Rapid bioluminescence method for bacteriuria screening

0095-1137/89/040716-05$02.00/0

CopyrightC 1989, AmericanSocietyfor Microbiology

Rapid

Bioluminescence Method for

Bacteriuria

Screening

MARIE T. PEZZLO,* VALERIE IGE, AMELIA P. WOOLARD, ELLENA M. PETERSON,

AND LUISM. DE LAMAZA

DepartmentofPathology, Division ofMedical Microbiology, University of California Irvine Medical Center,

101 City Drive, Orange, California 92668 Received 18August 1988/Accepted 3 January1989

Astudywasperformedtoevaluate the UTIscreen (Los AlamosDiagnostics, LosAlamos, N. Mex.),arapid

bioluminescencebacteriuriascreen. TheUTIscreenwascomparedwith threeotherrapidbacteriuriascreens: the Bac-T-Screen (Vitek Systems, Hazelwood, Mo.), an automated filtration device; the Chemstrip LN

(Boehringer Mannheim Diagnostics, BioDynamics, Indianapolis, Ind.), an enzyme dipstick; and the Gram stain. Asemiquantitative plate culturewasused asthereferencemethod. Ofthe 1,000 specimens tested,276

had colonycountsof >105CFU/ml bythe culturemethod. Of these, theUTIscreen detected 96% (265of276) using .5% ofthe integrated lightoutputofthe standard readingas a positive interpretive breakpoint, the

Bac-T-Screen detected 96% (266 of 276), the ChemstripLNdetected 90% (249of276), and the Gram stain detected 96% (264 of 276). Ofthe 214probable pathogensisolatedat >105 CFU/mI, theUTIscreendetected 95% (204 of 214),the Bac-T-Screendetected98%(210of214),theChemstripLNdetected 92% (198of214), and the Gramstain detected 98% (209of214). Thepredictivevalues ofnegativetestresultsat>105 CFU/ml for the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain were 98, 97, 93, and 98%,

respectively. The overallspecificitiesat>105CFU/mlfortheUTIscreen,theBac-T-Screen,theChemstrip LN, and the Gram stainwere70, 48, 51,and69%, respectively.Therewere532specimenswithcolonycountsof >103 CFU/ml, and of these, the UTIscreen,theBac-T-Screen, theChemstrip LN, and the Gram staindetected 72, 81, 76,and73%,respectively. Ofthe 249probable pathogensisolatedat>103CFU/ml,theUTIscreen,the Bac-T-Screen,theChemstrip LN,and the Gram stain detected91, 95, 89,and93%, respectively. Theoverall specificities at >103CFU/ml forthese methodswere79, 55, 57, and78%, respectively. Thecostpertestfor detection was approximately $1.00to $1.20 for the UTIscreen, the Bac-T-Screen, and the Gram stain and approximately $0.50for theChemstripLN. Overall,theUTIscreenisrapidandeasytoperform;itssensitivity compared favorably with thoseof otherscreening methods;ithadahigher specificity thanthe Bac-T-Screen andChemstrip LN; and it allowed forbatchingofspecimens.

Urine specimens represent the majority of samples

re-ceived in the clinical microbiology laboratory for culture. Thelaboratoryfaces thechallenge of rapidly identifying both positive and negative specimens. Although the semiquanti-tative plate culture method allows for the isolation and enumeration ofmost infectious agents, it does not provide for same-day reporting of negative specimens. For this

reason, rapid urine screening tests have been developed.

These tests notonlyprovideforrapid reporting of negative specimensbutalsohavethepotential of reducingthecostof patientmanagement.

A numberofrapidurine screenshavebeendescribed(12). Theseincludemicroscopic, enzymatic, filtration, and photo-metric methods. The most commonly used microscopic method is the Gram stain. As a urine screen, it is rapid,

reliable, and correlates with colony countsof>105CFU/ml (13, 22, 23). However, becausethe acceptable sensitivity of thismethod isat105CFU/ml, low-level bacteriuriamaynot bedetected. Furthermore,theaccuracyisgreatly dependent on theexpertise of the reader. Enzyme dipsticks have also been usedasrapid bacteriuriascreens. Although thesetests

are easy to perform, the overall sensitivity of these rapid

enzymedipsticksistoolow(.90%)tobe usedaloneasurine

screens (15, 25). The first generation of semiautomated bacteriuria screens include photometric methods which

re-quire growth of the organism for detection; therefore test

results are delayed (1 to 13 h). The second generation of

* Correspondingauthor.

semiautomated urine screens include bioluminescence and

filtration methods. These systems are more rapid (1 to 15 min)than thegrowthdetection methodsand results of both

compare favorably(2, 5, 6, 20, 21, 24, 25).

