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 22 Bacteroides spp., 4 Fusobacterium spp., 3 of 65 B. fragilis group isolates (95%) were correctly identi- Porphyromonas spp., Tissierella praeacuta, Wolinella sp., fied. Of the 25 Bacteroides melaninogenicus group organ- Capnocytophaga sp., and Mobiluncus sp. in the gram- isms, 24 (96%) were correctly identified (Table 1). Only one negative rod section. Clostridium clostridiiforme and Clos- organism was misidentified: a Bacteroides intermedius iso- tridium ramosum are included in both the gram-negative and late which was identified by the RapID-ANA II panel as a the gram-positive sections of the compendium (16). A total Bacteroides corporis isolate. Among 47 other Bacteroides of 24 Clostridium spp., 7 Actinomyces spp., 7 Lactobacillus spp., 45 isolates (96%) were identified, with 1 strain being spp., 3 Eubacterium spp., 2 Propionibacterium spp., Arach- misidentified and with 1 strain having an inadequate identi- nia propionica (Propionibacterium propionicus), Mobilun- fication. One Bacteroides oralis isolate was misidentified as cus sp., and Bifidobacterium sp. are included in the gram- Bacteroides oris. An inadequate identification was generated positive section. Eight Peptostreptococcus spp., two with one isolate ofBacteroides gracilis in which Bacteroides Streptococcus spp., Staphylococcus saccharolyticus, gracilis and Wolinella sp. were listed as the possible identi- Gemella morbillorum, and Veillonella sp. complete the cocci fications. All six Fusobacterium nucleatum isolates were section of the compendium (16). No attempt is made to identified to the genus level only, with the use of Gram stain differentiate among the species in the genera Capnocytoph- morphology, a lipase reaction, or both given as differential aga, Mobiluncus, Bifidobacterium, or Veillonella. It has tests. All isolates of Porphyromonas sp., Capnocytophaga recently been proposed that the moderately saccharolytic sp., and Mobiluncus sp. were correctly identified. One Bacteroides spp. that are inhibited by bile be classified in the isolate of Bacteroides zoogleoformans and two isolates of new genus Prevotella (25). This change has not yet been Fusobacterium gonidiaformans, which are not included in incorporated into the RapID-ANA II Code Compendium the RapID-ANA II data base, were called a Bacteroides (16), and as such, the genus designation Prevotella is not oralis group isolate and a Fusobacterium sp., respectively. used in this report. These were the only three organisms tested which were not Bacterial strains. The 300 organisms evaluated in the study in the RapID-ANA II data base and, as such, were not consisted of 259 clinical isolates and 41 stock strains. Clini- included in the statistical evaluation. Of the 152 anaerobic cal isolates were obtained from patient specimens submitted gram-negative bacilli tested, the RapID-ANA II panel iden- to the University of Illinois Hospital Anaerobic Bacteriology tified 131 isolates (86%) to the species level and an additional Laboratory and represented members of the genera Actino- 15 isolates (10%) to the genus level, with 4 strains (3%) being myces, Bacteroides, Bifidobacterium, Capnocytophaga, misidentified and 2 strains (1%) having inadequate identifi- Clostridium, Eubacterium, Fusobacterium, Gemella, Lacto- cations. bacillus, Mobiluncus, Peptostreptococcus, Porphyromonas, Table 2 presents RapID-ANA II panel results for spore- Propionibacterium, Staphylococcus, Streptococcus, and forming and nonsporeforming, anaerobic, gram-positive ba- Veillonella. Stock organisms included organisms from the cilli. Of 34 Clostridium spp. tested, 26 (76%) were correctly American Type Culture Collection (Rockville, Md.) and identified to the species level, including seven Clostridium clinical stock strains maintained as quality control organ- perfringens, five Clostridium ramosum, 5 Clostridium ca- VOL. 29, 1991 RapID-ANA II EVALUATION 459

TABLE 1. Identification of anaerobic gram-negative bacilli with the RapID-ANA II panel No. (%) of isolates:

OrganismT Identified to Identified to to Inadequate identification, Inadequate identification, Tested species levela genus levelb genus or spe- correct speciesd incorrect speciese Bacteroidesfragilis group 65 53 (81.5) 9 (13.8) 2 (3.1) 1 (1.5) B. fragilis 25 25 (100) B. distasonis 10 10 (100) B. thetaiotaomicron 10 10 (100) B. ovatus 10 1 (10) 7 (70) 2 (20)f B. vulgatus 5 5 (100) B. uniformis 5 2 (40) 2 (40) 1 (20) Bacteroides melaninogenicus group 25 24 (96) 1 (4)f B. intermedius 16 15 (94) 1 (6Yf B. melaninogenicus 8 8 (100) B. Ioescheii 1 1 (100) Other Bacteroides spp. 47 45 (96) 1 (2/ 1 (2) B. bivius 25 25 (100) B. buccae 11 11 (100) B. capillosus 3 3 (100) B. oralis 3 2 (67) 1 (33)f B. disiens 2 2 (100) B. ureolyticus 2 2 (100) B. gracilis 1 1 (100) Porphyromonas asaccharolytica 6 6 (100) Fusobacterium nucleatum 6 6 (100) Capnocytophaga spp. 2 2 (100) Mobiluncus spp. 1 1(100) Total 152 131 (86.2) 15 (9.8) 4 (2.6) 1 (0.7) 1 (0.7) aImplicit, satisfactory, or acceptable probability of identification to the species level. b Genus- or group-level identification with supplemental tests necessary for