acid metabolites of glucose fermentation icant anaerobic bacteria. Results obtained with the RapID- have traditionally marked the methods for the identification ANA II panel were compared with those obtained by the of anaerobic bacteria (13, 19). These conventional methods methods described by the Virginia Polytechnic Institute and are labor intensive, time consuming, expensive, and beyond State University, Blacksburg (13). the means of most clinical microbiology laboratories. Many (Part of this work was presented at the 90th Annual of the original commercial kit systems were dependent upon Meeting of the American Society for Microbiology [7].) the growth of the anaerobic organisms and therefore re- quired 24 to 48 h or more of anaerobic incubation before test results were generated (2). MATERIALS AND METHODS In recent years, kit systems have been developed for the RapID-ANA II panel. The RapID-ANA II panel has 10 identification of clinically relevant anaerobic bacteria that reaction wells molded into the periphery of a plastic dispos- are not growth dependent and that do not require anaerobic able tray with eight bifunctional wells to allow for a total of incubation. These systems are based on the detection of 18 biochemical reactions. The panel contains 16 chromoge- preformed bacterial enzymes (23) and include the RapID- nic substrate tests and 2 modified conventional tests. There ANA (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) (1, are nine nitrophenyl carbohydrate or phosphoester deriva- 3, 4, 6, 8-12, 15, 17, 20, 21, 28), the AN-Ident system tives which, when cleaved by enzymatic hydrolysis, release (Analytab Products) (4, 6, 12, 21, 27, 29), the Anaerobe yellow o- or p-nitrophenol. There are seven P-naphthylam- Identification card (Vitek Systems, Hazelwood, Mo.) (24), ide derivatives of amino acids which, upon enzymatic hy- the 2-h ABL system (Austin Biological Systems, Austin, drolysis, release free P-naphthylamine detected by the pres- Tex. [22]), the ATB 32A system (API System SA, La Balme ence of a purple color after addition of the RapID-ANA II les Grottes, Montalieu-Vercieu, France) (18), and the Micro- reagent. The RapID-ANA II reagent is a modified cinnamal- Scan system (American MicroScan, Sacramento, Calif.). dehyde reagent for detection of the amine. Modified conven- Innovative Diagnostic Systems, Inc., has revised the tional tests include urea hydrolysis and the formation of original RapID-ANA panel through the deletion of triphenyl indole. The Innova reagent for the detection of indole uses a tetrazolium reduction, rapid arginine dihydrolase, and modification of the spot indole reagent (1% p-dimethylami- trehalose. Three new substrates, namely, urea, p-nitrophe- nocinnamaldehyde with 10% hydrochloric acid). nyl-,3-D-disaccharide (BLTS), and p-nitrophenyl-a-L-arabi- The panel was inoculated by using a pure culture bacterial noside (aARA), have been added to the panel. The RapID- suspension, equivalent to that of a no. 3 McFarland turbidity ANA II panel and worksheets have been reconfigured for standard, prepared in the RapID inoculation fluid from 18 to ease of use, and the RapID-ANA II Code Compendium has 72 h of growth on an anaerobic blood plate. The panel was been revised with updated nomenclature and codes to coin- inoculated according to the directions of the manufacturer cide with the new panel. Evaluations of the newly configured and incubated aerobically at 35°C for 4 h. After 4 h, the first RapID-ANA II panels have been presented recently (5, 26). set of reactions was scored, followed by the addition of the RapID-ANA II reagent and the Innova spot indole reagent to the appropriate wells. A comparator card is available with * Corresponding author. the kit to facilitate test interpretation. Reactions were re- 457 458 CELIG AND SCHRECKENBERGER J. CLIN. MICROBIOL. corded, and a six-digit profile number was generated. Iden- isms. Stock organisms were subcultured onto agar media at tifications were obtained by using the RapID-ANA II Code least three times prior to testing with the RapID-ANA II Compendium (16) together with knowledge of the Gram stain panel and by conventional identification procedures. reaction, organism morphology, and the profile number. Conventional identification procedures included Gram The RapID-ANA II Code Compendium (16) is divided into staining, aerotolerance, growth on selective and differential three major sections: (i) gram-negative anaerobic bacilli, (ii) media (e.g., kanamycin-vancomycin-laked blood agar, Bac- gram-positive anaerobic bacilli, and (iii) anaerobic cocci. teroides bile esculin agar, egg yolk agar, peptone-yeast The compendium provides an organism identification or extract-glucose broth with bile), and gas-liquid chromatog- several possible identifications, along with the correspond- raphy. Biochemical tests and carbohydrate fermentation ing probability, biotype, and contraindicated test results. reactions were performed with prereduced, anaerobically Additional comments associated with the microcode or sterilized media (Carr-Scarborough Microbiologicals, Stone identification are printed beneath the differential informa- Mountain, Ga.) by methods described in the Virginia Poly- tion. Identification to the species level is associated with the technic Institute's Anaerobe Laboratory Manual (13) and in following levels of confidence. An "implicit" confidence the latest edition of Bergey's manual (14). Identifications by level indicates that the probability of the first choice is both conventional tests and with the RapID-ANA II panel >99.9% and that there are no contraindicated tests. "Satis- were performed in a blinded manner. factory" indicates a >95.0% probability and only minor contraindicated tests, while an "adequate" confidence level RESULTS indicates >95.0% probability of the first choice but contrain- dicated tests resulting in a fair degree of variation from the Table 1 shows the RapID-ANA II panel results for the ideal data base pattern. A "presumptive" identification anaerobic gram-negative bacilli. Of the 65 Bacteroides fra- indicates that there is a single major contraindicated test and gilis group isolates tested, the RapID-ANA II panel cor- the choice should be evaluated to confirm the identification. rectly identified 53 isolates (81%); all 25 Bacteroides fragilis "Questionable" indicates that the identification is inconsis- isolates were identified correctly. Nine B. fragilis group tent and a probability value is therefore not printed. "Inad- isolates (14%), including seven Bacteroides ovatus and two equate" identifications are generated when probability over- Bacteroides uniformis isolates, were identified to the genus laps occur. "Probability overlap" indicates that more than level only as Bacteroides fragilis group. Two isolates of one choice demonstrates a significant probability of at least Bacteroides ovatus were misidentified as Bacteroides theta- 5.0% and may be designated as being among the first two or iotaomicron, and one strain of Bacteroides uniformis gener- three choices listed. If the organisms are from the same ated an inadequate identification without the correct species genus, often a genus- or group-level identification is offered. included in the identification. When organisms correctly Taxa contained in the RapID-ANA II Code Compendium identified to the species or genus level only were included, 62 (16) include
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