The purpose of thisinvestigation wastoevaluatea

biolu-minescencemethod, theUTIscreen, andtocompareit with other rapidbacteriuriascreens: theBac-T-Screen, an auto-mated filtration method; the Chemstrip LN, an enzyme

dipstick method; and the Gram stain. In this study, these methods were evaluated at various colony counts in an

attempt to aid others in the selection ofa laboratory

ap-proach tourine screening.

MATERIALS ANDMETHODS

Specimens. Atotal of1,000 specimens which included 688 clean-voided and 312 catheterized urine specimens from both inpatients and outpatients submitted to the Medical Microbiology Laboratory at the University of California Irvine Medical Centerwere included in thisstudy. Patients receiving antimicrobial therapy were not excluded. Col-lected urinewas placed in asterile tube, refrigerated (4°C),

and processed within 8 h ofcollection.

Semiquantitative culture. A semiquantitative plate count

asdescribedby Clarridgeetal. (3)wasusedasthe reference

method. By using a calibrated platinum loop, a 0.001-ml sampleofawell-mixed urine specimenwasinoculated onto

a 5% sheep blood agar plate (BBL Microbiology Systems,

Cockeysville, Md.) and a biplate consisting ofMacConkey

agarand polymyxin B-nalidixic acid blood agar (Calscott,

Inc., Carson, Calif.). An additional 0.1 ml of urine was

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TABLE 1. Number and percentage of positive test results

Culture (CFU/ No.(%)positiveresultsby:

ml) (no.of

specimens) UTIscreen Bac-T-Screen ChemstripLN Gram stain

>105(276) 265 (96) 266 (96) 249 (90) 264(96) 1031-i1 (256) 116 (45) 164 (64) 156 (61) 124(48) 10'-103(175) 56 (32) 87 (50) 77 (44) 46(26) <10'(293) 44(15) 124 (42) 125 (43) 58(20)

inoculated ontoa5% sheepbloodagarplate. Cultureswere

incubated overnight at 35°C and examined for the number andtypes of organisms present. Organismsconsidered

con-taminants were diphtheroids, lactobacilli, viridans group

streptococci other than group D, and mixed cultures from

voided urine specimens.

UTIscreen. Urine specimenswere processed accordingto theinstructions of themanufacturer (Los Alamos Diagnos-tics, Los Alamos, N. Mex.). A sample of well-mixed urine (0.025 ml) was added to a tube containing dehydrated

somatic cell releasing agent. Additionally, 0.025 ml of the ATPstandard wasaddedtoaclean polystyrene tube (12 by

50 mm). The tubes were incubated for 15 min at room

temperature. Each tube was then placed into the specimen

well of the Luminometer 535 (Los Alamos Diagnostics), and bothluciferin-luciferasereagentandbacterial releasingagent

were added automatically. The integrated light output was

displayedonthe instrumentfront panelandalsorecordedas

partof theautomatic sequence. Inthisstudy,thedatawere

analyzed using 25% integrated lightoutput of the standard

as apositive test.

Bac-T-Screen. Urine specimens were processed with the

Bac-T-Screenaccordingto theinstructions ofthe manufac-turer (Vitek Systems, Hazelwood, Mo.). A 1-ml sample of well-mixed urine was added to the active barrel of the instrument.Thereagents,3 mlof urine diluent(14.5% acetic acid), 3ml of safraninOdye,andtwoadditions each of 3ml

ofdecolorizer(2.4%aceticacid),wereaddedautomatically. The filtercard was removed from the instrument whenthe

testcyclewas completed and placed in theDynadepthtest cardreader(VitekSystems). Apositivetestwasinterpreted as .4U above the negative control.

ChemstripLN.TheChemstripLN(BoehringerMannheim Diagnostics, BioDynamics, Indianapolis, Ind.) is a plastic

striptowhichareattachedreagentpapersforindicatingthe

presenceofleukocyteesteraseand nitrite in urine. The urine

specimenwasallowedtocometoroomtemperature priorto testing, and the plastic strip was dipped into the specimen

and immediately withdrawn to remove the excess urine.

Resultswerereadafter 2 min. Thecolor intensity ofthestrip wascoded withacolorguide provided by the manufacturer.

A positive leukocyte esterase was one that gave a purple

ranging fromatrace toa2+ intensity. Fornitrite, anypink was considered positive.

