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
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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.
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