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0095-1137/82/100668-05$02.00/0

Copyright©1982,American Society forMicrobiology

Rapid

Processing of Urine

Specimens

by

Urine Screening and

the AutoMicrobic

System

MANOHARWADKE,l2*CHRISTINE MCDONNELL,2ANDJOHN K. ASHTON'2

DepartmentofPathology, MuhlenbergHospital, Plainfield, New Jersey07061,1andUniversity of Medicine

andDentistry ofNewJersey-Rutgers Medical School, Piscataway, New Jersey088542

Received16April 1982/Accepted 14 July 1982

Atotalof1,500clean-voidedurine specimens were analyzed for the presence of

bacteria by urine screening with the Autobac 1 system. The specimens found

positive bythismethodwerefurther processed on the same day for identification andfor antimicrobial susceptibility testing on the AutoMicrobic system with the Enterobacteriaceae-plus Card and the General Susceptibility Card, respectively.

The inoculafor these tests were prepared from the centrifuged and washed growth

in the eugonic broth aspirated from the Autobac cuvette chambers. Of 1,500

specimens that were analyzed, 183 contained single isolates of gram-negative

bacilli.The results ofthese rapid procedures were compared with results for the same organisms isolated from urine specimens cultured by the conventional

method. The data showed92.3%agreementforidentification and a correlation of

93.6% for antibiotic susceptibility between the two procedures. It is concluded that gram-negative bacilli can be rapidly identified and tested for antimicrobial

susceptibility with ahigh degree ofaccuracy from the centrifuged eugonic broth

after urine screening. Thesefindings also suggest that the AutoMicrobic system

providesarapidandconvenientmethodfor same-day processing of positive urine

cultures when combined with the urine screening procedure.

Urinespecimens arethemostcommon

speci-mensprocessed by general hospital microbiolo-gy laboratories. Several physical and chemical

methodsaswellasdirectmicroscopy of stained

smearshave beendescribed fortherapid

detec-tion ofsignificantbacteriain thesespecimens

(2,

4, 5, 13, 14, 17, 18, 25). Recent studies have

shown that urine screening is a highly efficient

methodfor theearly detectionofbacteriuria and can be used for the rapid processing of large

numbers of urine specimens and thus decrease

theturnaround time(12, 16).

Semiautomated instruments such as Autobac

1 (General Diagnostics, Warner-Lambert Co.,

MorrisPlains, N.J.) and MS-2(Abbott

Diagnos-tics, Chicago, Ill.) have been employed to

per-form urine screening (7, 12, 15, 16). Several

studies have also reported that after urine

screening, the broth from thepositive specimens

can be rapidly processed for identification and

antimicrobial susceptibility testing of

Entero-bacteriaceae and thus permit the same-day

re-porting of urine cultures (9, 16). For instance,

Kelly et al. (16), using the Micro-ID system

(General Diagnostics, Warner-LambertCo.)for

enteric identification and Autobac 1 for urine

screening and antibiotic susceptibility testing,

have successfully completed the processing of

urinespecimenswith ahigh degree of accuracy.

The AutoMicrobic system (AMS; Vitek

Sys-tems, Inc., asubsidiary of McDonnell-Douglas,

Hazelwood, Mo.) is a highly automated

instru-mentwith avariety of applications (1, 6, 8, 10,

20, 22). Interpretation of biochemical and

sus-ceptibility tests is done automatically, eliminat-ingthe possibility of subjective errors. One of thedistinctivefeatures of the system is itsability

toprocessurine specimens directlyandto

iden-tify and quantitate the most common urinary

pathogens (8, 11, 20).TheAMS has beenshown

to perform in a highly cost-effective manner in

processing positive urine specimens; however,

its cost effectiveness for processing negative

urine specimensisquestionable (8, 20, 21).