Gram stain. A sample (0.01 ml) ofwell-mixed uncentri-fuged urinewasGramstained and examinedfor thepresence

orabsenceofleukocytes andmicroorganisms. Thecriterion

fora positive Gram stain was the presenceofone ormore

bacterialcellsandone or moreleukocytesperoilimmersion field, which has been reportedtocorrelate with-10' CFU/ ml and pyuria, respectively (3).

Predictivevalue. Predictive valueswerecalculated by the method of Ransohoff and Feinstein (17). The sensitivity, specificity, and predictive values ofpositive and negative testresults werecalculatedasfollows: sensitivity = TP/(TP

+ FN); specificity = TN/(TN + FP); predictive value ofa

positive test = TP/(TP + FP); and predictive value of a

negative test = TN/(TN + FN), where TPistrue-positive,

TN is true-negative, FP is false-positive, and FN is false-negative.

Time and cost analysis. An analysis ofcost per test was

doneby calculatingthecostof materials routinely used and technical time incurred in ourlaboratory. The average cost

perspecimenwas calculated by includingboth positive and

negative screen results. The cost of detection included materialcostsandtechnicaltime. Technicaltimewas

calcu-lated based on current College of American Pathologists workload units (4) when available or the average time required toprocess 20specimens by atest method.

RESULTS

Distribution oftestresults. A total of 1,000 clean-voided and catheterized urine specimens were evaluated. There were 276 (27.6%) specimens with colony counts of >10' CFU/ml by the standard semiquantitative plate culture method. Of these, the UTIscreen detected 265 (96%)

com-paredwith 266(96%) bytheBac-T-Screen,249(90%) bythe Chemstrip LN,and 264 (96%) by the Gram stain(Table 1). Overall,226(82%)weredetectedbyall methods,andatleast

onescreening methodwas positive for each specimen. There were 256(25.6%) specimens with colonycountsof 103 to 105 CFU/ml. Of these, the UTIscreen detected 116 (45%), while the Bac-T-Screen, Chemstrip LN, and Gram stain detected 164(64%), 156(61%), and 124(48%),

respec-tively (Table 1). The remaining 468 specimens had <103 CFU/ml; of these, 175 had colony counts of 101 to 103 CFU/ml,and theremaining293specimenshadnodetectable

growth(<101CFU/ml). Thedetectionratesbyeachmethod for these levels of bacteriuriaare shown in Table 1.

The sensitivities, specificities, and predictive values for thetestmethodsatvariouscolonycountsareshown inTable 2. Inadditiontoeach methodhavingasensitivity ofatleast

90%at >105 CFU/mI, thepredictive values ofnegative test

results rangedfrom93 to98%. When the interpretive

break-TABLE 2. Sensitivities, specificities, andpredictivevaluesfortestmethods Results(%)with method atcolonycount

Parameter UTIscreen Bac-T-Screen ChemstripLN Gramstain

>10 >103 >101 >105 >103 >lo0 >105 >103 >101 >105 >103 >101

Sensitivity 96 72 62 96 81 73 90 76 68 96 73 61

Specificity 70 79 85 48 55 58 51 57 57 69 78 80

Predictive value

Positive 55 79 91 42 67 81 40 67 79 54 79 88

Negative 98 71 48 97 72 47 93 68 43 98 72 46

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TABLE 3. Probablepathogens at >105 CFU/ml detected by the test methods

No.of isolates detected by': Organism (no.ofisolates)

UTIscreen Bac-T-Screen ChemstripLN Gramstain

Escherichiacoli(108) 107 108 105 105

Candidaspp.(21) 18 20 18 21

Klebsiella spp. (19) 19 19 15 19

Enterococcus spp. (20) 17 19 19 19

Pseudomonas spp. (10) 8 8 7 9

Proteus mirabilis (11) 10 il 10 il

Enterobacter spp. (5) 5 5 5 5

Coagulase-negative 7 7 7 7

staphylococci (7)

Staphylococcus aureus (5) 5 5 4 5

Streptococcus agalactiae (3) 3 3 3 3

Serratiamarcescens (2) 2 2 2 2

Providencia stuartii (2) 2 2 2

Citrobacterspp. (1) 1 1 1 1

" The percentages of all isolatesdetected were asfollows: UTlscreen,95:Bac-T-Screen,98:Chemstrip LN,92; andGramstain,98.

pointwasdecreased for all organisms to >103 CFU/ml, the Chemstrip LN. The sensitivities for all methods at >101

sensitivities forthetest methods ranged from 81 to72%and CFU/ml rangedfrom84 to91%.

