JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 457-462 Vol. 29, No. 30095-1137/91/030457-06$02.00/0Copyright ©D 1991, American Society for Microbiology

Clinical Evaluation of the RapID-ANA II Panel for Identification ofAnaerobic Bacteria

DEBORAH M. CELIG1* AND PAUL C. SCHRECKENBERGER" 2

Clinical Microbiology Laboratories, University of Illinois Hospital,' and Department of Pathology,2University 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 evaluatedby comparing the results obtained with that system with results obtained by the methods described by theVirginia Polytechnic Institute and State University. Three hundred anaerobic bacteria were tested, including259 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 theanaerobic cocci were correctly identified. When correct identifications to the genus and species levels werecompared, 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 otherpossible choices and extra tests were required for separation. A total of 9 isolates (3%) were misidentified bythe RapID-ANA II panel. Overall, the system was able to correctly identify 94% of all the isolates to the genuslevel and 87% of the isolates to the species level in 4 h by using aerobic incubation.

Time-consuming biochemical testing under strict anaero-bic conditions and gas-liquid chromatographic analysis ofshort-chain fatty acid metabolites of glucose fermentationhave traditionally marked the methods for the identificationof anaerobic bacteria (13, 19). These conventional methodsare labor intensive, time consuming, expensive, and beyondthe means of most clinical microbiology laboratories. Manyof the original commercial kit systems were dependent uponthe growth of the anaerobic organisms and therefore re-quired 24 to 48 h or more of anaerobic incubation before testresults were generated (2).

In recent years, kit systems have been developed for theidentification of clinically relevant anaerobic bacteria thatare not growth dependent and that do not require anaerobicincubation. These systems are based on the detection ofpreformed bacterial enzymes (23) and include the RapID-ANA (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) (1,3, 4, 6, 8-12, 15, 17, 20, 21, 28), the AN-Ident system(Analytab Products) (4, 6, 12, 21, 27, 29), the AnaerobeIdentification card (Vitek Systems, Hazelwood, Mo.) (24),the 2-h ABL system (Austin Biological Systems, Austin,Tex. [22]), the ATB 32A system (API System SA, La Balmeles Grottes, Montalieu-Vercieu, France) (18), and the Micro-Scan system (American MicroScan, Sacramento, Calif.).

Innovative Diagnostic Systems, Inc., has revised theoriginal RapID-ANA panel through the deletion of triphenyltetrazolium reduction, rapid arginine dihydrolase, andtrehalose. Three new substrates, namely, urea, p-nitrophe-nyl-,3-D-disaccharide (BLTS), and p-nitrophenyl-a-L-arabi-noside (aARA), have been added to the panel. The RapID-ANA II panel and worksheets have been reconfigured forease of use, and the RapID-ANA II Code Compendium hasbeen revised with updated nomenclature and codes to coin-cide with the new panel. Evaluations of the newly configuredRapID-ANA II panels have been presented recently (5, 26).

* Corresponding author.

The purpose of this study was to evaluate the accuracy ofthe RapID-ANA II for the identification of clinically signif-icant anaerobic bacteria. Results obtained with the RapID-ANA II panel were compared with those obtained by themethods described by the Virginia Polytechnic Institute andState University, Blacksburg (13).

(Part of this work was presented at the 90th AnnualMeeting of the American Society for Microbiology [7].)

MATERIALS AND METHODS

RapID-ANA II panel. The RapID-ANA II panel has 10reaction wells molded into the periphery of a plastic dispos-able tray with eight bifunctional wells to allow for a total of18 biochemical reactions. The panel contains 16 chromoge-nic substrate tests and 2 modified conventional tests. Thereare nine nitrophenyl carbohydrate or phosphoester deriva-tives which, when cleaved by enzymatic hydrolysis, releaseyellow o- or p-nitrophenol. There are seven P-naphthylam-ide derivatives of amino acids which, upon enzymatic hy-drolysis, release free P-naphthylamine detected by the pres-ence of a purple color after addition of the RapID-ANA IIreagent. The RapID-ANA II reagent is a modified cinnamal-dehyde reagent for detection of the amine. Modified conven-tional tests include urea hydrolysis and the formation ofindole. The Innova reagent for the detection of indole uses amodification of the spot indole reagent (1% p-dimethylami-nocinnamaldehyde with 10% hydrochloric acid).The panel was inoculated by using a pure culture bacterial

suspension, equivalent to that of a no. 3 McFarland turbiditystandard, prepared in the RapID inoculation fluid from 18 to72 h of growth on an anaerobic blood plate. The panel wasinoculated according to the directions of the manufacturerand incubated aerobically at 35°C for 4 h. After 4 h, the firstset of reactions was scored, followed by the addition of theRapID-ANA II reagent and the Innova spot indole reagent tothe appropriate wells. A comparator card is available withthe kit to facilitate test interpretation. Reactions were re-

