0095-1137/79/08-0226/05$02.00/0
Clinical
Laboratory Evaluation
of
Automated
Microbial
Detection/Identification System
in
Analysis
of Clinical Urine
Specimens
HENRY D.
ISENBERG,`*
THOMAS L. GAVAN,2 ALEXSONNENWIRTH,3 WELTON I. TAYLOR,4ANDJOHN A.WASHINGTON
II'
TheLong Island Jewish-Hillside MedicalCenter,NewHydePark,New York110421; Cleveland Clinic Foundation, Cleveland, Ohio 441062; JewishHospital of St.Louis, St.Louis,Missouri
631103;
St.MaryofNazarethHospital,Chicago, Illinois 606224; Mayo Clinic and MayoFoundation,Rochester, Minnesota559015
Received for
publication
7May 1979More than
4,000
clinical urinespecimens
were evaluated with an automatedmicrobial
detection/identification
system compared to a standardized manualanalysis and the routine modalities used in five peer-group laboratories. The
comparison indicates that the automated system recognizes the nine groups of
significantmicroorganismsinurinary tract infectionsinhospitalized
patients
withthe sameefficiency as a standardized manual method. The automated system's
ability
to enumerate the bacterialpopulations
in theoriginal
clinical specimenattained ahigh degreeofaccuracy.
Earlier papers
(1-4)
have described in detailthe
design
and function of the Auto MicrobicSystem
(AMS,
VitekSystems, Inc.),
itsap-proach to clinical
microbiology,
and its initialperformance
in several laboratories withseeded,
simulated
specimens
whichchallenged
the urineIdenti-Pak,
the firstavailable
testprocedure
which
permits
anautomated system to addressaclinical
specimen.
Thisreport summarizes theexperiences
of five clinicallaboratories
in whichclinical
urinespecimens
wereanalyzed by
theAMS, the routine procedures of
eachlaboratory,
and
anagreed-upon
standardizedmanual
method.
(Results
ofworkreported
here werepresentedinpart at the 78th Annual
Meeting
of theAmer-ican
Society
forMicrobiology,
Las Vegas,Ne-vada,
1978.)MATERIALS AND METHODS The participating laboratoriesincluded the clinical microbiology laboratories of the Mayo Clinic, Roch-ester, Minnesota; the Cleveland Clinic Foundation, Cleveland, Ohio;the Jewish Hospital ofSt. Louis, St. Louis, Mo.; the Long Island Jewish-Hillside Medical Center,NewHydePark, N.Y., andSt.Mary of Naza-rethHospital,Chicago,Ill.Theintroduction of
speci-mensintothe instrumentand themethods for evalu-atingnumbers andestablishing the generic and species groupsfor theprotista mostfrequently encountered in urine specimens ofpatients have been described in detailpreviously(1,3, 4).
All clinicalurinespecimensreceived in each labo-ratoryfor routine microbiological analysis were intro-duced into the AMS in the samemanner as previously
described (3). The specimens wereevaluated bythe samemanual procedure in all of the laboratories as reported earlier (3).
Briefly, these methods consisted ofintroducing200 /4 into the firstcompartmentof thespecimeninjector. A second 5-,ld amount of the urine specimen was introduced into the second compartment. The vol-umesweredilutedautomaticallywith 0.5% NaCland introducedthroughanevacuation step into theactual specimen cards (Identi-Pak). The latter were then introduced into the instrument for incubation and scanning. Theagreed-upon ("standard") evaluation of the urinespecimen consisted of seeding0.1mlof1:100 and 1:1,000 dilutions of the urine specimen on two blood agar plates and one MacConkey agar plate. Afterincubation, enumeration and further identifica-tionswerecarriedout. The twodilutions tested cor-responded to 1:1,000 and 1:10,000 dilutions of the original urine. In addition, each laboratory analyzed eachspecimeninaccordance withtheproceduresused routinelyinthatrespectivelaboratory. Table1 sum-marizes the different methods. No laboratory per-formed theanalysisof urinespecimens in exactly the same fashion. This table indicates that laboratory workup followed collection at relatively short inter-vals, that detrimental delay in processing was pre-ventedby refrigerationwhich did notexceed 24 h, that the bacterial densities were evaluated by different methods,andthatallparticipants used media to detect theEnterobacteriaceae.