from 73 to 61% at >101 CFU/ml. The specificities and The UTIscreen and the Gram stain hadthehighest

spec-predictive values of positive test results were lower for the ificities for all organisms, which ranged from69% at

>105

Bac-T-Screen and the Chemstrip LN because of the high CFU/ml to85%at

>101

CFU/ml(Table 2). The specificities

false-positiveresults by these methods. for theBac-T-Screen and the Chemstrip LN ranged from 48

Overall, there were 272probable pathogens isolatedfrom to 58% for colony counts of

>105

to

>10'

CFU/ml. The

157clean-voidedspecimens and 103 catheterizedspecimens. predictive value of a positive test increased by

approxi-Of these, 248 specimens had pure cultures of probable mately40%for each test method as the interpretive break-pathogens and the remaining 12 were catheterizedspecimens point of thereference method decreased from >105 to>101

with two probable pathogens. The most frequently isolated

CFU/ml,

whereas the

predictive

value of a negative test

probable pathogens included Escherichia coli (n = 130), decreased (Table 2).

Candida spp. (n = 30), Enterococcus spp. (n = 21), Klebsi- Costanalysis.The totalcostof

screening by

eachmethod

ellapneumoniae (n = 21),Pseudomonas spp. (n = 19), and was determined (Table 4). Although salaries differ from Proteus mirabilis (n = 16),accounting for themajority (79%) laboratory to laboratory, the cost per test was based on the

of isolates in this study. Of these, Pseudomonas spp., average salary ofthe technical personnelatour institution.

Enterococcus spp., and Candida spp. had the highest per- For

detection,

thecostof the testmethods rangedfrom$0.44

centage offalse-negative results for all the test methods. fortheChemstripLN to

$1.17

fortheGram staincompared

There were 214probable pathogens with colony counts of with

$1.63

for thereferenceculture method.However, when

>105

CFU/ml, 35 at

103

to

105

CFU/ml, and 23 at

101

to

103

the average cost for both positive and negative specimens

CFU/ml. wascalculated, the testmethods were more

expensive

than

Of the 214 probable pathogens at

>105

CFU/ml, the the reference method, ranging from

$0.12

higher for the

Bac-T-Screen and the Gram stain detected 98%, the UTI UTIscreen to $0.49 higher for the Bac-T-Screen, with one

screen detected95%, and theChemstrip LN detected92%, exception, theChemstrip LN, whichwas

$0.20

lower.

respectively (Table 3). There was no statistically significant

difference fordetection ofanyoftheprobable pathogens by DISCUSSION

the testmethods(P>0.1). When theinterpretive breakpoint

wasdecreased forprobable pathogensto

>103

CFU/ml,

the The purpose of urine screening is to improve patient

sensitivities for the UTIscreen, the Bac-T-Screen, and the managementby rapidly reporting negativeresults and

iden-Gram stain were at least

90%

compared with

85%

for the tifying those specimens which do not warrant culture.

Cri-TABLE 4. Costanalysisofrapidurine screens at>105CFU/ml

Cost($)/specimen by:

Determination Chemstrip

Culture method UTIscreen Bac-T-Screen LN Gramstain

Costof supplies 0.75a 0.75 0.75 0.20 0.05

Costoftechnical time" 0.88 0.22 0.33 0.22 1.12

Cost ofdetection 1.63 0.97 1.08 0.44 1.17

Avgcost/specimen, including 1.63 1.75 2.12 1.43 1.97

positiveandnegative screenresult

Difference fromculture +0.12 +0.49 -0.20 +0.34

a Includes5%sheepblood agar andbiplate of MacConkeyagarandpolymyxin-B-nalidixic acid bloodagar.

bEstimatedat$13.00/hassuming 4.0, 1.0, 1.5, 1.0,and 5.1 minpersampleforculture,UTIscreen,Bac-T-Screen,Chemstrip LN,and Gramstain,respectively.

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teria for

selection

of a rapid urine screen should include accuracy, reproducibility,

detection

time, ease of test per-formance, and low cost. Previous

evaluations

ofmicroscopic

and filtration methods have been favorable regarding sensi-tivities,

predictive

values of negative test results, and detec-tion times (10, 13, 14, 22, 23).

During recent years, the criterion of

2105

CFU/ml estab-lished by Kass (7) as the

definition

of a urinary tract infection has been challenged. Studies by Latham etal.(8) and Stamm et

al.