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corded, and a six-digit profile number was generated. Iden-tifications were obtained by using the RapID-ANA II CodeCompendium (16) together with knowledge of the Gram stainreaction, organism morphology, and the profile number.The RapID-ANA II Code Compendium (16) is divided into

three major sections: (i) gram-negative anaerobic bacilli, (ii)gram-positive anaerobic bacilli, and (iii) anaerobic cocci.The compendium provides an organism identification orseveral possible identifications, along with the correspond-ing probability, biotype, and contraindicated test results.Additional comments associated with the microcode oridentification are printed beneath the differential informa-tion. Identification to the species level is associated with thefollowing levels of confidence. An "implicit" confidencelevel indicates that the probability of the first choice is>99.9% and that there are no contraindicated tests. "Satis-factory" indicates a >95.0% probability and only minorcontraindicated tests, while an "adequate" confidence levelindicates >95.0% probability of the first choice but contrain-dicated tests resulting in a fair degree of variation from theideal data base pattern. A "presumptive" identificationindicates that there is a single major contraindicated test andthe choice should be evaluated to confirm the identification."Questionable" indicates that the identification is inconsis-tent and a probability value is therefore not printed. "Inad-equate" identifications are generated when probability over-laps occur. "Probability overlap" indicates that more thanone choice demonstrates a significant probability of at least5.0% and may be designated as being among the first two orthree choices listed. If the organisms are from the samegenus, often a genus- or group-level identification is offered.Taxa contained in the RapID-ANA II Code Compendium

(16) include 22 Bacteroides spp., 4 Fusobacterium spp., 3Porphyromonas spp., Tissierella praeacuta, Wolinella sp.,Capnocytophaga sp., and Mobiluncus sp. in the gram-negative rod section. Clostridium clostridiiforme and Clos-tridium ramosum are included in both the gram-negative andthe gram-positive sections of the compendium (16). A totalof 24 Clostridium spp., 7 Actinomyces spp., 7 Lactobacillusspp., 3 Eubacterium spp., 2 Propionibacterium spp., Arach-nia propionica (Propionibacterium propionicus), Mobilun-cus sp., and Bifidobacterium sp. are included in the gram-positive section. Eight Peptostreptococcus spp., twoStreptococcus spp., Staphylococcus saccharolyticus,Gemella morbillorum, and Veillonella sp. complete the coccisection of the compendium (16). No attempt is made todifferentiate among the species in the genera Capnocytoph-aga, Mobiluncus, Bifidobacterium, or Veillonella. It hasrecently been proposed that the moderately saccharolyticBacteroides spp. that are inhibited by bile be classified in thenew genus Prevotella (25). This change has not yet beenincorporated into the RapID-ANA II Code Compendium(16), and as such, the genus designation Prevotella is notused in this report.

Bacterial strains. The 300 organisms evaluated in the studyconsisted of 259 clinical isolates and 41 stock strains. Clini-cal isolates were obtained from patient specimens submittedto the University of Illinois Hospital Anaerobic BacteriologyLaboratory and represented members of the genera Actino-myces, Bacteroides, Bifidobacterium, Capnocytophaga,Clostridium, Eubacterium, Fusobacterium, Gemella, Lacto-bacillus, Mobiluncus, Peptostreptococcus, Porphyromonas,Propionibacterium, Staphylococcus, Streptococcus, andVeillonella. Stock organisms included organisms from theAmerican Type Culture Collection (Rockville, Md.) andclinical stock strains maintained as quality control organ-

isms. Stock organisms were subcultured onto agar media atleast three times prior to testing with the RapID-ANA IIpanel and by conventional identification procedures.