The procedure described by Kelly et al. (16)

for therapidprocessing of urinespecimensuses

Micro-ID for identification and the Autobac 1

systemforsusceptibility testing. Thesesystems

are not fully automated and require the actual

presence ofa technician for reading and

inter-preting the results. The present study was

un-dertaken toevaluate the AMSforrapid

identifi-cation andantimicrobial susceptibilitytestingof

gram-negative bacilli directly from the eugonic

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RAPID PROCESSING OF URINE SPECIMENS 669

TABLE 1. AccuracyofAMS-EBC+ testsfor rapid identification ofgram-negativebacilli in 8 h from

positiveurinespecimens

No.ofstrains identified

Organism by method %

Agreementa Conventional Rapid

Escherichia 122 115 94.3

coli

Klebsiella 20 18 90.0

pneumoniae

Proteus 14 13 92.9

mirabilis

Pseudomonas 9 7 77.8

aeruginosa

Klebsiella 3 3 100.0

oxytocab

Citrobacter 6 4 66.7

freundii

Enterobacter 3 3 100.0

aerogenes

Enterobacter 2 2 100.0

cloacae

Providencia 1 1 100.0

sp.

Proteus 1 1 100.0

vulgaris

Morganella 2 2 100.0

morganii

aAverage percent agreement, 92.3.

bK.oxytoca wasidentifiedbyperforming the indole test onthepositivecontrol well of the EBC+ after the identificationontheAMSwascompleted.

broth ofpositive urine samples screenedonthe Autobac I system. The results of these studies were compared with results for the same

orga-nisms on the AMSfollowingthe standard

isola-tiontechnique.

MATERIALS AND METHODS

A total of 1,500 randomly selected clean-voided urinespecimenswereanalyzedsimultaneously bythe conventional and rapid methods described below.

Bladder, catheter, and suprapubic urine specimens

were excluded.Allurine specimens were refrigerated

uponreceiptin thelaboratoryand were batch tested.

Conventional method.Allurinespecimenswere ana-lyzed by streaking 0.001 ml of the specimen with a calibrated loop on 5% sheep blood agar, colistin-nalidixic acidagar,andMacConkey agar. The colony

countwasdetermined after 18 to 24 hof incubation (3).

Identification ofgram-negative bacilli and

antimicrobi-alsusceptibility testing were performed on all

signifi-cantisolates on the AMS as described later, according totheinstructions of the manufacturer.

Rapid method. (i) Urine screening. Urinescreening was performed with the Autobac 1 system as de-scribedpreviously (16). Samples having a voltage drop

>0.5after5h ofincubation werefurther processed for

rapid identification and antibiotic susceptibility testing asdescribed below.Specimens yielding voltage drops

between 0.2 and 0.5 generally produced insufficient inocula, mainly because of the low concentration of bacteria (<10 colony-formingunits perml), and thus were cultured only by thestandard method.

(ii) Inoculum preparation.Inoculawerepreparedfor rapid identification and antimicrobial susceptibility testing by a procedure similarto thatdescribed else-where, with slight modification (16). After urine screening, the broth from eachpositivechamberof the Autobac cuvette was aspirated with a sterile Pasteur pipette into a 5-ml sterile plasticdisposable tube. A drop of the specimen was alsodispensed on a clean glass slide for Gram stain to check thepurity. Speci-mensshowingonlygram-negative bacilliwerefurther processed. The brothwascentrifugedat approximate-ly1,000xgfor 10 min. Thesupernatant was discarded by carefully inverting the tube, and the pellet was suspended, washed with 0.45% saline, and centri-fuged. The process wasrepeatedto removeanytraces ofeugonic broth. After finalcentrifugation,thepellet was tested forcytochrome oxidaseactivity with Path-oTec strips (General Diagnostics, Warner-Lambert Co.) asrequired in the AMS identification procedure. The pellet was then suspended in 0.45% saline to achieve turbidity equivalent to the McFarland stan-dard1. This inoculumwasused for identification and antimicrobial susceptibilitytesting.

Identification andantimicrobialsusceptibility testing. Identification and antimicrobial susceptibility testing were performed with the Enterobacteriaceae-plus Card (EBC+) and General Susceptibility Card, re-spectively, ontheAMSaccordingtotheinstructions of the manufacturer. A loopful of the standardized suspension was inoculated on 5% sheep blood agar and incubated ovemight to check the purity of the inoculum.

