Vol. 22, No. 2JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1985, p. 176-1810095-1137/85/080176-06$02.00/0Copyright © 1985, American Society for Microbiology

Comparison of the AutoMicrobic System, Acridine Orange-StainedSmears, and Gram-Stained Smears in Detecting Bacteriuria

BENJAMIN A. LIPSKY,l,2* JAMES J. PLORDE,"2'3'4 FRED C. TENOVER,3'4 AND FRANK P. BRANCATO5Departments of Medicine,' and Laboratory Medicine,3 Microbiology, University of Washington School of Medicine, and

Medical2 and Laboratory4 Services, Veterans Administration Medical Center, Seattle, Washington 98108

Received 28 January 1985/Accepted 16 April 1985

We compared the accuracy of the Gram-stained smear, the acridine orange-stained smear, and theAutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) in screening for bacteriuria, as detected byconventional cultures. For 1,024 clinical specimens, results with the acridine orange-stained smear and theGram-stained smear were very similar. When read for the presence of one or more microorganisms orleukocytes per 20 oil immersion fields, both smears were highly sensitive (92.1 and 93.3%, respectively) andmoderately specific (70.0 and 61.7%, respectively). Sensitivity was greater for specimens yielding i105 CFU/ml(96.1 and 98.9%, respectively) than for those with 103 to 104 CFU/ml (81.4 and 78.0%, respectively).Preliminary classification based upon the tinctorial and morphological characteristics of the Gram-stainedsmear was compatible with culture results in nearly all cases. The accuracy of the Gram-stained smears wasnot influenced by special cleaning of the microscopic slides, or the level of expertise of the microscopist. For 715specimens, the sensitivity of the AMS in detecting bacteriuria (91.5%) was very similar to that of the stainedsmears (92.1 and 95.7%, respectively), but the specificity was significantly higher (83.2% versus 42.6 and70.0%). Detection of microorganisms by the AMS took an average of 6.3 3.0 h. These data suggest that theGram-stained smear is easily interpreted, very sensitive, acceptably specific, and still the optimal rapid methodfor screening for bacteriuria in most clinical microbiology laboratories.

Detecting bacteriuria is the most frequent task undertakenby clinical microbiology laboratories (10), often accountingfor more than half their workload (S. M. Hoyt and P. D.Ellner, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C8, p.313). Quantitative urine culture, the conventional procedureemployed for this purpose, requires at least 18 h. Since thegreat majority of these cultures are negative, this detectiontechnique is time-consuming and expensive. Accordingly,methods have long been sought that would more expedi-tiously distinguish sterile from infected specimens. Theearliest technique used was microscopic examination ofstained or unstained urine for the presence of bacteria andleukocytes. Later, both growth-dependent and growth-independent chemical, mechanical, and electrical methodswere developed to screen for bacteriuria (12, 32-34, 38, 45).

In the past few years, several automated systems havebeen marketed that are capable of rapidly detecting bacteriain urine specimens. One of the most promising is theAutoMicrobic system (AMS; Indenti-Pak, Vitek Systems,Inc., Hazelwood, Mo.), a device which not only detects andenumerates, but identifies the commonest uropathogenswithin 13 h (1). Although these systems possess the advan-tages inherent in automation, they are more expensive andless accurate than standard cultural methods (33). Recently,preliminary reports have suggested that urine smears stainedwith acridine orange may be more accurate than Gram-stained smears (7, 18; M. A. Bartelt, Abstr. Annu. Meet.Am. Soc. Microbiol. 1983, C2, p. 312). How well these andother new techniques compare with screening methodspresently in use is, however, still under assessment. To thisend, we compared the results obtained by the oldest urine-screening technique (Gram-stained smear) with those ob-tained by a newer microscopic method (acridine orange-stained smear) and an automated method (AMS). We also

* Corresponding author.

evaluated the influence of the experience level of the micros-copist and the effect of using highly cleaned glass slides onthe sensitivity and specificity of the Gram-stained smear.