Conventional identification procedures included Gramstaining, aerotolerance, growth on selective and differentialmedia (e.g., kanamycin-vancomycin-laked blood agar, Bac-teroides bile esculin agar, egg yolk agar, peptone-yeastextract-glucose broth with bile), and gas-liquid chromatog-raphy. Biochemical tests and carbohydrate fermentationreactions were performed with prereduced, anaerobicallysterilized media (Carr-Scarborough Microbiologicals, StoneMountain, Ga.) by methods described in the Virginia Poly-technic Institute's Anaerobe Laboratory Manual (13) and inthe latest edition of Bergey's manual (14). Identifications byboth conventional tests and with the RapID-ANA II panelwere performed in a blinded manner.

RESULTS

Table 1 shows the RapID-ANA II panel results for theanaerobic gram-negative bacilli. Of the 65 Bacteroides fra-gilis group isolates tested, the RapID-ANA II panel cor-rectly identified 53 isolates (81%); all 25 Bacteroides fragilisisolates were identified correctly. Nine B. fragilis groupisolates (14%), including seven Bacteroides ovatus and twoBacteroides uniformis isolates, were identified to the genuslevel only as Bacteroides fragilis group. Two isolates ofBacteroides ovatus were misidentified as Bacteroides theta-iotaomicron, and one strain of Bacteroides uniformis gener-ated an inadequate identification without the correct speciesincluded in the identification. When organisms correctlyidentified to the species or genus level only were included, 62of 65 B. fragilis group isolates (95%) were correctly identi-fied. Of the 25 Bacteroides melaninogenicus group organ-isms, 24 (96%) were correctly identified (Table 1). Only oneorganism was misidentified: a Bacteroides intermedius iso-late which was identified by the RapID-ANA II panel as aBacteroides corporis isolate. Among 47 other Bacteroidesspp., 45 isolates (96%) were identified, with 1 strain beingmisidentified and with 1 strain having an inadequate identi-fication. One Bacteroides oralis isolate was misidentified asBacteroides oris. An inadequate identification was generatedwith one isolate of Bacteroides gracilis in which Bacteroidesgracilis and Wolinella sp. were listed as the possible identi-fications. All six Fusobacterium nucleatum isolates wereidentified to the genus level only, with the use of Gram stainmorphology, a lipase reaction, or both given as differentialtests. All isolates of Porphyromonas sp., Capnocytophagasp., and Mobiluncus sp. were correctly identified. Oneisolate of Bacteroides zoogleoformans and two isolates ofFusobacterium gonidiaformans, which are not included inthe RapID-ANA II data base, were called a Bacteroidesoralis group isolate and a Fusobacterium sp., respectively.These were the only three organisms tested which were notin the RapID-ANA II data base and, as such, were notincluded in the statistical evaluation. Of the 152 anaerobicgram-negative bacilli tested, the RapID-ANA II panel iden-tified 131 isolates (86%) to the species level and an additional15 isolates (10%) to the genus level, with 4 strains (3%) beingmisidentified and 2 strains (1%) having inadequate identifi-cations.

Table 2 presents RapID-ANA II panel results for spore-forming and nonsporeforming, anaerobic, gram-positive ba-cilli. Of 34 Clostridium spp. tested, 26 (76%) were correctlyidentified to the species level, including seven Clostridiumperfringens, five Clostridium ramosum, 5 Clostridium ca-

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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)fB. vulgatus 5 5 (100)B. uniformis 5 2 (40) 2 (40) 1 (20)

Bacteroides melaninogenicus group 25 24 (96) 1 (4)fB. intermedius 16 15 (94) 1 (6YfB. 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)fB. 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, 1Clostridium clostridiiforme, and 1 Clostridium paraputri-ficum isolate. Two Clostridium difficile isolates were cor-rectly identified, but one was misidentified as an Eubacte-rium limosum isolate. Of three Clostridium innocuumisolates tested, one was correctly identified, one yielded anidentification of Clostridium spp. in which the correct iden-tification was not among the listed choices, and one wasincorrectly identified as a Clostridium subterminale. Of twoClostridium sporogenes isolates tested, both yielded identi-fications of Clostridium spp., with the choice being betweenClostridium difficile and Clostridium sporogenes. All threeisolates of Clostridium tertium generated a genus-level iden-tification of Clostridium spp., with the correct choice beingamong the three possible species and a definitive test ofaerobic growth to differentiate the species.Only 2 of 23 isolates of propionibacteria tested gave