RESULTS

A total of 1,500 urine specimens were

ana-lyzedby urinescreening in this study. Of these, 235weredetectedaspositive after 5hof incuba-tion.Atotalof208specimens hadavoltagedrop 20.5,including201 specimensthatyielded only gram-negative bacilli upon Gram stain and also showed acolony count ofgreater than 100,000

per mlby the standard plate method. The other

seven specimens containedgram-positive cocci

with>105colony-forming units perml detected by both Gram stain and culture and were not processed on the AMS. Of the 201 specimens with gram-negative bacilli, 183 yielded single isolates, and 18 contained two or more gram-negative species. The remaining 27 specimens positive by urine screeningthat were excluded

in thisstudywere asfollows. (i) Twelve

speci-mens had a voltage drop <0.5 and contained

either gram-negative bacilli or gram-positive

bacteria singly orinmixture. (ii) Fifteen

speci-mens with a voltage drop <0.5 were

false-positiveandyielded nogrowth uponculture.

Identification oforganisms. Table 1 shows the

correlation between the rapid method and the VOL.16,1982

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TABLE 2. Correlation of antimicrobial susceptibility tests between the rapid and the conventional methods

ontheAMS'

%Discrepancy'

...

~~~~~~~~~~~~~~~~~~~~~~~~~~Interpretive

Antimicrobialagent Very

major Major Minor agreement

Amikacin 0.0 (0) 0.0 (0) 1.1 (2) 98.9

Ampicillin 1.1 (2) 1.6 (3) 4.9 (9) 92.4

Cefamandole 0.5 (1) 0.5 (1) 2.7 (5) 96.3

Carbenicillin 0.5 (1) 1.6 (3) 4.3 (8) 93.6

Cephalothin 1.1 (2) 1.6 (3) 3.2 (6) 94.1

Chloramphenicol 0.0 (0) 0.0 (0) 1.6 (3) 98.4

Gentamicin 0.0 (0) 0.0 (0) 1.1 (2) 98.9

Kanamycin 0.0 (0) 1.1 (2) 3.8 (7) 95.1

Tetracycline 0.0 (0) 2.7 (5) 7.1 (13) 90.2

Tobramycin 0.0 (0) 0.0 (0) 2.7 (5) 97.3

Cefoxitin 0.5 (1) 1.1 (2) 2.2 (4) 96.2

Sulfamethoxazole- 0.0 (0) 1.6 (3) 6.0 (11) 92.4

trimethoprim

Nitrofurantoin 2.2 (4) 10.9 (20) 14.2 (26) 72.7

Avg 0.45 1.7 4.2 93.6

a Atotal of183strains ofgram-negativebacilli were tested.

bNumbers inparentheses indicatenumbers of strains showingdiscrepancies.

standard method for each organism. An excel-lent overall correlation of 92.3% was obtained

for identification ofgram-negative bacilli.

Esch-erichia coli, asexpected, was the predominant

organism and was correctly identified (94.3%)

by the rapid method within 8 h after urine

screening. A somewhat lower correlation of

66.7%wasnoted forCitrobacterfreundii. Inthe

present study, Pseudomonas aeruginosa also

yielded a lower correlation (77.8%) at the spe-cies level.

The 18 specimens with two or more

gram-negative species, as confirmed by the purity checkperformed on blood agar plates, yielded

false identification or mixed susceptibility

re-sultson the AMS.

Atotalof14strainswereincorrectly identified bytherapid method. Fivestrains ofE.coliwere

misidentifiedasArizonahinshawii owingto

pos-itive malonate and citrate reactions, and two

strains were misidentified as Shigella sp. Two

strains ofKlebsiella pneumoniae were

misiden-tified as Enterobacter aerogenes owing to

posi-tive ornithine decarboxylase reactions. The

re-maining misidentifications occurred with two

strains each of Citrobacter and Pseudomonas

and one strain ofProteus. No one biochemical

reactionwasregularlyresponsible for

misidenti-fication. Although all nine strains ofP.

aerugin-osawereidentifiedaccuratelyatthe genuslevel,

two strains were misidentifiedasPseudomonas

fluorescensin the present study.

Resultsofantibiotic susceptibility testing.

Dis-crepanciesfor each antibioticwereevaluatedas

very major, major, or minor as defined

else-where (19). The present data show excellent

agreement between the two procedures (Table

2). The agreement ranged from72.7%for

nitro-furantointo98.9% foramikacin andgentamicin.

The average occurrence of major discrepancies

was 1.7%; minor ones occurred at a rate of

4.2%. A totalof11 strains outof183 exhibited

very majordiscrepancies with some antibiotics.

The dataalso indicate that nitrofurantoin

exhib-ited the largest percentages of major and minor

discrepanciesamongtheantibiotics tested, 10.9 and 14.2%, respectively. Analysis of the

