Rapid Bioluminescence Method for Bacteriuria Screening MARIE T

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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1989, p. 716-720 Vol. 27, No. 4 0095-1137/89/040716-05$02.00/0 Copyright C 1989, American Society for Microbiology Rapid Bioluminescence Method for Bacteriuria Screening MARIE T. PEZZLO,* VALERIE IGE, AMELIA P. WOOLARD, ELLENA M. PETERSON, AND LUIS M. DE LA MAZA Department of Pathology, Division of Medical Microbiology, University of California Irvine Medical Center, 101 City Drive, Orange, California 92668 Received 18 August 1988/Accepted 3 January 1989

A study was performed to evaluate the UTIscreen (Los Alamos Diagnostics, Los Alamos, N. Mex.), a rapid bioluminescence bacteriuria screen. The UTIscreen was compared with three other rapid bacteriuria screens: 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. A semiquantitative plate culture was used as the reference method. Of the 1,000 specimens tested, 276 had colony counts of >105 CFU/ml by the culture method. Of these, the UTIscreen detected 96% (265 of 276) using .5% of the integrated light output of the standard reading as a positive interpretive breakpoint, the Bac-T-Screen detected 96% (266 of 276), the Chemstrip LN detected 90% (249 of 276), and the Gram stain detected 96% (264 of 276). Of the 214 probable pathogens isolated at >105 CFU/mI, the UTIscreen detected 95% (204 of 214), the Bac-T-Screen detected 98% (210 of 214), the Chemstrip LN detected 92% (198 of 214), and the Gram stain detected 98% (209 of 214). The predictive values of negative test results at >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 overall specificities at > 105 CFU/ml for the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain were 70, 48, 51, and 69%, respectively. There were 532 specimens with colony counts of >103 CFU/ml, and of these, the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain detected 72, 81, 76, and 73%, respectively. Of the 249 probable pathogens isolated at >103 CFU/ml, the UTIscreen, the Bac-T-Screen, the Chemstrip LN, and the Gram stain detected 91, 95, 89, and 93%, respectively. The overall specificities at >103 CFU/ml for these methods were 79, 55, 57, and 78%, respectively. The cost per test for detection was approximately $1.00 to $1.20 for the UTIscreen, the Bac-T-Screen, and the Gram stain and approximately $0.50 for the Chemstrip LN. Overall, the UTIscreen is rapid and easy to perform; its sensitivity compared favorably with those of other screening methods; it had a higher specificity than the Bac-T-Screen and Chemstrip LN; and it allowed for batching of specimens.

Urine specimens represent the majority of samples re- semiautomated urine screens include bioluminescence and ceived in the clinical microbiology laboratory for culture. filtration methods. These systems are more rapid (1 to 15 The laboratory faces the challenge of rapidly identifying both min) than the growth detection methods and results of both positive and negative specimens. Although the semiquanti- compare favorably (2, 5, 6, 20, 21, 24, 25). tative plate culture method allows for the isolation and The purpose of this investigation was to evaluate a biolu- enumeration of most infectious agents, it does not provide minescence method, the UTIscreen, and to compare it with for same-day reporting of negative specimens. For this other rapid bacteriuria screens: the Bac-T-Screen, an auto- reason, rapid urine screening tests have been developed. mated filtration method; the Chemstrip LN, an enzyme These tests not only provide for rapid reporting of negative dipstick method; and the Gram stain. In this study, these specimens but also have the potential of reducing the cost of methods were evaluated at various colony counts in an patient management. attempt to aid others in the selection of a laboratory ap- A number of rapid urine screens have been described (12). proach to urine screening. These include microscopic, enzymatic, filtration, and photometric methods. The most commonly used microscopic MATERIALS AND METHODS method is the Gram stain. As a urine screen, it is rapid, Specimens. A total of 1,000 specimens which included 688 reliable, and correlates with colony counts of >105 CFU/ml clean-voided and 312 catheterized urine specimens from (13, 22, 23). However, because the acceptable sensitivity of both inpatients and outpatients submitted to the Medical this method is at 105 CFU/ml, low-level bacteriuria may not Microbiology Laboratory at the University of California be detected. Furthermore, the accuracy is greatly dependent Irvine Medical Center were included in this study. Patients on the expertise of the reader. Enzyme dipsticks have also receiving antimicrobial therapy were not excluded. Col- been used as rapid bacteriuria screens. Although these tests lected urine was placed in a sterile tube, refrigerated (4°C), are easy to perform, the overall sensitivity of these rapid and processed within 8 h of collection. enzyme dipsticks is too low (.90%) to be used alone as urine Semiquantitative culture. A semiquantitative plate count screens (15, 25). The first generation of semiautomated as described by Clarridge et al. (3) was used as the reference bacteriuria screens include photometric methods which re- method. By using a calibrated platinum loop, a 0.001-ml quire growth of the organism for detection; therefore test sample of a well-mixed urine specimen was inoculated onto results are delayed (1 to 13 h). The second generation of a 5% sheep blood agar plate (BBL Microbiology Systems, Cockeysville, Md.) and a biplate consisting of MacConkey agar and polymyxin B-nalidixic acid blood agar (Calscott, * Corresponding author. Inc., Carson, Calif.). An additional 0.1 ml of urine was 716 VOL. 27, 1989 RAPID BACTERIURIA SCREENING BY BIOLUMINESCENCE 717

