Comparative Evaluation of the Limulus Assay and the Direct Gram Stain for Detection of Significant Bacteriuria

Comparative Evaluation of the Limulus Assay and the Direct for Detection of Significant Bacteriuria JAMES H. JORGENSEN, PH.D., AND PAMELA M. JONES, M.T. (ASCP) Departments of Pathology and Microbiology, The University of Texas Health Science Center at San Antonio and the Bexar County Hospital, San Antonio, Texas 88 ABSTRACT Jorgensen, James H., and Jones, Pamela M.: Comparative evaluation of the Limulus assay and the direct Gram stain for detection of significant bacteriuria. Am. J. Clin. Pathol. : -8, 9. A double-blind study comparing the Limulus in-vitro endotoxin assay with the direct Gram stain of uncentrifuged urine for detection of significant bacteriuria was performed. One-thousand seventy-seven urine specimens were examined by the two methods and the results compared with results of quantitative urine cultures. Two hundred three samples produced growth of > organisms per ml. urine. The Limulus assay detected 8.% of these specimens, and 98.8% of urines that contained > Gram-negative bacilli per ml. The Gram stain procedure detected only 9.% of urines containing > organisms per ml. and.% of specimens with > Gram-negative bacteria per ml. urine. The Limulus assay demonstrated both greater sensitivity and greater specificity than the Gram stain procedure. Moreover, the Limulus test is much less susceptible to errors of interpretation than methods involving microscopy. (Key words: Endotoxin in urine; Detection of bacteriuria; Gram stain for bacteriuria; Limulus assay for bacteriuria.) IN GENERAL, there have been three separate approaches to development of new screening tests for the detection of significant bacteriuria. One approach has been the introduction of miniaturized inexpensive culture devices., However, these devices still require a traditional culture incubation period of 8- hours. A second approach has been the detection of bacterial products or enzymes in urine by chemical tests such as the Griess nitrate Received June, 9, revised July, 9; accepted July, 9. Supported in part by NIH-General Research Support Grant # SRR-. Address reprint requests to Dr. Jorgensen. reductase test or the measurement of tetrazolium reductase. These tests have the advantage of providing more rapid results, but are often much less reliable. ' 8 A third approach has been the visualization of bacteria in urinary sediment by use of either the phase-contrast microscope or a Gram stain of fresh uncentrifuged urine. In particular, the Gram stain has been reported to correlate with significant colony counts in -9% of cases. ', However, considerable judgment must often be applied to the estimation of the number of organisms visualized in Gram-stained smears. Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8

January 9 DETECTION OF BACTERIURIA The Limulus in-vitro assay is presently the most sensitive method available for the detection of endotoxin, and can be applied easily to the sampling of a variety of biologic fluids.,8 A previous study of a limited number of selected patients indicated that the Limulus test could be applied to the examination of urine for the detection of endotoxin associated with Gram-negative bacteria. 9 Based on this preliminary study, the Limulus test appeared to be a promising screening test for significant bacteriuria. The purpose of the present investigation was to compare, by use of a double-blind study, the Limulus assay and the direct Gram stain for rapid detection of significant bacteriuria in an expanded, unselected patient group. Table. Criteria for Evaluation of Gram Stains of Urinary Sediment WBC's/Field Classification scheme for objective > -9 8- - < Cells/Field Classification leukocytes seen with x + + + + Neg. Classification Classification scheme for bacteria seen with 9 x oil immersion objective > -9 - < + + + + Neg. Materials and Methods Patients Surveyed The patients in this study were those individuals whose urine specimens were submitted to the Microbial Pathology Laboratory of the Bexar County Hospital for routine quantitative bacteriologic culture. They included hospitalized patients and patients seen in one of more than outpatient clinics. In most cases, cleanvoided midstream urine was collected; in certain other cases catheterized specimens were obtained. Every effort was made to process urine specimens within one hour of collection, or they were refrigerated to assure the accuracy of quantitation of bacteria in the sample. Culture Methods Urine specimens were cultured by use of a volume-calibrated platinum bacteriologic loop calibrated to deliver. ml. of urine. 8 Samples were streaked onto one plate each of.% sheep blood agar (Hyland) and MacConkey agar (Hyland). Cultures were incubated at C. and examined at and 8 hours. Performance of Limulus Assay Limulus lysate was prepared according to methods previously described. Lysate batches used in this study could detect as little as ng. per ml. of Escherichia coli :B endotoxin (Boivin extract, Difco Laboratories). Prior to performance of the Limulus assay, urine samples were diluted : in pyrogen-free saline solution (Travenol Laboratories). A.-ml volume of the diluted urine was added to a.-ml. volume of Limulus lysate in pyrogen-free disposable glass test tubes (Corning Glass Works). The reaction mixture was then incubated for hours at C. A Limulus assay was considered positive only if a + or + reaction was obtained following the incubation period. Therefore, based on the lysate sensitivity ( ng. per ml.) and the urine dilution factor (:), an equivalent endotoxin level of ng. per ml. or more was considered "positive" in this study. Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8