dis-crepancies amonggram-negative bacilli by gen-eraandby antibiotics indicatesthat the distribu-tion was fairly uniform and not peculiar to a

single genusorantibiotic.

DISCUSSION

Results ofthe presentstudy demonstrate that more than92% ofgram-negative bacilli can be

accurately identified at the species level in 8 h from eugonic broth after urine screening.

Simi-larly, the data showan excellentcorrelation of

93.6% for antibiotic susceptibility results

be-tween the rapid and the conventional AMS

methods. Thus,mosturinetests canbe

complet-ed and reported within 24 h afterreceipt in the

laboratory. The high accuracy of 92.3% for

identification and a correlation of 93.6% for

susceptibility results for gram-negative bacilli

were obtained from those urine specimens that

contained only single gram-negative isolates.

Such urine specimens accounted for 77.9% of

thetotalpositive specimens in this study.

Therapid identificationof Enterobacteriaceae

obtained directly from positive clinical

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RAPID PROCESSING OF URINE SPECIMENS 671

mens such as urine and blood specimens has been reportedby several investigators (8, 9, 16,

22, 24). For instance, Kelly et al. (16), using

Micro-ID for identification and Autobac 1 for

susceptibility testing, successfully processed positive urine cultures afterurine screening. In

theirstudy,anaccuracyof 95%for identification andacorrelation of94%forantibiotic

suscepti-bility tests ofEnterobacteriaceae between the

rapid andthe standard methods wereobtained. The identification of Enterobacteriaceae

ob-tained directly from clinical specimens such as

blood specimens is generally confirmed by a

definitive identification froman isolatedcolony.

This may not benecessary for the urine

speci-mens, as observed in the present study and by

others (16), unless erroneous results are

ob-tained.

Inthepresent study, 92.3% of 183 strains of

gram-negative bacilli were correctly classified by therapid method (Table 1). This is

compara-bletothe dataofKellyetal. (16). However,use

ofboth Micro-ID and the Autobac 1 system as

employed in theirstudyrequires the actual pres-ence ofa technician to read and interpret the

results. Many clinicallaboratories may nothave extendedshift hours. Thisdisadvantageis

over-comebytheuseofafullyautomatedinstrument

such as the AMS. The benefit and distinct

advantage of the AMS is that the interpretation

ofbiochemical andsusceptibility resultsis done

automatically, thus eliminating the needfor the actualpresenceofatechnician and the

possibili-tyofsubjectiveerror.

TheMicro-IDsystemisnotdesignedto

identi-fy oxidase-positive or glucose-nonfermentative

gram-negative bacilli. Such limitations of the

system have been discussed elsewhere (16).

However, the AMS can be used for routine identification ofcommon glucose-nonfermenta-tivebacilli withahighdegree ofaccuracy (23). A totalof14strainsweremisidentifiedby the

rapidmethod in thepresentstudy. Misidentifica-tion of some Enterobacteriaceae has been knownto occurowingtooverloading the EBC+

withanimproperly standardized inoculum

(per-sonalcommunication from VitekSystems, Inc.).

Similarly, preliminary studies conducted in our

laboratory have indicated that false-positive

re-actions may occur owing to traces of eugonic

broth present in the inoculum if the bacterial

suspensionis not washed repeatedly. Such

oc-currencesoffalse-positive reactions inthe AMS

have been noted by others (6, 22). Kelly et al.

(16) have shown that careful washing of the bacterialsuspension resultsin theelimination of

mostofthefalse reactions.We used aprocedure

forinoculum preparation similar to that ofthe

aboveworkers, whichmay accountforthe low

rateof misidentification by theAMS.

In the present study, 18 specimens (10%)

yieldedtwo ormoreorganisms in thecultureor were contaminated. These cultures, although

considered pureby Gram stain, yieldedtwo or

moregram-negative speciesin thepurity check

plate.In all of the cases, an erroneous

antimicro-bial susceptibility pattern and noidentification,

questionable identity, or an octal code with a