TABLE 1. Number and percentage of positive test results ranging from a trace to a 2+ intensity. For nitrite, any pink was considered positive. Culture (CFU/ No. (%) positive results by: ml) (no. of Gram stain. A sample (0.01 ml) of well-mixed uncentri- specimens) UTIscreen Bac-T-Screen Chemstrip LN Gram stain fuged urine was Gram stained and examined for the presence or absence of leukocytes and microorganisms. The criterion (90) 264 (96) >105 (276) 265 (96) 266 (96) 249 for a positive Gram stain was the presence of one or more 1031-i1 (256) 116 (45) 164 (64) 156 (61) 124 (48) 10'-103 (175) 56 (32) 87 (50) 77 (44) 46 (26) bacterial cells and one or more leukocytes per oil immersion <10' (293) 44 (15) 124 (42) 125 (43) 58 (20) field, which has been reported to correlate with -10' CFU/ ml and pyuria, respectively (3). Predictive value. Predictive values were calculated by the method of Ransohoff and Feinstein (17). The sensitivity, inoculated onto a 5% sheep blood agar plate. Cultures were specificity, and predictive values of positive and negative incubated overnight at 35°C and examined for the number test results were calculated as follows: sensitivity = TP/(TP and types of organisms present. Organisms considered con- + FN); specificity = TN/(TN + FP); predictive value of a taminants were diphtheroids, lactobacilli, viridans group positive test = TP/(TP + FP); and predictive value of a streptococci other than group D, and mixed cultures from negative test = TN/(TN + FN), where TP is true-positive, voided urine specimens. TN is true-negative, FP is false-positive, and FN is false- UTIscreen. Urine specimens were processed according to negative. the instructions of the manufacturer (Los Alamos Diagnos- Time and cost analysis. An analysis of cost per test was tics, Los Alamos, N. Mex.). A sample of well-mixed urine done by calculating the cost of materials routinely used and (0.025 ml) was added to a tube containing dehydrated technical time incurred in our laboratory. The average cost somatic cell releasing agent. Additionally, 0.025 ml of the per specimen was calculated by including both positive and ATP standard was added to a clean polystyrene tube (12 by negative screen results. The cost of detection included 50 mm). The tubes were incubated for 15 min at room material costs and technical time. Technical time was calcu- temperature. Each tube was then placed into the specimen lated based on current College of American Pathologists well of the Luminometer 535 (Los Alamos Diagnostics), and workload units (4) when available or the average time both luciferin-luciferase reagent and bacterial releasing agent required to process 20 specimens by a test method. were added automatically. The integrated light output was displayed on the instrument front panel and also recorded as RESULTS part of the automatic sequence. In this study, the data were analyzed using 25% integrated light output of the standard Distribution of test results. A total of 1,000 clean-voided as a positive test. and catheterized urine specimens were evaluated. There Bac-T-Screen. Urine specimens were processed with the were 276 (27.6%) specimens with colony counts of >10' Bac-T-Screen according to the instructions of the manufac- CFU/ml by the standard semiquantitative plate culture turer (Vitek Systems, Hazelwood, Mo.). A 1-ml sample of method. Of these, the UTIscreen detected 265 (96%) com- well-mixed urine was added to the active barrel of the pared with 266 (96%) by the Bac-T-Screen, 249 (90%) by the instrument. The reagents, 3 ml of urine diluent (14.5% acetic Chemstrip LN, and 264 (96%) by the Gram stain (Table 1). acid), 3 ml of safranin O dye, and two additions each of 3 ml Overall, 226 (82%) were detected by all methods, and at least of decolorizer (2.4% acetic acid), were added automatically. one screening method was positive for each specimen. The filter card was removed from the instrument when the There were 256 (25.6%) specimens with colony counts of test cycle was completed and placed in the Dynadepth test 103 to 105 CFU/ml. Of these, the UTIscreen detected 116 card reader (Vitek Systems). A positive test was interpreted (45%), while the Bac-T-Screen, Chemstrip LN, and Gram as .4 U above the negative control. stain detected 164 (64%), 156 (61%), and 124 (48%), respec- Chemstrip LN. The Chemstrip LN (Boehringer Mannheim tively (Table 1). The remaining 468 specimens had <103 Diagnostics, BioDynamics, Indianapolis, Ind.) is a plastic CFU/ml; of these, 175 had colony counts of 101 to 103 strip to which are attached reagent papers for indicating the CFU/ml, and the remaining 293 specimens had no detectable presence of leukocyte esterase and nitrite in urine. The urine growth (<101 CFU/ml). The detection rates by each method specimen was allowed to come to room temperature prior to for these levels of bacteriuria are shown in Table 1. testing, and the plastic strip was dipped into the specimen The sensitivities, specificities, and predictive values for and immediately withdrawn to remove the excess urine. the test methods at various colony counts are shown in Table Results were read after 2 min. The color intensity of the strip 2. In addition to each method having a sensitivity of at least was coded with a color guide provided by the manufacturer. 90% at >105 CFU/mI, the predictive values of negative test A positive leukocyte esterase was one that gave a purple results ranged from 93 to 98%. When the interpretive break-