JORGENSEN AND JONES A.J.C.P. Vol. Table. Comparison of Limulus Assay and Results on Urine Specimens Containing > Organisms per ml. Table. Comparison of Limulus Assay and Results on Urine Specimens Containing Organisms per ml. Organism Total No. Limulus Organism Total No. ] Limulus Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Proteus mirabilis Enterobacler aerogenes Enterobacter cloacae Serralia tnarcescens Providencia spp. Herellea vaginocola Citrobacter freundii Staphylococcus aureus Group ) Streptococcus Group B Streptococcus Streptococcus viridaus Candida albicans GNR'st or >GNR's CP'st 8 or >CP's GNR + GP Mull. GNR's + GP's 9 8 9 8 8 (9.9%) (.%) (,%) (.%) (.%) (,%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) 8 (8.%) (8.%) (8.%) (8.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa Proteus spp. Misc. GNR'st Group D Streptococcus Staphylococcus epidermidis Group B Streptococcus Streptococcus viridans Candida albicans GNR's or >GNR's GP'st or >GP's GNR+GP Mult. GNR's + GP's TOTAL 8 (.8%) (.%) (.%) (.%) (8.%) (.%) (.8%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.%) (.8%) * Percentages indicate % of total number positive. t GNR = Grain-negative rod: GP = Gram-positive organism. TOTAL (8.%) * Percentages indicate <c of total number positive. t GNR = Gram-negative rod: GP = Gram-positive organism. (9.%) of Urinary Sediment Gram stains were prepared in the usual manner following transfer of a -mm. bacteriologic loopful of uncentrifuged urine to a glass slide and allowing it to air dry. Smears were examined with the high-power objective ( x) for the presence of leukocytes, followed by examination for bacteria using the oil-immersion lens (9 X). The results of examination of ten successive fields were recorded based on the criteria outlined in Table. Smears were considered "positive" if they could be classified as + or greater. Definition of Significant Bacteriuria The criterion for "significant bacteriuria" used in this study was a colony count of > organisms per ml. urine, as defined by Kass." Table. Correlation of and Culture Results (> Organisms per ml.)* Partly Organisms Correct Incorrect Correct GNR's GPC's GPR's GN + GP Yeast * GNR = Gram-negative rod; GPC = Gram-positive coccus; GPR = Gram-positive rod. Controlling Bias A double-blind protocol was employed to minimize bias in comparing the results of Limulus assay and the Gram stain with results of quantitative cultures. The urine cultures were performed in the usual fashion by the routine clinical laboratory technologists. Results of Limulus tests and Gram stains were read and recorded separately by the two authors prior to the availability of the culture results. Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8

