Jourmd of Microbiological Methods, 16 ( i 992) ! 81 194 ~ 1992 Elsevier Science Publishers B.V. All rights reserved 0167 7012:92505.00

181

M I M ET 00526

Rapid identification and susceptibility of routine urine isolates with the COBAS MICRO

semiautomated system

Paul Nemes and Martin Altwegg Department of Medical Microbiology, University of Zt~rich, Zz~rich. Swit-erland

(Received 22 December 1991 accepted 14 May 1992)

Summary

The COBAS MICRO semiautomated micromethod system for bacterial susceptibility testing and identification within either 5 or 18 h was evaluated against conventional methods. We tested its performance and practicability for the short incubation period under routine operating conditions. The evaluation was done by using 268 recent isolates (162 Enterobacteriaceae, 34 nonfermenters and 72 gram- positive cocci) and from urine specimens and 6 (methicillin-resistant Staphylococcus aureus) stock strains. Susceptibility tests were carried out with a total of 22 user-selectable antimicrobials and compared with the Kirby-Bauer disk diffusion method. For Enterobacteriaceae, the rate of essential and full agreement between COBAS MICRO and reference antibiograms was 98.9% and 95.5%, respectively: for nonfermenters, it was 96.9% and 91.8%; and for gram-positive cocci, 97.7% and 95.1%. Enteric and nonfermentative gram-negative bacilli representing 31 species but not gram-positive cocci were included for identification. Of 196 strains, 192 (97.4%) were correctly identified either at the species or at the genus level, with 27 (13.8%) requiring extra tests for complete identification. A delay of results occurred in only 15 (7.7%) of isolates. Only i (0.06%) of the Enterobacteriaceae, but 4 (12.3%) of the nonfermenters were misidentified at the species level. The actual COBAS MICRO system represents a very good alternative to conventional identification and susceptibility testing techniques for a laboratory that does not desire a totally automated but rather a flexible and easy-to-use system.

Key words: COBAS MICRO system; Rapid identification; Susceptibility testing; Urine isolate

Introduction

Urinary tract infections are the most prevalent infections of adults for which antimicrobials are used. Infections occasionally result in protracted illness and serious disease, including sepsis and death. Antimicrobial agents are usually adminis- tered empirically after urine cultures are taken. If a less toxic or costly drug can be

Correspondence to: P. Nemes, Medizinisch-Diagnostische Laboratorien, Nordstrasse 44, D-4000 Diisseldorf 30, Germany.

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substituted or if early results suggest resistance to the antibiotic chosen, therapy could be appropriately changed. Therefore, same-day results can be of substantial clinical benefit for the diagnosis and treatment of urinary tract infections. For rapid identification and susceptibility testing of such isolates an increasing number of diagnostic microbiology laboratories are willing to use automated computerized systems. Examination of cultured urine dip-slides represents a substantial propor- tion of the workload in our bacteriological laboratory and, consequently, a fully or partially automated system which also reduces hands-on time would be very wel- come.

The earlier totally automated prototype, the COBAS BACT, revealed some major problems both in identification and susceptibility testing accuracy. The new semi- automated instrument, now called COBAS MICRO, has an updated software, improved antibiotic disks awl changed amounts of antibiotic per disk. We have evaluated the performance of the available instrument to urine cultures with respect to its accuracy and applicability in a clinical microbiology laboratory.

Materials and Methods

Strains Two hundred sixty-eight strains (162 Enterobacteriaceae, 72 gram-positive cocci

and 34 nonfermenters) were isolated from dip-slides inoculated with clean-catch and catheter urine specimens submitted to our diagnostic laboratory. The number of strains of each group reflects approximately the frequency with which such strains are found in urinary tract infections; however, the proportion of Klebsiella species and Escherichia coli strains was reduced in favor of a broader panel. To test the ability of the system in differentiating resistant strains from susceptible isolates we limited the number of the fully sensitive E. coli strains and added 6 methicillin-resistant Staphylococcus aureus stock strains.