TABLE 2. Sensitivities, specificities, and predictive values for test methods Results (%) with method at colony count Parameter UTIscreen Bac-T-Screen Chemstrip LN Gram stain >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 46 718 PEZZLO ET AL. J. CLIN. MICROBIOL.

TABLE 3. Probable pathogens at >105 CFU/ml detected by the test methods No. of isolates detected by': Organism (no. of isolates) UTIscreen Bac-T-Screen Chemstrip LN Gram stain Escherichia coli (108) 107 108 105 105 Candida spp. (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 Serratia marcescens (2) 2 2 2 2 Providencia stuartii (2) 2 2 2 Citrobacter spp. (1) 1 1 1 1 " The percentages of all isolates detected were as follows: UTlscreen, 95: Bac-T-Screen, 98: Chemstrip LN, 92; and Gram stain, 98. point was decreased for all organisms to >103 CFU/ml, the Chemstrip LN. The sensitivities for all methods at >101 sensitivities for the test methods ranged from 81 to 72% and CFU/ml ranged from 84 to 91%. from 73 to 61% at >101 CFU/ml. The specificities and The UTIscreen and the Gram stain had the highest spec- predictive values of positive test results were lower for the ificities for all organisms, which ranged from 69% at >105 Bac-T-Screen and the Chemstrip LN because of the high CFU/ml to 85% at >101 CFU/ml (Table 2). The specificities false-positive results by these methods. for the Bac-T-Screen and the Chemstrip LN ranged from 48 Overall, there were 272 probable pathogens isolated from to 58% for colony counts of >105 to >10' CFU/ml. The 157 clean-voided specimens and 103 catheterized specimens. predictive value of a positive test increased by approxi- Of these, 248 specimens had pure cultures of probable mately 40% for each test method as the interpretive break- pathogens and the remaining 12 were catheterized specimens point of the reference 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- Cost analysis. The total cost of screening by each method ella pneumoniae (n = 21), Pseudomonas spp. (n = 19), and was determined (Table 4). Although salaries differ from Proteus mirabilis (n = 16), accounting for the majority (79%) laboratory to laboratory, the cost per test was based on the of isolates in this study. Of these, Pseudomonas spp., average salary of the technical personnel at our institution. Enterococcus spp., and Candida spp. had the highest per- For detection, the cost ofthe test methods ranged from $0.44 centage of false-negative results for all the test methods. for the Chemstrip LN to $1.17 for the Gram stain compared There were 214 probable pathogens with colony counts of with $1.63 for the reference culture 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. was calculated, the test methods 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 detected 95%, and the Chemstrip LN detected 92%, exception, the Chemstrip LN, which was $0.20 lower. respectively (Table 3). There was no statistically significant difference for detection of any of the probable pathogens by DISCUSSION the test methods (P > 0.1). When the interpretive breakpoint was decreased for probable pathogens to >103 CFU/ml, the The purpose of urine screening is to improve patient sensitivities for the UTIscreen, the Bac-T-Screen, and the management by rapidly reporting negative results and iden- Gram stain were at least 90% compared with 85% for the tifying those specimens which do not warrant culture. Cri-