MATERIALS AND METHODS

Specimen collection. All urine specimens submitted forculture to the Seattle Veterans Administration Medical Cen-ter clinical microbiology laboratory during the day-shift hourswere included in this study, if received within 2 h of collec-tion. Although some specimens were obtained by urethralcatheterization and suprapubic aspiration, the great majoritywere collected by the clean-catch midstream void procedure.Submitted specimens came from both ambulatory and hos-pitalized patients, about 90% ofwhom were men. Specimensfrom patients receiving antimicrobial agents were included inthe study.

Culture and smear preparation. Urine was inoculated ontotwo Columbia blood agar-MacConkey agar biplates (Pre-pared Media Laboratories, Renton, Wash.) with platinumquantitative inoculating loops. Both media of one plate werestreaked with 0.01 ml of urine, while those of the otherreceived 0.001 ml. The plates were incubated in a humidifiedatmosphere at 35°C for at least 18 h. A 0.01-ml drop of eachurine specimen was placed on each of three separate glassmicroscope slides (Rite-on precleaned microscope slides;Clay Adams, Oxnard, Calif.). A Gram-stained smear (bystandard methods, using Hucker's crystal violet), and anacridine orange (Vacutainer type; Becton Dickinson andCo., Paramus, N.J.)-stained smear (stained for 2 min, rinsedwith tap water, and air dried) (24) were prepared on standardslides taken directly from the package. A second Gram-stained smear was prepared on a specially cleaned slide,which had been washed in a mild detergent (Labtone;V.W.R. Scientific, Inc., San Francisco, Calif.), rinsed twicein distilled water, and then rinsed in 95% ethanol. All threesmears were prepared and coded by a laboratory technician

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who, as time permitted, also inoculated specimens into theAMS urine cards (lot no. A5C) as described in the directionsof the manufacturer.Smear reading and interpretation. The technician who read

the Gramr-stained smears had had no microbiology labora-tory experience before bein& employed by our laboratory 6months before the start of this study. He was unaware of thepurpose of the study and read all of the cleaned Gram-stained slides as they were prepared. Both the cleaned andthe standard Gram-stained slides were later read blindly by amicrobiologist (F.P.B.) who is a recognized expert in Gramstain interpretation (5, 14). The two slides of each set wereoverlaid with immersion oit and arranged in random order toensure that the microbiologist was unaware of which type ofslide he was reading. Gram-stained smears were read underoil immersion (x 1,000) with a Zeiss RA microscope. Theacridine orange-stained smear was read by a secondmicrobiologist (F.C.T.) using an epi-illuminated Zeiss RAmicroscope fitted with a x63 dry objective (total magnifica-tion, x630).The three readers were instructed to view 20 microscopic

fields and to spend no more than 60 s per slide. Bacteria,yeast, and leukocytes (WBCs) were quantitated as thenumber per field. Results were recorded on separate logsheets and çollated with results of the conventional cultureand AMS by two nonreaders.

Culture interpretation. Growth on standard cultures wasrounded off to the nearest logarithm (e.g., 10- CFU/ml = 5 x104 to 4 x 105 CFU/ml). Based on previous studies from ourlaboratory (B.A. Lipsky, R. Ireton, R. Berger, and S. Fihn,Program Abstr. Intersci. Conf. Antimicrob. Agents Chemo-ther. 23rd, Las Vegas, Nev., abstr. no. 514, 1983), cultureresults were interpreted as follows. Specimens resulting ingrowth of <102 CFU/ml of urine were considered negative.Those growing 2103 CFU/ml with three or more colonialtypes were considered to represent mixed normal flora (i.e.,they were presumed to have been contaminated duringcollection). Cultures with 2103 CFU/ml with one or twospecies were considered positive. AUl organisms were identi-fied to the species level, except nonhemolytic streptococci(which were classified only as enterococci or nonenterococ-ci), coagulase-negative staphylococci (which were classifiedas Staphylococcus saprophyticus or non-S. saprophyticus),and yeasts (which were identified as Candida albicans ornon-C. albicans). Beta-hemolytic streptococci were groupedby the Streptex Kit (Wellcome Diagnostics, Bechenham,England). Gram-negative bacilli were identified by conven-tional tube media by the scheme of Edwards and Ewing (11),The AMS reports specimens as negative, positive with