Identification and susceptibility tests were performed with a total of 196 Enterobacteriaceae and nonfermenters including: i0 Citrobacter freundii, 7 C. diversus, 54 E. coli, 6 Enterobacter aerogenes, 1 E. asburiae, 9 E. cloacae, 8 Klebsiella oxytoca, 1 K. ozaenae, 13 K. pneumoniae, 8 Morganella morganii, 8 Proteus mirabilis, 8 P. vulgaris, 1 Providencia alca!ifaciens, 2 P. rettgeri, 8 P. stuartii, 1 Serratia liquefaciens, 12 S. marcescens, 1 S. odorifera, 1 Acinetobacter anitratus, 7 A. baumannii, 15 Pseudomonas aeruginosa, 2 P. cepacia, 1 P. fluorescens, 1 P. putida, 1 P. stutzeri, 6 Xanthomonas maltophilia and 5 rare urine isolates, one strain of each (Aeromonas caviae, Edwardsiella tarda, Hafnia alvei, Salmonella typhimurium and Yersinia enterocolitica).

Susceptibility tests only were performed on gram-positive cocci: 8 Enterococcus spp., 21 Staphylococcus aureus (13 methicillin-sensitive and 8 methiciUin-resistant) and 28 coagulase-negative staphylococci (including 9 Staphylococcus saprophyticus). Six of the 8 methicillin-resistant staphylococci strains were from clinical non-urine stock cultures.

Isolated colonies growing on 'Urotube' dip-slides (Roche Diagnostics, Basel, Switzerland) or on their subcultures to MacConkey or Columbia CNA (BBL, Microbiology Systems, Cockeysville, MD) agar plates were suspended directly in 4

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ml physiological saline solution. With the 'Cobas r,,~,.h,~t.' ,,,~,,h,~n,,..,..,,... the ~L JL JL ql~,J ~ I Z "~ ' ~ ' .1~ l 1141, ]~_.v l l ~11=,, I I . ,w 1 1 1%=,, L lk,,l 1

inoculum was adjusted to a McFarland standard of 1.0. The ~am, • ~,,~,-,,~,,~i,. ,,,~ used for the conventional and the instrumented COBAS MICRO identification and susceptibility tests.

Conventional susceptibility testing and identification Standardized disk agar diffusion susceptibility tests were performed according to

NCCLS recommendations [1] on Mueller-Hinton Agar (BBL). Results were recorded as 'susceptible', 'intermediate' and 'resi¢,ant'. Conventional identification was made

• • r ' ~ 1 A . . Z . , Z : . : ~ _ - I ,,,;,h,, ,,., an In-house set ,,,"r o,̂ °" ,..~,oA,,,,,,.,~ .~oA;...,,.u,., and l I t..;....t..~....;....to,u,..~,,,,,.a, reactions t/-j. t-~utaitnotiiai API 20 E or NE (API S.A., La Balroe-Les Grottes, France) was performed when: (i) the conventional identification results were inconclusive; (ii) a low-likelihood COBAS MICRO identification result occurred; (iii) conventional and COBAS MICRO iden t i f i ca t ions disagreed. If the d i sc repancy pers is ted an APt 50 for Enterobacteriaceae, and confirmatory conventional tests for nonfermenters accord- ing to Gilardi [3] were used.

Commercial susceptibility testing and ident~'cation The COBAS MICRO instrument (Roche Diagnostics, Basel, Switzerland) is a

semiautomated system for susceptibility and identification analysis of clinically significant bacterial isolates. It consists of two separate round multicompartemented polystyrene rotors, one for susceptibility testing aad one for identification, with 16 to 33 peripheral wells and a single compact unit with a centrifuge, spectrophotometer, keyboard, display and thermoprinter. This system uses a battery of dehydrated substrates for identification and paper-elution disks to which a liquid inoculum is added for susceptibility testing. The manually prepared inoculum is distributed by centrifugation to each rotor chamber. After external incubation the rotor is placed into the centrifugal station of the instrument where a spectrophotometer determines colorimetric and turbidimetric changes. These values are evaluated automatically by comparing the rotor data with the internal data base. Organism identification choices, percent likelihood values, biochemical reactions and comments on identifi- cation quality, atypical reactions and suggestions for supplementary tests are printed out after final reading.