Results of the instrument analysiswere recorded
automatically byatape cassette in each instrument.
These cassettes along with the findings of analyses achievedbystandard and routine methods were intro-duced into the computer based in the McDonnell-Douglas Corp. Comparisons of the findings were ac-complished bythis means.
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AUTOMATED URINE SPECIMEN ANALYSIS SYSTEM 227
TABLE 1. Routine methods
ofparticipant
laboratoriesMethod No. of
labora-toriesusing
Lengthof time before workup:
Lessthan 30 min ... ... 2
Lessthan2h ... 2
Unknown ... ... 1
Refrigerated specimens ... ... 3
Dilutionsperformed, samples cultured .... 2
Calibrated loop -0.001ml ... 1
-0.01 ml ... 2
Media: Blood agar ... 4
Separatemedium for detection of Enterobacteriaceae ... ... 5
Separatemedium for detection of enterococci... 2
Asaresult of thefillingmodebyvacuumand the
natureof the original lyophilized materialwithin each
well of the urine Identi-Pak cards, a number of air bubbles appeared occasionally within some of the wells. These bubbles, recognized initially by the in-strumentasmicrobialgrowth,wererecorded
mistak-enly bythe machineaspositive. Inspectionby
tech-nologistsatthe conclusion of therun indicated the
lackof microbialpresenceinwellsrecognizedas
posi-tive because of the bubbles. These observationswere
duly recorded,followedbymanualanalysisofthewell
contents,and consideredduringtheevaluation. Even-tually,adiscriminatingscanner wasinstalled insome
of the instruments and usedby several of the peer
groupmembers. Thisscannerdifferentiates between
air bubbles and microorganisms in the enrichment
wellsofthe urine Identi-Pak card.
The sensitivity of the AMS was established by
multiplying the positive reactions recorded by the instrument, as confirmed by the standard, manual
analysis, by100 anddividing bythesumof thetruly positive specimens plusfalse-negative specimens, i.e., specimensnotidentifiedorrecognizedcorrectlybythe
AMS but determined viathemanualmethod. Speci-ficity,onthe otherhand,wasdefinedastheorganisms
designatedasnegative bythemachine(confirmed by standardlaboratory tests), multiplied by 100,divided
bythesumof thenegativeidentifications confirmed by the machine, plus those organisms identified as
belongingtoaspecific categoryinthe AMS but not
confirmed in standardlaboratory testing.
To avoid misunderstanding, the original machine
without the bubble scannerisreferredtoasthe pro-totypeAMS;thefindingsobtained with thescanner
attachedarereferredtoasevaluation with the
scan-ner-improvedAMS.
RESULTS
Table2 shows theperformanceof the
proto-type AMS in thepeergrouplaboratories.
Spec-ificity ranged from 94 to 99% for4,184 clinical
urine samples. Sensitivity, on the other hand,
didnotachieve similarlevels,
prmnarily
becauseofthe aforementioned problemwith the
forma-tionof bubbles in the wells. Pseudomonas
de-tection was only 75%; staphylococci were
re-covered ata 82% level, the
Klebsiella-Entero-bactergroup and Serratia marcescensat87%,
yeast at 88%, Citrobacter
freundlii
at 89%, andtheremainingbacteria at94%.
Theintroduction of thescanner(Table3)led
toimprovement in thespecificity of the
instru-ment, with the recognition accuracy of
impor-tantgroupsbeinggreaterthan 96.8%. The sen-sitivity of the urine Identi-Pak, namely its ability
torecognize thepresenceofaspecific organism
oradefinedgroup oforganisms, wasenhanced
considerably for allcategoriesexceptS.
marces-cens. Subsequent to this collaborative study,
investigations in laboratories oftwoparticipants
(H.D.I., T.L.G.) have demonstrated that
recog-nitionthreshold adjustments in the AMS
soft-wareand fine-tuning of the enrichmentmedium
constituents have brought the sensitivity
capa-bility of the AMS in properly identifying
Ser-ratia, Pseudomonas, and staphylococcitolevels
inexcessof 90%.