inadequate identifications in which the correct identificationwas included in the choices. Both strains were indole-negative strains of Propionibacterium acnes. One isolate ofEubacterium lentum was incorrectly identified as Clostrid-ium hastiforme, one isolate was given an inadequate identi-fication in which the correct identification was among thoselisted, and five isolates were correctly identified to the

species level. Three isolates of Lactobacillus catenaformewere correctly identified to species level but only one of twoisolates of Lactobacillus acidophilus was correctly identifiedwith the second isolate being misidentified as a Lactobacilluscatenaforme. Isolates of the genus Bifidobacterium wereidentified only as Bifidobacterium spp. in the RapID-ANA IIsystem. Two of three isolates were correctly identified. Theremaining strain was given an inadequate identification, withthe correct identification being among the choices. Oneisolate of Actinomyces odontolyticus was incorrectly identi-fied as Clostridium hastiforme, while one strain was calledActinomyces sp., with the correct species being among thepossible choices.

Table 3 summarizes the RapID-ANA II panel results forthe anaerobic cocci. A total of 53 of the peptostreptococci(96%) were correctly identified to the species level. TwoPeptostreptococcus prevotii isolates produced inadequateidentifications in which the correct identification was amongthe choices. All isolates of Peptostreptococcus asaccharo-lyticus, Peptostreptococcus anaerobius, Peptostreptococ-cus magnus, Peptostreptococcus micros, and Peptostrep-tococcus tetradius were correctly identified. One Gemellamorbillorum, seven Streptococcus intermedius, one Staph-

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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)bC. 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. werealso correctly identified.

DISCUSSION

In this study, the RapID-ANA II system was evaluated forits ability to identify a wide variety of anaerobic bacteriarecovered in the clinical laboratory. After familiarizationwith the various types of color reactions produced byorganisms in the various substrates, the interpretation ofmost of the tests on the panel was not difficult. The yellowcolors resulting from the o- or p-nitrophenol reactions andthe dark pink to purple color from the ,3-naphthylaminereactions were generally clear-cut. Both the urea and indolereactions were easily interpreted.

In reviewing the initial studies performed on the originalRapID-ANA panel, the Anldent panel and the Vitek ANIcard, widely varying percentages of accuracy have beenreported. In previous studies performed on the first RapID-ANA system, 59 to 92% of anaerobic gram-negative bacilli,66 to 100% of clostridia, 50 to 100% of nonsporeforminggram-positive bacilli, and 78 to 100% of anaerobic cocci werecorrectly identified by the system compared with correctidentifications by various conventional methods (1, 3, 4, 6,8-12, 15, 17, 20, 21, 28). The wide variations in performanceof these systems are related to the numbers, types, andsources (e.g., stock strains, human clinical isolates, andveterinary strains) of organisms tested, whether the addi-tional tests suggested by the systems' computer-generated

TABLE 3. Identification of anaerobic gram-positive and gram-negative cocci with the RapID-ANA II panel'

No. (%) of isolates:

Organism Identified InadequateTested to. identification,species correct specieslevel

Peptostreptococcus spp. 55 53 (96) 2 (4)P. asaccharolyticus 24 24 (100)P. magnus 13 13 (100)P. anaerobius 7 7 (100)P. micros 6 6 (100)P. tetradius 3 3 (100)P. prevotii 2 2 (100)

Staphylococcus 1 1 (100)saccharolyticus

Streptococcus 7 7 (100)intermedius

Gemella morbillorum 1 1 (100)

Veillonella sp. 9 9 (100)

Total 73 71 (97.3) 2 (2.7)

a See text and footnotes to Table 1 for explanations of column heads. Therewere no organisms identified to only the genus level, misidentified to the genusor species level, or inadequate identification and with the correct species notbeing among the species listed.