<105 CFU/ml, or positive with -105 CFU/ml. Up to threedifferent microbial species are reported per specimen. Mi-croorganisms which cannot be identified are so reported.The time required to identify a positive result is recorded.

Statistical methods. Statistical tests were performed onlyon the overall results of sensitivity and specificity betweenthe various screening methods. These were evaluated byCochran's Q and related statistiçs (13).

RESULTS

Between 1 March 1982 and 1 Marçh 1983, 1,150 clinicalurine specimens were processed for this study. Completeresults for all three smear methods (Gram-stained smearsread by the technician, standard and cleaned Gram-stainedsmears read by the microbiologist, and acridine orange-stained smears) were available for 1,024 specimens. Results

for the AMS were available for 832 specimens, 715 of whichalso had smears prepared by all three methods.Of the 1,024 sets of specimens with complete smear

results, 640 (62.5%) were culture negative. Mixed growth(i.e., three or more colony types) was found in 143 (14.0%) ofthe specimens; only 26 (18.2%) of these 143 grew -10CFU/ml. Cultures of 241 specimens were positive; the greatmajority (83.9%) yielded a single microbial species. Faculta-tive or aerobic gram-negative bacilli constituted 79.0% of thepositive cultures, facultative gram-positive cocci constituted23.6%, yeasts constituted 2.6%, and the rest were miscel-laneous bacterial species. When specimens were analyzedby miçrobial species, there were no appreciable differencesin the proportion of specimens resulting in the growth of highcolony counts (2105 CFU/ml) versus low colony çounts (103to 104 CFU/ml)'.Comparison of the stain methods: (i) specially cleaned slides

versus glss slides. The sensitivitiçs and specificities of theGram-stained smears made on the standard and speciallycleaned glass slides were nearly identical for bacteria andpyuria regardless of the microorganism type or colonycount. Therefore, in further comparisons, only the resultsobtained with the specially cleaned slides were used.

(il) Detection of nuvcroorganisms. There were only smalldifferences in the readings of the Gram-stained smears formicroorganisms by the expert microbiologist and the rela-tively inexperienced technician (Table 1). For both observ-ers, the sensitivity of the smear was just over 40% for thelow-colony-count specimens and about 90% for the high-colony-count specimens; the specificity of the smear wasjust over 90%. The sensitivity (at high and low colonycounts) and specificity of the acridine orange-stained smearwere nearly identical to those of the Gram-stained smears.

TABLE 1. Comparison of the sensitivities and specificities of thethree stained-smear methods for detecting nnicroorganisms and

WBC(s in 1,024 urine specimensSensitivity (%)b for thefollowing colony count

Critçrion,I method, and reader (CFU/ml): Specificityc103-104 1lo' .ij03 %

MicroorganismsGram stain

Technician 40.7 90.7 78.4 90.6Microbiologist 44.1 89.0 78.0 94.4

Acridine orange stain 44.1 92.9 80.9 91.9

WBCGram stain

Technician 76.3 90.7 87.1 67.2Microbiologist 84.7 94.1 91.7 47.1

Acridine orange stain 67.8 73.1 71.8 75,0

Microorganisms or WBCGram stain

Technician 78.0 98.9 93.8 61.3Microbiologist 88.1 98.4 95.9 45.8

Acridine orange stain 81.4 96.1 92.5 70.0

a A positive result was çiefined as 21 microorganism or WBC (depending onçriterion) seen in 20 OIFs.

b Percent positive cultures with positive smears. Mixed cultures (i.e" thosegrowing -103 CFUIml with 23 colonial types) were excluded.