Table4shows the performance of the
individ-ual laboratories inthe original evaluation with
the prototype AMS. The results indicate that
notallparticipants
encountered each
andeveryorganism that can be detected by the
Identi-Pak. Otherdifferencesweredistributed ina
ran-TABLE 2. Collaborative analysis of clinical specimens withprototypeAMS'
OrganismOrganism ilateNo. Ses-tivity Speci-ficity
Pseudomonasaeruginosa 124 75 96
Proteus sp. 233 94 97
C.freundii 19 89 99
S.marcescens 15 87 98
E.coli 608 94 99
Klebsiella-Enterobacter 232 87 98
group
Yeast 72 88 98
Enterococci 358 94 95
Staphylococcusaureus 22 82 94
Enumeration 91 95
Standard manual evaluation served as control.
Sensitivitywascalculatedas: {True(+)/[True (+) +
False(-)]} x100.Specificitywascalculatedas:[True
(-)x100]/[True (-)+False (+)]. Of4,184specimens,
1,683 (above) hadpositiveresults and2,501 had
neg-ativeresults;1,195specimens(29%)yieldedorganisms innumbers >70,000colony-forming units
(CFU)
per mlby both the standard method and AMS.Thestan-dard method detected
microorganisms
in numbers <70,000CFU/mlin 274(6%)additionalspecimens;the AMS detected numbers<70,000CFU/ml
in488(11%)
additionalspecimens.10,1979
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domfashion; therewas not one
laboratory
whichperformed
poorly
ascompared
to the others.Theability of each
laboratory
toanalyze
mono-microbic andpolymicrobic seeded samples
sat-isfactorily with the prototype instrument was
predicated,
to adegree,
on visualinspection
ofeach card at the
completion
ofincubation,
therecording of air
bubbles, and,
insomeinstances,
cultural
analyses
ofthose wells whichdisplayed
bubbles
(3).
A similar evaluation of resultsob-tained with protoype instruments in this
inves-tigation
resolvedmostdifferences in thepartic-TABLE 3. Collaborativeanalysis of clinical specimenswithscanner-improved AMS'
No. iso- Sensitiv-
Specific-Organism lated ity ity
P. aeruginosa 37
85.7b
99.4Proteussp. 106 96.2 97.7
C.freundii 16 100.0 99.7
S.marcescens 4 33.3b 99.4
E.coli 333 92.4 98.3
Klebsiella-Enterobacter 102 95.7 98.7
group
Yeast 33 100.0 99.3
Enterococci 181 94.7 98.3
S.aureus 17 87.5 96.8
Enumeration 95.4 94.7
aStandard manual evaluation served as control. Sensitivity and specificity are asdefined inthe foot-note of Table 2. Of 1,773 specimens, distinct and differentfromspecimens describedinTable2, 829had positive results and304had negative results.A total of 640specimens (36%)yielded organismsinnumbers ,>70,000CFU/mlby the standard methodand AMS. The standard method detected microorganisms in numbers <70,000 CFU/ml in 569 (32%) additional specimens; the AMS detected numbers<70,000CFU/ ml in 829(47%) additional specimens.
'Now
improved by adjustmentsofenrichment mi-lieu.ipating laboratories. As mentioned above, the
use of the scanner-improved AMS avoids this
problem entirely.
TheabilityoftheprototypeAMSto
enumer-atethemicrobialdensitiesin clinical urine
sam-ples exceeded 90% in this trial. Thefive wells
separately inoculated from the injector reservoir
receiving 5,l of the original specimen provided
an acceptable account of the colony-forming
units (CFU) in the original specimen, i.e.,more
thanorless than70,000 CFU/ml.