resolution of species. " Misidentification at the genus level. d Inadequate identification; correct species included among species listed; additional tests necessary. eInadequate identification; correct species not among species listed. f Misidentification at the species level; correct genus identification. daveris, 2 Clostridium sordellii, 2 Clostridium septicum, 1 species level. Three isolates of Lactobacillus catenaforme Clostridium clostridiiforme, and 1 Clostridium paraputri- were correctly identified to species level but only one of two ficum isolate. Two Clostridium difficile isolates were cor- isolates of Lactobacillus acidophilus was correctly identified rectly identified, but one was misidentified as an Eubacte- with the second isolate being misidentified as a Lactobacillus rium limosum isolate. Of three Clostridium innocuum catenaforme. Isolates of the genus Bifidobacterium were isolates tested, one was correctly identified, one yielded an identified only as Bifidobacterium spp. in the RapID-ANA II identification of Clostridium spp. in which the correct iden- system. Two of three isolates were correctly identified. The tification was not among the listed choices, and one was remaining strain was given an inadequate identification, with incorrectly identified as a Clostridium subterminale. Of two the correct identification being among the choices. One Clostridium sporogenes isolates tested, both yielded identi- identi- fications of Clostridium spp., with the choice being between isolate of Actinomyces odontolyticus was incorrectly Clostridium difficile and Clostridium sporogenes. All three fied as Clostridium hastiforme, while one strain was called isolates of Clostridium tertium generated a genus-level iden- Actinomyces sp., with the correct species being among the tification of Clostridium spp., with the correct choice being possible choices. among the three possible species and a definitive test of Table 3 summarizes the RapID-ANA II panel results for aerobic growth to differentiate the species. the anaerobic cocci. A total of 53 of the peptostreptococci Only 2 of 23 isolates of propionibacteria tested gave (96%) were correctly identified to the species level. Two inadequate identifications in which the correct identification Peptostreptococcus prevotii isolates produced inadequate was included in the choices. Both strains were indole- identifications in which the correct identification was among negative strains of Propionibacterium acnes. One isolate of the choices. All isolates of Peptostreptococcus asaccharo- Eubacterium lentum was incorrectly identified as Clostrid- lyticus, Peptostreptococcus anaerobius, Peptostreptococ- ium hastiforme, one isolate was given an inadequate identi- cus magnus, Peptostreptococcus micros, and Peptostrep- fication in which the correct identification was among those tococcus tetradius were correctly identified. One Gemella listed, and five isolates were correctly identified to the morbillorum, seven Streptococcus intermedius, one Staph- 460 CELIG AND SCHRECKENBERGER J. CLIN. MICROBIOL.

TABLE 2. Identification of anaerobic sporeforming and nonsporeforming, gram-positive bacilli with the RapID-ANA II panel' No. (%) of isolates: Organism Identified to Identified to Misidentified Inadequate identification, Tested species genulev to genus or correct species level genus level species level correct species Clostridium spp. 34 26 (76) 6 (18) 2 (6) C. perfringens 7 7 (100) C. ramosum 5 5 (100) C. cadaveris 5 5 (100) C. difficile 3 2 (67) 1 (33) C. innocuum 3 1 (33) 1 (33) 1 (33)b C. tertium 3 3 (100) C. septicum 2 2 (100) C. sordellii 2 2 (100) C. sporogenes 2 2 (100) C. clostridiiforme 1 1 (100) C. paraputrificum 1 1 (100) Other nonsporeforming spp. 41 33 (81) 1 (2) 3 (7) 4 (10) Propionibacterium acnes 23 21(91) 2 (9) Eubacterium lentum 7 5 (71) 1 (14) 1 (14) Lactobacillus catenaforme 3 3 (100) Lactobacillus acidophilus 2 1 (50) 1 (50) Bifidobacterium spp. 3 2 (67) 1 (33) Actinomyces odontolyticus 2 1 (50) 1 (50) Actinomyces israelii 1 1 (100) Total 75 59 (78.7) 7 (9.3) 5 (6.7) 4 (5.3) a See text and footnotes to Table 1 for explanations of column heads. There were no organisms with inadequate identifications and with the correct species not being among the species listed. b Misidentification at the species level; correct genus identification. ylococcus saccharolyticus, and nine Veillonella spp. were TABLE 3. Identification of anaerobic gram-positive and gram- also correctly identified. negative cocci with the RapID-ANA II panel' No. (%) of isolates: DISCUSSION Organism Identified Inadequate In this study, the RapID-ANA II system was evaluated for Tested to. species identification, its ability to identify a wide variety of anaerobic bacteria level correct species recovered in the clinical laboratory. After familiarization with the various types of color reactions produced by Peptostreptococcus spp. 55 53 (96) 2 (4) organisms in the various substrates, the interpretation of P. asaccharolyticus 24 24 (100) most of the tests on the panel was not difficult. The yellow P. magnus 13 13 (100) P. anaerobius 7 7 colors resulting from the o- or p-nitrophenol reactions and (100) P. micros 6 6 (100) the dark pink to purple color from the ,3-naphthylamine P. tetradius 3 3 (100) reactions were generally clear-cut. Both the urea and indole P. prevotii 2 2 (100) reactions were easily interpreted. In reviewing the initial studies performed on the original Staphylococcus 1 1 (100) RapID-ANA panel, the Anldent panel and the Vitek ANI saccharolyticus card, widely varying percentages of accuracy have been reported. In previous studies performed on the first RapID- Streptococcus 7 7 (100) ANA system, 59 to 92% of anaerobic gram-negative bacilli, intermedius 66 to 100% of clostridia, 50 to 100% of nonsporeforming Gemella morbillorum 1 1 (100) gram-positive bacilli, and 78 to 100% of anaerobic cocci were correctly identified by the system compared with correct Veillonella sp. 9 9 (100) identifications by various conventional methods (1, 3, 4, 6, 8-12, 15, 17, 20, 21, 28). The wide variations in performance Total 73 71 (97.3) 2 (2.7) of these systems are related to the numbers, types, and sources (e.g., stock strains, human a See text and footnotes to Table 1 for explanations of column heads. There clinical isolates, and were no organisms identified to only the genus level, misidentified to the genus veterinary strains) of organisms tested, whether the addi- or species level, or inadequate identification and with the correct species not tional tests suggested by the systems' computer-generated being among the species listed. VOL. 29, 1991 RapID-ANA II EVALUATION 461 codes were included as a part of the "rapid" identification reactions was not considered to be a problem in this study. procedure, and whether genus-level identifications were Referral to the color reaction card available from the manu- considered as correct identifications. In this evaluation, the facturer helped in resolving color interpretations. In this RapID-ANA II system correctly identified 131 of 152 anaer- study, all tests were read by one person, eliminating vari- obic gram-negative bacilli (86.2%), 26 of 34 Clostridium spp. ability in test result interpretations by different personnel. In (76.5%), 33 of 41 nonsporeforming, gram-positive bacilli the study by Karachewski et al. (17), 23% of code numbers (80.5%), and 71 of 73 anaerobic cocci (97.3%). When genus- generated were not listed in the Code Compendium. In this level identifications are included, 146 of 152 anaerobic study, all code numbers were listed in the updated version of gram-negative bacilli (96.1%), 32 of 34 Clostridium spp. the Code Compendium (16). (94.1%), 34 of 41 anaerobic, nonsporeforming bacilli The RapID-ANA II system performed well in identifying (82.9%), and 71 of 73 anaerobic cocci (97.3%) were correctly certain Clostridium spp., particularly Clostridium perfrin- identified. Seven additional isolates produced inadequate gens (100%), and Propionibacterium spp. (91%), but it identifications in which the correct identification was among performed less well with other species of gram-positive the listed choices and additional instructions were given for bacilli and needs to be tested with larger numbers of organ- further definitive testing. isms in order to fully evaluate the system's performance with The addition of urea to the RapID-ANA II panel aided in this group of organisms. The addition of the updated data the identification of Bacteroides ureolyticus, Clostridium base, updated nomenclature, new code sheets, and the sordellii, Actinomyces naeslundii, Actinomyces viscosus, substitution of three tests has enhanced the system and leads and Peptostreptococcus tetradius. The other two new sub- to improved overall performance. strates (BLTS and aARA) assisted in differentiating some of In summary, we found the RapID-ANA II panel to be an the Bacteroides and Clostridium spp. The deletion of acceptable rapid test system for identifying most of the trehalose from the RapID-ANA II test panel increased the clinically significant anaerobic bacteria tested in this study. difficulty of differentiating between Bacteroides ovatus and The decrease in the amount of labor involved in the identi- Bacteroides uniformis. There was no single test in the fication of anaerobic organisms and the more rapid definitive RapID-ANA II system that separated these two species. identification help to provide a more timely diagnostic ser- Consequently, 7 of 10 Bacteroides ovatus isolates were vice in clinical anaerobic bacteriology laboratories. identified to the group level only as the Bacteroides fragilis group, with Bacteroides ovatus and Bacteroides uniformis ACKNOWLEDGMENTS being possible identifications of the organism. Two addi- tional strains of Bacteroides ovatus were incorrectly identi- We thank Gail Richardson, Judy Schermond, Rebecca Shone, fied as Bacteroides thetaiotaomicron because of positive Hazel Livingston, and Theresa Hutchen for assistance in performing arginine reactions in the RapID-ANA II panel. The system the reference identifications. used arginine and serine as key tests in separating Bacteroi- REFERENCES des thetaiotaomicron (usually positive) from Bacteroides 1. Adney, W. S., and R. L. 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