C Percent negative cultures with negative smears for cultures with colonycounts of s210 CFU/ml.

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(iii) Detection of WBCs. Pyuria (defined as -1 WBC per 20oil immersion fields [OIFs]) detected by the technician onthe Gram-stained smear had a sensitivity of 76.3% for thelow-colony-count cultures and 90.7% for the high-colony-count cultures; the specificity was 67.2% (Table 1). Thesensitivity of the readings by the microbiologist was some-what greater (84.7 and 94.1%, respectively), but the speci-ficity was substantially less (47.1%). The specificity of thereadings by the technician increased when pyuria was de-fined as 21 WBC per OIF (95.6%) or 210 WBCs per OIF(99.3%), but the sensitivity for high colony count cultureswas disproportionately reduced (to 35.1 and 8.8%, respec-tively). Compared with the Gram stain, the sensitivity of theacridine orange stain for pyuria was appreciably less for bothlow-colony-count (67.8%) and high-colony-count (73.1%)cultures, whereas the specificity was greater (75.0%).The relationship between the degree of pyuria on smears

and the microorganism isolated on cultures was weak.Specifically, the proportion of specimens with >1 WBC perOIF was not appreciably different for those growing Proteus(57.1%) or Pseudomonas (38.1%) species than for thosegrowing other gram-negative bacilli (42.2%), although it wassomewhat lower for those growing gram-positive cocci(24.2%).

(iv) Detection of microorganisms or WBCs. When thedetection of one or more microorganisms or WBCs in 20OIFs (as compared to either finding alone) was considered apositive smear, the sensitivity in detecting urines yielding2105 CFU/ml on culture was substantially increased for boththe Gram-stained (98.9%) and acridine orange-stained(96.1%) smears. This definition leads to only a moderatedecrease in the specificity of the smears (to 61.3 and 70.0%,respectively).

(v) Effect of double readings of Gram-stained smear. Toassess whether separate readings of the Gram-stained smearby two observers would increase its sensitivity, a positivereading by either the technician or microbiologist was tabu-lated as a positive result. This strategy increased the sensi-tivity of the smear (for detection of microorganisms orpyuria) from 93.3 to 97.9% for all positive cultures and to100% for specimens yielding i105 CFU/ml.Comparison of AMS and standard culture results: (i) evalu-

ation of the AMS as a screening technique for bacteriuria. Theresults obtained with 832 specimens processed by the AMS

TABLE 2. Results of 832 urine specimens processed by AMScompared with those obtained by conventional culture"

No. of the following results obtained byStandard culture AMS: Total %result(CFU/ml)Toa%

Negative Mixed" Unidentified' Positive"Negative ('102) 439 4 21 63 63.3

Mixed 14.8103-104 48 1 18 302-105 4 8 4 10

Positive 21.9103-104 13 1 4 20::>105 1 27 6 110

Total % 60.7 4.9 6.4 28.0

'See the text for definitions.Three or more isolates detected at any level of growth.

'Growth was detected in AMS, but the isolate was not identified.dOne or two isolates detected at any level of growth.

TABLE 3. Accuracy of various methods compared with standardculture in screening for bacteriuria on 715 specimens'

Methodad reader Sensi- Speci- Positive NegativeMethod and reader tivity (%)b ficity (%)' predictive predictivevalue (% value (%

Gram stainedTechnician 93.3 61.7 47.3 96.1Microbiologist 95.7 42.6 38.1 96.4

Acridine orange stain 92.1 70.0 53.2 96.0

AMS 91.5 83.3 67.0 96.4" Mixed cultures (i.e., those with 2103 CFU/ml and -3 colonial types)

excluded. See the text for definitions of positive and negative results andpositive and negative predictive values.