The identification rotor contains 31 biochemical tests for the identification of 102 different species of Enterobacteriaceae and nonfermenters. The reactions include substrate utilization, carbohydrate-dependent acidification, enzymatic tests and growth in the presence of polymixin B and cetrimide. No additional reagents are necessary to read the reactions. Observations made during the preliminary culture, like growth on MacConkey agar, hemolysis, pigment production, swarming, or motility can be entered into the data evaluation system and will be taken into consideration during the calculations.

The susceptibility test rotor allows the simultaneous testing of 15 user-selected antimicrobial agents (from a panel of 60). Use of the COBAS MICRO system for susceptibility testing has been described previously [4]. The instructions provided by the manufacturer were followed in detail. Nonfermenters were incubated overnight at 29°C whereas test rotors with Enterobacteriaceae and gram-positive strains were read

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after 5 h of incubation at 37C. The antimicrobial agents tested correspooaed to th ~se used in the routine disk diffusion method with urine pathogens. Colistin resistance was examined mainly with respect to the ability to differenti:tte gram-negative bacteria, and novobiocin resistance to identify S. saprophyticus.

Data anah'sis Susceptibilio' testing. The results of the COBAS MICRO susceptibility tests were compared with those of the agar disk diffusion technique by using the criteria suggested by Sherris and Ryan [5]. Discrepancies were categorized as very major (susceptible by COBAS MICRO and resistant by the reference method), major (resistant by COBAS MICRO and susceptible by the reference method), and minor (involving an intermediate result). Agreement between the two methods was defined as full (both methods giving the same results) or essentiz! (only very major and major discrepancies were considered errors). Data were analyzed f~r individual organism- antibiotic combinations, overall agreement ior ~ach drug with all organisms tested, and overall agreement for each organism group with all drugs tested. Results are presented in this manner to highlight the organism-antibiotics combinations that were problematical. Additionally, proportions of resistant strains to particular antibiotics have been calculated.

Identification. A COBAS MICRO identification printout provides a series of comments regarding the level of probability of a given organism's code number. This was accompanied by a 'normalized likelihood' (NL) value, expressed as a percentage, which indicates the extent of conformity between the reaction pattern of the test organism and the data base matrix. Another parameter, the 'modal likelihood fraction' (MLF) is depending on the deviation from the ideal profile for that species, caused by atypical or rarely encountered reactions. This value is calculated by dividing the measured likelihood by the lnaximum possible likelihood. The combination of NL and MLF values leads to a verbal c o m i n e n t ~mooa~tino thP . . . . . . . . . . . . . ~ . . . . . . ~ ~. l A , . . ,

confidence of identifications, graded in 6 different categories: excellent, very good, good, acceptable, poor, and low selectivity. The result was considered correct if the only available or the first choice identification agreed with that of the reference method and was rated with at !cast 'acceptable identification'. The strain w~s regarded as correctly identified but requiring additional tests if the correct species was listed within a spectrura of choices with 'poor' or 'low selectivity' identification quality. Definitive ideotitication in this category always required additional testing (as prompted by the instrument) to pinpoint the correct result. Misidentification was defined as incorrect high-confidence ('very good', 'good' or 'acceptable') identifi,:atieiL Earlier comments such as 'not identified' or 'not in the data base' had beo, eliminated in the updated software and, therefore, a spectrum of possible ide~:dfications was always available.

Results

During the period of the study less than 2% of the tests had to be repeated, mainly because of insufficient growth control or inadequate turbidity of the inoculum, the

185

latter resulting in a rejection of the rotor. No system failures for mechanical or other reasons occurred.