DISCUSSION
The studies with clinical specimens indicate
that the AMS evaluates actual clinical
speci-mens with the same accuracy manifested with
seeded specimens (3).Theinstrumentiscapable
of recognizing with efficiency the most
impor-tantand mostcommonmicroorganisms isolated
fromurinarytractinfections encountered in
pa-tients. The instrument is capable ofperforming
these tasks withaminimumofintervention by
technical personnel and it reportsthe findings
withanacceptableaccuracy.Since the operator
has thecapabilityofrequesting the machineto
report ad lib, the time to detect a significant
microbialorganisminany one urine specimenis
limitedonly by theinitial numberandthe
sen-sitivitythresholdincorporatedinthesoftwareof
the instrumentation. However, the total
incu-bation of 13 h is required to conclude that a
culture isnegative.Furthermore, the instrument
provides theopportunitytoconfirmthe
identi-ficationof each ofthe organismsas well asthe
chancetosubmit theorganisms within thewells
ofthe card to furtherbiochemical or biological
testing. This taskcanbe performedeasilywith
a 10-M1 pipette. Several studies reported by
Isenbergetal.attheInterscienceConferenceon
AntimicrobialAgents andChemotherapy, 1977,
TABLE 4. Individuallaboratoryresultsof clinical specimen evaluation withprototypeAMSlaboratory
Laboratory
Organism A B C D E
SENSa SPEC SENS SPEC SENS SPEC SENS SPEC SENS SPEC
P.aeruginosa 76.4 98.6 65.6 98.4 80 99.2 100 97.1 71.4 99.3
Proteussp. 97 93.7 95 93.8 82.2 97.4 91.6 93.8 90 94.5
C.freundii 33.3 98.8 50 99.1 - 99.7 - 96.9 100 98.3
S. marcescens 50.0 99 - 98.2 66.6 99.6 100 98.6 - 98.9
E.coli 94 90 97.8 98.3 88.3 88.8 92.3 94.2 91.2 91.5
Klebsiella-Enterobacter 89.1 95 77.4 98.4 96.5 99.3 100 95.9 91.8 97.0
group
Yeast 100 99.8 - 96.5 100 99 83.3 98.6 93.3 99.4
Enterococci 93.4 92.6 92.3 93.5 81.4 93.7 79.1 97.5 86.9 89.9
S. aureus 100 97.6 100 93.9 - 98.2 100 94.8 85.7 95.3
Enumeration 87.9 96.1 95 69.5 81.4 96.7 93 92.5 92.3 89.3
aSENS, Sensitivity;SPEC,
specificity.
Sensitivity andspecificity
are asdefined in the footnote of Table 2.on February 7, 2020 by guest
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AUTOMATED URINE SPECIMEN ANALYSIS SYSTEM 229
utilized this mode of
entry toestablish antibiotic
susceptibility
profiles for
Escherichia coli. They
indicated
easeof
performance and the capability
of
further analysis by
eithermanual
orauto-mated
methodologies.
The
early experience with
thebubbles
led to animproved
addition to thedetecting
mecha-nisms of the
device. Whereas the presence
of suchphysical expressions
offilling
aredepend-ent
in
part onthe
efficacy of the
vacuumsystemsupplied
inthe
filling station, it
issignificant
that
by the introduction of
ascanning
detector
the
automated
systemacquired the capability
todifferentiate between the
artificially
accumulat-ing air bubbles and the turbidity and color
changes produced by the
presenceof
microbial
particles
inspecific wells.
Initially, it
wasthought that this
multilabor-atory
collaborative
investigation
would provide
the
opportunity
tocompare the routine
micro-biological analysis of urine performed
in eachlaboratory with
themethod
whichserved as themanual standard
for AMS
performance.
Thelatter approach used materials produced
at asingle
source andanalyzed several dilutions
ofeach
specimen in
aprescribed fashion
followed
by each
participant
(3).
Any
microorganism
iso-lated
in anydilution
wasidentified and reported,
i.e.,
nojudgment concerning the clinical finding
was
exercised.
On the other hand, the routine
methods of each
laboratory
wereconstrained
by
guidelines such
asthe
significant
number of
CFU
leading
tofurther
studies, the acceptability of
aspecimen
yielding
morethan
twomicrobial
spe-cies, classification of
specimens
onthe basis of
CFU/ml, the
identity
of the
microorganism
iso-lated, and the available history of patients.
These
judgmental considerations made
amean-ingful comparison between the
twomanual
mo-dalities
impossible.