b None of the differences in sensitivity between the methods is statisticallysignificant (P . 0.2)

' All of the differences in specificity between the methods are statisticallysignificant (P < 0.001).

and conventional culture are compared in Table 2. Amongthe positive cultures, the AMS reported growth in 25 (65.8%)of 38 cultures with 103 to 104 CFU/ml and in 143 (99.3%) of144 cultures with i105 CFU/ml. Thus, the overall sensitivitywas 92.3% (168 of 182). For all specimens growing i105CFU/ml (i.e., both positive and mixed cultures), the sensitiv-ity of the AMS was 97.1%. Interestingly, the AMS detectedgrowth in only 71 (58%) of 123 cultures yielding mixed floraat any colony count. For the 527 specimens with negativecultures, the AMS reported growth in 88 (16.7%), giving aspecificity of 83.3%. When all cultures yielding <105CFU/ml were considered negative, the specificity of theAMS was 75.1%.The results with the AMS are compared with those of the

Gram-stained and acridine orange-stained smears in Table 3.Although similar definitions were used for these admittedlydifferent methods, the results for all were remarkably alike.The most important results, in terms of the value of thesemethods as screening tests, are the sensitivity (92.1 to95.7%) and negative predictive value (96.1 to 96.4%). Therewere no statistically significant differences between any ofthese values (Cochran's Q = 4.60 with three degrees offreedom; P 2 0.20). The specificities varied more, rangingfrom 42.6% for the reading of the Gram-stained smear by themicrobiologist to 83.3% for the AMS. Each of these values issignificantly different from the others (Cochran's Q = 133with one degree of freedom; P 2 0.001). Positive predictivevalues were low (38.1 to 67.0%), paralleling the specificities.

(il) Evaluation of time to positive results with the AMSsystem. The elapsed time to a positive result was recordedfor 158 of the 182 specimens. The range in times was 1 to 13h, with a mean time for all specimens of 6.3 ± 3.0 (standarddeviation) h. The differences in times for the various classesof organisms were relatively small.

DISCUSSIONGram-stained smear. Among the techniques devised to

rapidly detect significant bacteriuria, microscopic examina-tion of urine for microorganisms and WBCs has been themost extensively evaluated (2, 6, 10, 14-16, 21, 23, 25, 26,28, 31, 34, 36, 37, 41, 42, 44, 45). Definitions of a positivemicroscopic examination have varied, but most authors usedthe detection of 21 microorganism per OIF for visiblebacteriuria, and 210 WBCs per high-power field for pyuria.Examination of the sediment of centrifuged urine has notusually yielded better results than examination of an uncen-

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trifuged specimen (2, 10, 15, 23, 26, 28), but most reportssuggest that staining the urine smear improves the reliabilityof the results over those obtained with direct, unstainedsmears (14, 22, 32).

Previous studies have shown that the Gram-stained smearcan detect microorganisms in about 88% (range, 52 to 100%)and WBCs in about 65% (range, 25 to 95%) of specimensyielding 105 CFU/ml on culture. The specificities of apositive smear for bacteria and WBCs have averaged 89%(range, 52 to 100%o) and 81% (range, 41 to 97%), respec-tively. Results become progressively poorer as the colonycount of the specimen decreases (2, 6, 15, 19, 21, 23, 26, 34,42). Combining the observations of pyuria and microorgan-isms has been reported to improve the sensitivity of micro-scopic examination (6, 36, 37).The results of the readings of the Gram-stained smears by