Susceptibility testing results Two hundred seventy-four strains were included in the evaluation. Overall, 3998

antibiotic-organism combinations were tested• Full agreement reached 93.6% and essential agreement reached 97.8%. Minor discrepancies were found in 125 (3%)of the tests, major discrepancies were found in 40 (1%) of the tests, and very major discrepancies were found in only 12 (0.3%) of the tests.

One hundred sixty-two Enterobacteriaceae were tested against 15 antibiotics (Table 1). Overall, essential agreement between the methods was 98.9%, whereas full agreement varied between 88.3% for cefamandole and 99.4% for ceftazidime, with an average of 95.5%. Minor discrepancies were observed more often with the earlier generation cephalosporins (cephalothin and cefamandole) than with other antibiotics. Very major discrepancies were associated with individual drug-strain combinations like ampicillin/Enterobacter species and piperacillin/Klebsiella spe- cies. Of the 14 therapeutically useful drugs tested, ampicillin, cephalothin, cefaman- dole, and piperacillin were responsible for 56% of the discrepancies. When analyzed by organism group, full agreement was minimally 93.6% (Pmvidencia species). As a function of the specific antimicrobial agent tested, only cefamandole showed full agreement percentages below 90%. Used for diagnostic purposes, colistin showed full agreement with the disk diffusion test when a zone diameter of more than 11 mm was regarded as sensitive.

For the 34 nonfermenters, susceptibility results are compared in Table 2. On average, essential agreement was observed in 96.9% of the tests and full agreement in 91.8%. By drug, a very low full agreement percentage (73.5%) was observed with piperacillin, caused by major discrepancies in the combination with all X. maltophilia strains tested. Another critical drug-species combination was ceftriaxone in connec- t-;,,-~ . . . . ; t l . , D . . . . 2 , 1 IlK7 UIIK7 d l l U U l l l y V u I . y t l O l l W l L I I • 3 U t 4 L , I O f g l O f l U 3 ~ . J ~ L . , ] ( ~ 3 . " I r k . . . . . . J _ _ ! . . . . . . . . " _ _ _ majo~ discrepancy resulted from testing chloramphenicol with Pseudomonas species.

Analysis of susceptibility tests with the 78 gram-positive cocci is presented in Table 3. The overall essential and full agreement was good with 97.7% and 95.1% respectively, but poor results were seen for the combinations ampicillin and oxacil- lin/S, saprophyticus, norfloxacin/Enterococcus spp. and erythromycin/S, aureus. Three of a total of four very major discrepancies involved the trimethoprim-sulfa- methoxazole/coagulase-negative staphylococci combination. No problems were en- countered with methicillin-resistant S. aureus strains. Novobiocin resistance for separating S. saprophyticus from other coagulase-negative staphylococci was accu- rately detected with user-defined limits, since this agent was not included in the actual data base by the manufacturer.

Identification results Overall, the COBAS MICRO system correctly identified at the genus and species

level 97.4% of the 196 strains of Enterobacteriaceae and nonfermenters. However, the manner in which these identification levels were obtained varied depending on the type of organism being tested. As shown in Table 4, the accuracy of identification for

o~

TABLE !

Comparison of antibiotic susceptibility testing with COBAS MICRO and Kirby-Bauer for Enterobacteriaceae

Antibiotic Citrobacter Escherichia Enterobacter Khbsiella Morganella Proteus Providencta Serratia 'rare' Total spp. coli spp. sp~?. morganii spp. spp. spp. spp.