(18) found

.102

CFU/ml to be a better predictor of infection in symptomatic women. Lipsky et al. (9) and Musher et al. (11) recommended that

2103

CFU/ml be used when evaluating voided urine specimens from men. Stark and Maki (19) found that

.102

CFU/ml was a more valid index when patients were catheterized and had urinary symptoms or were immunosuppressed. Pfaller et al. (16) studied a random patient population and also found that lower count cultures most accurately identified urine speci-mens from infected patients. Because of the findings of these investigators, the rapid screens described here were evalu-ated at various colony counts.

Previous

evaluations employing

bioluminescence assays for urine screening have demonstrated sensitivities of.85%

at

>i04

CFU/ml,

with

predictive

values of negative test

results

of -95% (2, 5, 6, 20, 21, 24, 25). The overall

sensitivityat

>105

CFU/ml

for the UTIscreen in this study

was 96% when a

25%

integrated light output was used as the positive breakpoint. At this interpretive breakpoint, the

UTIscreen

was as sensitive as the Bac-T-Screen and the Gram stain.

All

three methods had sensitivities of at least

90% at

103

CFU/ml

for probable pathogens and 84% at 10'

CFU/ml.

The UTIscreen missed Candida spp. and Enterococcus

spp. more frequently than the Bac-T-Screen and the Gram stain did. A possible explanation may be that some strains of bacteria and yeast had lower ATP levels in spite of high colony counts (21). Another explanation may be that the bacterial releasing agent did not sufficiently lyse the cell walls of these gram-positive organisms (24). These factors may have contributed to a lower UTIscreen sensitivity for these species, which were detected by the other screening methods.

The major

difference

between the semiautomated meth-ods, theUTIscreenand the Bac-T-Screen, is in the specific-ities of these methods. The specificity of the UTIscreen at

>105

CFU/ml was higher than that of the Bac-T-Screen (70 versus 48%); however, the sensitivities were the same (96%). Although the specificities were increased in all in-stances by lowering the interpretive breakpoint to either

103

or10'

CFU/ml,

the specificity of the Bac-T-Screen remained lower than that of theUTIscreen. A possible explanation for decreased specificities by the test methods is that low-level bacteriuria may not be detected by the culture method. Also, it has been reported that the culture method may have an error rate of as high as 50%(1). Another contributing factor may have been the presence of antimicrobial agents which inhibit microbial growth but do not inhibit detection by bioluminescence or filtration (6). The Bac-T-Screen detects leukocytes as well as bacteria (14). The trapping of leuko-cytes along with other cells (i.e., squamous epithelial cells) by the filter card probably accounts for the lower Bac-T-Screen specificity. This may also account for lower spec-ificities by the UTIscreen at the higher colony counts. If the somatic releasing agent does not destroy all of the somatic

cell ATP, it will be detected by the luminometer. Macro-scopically bloody urine specimens have been reported to be

positive by bioluminescence (2). The specificities of the

UTIscreen

atvarious colonycounts are acceptable,

consid-ering that the main purpose of a urine screen is to rapidly

identify

negative

specimens whilereliably detectingpositive

specimens.

In this study, the UTIscreen results were similar to the

Gram stain

results

regarding sensitivity, specificity, and

predictive

values at various colony counts. Although the Gram stain comparesfavorably with other urine screens and

test interpretation is subjective, it requires more labor time

to perform than the UTIscreen, therefore resulting in a higher cost per test. In addition, it can be a tedious proce-dure because mosturine specimens are negative. However, it can give a preliminary tentative identification of the

organism group and the presence ofleukocytes.

Both the UTIscreenand the Bac-T-Screen employ instru-mentation; therefore, interpretation of test results is objec-tive, whereas the Gram stain interpretation is subjective, requinng technical expertise. Another difference between the instrument methods is the volume of urine required to perform the tests; 0.025mlfor the UTIscreen compared with 1.0 ml for the Bac-T-Screen.

The ease of performance is an additional advantageof the

UTIscreen.

Once the 0.025 ml of urine is added to the tube

containing the dehydrated somatic cell releasing agent and

the tube is allowed to incubate at room temperature for 10 min, technical time required to complete the test is minimal (15 s). Reagents are added and results are printed

automat-ically. Specimens can be easily batched, whereas the other

methods require individual handling. Cost is an important

consideration and the UTIscreen compared closely with the

culture method in this study when both positive and negative test results were considered.

In conclusion, the UTIscreen compared favorably with otherscreening methods with respect to sensitivity, predic-tive value of a negapredic-tive test, ease ofperformance, and cost. The advantages of this rapid urine screen include theability to batch test, objectiveinterpretation, higher specificity than the enzyme dipstick or the colorimetric filtration methods, and same-day results with cost similar to culture.

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