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RapID-ANA II EVALUATION 461

codes were included as a part of the "rapid" identificationprocedure, and whether genus-level identifications wereconsidered as correct identifications. In this evaluation, theRapID-ANA II system correctly identified 131 of 152 anaer-obic gram-negative bacilli (86.2%), 26 of 34 Clostridium spp.(76.5%), 33 of 41 nonsporeforming, gram-positive bacilli(80.5%), and 71 of 73 anaerobic cocci (97.3%). When genus-level identifications are included, 146 of 152 anaerobicgram-negative bacilli (96.1%), 32 of 34 Clostridium spp.(94.1%), 34 of 41 anaerobic, nonsporeforming bacilli(82.9%), and 71 of 73 anaerobic cocci (97.3%) were correctlyidentified. Seven additional isolates produced inadequateidentifications in which the correct identification was amongthe listed choices and additional instructions were given forfurther definitive testing.The addition of urea to the RapID-ANA II panel aided in

the identification of Bacteroides ureolyticus, Clostridiumsordellii, Actinomyces naeslundii, Actinomyces viscosus,and Peptostreptococcus tetradius. The other two new sub-strates (BLTS and aARA) assisted in differentiating some ofthe Bacteroides and Clostridium spp. The deletion oftrehalose from the RapID-ANA II test panel increased thedifficulty of differentiating between Bacteroides ovatus andBacteroides uniformis. There was no single test in theRapID-ANA II system that separated these two species.Consequently, 7 of 10 Bacteroides ovatus isolates wereidentified to the group level only as the Bacteroides fragilisgroup, with Bacteroides ovatus and Bacteroides uniformisbeing possible identifications of the organism. Two addi-tional strains of Bacteroides ovatus were incorrectly identi-fied as Bacteroides thetaiotaomicron because of positivearginine reactions in the RapID-ANA II panel. The systemused arginine and serine as key tests in separating Bacteroi-des thetaiotaomicron (usually positive) from Bacteroidesovatus and Bacteroides uniformis (usually negative). TheRapID-ANA II data base shows that only 11% of Bacteroi-des ovatus isolates should be positive for arginine.Two strains of Bacteroides uniformis were also identified

to the group level only as the Bacteroidesfragilis group, withBacteroides ovatus and Bacteroides uniformis being thesuggested identifications. This was due to a positive reactionwith aARA. Bacteroides uniformis is listed as 81% positivefor aARA and Bacteroides ovatus is listed as 95% positivefor aARA in the RapID-ANA II data base. When this test isnegative, the isolate keys out as Bacteroides uniformis.However, when the test is positive, as occurred with two ofour isolates, the organism keys out as a Bacteroides fragilisgroup isolate. One additional strain keyed out as an inade-quate identification, with Bacteroides eggerthii and Bac-teroides ovatus being the possible choices. The problem herewas traced to a negative L-fucoside reaction (Bacteroidesuniformis is 95% positive) and a positive glycine reaction(Bacteroides uniformis is 12% positive).

Since the indole-positive strains of the Bacteroidesfragilisgroup are routinely more resistant to a greater number ofantibiotics, it is suggested that when reporting the group-level identification, the report should be amended to statethat the organism is one of the indole-positive strains.

Various problems were listed in previously publishedreports that reviewed the first RapID-ANA system. A majorproblem cited in several reports addressed the interpretationof the aryl and amide color reactions, variability in test resultinterpretations by different personnel, and the initial color inthe wells becoming obscured by the addition of the second-ary reagents (6, 9, 17). The last concern still applies to theRapID-ANA II system; however, interpretation of the color

reactions was not considered to be a problem in this study.Referral to the color reaction card available from the manu-facturer helped in resolving color interpretations. In thisstudy, all tests were read by one person, eliminating vari-ability in test result interpretations by different personnel. Inthe study by Karachewski et al. (17), 23% of code numbersgenerated were not listed in the Code Compendium. In thisstudy, all code numbers were listed in the updated version ofthe Code Compendium (16).The RapID-ANA II system performed well in identifying

certain Clostridium spp., particularly Clostridium perfrin-gens (100%), and Propionibacterium spp. (91%), but itperformed less well with other species of gram-positivebacilli and needs to be tested with larger numbers of organ-isms in order to fully evaluate the system's performance withthis group of organisms. The addition of the updated database, updated nomenclature, new code sheets, and thesubstitution of three tests has enhanced the system and leadsto improved overall performance.

In summary, we found the RapID-ANA II panel to be anacceptable rapid test system for identifying most of theclinically significant anaerobic bacteria tested in this study.The decrease in the amount of labor involved in the identi-fication of anaerobic organisms and the more rapid definitiveidentification help to provide a more timely diagnostic ser-vice in clinical anaerobic bacteriology laboratories.

ACKNOWLEDGMENTS

We thank Gail Richardson, Judy Schermond, Rebecca Shone,Hazel Livingston, and Theresa Hutchen for assistance in performingthe reference identifications.

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