No. of strains 17 54 16 22 8 16 11 13 5 162

Resistant (%) Agreement by antibiotic EA (%) FA (%)

Amoxiciilin/ 0/0/0 ~ 7/0/0 i/0/0 1/0/0 0/0/0 0/0/0 0/0/0 0/O/0 0/0/0 9/0/0 39.5 100 94.4 clavulanate

Ampicillin i/0/0 3/0/0 0/0/3 1/0/0 0/0/0 0/0/0 2/0/0 1/0/0 0/0/0 8/0/3 72.2 98.1 93.2 Cephalothin 2/1/0 8/0/0 0/0/0 2/'0/0 0/0/0 .0/0 1/0/0 0/0/0 0/0/0 14/1/0 45.1 99.4 90.7 Cefamandole 0/0/0 3/1/0 !/0/0 2/0/0 1/2/0 3/0/0 0/0/0 5/1/0 0/0/0 15/4/0 19.1 97.5 88.3 Chloramphenicol 1/1/0 1/0/0 |/0/0 1/0/0 0/0/0 1/0/0 0/0/0 0/0/0 0/0/0 5/1/0 25.7 99.4 96.3 Netilmicin 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 2/0/1 1", 0 0/0/0 3/0/1 3.7 98.1 96.3 Norfloxacin 0/0/0 2/0/0 0/0/0 0/0/0 0/0/0 0/0/0 1/0/0 0/0/0 0/0/0 3/0/0 4.9 100 96.9 Piperacillin 1/0/0 i/0/0 |/0/0 !/0/3 0/0/0 1/0/0 0/0/0 0/0/0 1/1/0 6/1/3 22.2 97.5 93.8 Trimethoprim/ 0/0/0 2/0/0 1/0/0 0/0/0 0/0/0 0/0/0 1/0/0 0/0/0 0/0/0 4/0/0 22.8 100 95.1

sulfamethoxazole Tetracyclin 1/0/0 1/0/0 !/0/0 0/0/0 0/0/0 3/0/0 0/0/0 1/0/0 0/0/0 7/0/0 36.4 100 95.7 Tobramycin 0/0/0 2/0/0 0/0/0 0/0/0 0/0/0 0/0/0 1/0/0 1/0/0 0/0/0 4/0/0 3.7 100 97.5 Ceftazidime 0/0/0 0/0/0 0/0/0 1/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 1/0/0 1.8 100 99.4 Ceftriaxone 1/0/0 0/0/0 0/0/0 1/0/0 0/0/0 0/0/0 0/0/0 0/0/0 0/0/0 2/0/0 3.7 100 98.8 Imipenem 1/0/0 0/0/0 0/0/0 0/0/0 0/0/0 2/2/0 0/0/0 0/0/0 0/0/0 3/2/0 1.8 98.8 97.5

Total 8/2/0 30/1/0 6/0/3 10/0/3 1/2/0 11/2/0 8/0/1 9/0/0 1/0/0 84/9/7 21.5

Agreement 99. I 99.9 98.6 99 98.2 99.1 98.1 99.4 98.6 by organism EA ~%)

Agreement 95.7 95.8 96.1 95.6 97.4 94 93,6 94.4 97.1 by organism FA (%)

98.9

95.5

a Numbers indicate number of minor/major/very major discrepancies. EA, essential agreement, FA, full agreement.

187

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T A B L E 3

Compar i son of ant ibiot ic susceptibil i ty testing with C O B A S M I C R O and Kirby-Bauer lbr gram-pos i t ive cocci

Antibiotic Entrro( occi Staph.vh,(o,'('u.~ aureu:;

Melhicitlin Mcthicillin sensitive resistant

No. of strains I~ 13 8

Staphyh,coccus Staph.vlo('occu.~ Total coagulase negative sapr<q~hyticus

19 9 78

Resistant (%) Agreement by antibiotic EA (%) FA I%)