verylow probability were obtained. The results

in such cases may be withheld pendingfurther

work-up. The rapid processing of such

speci-mens as described herein does not result in a

loss of time since purecolonies can be obtained

from the purity check plate on the next day and further testing can be performed as needed.

An excellent agreement (average, 93.6%) in the susceptibility pattern for gram-negative

ba-cilli between the rapid and the conventional methods on the AMS was obtained. Although

some discrepancies occurred, their incidence

was relatively low for allthe organisms tested.

Inthepresentstudy, noneof the antibiotics and

no genus yielded consistent discrepancies, ex-ceptfornitrofurantoin. Thereasonsfor thepoor

correlation between the two procedures for ni-trofurantoinare notfully understood, andno

at-tempt wasmade heretoresolve the discrepancy.

The specificity, reliability, and sensitivity of the AMS for theidentification of urinary

patho-gens by using the urine card have been amply documented (8, 11, 20). A number of workers have alsoperformedcostanalysis studies of the AMS and the conventional method (8, 20, 21). Theirfindings suggestedthatthe AMS provides considerable savings in technician timeand

de-creasesturnaround time. Thecostisparticularly

welljustified in processingpositive culture

spec-imens. However,preliminary studies performed inourlaboratoryindicatethat the cost

effective-ness for processing negative urine specimens

directlyonthe AMS ishighly questionable. Itis general experience that the majority of urine specimens submitted to laboratories are either sterile or have an insignificant colony count.

Therefore, urine screening offers an advantage

in thatnegative urine specimenscanbe reported assuch, andonly presumptively positive speci-mensrequire further processing. This results in substantial reductions in both laborand

materi-als for the processing of urine specimens and

alsopermitsreporting ofthe results within24 h

afterreceipt ofthe specimens in the laboratory, asobserved inthe present study.

Inconclusion, thepresentstudy demonstrates

thattheAMS canaccuratelyandrapidly

identi-fy,andperform antimicrobial susceptibilitytests on, gram-negative bacilli obtained from the

eu-gonic broth after urine screening.

Enterobac-teriaceae and P. aeruginosa were correctly

identified in 8 h by the rapid method with

VOL. 16,1982

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EBC+. Similarly, a correlation of 93.6% was

obtained for the susceptibility patterns by the

rapid method. These findings suggest that the

combination of urine screening by Autobac 1

and identification and susceptibility testing by

the AMS can be efficiently used for processing

and reporting urine specimens in a rapid and

cost-efficient manner.

LITERATURECITED

1. Aldridge, C., P. W. Jones, S. Gibson, J. Lanham, M. Meyer, R. Vannest, and R. Charles. 1977. Automated microbiological detection/identification system. J. Clin. Microbiol. 6:406-413.

2. Alexander, D. N., G. M.Ederer,andJ. M. Matsen. 1976.

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3. Barry, A. L., P. B. Smith, and M. Turck. 1975.Cumitech

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AutoMicrobic System Enterobacteriaceae Biochemical Card. J.Clin. Microbiol.14:370-375.

7. Godowski, K. C., and W. F.Hodges.1981.Accuracyand

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13. Jorgensen, J. H., and P. M. Jones. 1975. Comparative evaluation ofthe Limulus assay and thedirect Gram stain for detection of significant bacteriuria. Am. J. Clin. Pathol. 63:142-148.

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17. Lamb, V. A., H. P. Dalton, and J. R. Wilkins. 1976. Electrochemical method for the early detection of uri-nary-tractinfections.Am.J.Clin. Pathol.66:91-95. 18. Lewis, J. F., and J. Alexander. 1976. Microscopy of

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Maza.1982.Screening ofurine cultures by three

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with the AutoMicrobic System. J. Clin. Microbiol. 15:260-264.

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