our laboratory technician were similar to those reported inmost previous studies: he saw microorganisms in 90.7% ofcultures yielding -105 CFU/ml but in only 40.7% of thosewith 103 to 104 CFU/ml (Table 1). The high specificity(90.6%) is perhaps surprising, since we chose a less restric-tive definition of a positive smear (i.e., any microorganismsin 20 microscopic fields) than most authors. There was norelationship between the Gram-stain characteristics of amicroorganism or the number of isolates on the standardculture and the likelihood of detecting bacteria on the smear.The definition we used for pyuria (i.e., 21 WBC seen in the20 fields examined) helps explain its surprisingly high sensi-tivity (76.3 to 90.7%) and specificity (67.2%). The level ofpyuria was related to the colony count on culture but not tothe species of bacteria. The markedly lower incidence ofpyuria in mixed specimens is consistent with the hypothesisthat most of these represent specimen contamination ratherthan true urinary tract infection.When the presence of either microorganisms or WBCs on

the Gram-stained smear is considered a positive result, thesensitivity of the smear is excellent (98.9%) for detecting allspecimens with 2105 CFU/ml. Even culture-positive speci-mens growing only i03 to i04 CFU/ml were detected in78.0% of such cases. Combination of the readings by thetechnician and the microbiologist demonstrated that inde-pendent double examinations further increased the sensitiv-ity of the Gram-stained smear to nearly 100%. Similarfindings have been reported for other microscopic examina-tions (27). This procedure would only add 1 min to theprocessing time of the smear and might substantially im-prove its sensitivity in some laboratories.

SpeciaUy cleaned slides versus standard glass slides. Wepostulated that the reason some specimens with .210CFU/ml have negative Gram-stained smears is that organ-isms and WBCs may wash off the glass slide during thestaining procedure. Removing any surface film that mightlessen adhesion of cells to the slide could, therefore, result ingreater smear sensitivity. There were, however, no substan-tial differences in the results of Gram-stained smears pre-pared on the specially cleaned slides versus the standardslides. This technique, therefore, appears to be of no benefit.

Technician versus microbiologist. The major argumentsadvanced against using Gram-stained smears to screen forbacteriuria have been that their examination is time consum-ing and that their interpretation requires considerable skill(9, 17, 21, 29, 37). This contention is not supported by ourexperience. Compared with those of the expert microbiolo-gist, the readings of the less experienced technician were assensitive and somewhat more specific (owing to fewer false-positive readings of low-level pyuria). This result suggests

that proficiency in reading Gram-stained smears of urine isgained quickly and not substantially improved upon withexperience.

Acridine orange-stained smear. Only three studies haveevaluated the usefulness of acridine orange-stained smearsin screening for bacteriuria (7, 18; M. A. Bartelt, Abstr.Annu. Meet. Am. Soc. Microbiol. 1983, C2, p. 312). Inthese, the sensitivities ranged from 92 to 98%, and thespecificities ranged from 59 to 87%. In the two studies inwhich they were compared, the results differed only slightlyfrom those obtained concurrently with Gram-stainedsmears. In our study, the sensitivity of the acridine orangestain was nearly identical to that of the Gram stain fordetection of microorganisms or pyuria (Table 1). In theoverall comparison (Table 3), there was a small difference inspecificity between the methods which did, however, reachstatistical significance. Both stains were about 96% accuratein detecting the morphological characteristics (i.e., cocciversus bacilli) of the bacteria. The acridine orange stain doesnot, of course, distinguish gram-positive from gram-negativeorganisms, a diagnostically useful classification. In view ofthe diagnostic accuracy of the Gram stain evaluated by evena relatively inexperienced reader and the expensive equip-ment required for reading a fluorescent stain, the acridineorange stain appears to offer little advantage over the Gramstain in screening for bacteriuria.AMS. When used to screen urine specimens, the AMS is