Amoxiciilin 0 0 ()" 0 0 0 00 () 0 0 0 02/0 0/20 clavulanate

Ampicillin 0 0 0 0 1 0 0 0 0 0 0 1 0:50 0/6 il Chloramphenicol 0 1 0 0 0 0 () 0 0 0 0 0 0/10 0/2/0 Ciprofioxacin 0 0 0 0 0 0 0 0 0 4 ¢10 0/00 4'0'0 Erythrom~cin 0 0 0 ! 20 O 1 0 1 0 0 1:0:0 33/0 Netihnicin 0 0 0 0 0 0 0 0 0 0 0 0 0/0/0 0/00 Norfloxacin 8 0 0 0 0 0 0 0 0 2 0 0 0:0'0 10/0/0 Oxaciilin 0 0 0 0 0 0 0 0 0 2 0 0 32 '0 5:2(I Trimethoprim 0 0 0 0 0 0 (10 0 0/0 3 0/00 0/03

suilamethoxt, zolc Tobramvcin 0 "~ 0 0 0 0 0 0 0 4 0 0 fi i~,fJ .. . . . . ,.~/ "£,11

Fusidic acid 10 0 0 0 0 0 0 0 0 0 0 0/0/0 I/0/0 Rifampicm 2 0 0 0 0 0 0 0 0 0 0 0 00/O 2/0/0 Vancomycin 0 0 0 0 0 0 0 0 0 0 0 0 00/0 0/0/0

15.4 97.4 97.4

46.1 91 91 16.7 97.4 97.4 ! ! .6 ! 00 94.9 24.4 96.2 92.3

2.6 100 100 I1.6 100 87.2 26.9 97.4 91 !! .6 96.2 96 2

i2.8 97.4 92.3 20.5 100 98.7

2.6 100 97.4 0 1 O0 1 O0

Total 1 1 3 0 ! 30 0 1 0 13 0 4 4/10,'0 20/1714 15.6

Agreement by 95.2 98.2 99. I organism EA ('!.o)

Agreement by 86.6 97.6 99. i organism FA (%)

98.4 91.5

93.2 88

97.7

95.1

N u m b e r s indicate n u m b e r of minor , major , very ma jo r discrepancies. EA, essential agreement . FA, full agreement .

T A B L E 4

Ident i f ica t ion of en te robac te r iaceae with the C O B A S M I C R O s)~tem

Species no. (%) Correctly identified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

To specics Within a spectrum Suggested additional levcl ot choices tests

To genus level

Misidentified

To., ~ecies ieve!

Biochemical tests responsible

Citrohacter spp. ! 7 (10)

E. coli 54 (33)

Enterohacter spp. 16 (10)

Kh, hsiella spp. 22 (14~

16 (99) 1 (I) H2S

51 (94) 3 (6) (.]as, H2S, motility

12 (75) 4 (25) Gelatinase, motility

16 (73) 6 (27) Glucose fermentation at 10 C, Vogcs-Proskauer

Morganella morganii 8 (5) 8 (1(;0}

Proteus spp. 16 (10) 11 (87)

ProvMencia spp. 11 (7) 5 (46)

Serratia spp. 13 (8) 12 (100)

"Rare" species 5 (3) 5 (100)

Total 162 136 (84.4)

5 (13) (,]as, H,S

Jr 3 {27) ! henylalanine dearninase

22 (13.6)

3 (55)

3 (1.9)

liquefaciens instead of odorifera

1 (0.06)

Indol positive; arabinose, ribose and xylose v-gative

Number s in parentheses indicate percent. Fo r exp lana t ion o f categories, see text.

O C

T A B L E 5

Ident i f icat ion of nonfe rmente r s with the C O B A S M I C R O system

Species no. (%) Correctly identified

To species Within a spectrum Suggested additional level of choices tests

To gcnus level

Misidentified

To species level

Biochemical tests responsible

Acinetobacter aniirct~s 1 (3) 1 (100) baumannii 7 (21) 3 (:13) 2 (29) none suggested

Pseudomonas aeruginosa 15 (44) 9 (60) 3 (20) growth at 42°C

cepacia 2 (6) 2 (100) fluorescen~ 1 (3) 1 (100) putida 1 (3) 1 (100) stutzeri 1 (3) 1 (100)

Xanthomonas maltophilia 6 (17) 6 (100)

Total 34 24 (69.7) 5 (15.2)

N u m b e r s in parentheses indicate percent. Fo r explanatio:n o f categories, see text.