By
far,
the
mostfrequent
difference
arosewhen the routine clinical
ap-proach did
not pursueidentification of
aniso-lated
protist further because it
was presentin
the
specimen
innumbers
less than the
accepted
criterion.
This
appeared
as anegative result
when
compared
tothe standard manual
method,
which
identified all
organisms
regardless
of
num-ber in
accordance
with itsexpressed
purposeasa
manual control
of the
enumerating
andrec-ognition functions of the
AMS.
It mustbe
emphasized
aswell
that theability
of
theinstrument
tocategorize
themicroorga-nisms ina
specimen
isindependent
of thenum-ber
ofparticles established
by
thenonspecific
enumeration
wells.
Thelatter
isa 1:10 dilutionof
theinoculum introduced
into thedifferentiat-ing
wells. Theability
of eachorganism
togrowin its
specific
compartment isinfluenced,
ofcourse,
by the original number present and the
time
allowed to reach apreset
threshold
by
multiplication. Thus,
countsindicating
amicro-bialload of less than
70,000
donotinterfere
withthe
ability
ofthe instrument
toreport
thepres-ence of certain
bacteria
within aspecimen. It
must also be
kept
inmind
thatthe
time/CFU
ratiois
different for each well.
Theenrichment-selective media
employed
do notpermit the
same number of
multiplications for each
orga-nism in its
specific
well inthe
sametime
interval.
Thus,
thetimereport
doesnotnecessarily
indi-cate
the number
ofparticles present.
Instead,
final counts in
the
enumeration wells
mustbe
taken
into
consideration.
Thislack of
distin-guishing between
componentmicroorganisms
in
a
polymicrobic specimen may represent
alimi-tation ofthe
AMS
in itspresent
configuration.
Studies
now in progress aredesigned
toalleviate
this
problem.
In a
previous study (3), the
ability
of otherorganisms
to growand
manifest
withinthe
urineIdenti-Pak has been evaluated. Organisms rarely
encountered in clinical urine
specimens do
notproliferate in the
enrichment-selective
compart-ments
for
specific organisms. The growth control
medium, provided
in aspecific compartment
within the card and
identical
tothe
medium
employed in the enumeration
wells, will permit
the
growth of
organisms other than those
spe-cifically separated
inthe AMS. The AMS
re-ports
the
presenceof such
organisms
as anuni-dentified
microorganism. Aspiration of the
growth
control
well contentsand
analysis by
manual
techniques
canbe used for their
identi-fication.
The
AMS is
the first automated procedure
which
addresses clinical specimens
directly (2);
it
detects, enumerates, and identifies the
mostfrequent and
significant bacteria and yeasts
commonly found in urine in
afully automated
mode.
Heretofore, instrumentation has, for the
mostpart,
provided
amechanized
approach
tothe
postisolation functions of the clinical
micro-biology
laboratory.
Whereasthe AMS
proce-dures described
sofar
apply
only
tothe
analysis
of urine
specimens,
other
applications,
such
asantibiotic
susceptibilities,
biochemical
testsfor
identification,
the determination of minimal
in-hibitory
concentrations,
aswell
asother typesof
clinical
laboratory specimens,
areunderactive
investigation and should aid in
bringing
this
capability
tomany of theroutiih.-
procedures
of
the
clinical
microbiology
laboratory.
LITERATURE CITED
1. Aldridge, C., P. W. Jones, S.Gibson,J.Lanham,M. Meyer,R.Vannest, andR.Charles.1977.Automated 10,
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microbiologicaldetection/identification system. J.Clin. Microbiol. 6:406-413.
2.Isenberg, H. D., and J. D. MacLowry. 1976. Automated methods and data handling in bacteriology. Annu. Rev. Microbiol. 30:483-505.
3. Smith,P.B., T. L.Gavan, H. D. Isenberg, A. Sonnen-wirth, W. I. Taylor, J. A. Washington II, and A.
Balows. 1978.Multi-laboratory evaluation ofan auto-mated microbial detection/identification system. J. Clin. Microbiol. 8:657-666.
4. Sonnenwirth,A.C.1977.Preprototypeofanautomated microbial detectionand identification system:a devel-opmentalinvestigation. J. Clin. Microbiol. 6:400-405.