extremely sensitive for positive cultures growing 2î0oCFU/ml (99.3%) but less so for those growing 10 to 104CFU/ml (65.8%) (Table 2). For unknown reasons, the sen-sitivity for mixed cultures is considerably less than that forpositive cultures: 84.6% for those growing 2105 CFU/ml and49.4% for those growing 103 to 104 CFU/ml. The AMS, infact, failed to correctly identify the great majority of speci-mens-growing mixed flora on conventional cultures (Table2). For all positive cultures, the sensitivity of the AMS(91.5%) is very similar to those ofthe stained smears, but thespecificity (83.3%) is significantly better (Table 3). Ourresults with the AMS, even with the less restrictive defini-tion of a positive culture (i.e.,-103 CFU/ml), are similar tothose of other studies in which this device was used for urinespecimens. Compared with standard cultures, sensitivitiesfor significant bacteriuria (usually defined as .105 CFU/ml)have ranged from 83 to 99%, and specificities have rangedfrom 55 to 99% (1, 3, 4, 16, 20, 25, 30, 32, 33, 39, 40, 43, 44).The time required for a positive result to be reported by

the AMS varied from 1 to 13 h and averaged about 6 h. Thisdelay reduces the value of this procedure as a screening test.Correlations between the time to a positive AMS result andthe class of microorganism isolated or the colony count onculture were minor. Because the AMS manufacturers havechanged the method by which positive specimens are quan-titated, we compared the outcome of quantitationi by the old(enumeration) and new (time-to-positivity) methods with theresults of standard cultures. The differences in results be-tween these methods were small and slightly favored the oldmethod.Almost 30 years ago, Kass wrote concerning urine speci-

mens that cliniciansas have long depended upon the Gramstain as a screening device to distinguish infection fromcontamination, and its use is amply justified . .. " (22).None of the many new methods promoted for screeningurine culture specimens since that time have proven to bemore useful than the Gram-stained smear. Furthermore,unlike the Gram-stained smear, they provide no informationon the likely etiological agent of infection. Among the

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several automated systems now available for bacteriuriadetection, the AMS has performed as well as any (32).

In our experience, the AMS was quite reliable, but it isprobably too slow and expensive for use as a screeningdevice. The Gram-stained smear was, surprisingly, about as

sensitive as the AMS, although considerably less specific.The specificity of the stained smears was certainly artificiallylowered by smear-positive specimens rendered culture nega-tive by inhibitory substances in the urine (e.g., povidone-iodine, antimicrobial agents) (8). Nevertheless, even whenthe reading is performed by a relatively inexperienced andbusy technician, less than a minute spent reading the smearfor bacteria and WBCs allows a highly accurate prediction ofthe result of a standard urine culture. Furthermore, withexperience, a microscopist can usually predict the class (andin many instances the genus) of the infecting organismss.Contrary to the opinion of others (9, 10, 35), we found theGram-stained smear was as accurate for gram-positive mi-croorganisms as for gram-negative microorganisms. The useof specially leaned glass slides did not improve the sensitiv-ity (or specificity) of the Gram-stained smear. The acridineorange stain was no better than the Gram stain in screening

for bacteriuria.Few studies of the Gram-stained smear of urine specimens

have been performed in predominantly male populations.Qur results, like those ofPerry et al. (31), are very encourag-

ing. To maximize the sensitivity of this procedure when usedas a screening test, we recommend that the sighting of any

bacteria or WBCs in 20 OIFs be considered a positive smear.

With these criteria, less than 2% of specimens with 2105CFU/ml will fail to be detected by the Gram-stained smear.Although the specificity of the smear is about 60% by thesecriteria, the number of urine specimens most clinical micro-biology laboratories would need to culture would still de-crease by about 50% (i.e., 80% of submitted specimens are

culture negative, and 60% of these would be microscopicallynegative). Considering the universal availability, rapidity,ease ofinterpretation, and very low cost ofthe Gram-stainedsmear, we believe that it is still the procedure of choice forscreening for bacteriuria.

ACKNOWLEDGMENTS

We thank Jo Anne Gates and Larry Carlson for their technical andcomputer assistance. We also thank Thomas D. Koepsell for adviceon statistical methods, the Seattle Veterans Administration MedicalCenter microbiology technicians for processing the smears andcultures, and Bi-Lan Chiong and Ruth Hamada for preparing themanuscript.

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