1(14)

I (3.0)

! (14) anitratus

3 (20); 2 putida, I fluorescens

4(12.1)

Galactose positive; indol and ornithine negative

Benzoate positive; arabinose, galactose, malon:,te and mannose negative

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Enterobacteriaceae is remarkable, with only one misidentification at the species level. S. odor~fera was incorrectly identified as S. liqm:/'aciens due to four incorrect single reactions. In 22 (13.6%) cases of lower identification levels, a spectrum of choices was available. To pinpoint the correct strain one or more confirmatory tests had to be performed additionally. The tests recommended most frequently were rapid and/or easy to perform ones such as motility, gas or H2S production. Fifteen (7.7%) additional overnight tests caused delays in obtaining correct identification results (data not showa).

For the 34 nonfermenter strains (Table 5) misidentification was observed in 4 cases '12.1%), inel~lding nnc A h q ~ J m m ~ . i i ~tr 'a ln iHontlf~oA a s A .~,.;t,..-,t . . . . . A ~ p

aeruginosa, two identified as P..fluorescens and one as P. putida.

Discussion

Urines are among the most common specimens processed in a clinical microbiol- ogy laboratory. Most of the instrumented systems for identification and susceptibility testing require isolated colonies after ovf. night incubation of the specimen. When a dip-slide is used as a transport medium, bacterial growth may be present at the time of its arrival to the laboratory. By rapid organism identification and susceptibility testing, the COBAS MICRO system offers the potential for "same-day" result reporting.

In order to carry out an evaluation under routine conditions prevailing in our laboratory we compared the COBAS MICRO system with conventional identifica- tion and susceptibility testing methods. The selection of these routine techniques can be justified since previous evaluations could demonstrate that disk diffusion results do correlate well with minimal inhibitory concentration determined by a standard broth dilution method [6,8], and the identification-accuracy of our homemade tubed media [2] and commercial API strips [9] are well documented. Although the use of stock cultures is often necessary to challenge a test system, the hallmark of a system is to test flesh isolates as they are recovered from clinical materials. Our evaluation included a limited number of strains, but care was taken to evaluate the system with a broad panel of various organisms with clinically important resistance patterns.

Compared with published results for other automated instruments [10-13], sus- ceptibility testing performance of the COBAS MICRO system was favorable. Few data have been reported for the updated system. One evaluation [4] using agar dilution as the reference method for susceptibility testing of routine gram-positive rods and gram-positive cocci against 4 antibiotics showed an overall full agreement of 90.2% as compared to the slightly better 93.6% in this study. The results of the present evaluation are significantly better than for its predecessor, the fully auto- mated COBAS BACT system, which had also been evaluated in our laboratory [6]. Essential agreement increased from 95.7% to 97.8% and full agreement from 87.9% to 93.6%. Some of the major problems, especially the failure to detect methicillin- resistant S. aureus and the poor performance in testing Pseudomonas species, could be resolved with the current instrument.

Thornsberry [14] suggested that an automated susceptibility test should provide 90% or greater agreement with reference methods and no greater than 5% major and

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very naajor discrepancies when testing randomly by selected clinical isolates. These proposals and even the more strict criteria from Sherris and Ryan [5] were fulfilled in our eval0ation of the COBAS MICRO system: less than 5% overall errors for random clinical isolates and a maximum of 1.5%, very major errors for each species tested. The greatest impact on the clinical outcome of an infectious disease has a strain reported as 'sensitive" which, in fact, is resistant (very major discrepancy), especially when the antimicrobial agent was considered the drug of choice for therapy. Fortunately, such very major discrepancies occurred in 12 cases only, with 6 antibiotic-organism combinations being clinically relevant (piperacillin/Klebsiella species and ampicillin/Enterobacter species). The latter combination might be less important, because of of the frequent resistance of Enterobacter spp. against ampi- cillin, which is why it should not be used clinically in the first place. These problems and the laigh rate of major discrepancies with ceftriaxone, another fl-lactam anti- biotic, with Pseudomonas species may result from the insufficient incubation period for expression of drug-modifying enzymes. None of the antimicrobial agents tested had sucla an unacceptably low agreement with the conventional disk diffusion technique thus, we would discourage to test this combination or to report it to the physician.