TABLE 4. Cost analysis of rapid urine screens at >105 CFU/ml Cost ($)/specimen by: Determination Chemstrip Culture method UTIscreen Bac-T-Screen LN Gram stain Cost of supplies 0.75a 0.75 0.75 0.20 0.05 Cost of technical time" 0.88 0.22 0.33 0.22 1.12 Cost of detection 1.63 0.97 1.08 0.44 1.17 Avg cost/specimen, including 1.63 1.75 2.12 1.43 1.97 positive and negative screen result Difference from culture +0.12 +0.49 -0.20 +0.34 a Includes 5% sheep blood agar and biplate of MacConkey agar and polymyxin-B-nalidixic acid blood agar. b Estimated at $13.00/h assuming 4.0, 1.0, 1.5, 1.0, and 5.1 min per sample for culture, UTIscreen, Bac-T-Screen, Chemstrip LN, and Gram stain, respectively. VOL. 27, 1989 RAPID BACTERIURIA SCREENING BY BIOLUMINESCENCE 719 teria for selection of a rapid urine screen should include positive by bioluminescence (2). The specificities of the accuracy, reproducibility, detection time, ease of test per- UTIscreen at various colony counts are acceptable, consid- formance, and low cost. Previous evaluations of microscopic ering that the main purpose of a urine screen is to rapidly and filtration methods have been favorable regarding sensi- identify negative specimens while reliably detecting positive tivities, predictive values of negative test results, and detec- specimens. tion times (10, 13, 14, 22, 23). In this study, the UTIscreen results were similar to the During recent years, the criterion of 2105 CFU/ml estab- Gram stain results regarding sensitivity, specificity, and lished by Kass (7) as the definition of a urinary tract infection predictive values at various colony counts. Although the has been challenged. Studies by Latham et al. (8) and Stamm Gram stain compares favorably with other urine screens and et al. (18) found .102 CFU/ml to be a better predictor of test interpretation is subjective, it requires more labor time infection in symptomatic women. Lipsky et al. (9) and to perform than the UTIscreen, therefore resulting in a Musher et al. (11) recommended that 2103 CFU/ml be used higher cost per test. In addition, it can be a tedious proce- when evaluating voided urine specimens from men. Stark dure because most urine specimens are negative. However, and Maki (19) found that .102 CFU/ml was a more valid it can give a preliminary tentative identification of the index when patients were catheterized and had urinary organism group and the presence of leukocytes. symptoms or were immunosuppressed. Pfaller et al. (16) Both the UTIscreen and the Bac-T-Screen employ instru- studied a random patient population and also found that mentation; therefore, interpretation of test results is objec- lower count cultures most accurately identified urine speci- tive, whereas the Gram stain interpretation is subjective, mens from infected patients. Because of the findings of these requinng technical expertise. Another difference between investigators, the rapid screens described here were evalu- the instrument methods is the volume of urine required to ated at various colony counts. perform the tests; 0.025 ml for the UTIscreen compared with Previous evaluations employing bioluminescence assays 1.0 ml for the Bac-T-Screen. for urine screening have demonstrated sensitivities of .85% The ease of performance is an additional advantage of the at >i04 CFU/ml, with predictive values of negative test UTIscreen. Once the 0.025 ml of urine is added to the tube results of -95% (2, 5, 6, 20, 21, 24, 25). The overall containing the dehydrated somatic cell releasing agent and sensitivity at >105 CFU/ml for the UTIscreen in this study the tube is allowed to incubate at room temperature for 10 was 96% when a25% integrated light output was used as the min, technical time required to complete the test is minimal positive breakpoint. At this interpretive breakpoint, the (15 s). Reagents are added and results are printed automat- UTIscreen was as sensitive as the Bac-T-Screen and the ically. Specimens can be easily batched, whereas the other Gram stain. All three methods had sensitivities of at least methods require individual handling. Cost is an important 90% at 103 CFU/ml for probable pathogens and 84% at 10' consideration and the UTIscreen compared closely with the CFU/ml. culture method in this study when both positive and negative The UTIscreen missed Candida spp. and Enterococcus test results were considered. spp. more frequently than the Bac-T-Screen and the Gram In conclusion, the UTIscreen compared favorably with stain did. A possible explanation may be that some strains of other screening methods with respect to sensitivity, predic- bacteria and yeast had lower ATP levels in spite of high tive value of a negative test, ease of performance, and cost. colony counts (21). Another explanation may be that the The advantages of this rapid urine screen include the ability bacterial releasing agent did not sufficiently lyse the cell to batch test, objective interpretation, higher specificity than walls of these gram-positive organisms (24). These factors the enzyme dipstick or the colorimetric filtration methods, may have contributed to a lower UTIscreen sensitivity for and same-day results with cost similar to culture. these species, which were detected by the other screening methods. LITERATURE CITED The major difference between the semiautomated meth- 1. Albers, A. C., and R. D. Fletcher. 1983. Accuracy of calibrated- loop transfer. J. Clin. Microbiol. 18:40-42. ods, the UTIscreen and the Bac-T-Screen, is in the specific- 2. Bixier-Forell, E., M. A. Bertram, and D. A. Bruckner. 1985. ities of these methods. The specificity of the UTIscreen at Clinical evaluation of three rapid methods for the detection of >105 CFU/ml was higher than that of the Bac-T-Screen (70 significant bacteriuria. J. Clin. Microbiol. 22:62-67. versus 48%); however, the sensitivities were the same 3. Clarridge, J. E., M. T. Pezzlo, and K. L. Vosti. 1987. Cumitech (96%). 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