The overall identification rate (97.4%) of the COBAS MICRO is at least similar to that obtained with other instrumented systems [11,13-16]. Most of the systems proved to be more apt at identifying Enterobacteriaceae than the slow-growing nonferrnenters. Therefore, we recommend the longer incubation period of 18 h, both ['or identification and susceptibility testing of the nonfermenters. Three more serious Pseuctomonas misidentifications on species level were omittable when growth at 42°C ,~'as considered for exact identification.

Apart from the accuracy of the results obtained with the COBAS MICRO, some praclical points may be relevant. Inoculation of the rotors may need some time and attent~,3n because the density of the suspension is critical. If it is too low or too high the rotor may not be accepted for reading. Identification but not susceptibility testing of no,fermentative gram-negative rods and Enterobacteriaceae is possible with a single rotor. Consequently, an oxidase-negative glucose-nonfermenting organism e.g. X. mal~ophilia could be placed in the false susceptibility rotor and the test would have to be repeated. Performing identification on inocula prepared from single colonies is possible but results are available only after overnight incubation.

When a profile code was associated with a strong probability and (either 'ex- cellent', 'very good', or 'acceptable' identification), the first-choice organism was found to be accurate and the suggested supplemental tests could be ignored. How- ever, 13.8% of the organisms included in our study required some form of supple- mental testing, most often overnight conventional tube biochemical testing. The degree to which a 'same day' system does not require supplemental overnight testing is a meastare of its "same-day' applicability. The practical utility of a rapid test system is diminished if more than 10% of responses are equivocal and therefore require additional confirmatory tests [17]. To circumvent the costly time taken to incubate the required supplemental tests for definitive identification, like motility or growth at 42°C, the need should be anticipated by performing the tests initially. In 7.7% of our strains identification was delayed until the next day. This is still an acceptable rate for

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a rapid system and is comparable with other automated systems [I 5]. The computer program that drives the COBAS MICRO system is user-friendly,

allows the technologist to check results, to add external results, and to rccalculate. Technologists' time for interpretation and recording of results can be substantially reduced by the computerized functions provided by the instrument. The automatiza- tion of these functions also reduces human errors. The only non-automated proce- dures are: preparation of the inoculum, loading of the rotor, its transfer into ,t...~,,~ incubator and finally, transfer of incubated rotors to the reader. While the system as a whole is easy to use, a few minor adjustments in the program would enhance its k " l ~ . . : 1L..: ! : . . . . i~CXlUi.ty. Until now, rotors ---'J " " ' "" couiu be read only at fixed time intervals. ! ne next software version will enable the reading independently from the duration of incuba- tion (information provided by the manufacturer).

Based on the above results the following conclusions can be drawn. Identification and susceptibility testing with the COBAS MICRO system in 5 h allows "same-day' reporting within the technologists' and physicians' working hours. Thus, it can be considered a rapid system. Compared with conventional methods, it demonstrated a high level of accuracy when used for common urine pathogens. A near, single rotor available in the near future for simultaneous susceptibility testing and identification of urine isolates promises to become an economical alternative to the present configuration with separate rotors. Availability of further rotors for testing anae- robes, gram-positive rods, fastidious gram-negative bacilli and yeasts would be advantageous for clinical laboratories.

Acknowledgement

We gratefully acknowledge Alexander von Graevenitz for carefully reviewing the manuscript.

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