January 9 DETECTION OF BACTERIURA Calculation of Sensitivity and Specificity The definitions of sensitivity and specificity are those used by the World Health Organization 9 : sensitivity = diseased persons with positive test/all persons in population with disease; specificity = non diseased persons with negative test/all persons in population without disease. Results One thousand seventy-seven (,) urine specimens were examined. Two hundred three () of the specimens contained more than, organisms per ml. urine, while contained between, and, per ml. Totals of 9 positive Limulus assays and 9 positive Gram stains were obtained. One hundred forty-six () of specimens containing > organisms per ml. produced grwoth of pure cultures of the organisms shown in Table, with Escherichia coli being the most frequent isolate. One hundred and thirty-two () of these specimens had positive Limulus tests (/ or 9.%) and had positive Gram stains (/ or.%). Fifty-six () of the urines containing > organisms per ml. comprised mixed cultures of two or more organisms. Mixed cultures of Gram-negative rods totaling > per ml. were detected in all cases by the Limulus assay, while only of were detected by the Gram stain. However, more false-negative results were obtained with the Limulus test than with the Gram stain procedure with mixed cultures comprised chiefly of Grampositive organisms. Table describes results of Limulus assays and Gram stains of urine specimens containing s organisms per ml. Of the specimens, (.%) yielded positive Limulus tests, while only specimens (.8%) were recorded as positive using the direct Gram stain. Table. Correlation of Pyuria* with Bacteriuriaf WBC's + + + + Total positive Negative Culture > per ml. 8 Culture - per ml. Culture < per ml. * Pyuria defined as or more WBC's per high-power field of uncentrifuged Grani-slrained urine. t Bacteriuria defined as >. organisms per nil. urine. The correlation between culture results and organisms visualized on positive Gram stains is outlined in Table. Results of Gram stains and cultures were reviewed to determine whether a specimen that grew > E. coli had a corresponding Gram stain report indicating that Gram-negative rods were seen, as opposed to diphtheroid-like organisms or Gram-positive cocci, for example. Therefore, Gram stain results were graded either correct, incorrect, or partly correct (in the case of some mixed cultures). It should be noted that proportionately more incorrect responses were recorded with Gram-positive cocci and rods than with Gram-negative bacilli. An attempt was also made to correlate the presence of pyuria, as determined in this study, with significant bacteriuria (Table ). Twenty-five () per cent (/ ) of specimens with > organisms per ml. had significant numbers of leukocytes visualized on the Gram-stained smear. Only 9% (/) of specimens containing s organisms per ml. had significant numbers of leukocytes. Less than % (/) of specimens with < per ml. had pyuria. The overall correlation of quantitative urine cultures with Limulus assays and Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8

JORGENSEN AND JONES A.J.C.P. Vol. Table. Overall Correlation of Urine Cultures with Limulus Assays and s Agreement with Culture Limulus Assay Samples with > s 8.% 9.% organisms per ml. (/) (/) Samples with > s 98.8%.% GNR'sperml.* (9/) (/) Overall ability to classify urine specimens correctly as < s per ml. 9.% 88.% or>io'perml. (,/.) (9/,) * GNR = Gram-negative rod. Gram stains is summarized in Table. The Limulus assay detected 8.% of urine specimens containing > organisms per ml., while the Gram stain detected only 9.%. In the case of urines containing > Gram-negative bacilli per ml., the Limulus assay detected 98.8%, while.% were detected by the Gram stain. The overall ability to classify urine specimens correctly as containing either < or > per ml. indicates that the Limulus assay correctly classified 9.% of all urines, whereas the Gram stain method accurately classified 88.% of the samples. Table indicates the results of calculations of the sensitivity and specificity of each test. The Limulus assay has a sensitivity of 8.% and a specificity of 9.%. The Gram stain procedure has a sensitivity of 9.% and a specificity of 9.8%. Discussion The need for periodic screening of certaia high-risk groups such as pregnant women, school girls, and diabetics for the presence of significant bacteriuria has been well established., The pour-plate quantitative culture has been accepted as the most accurate laboratory procedure available for documenting bacteriuria. A streak plate made with a calibrated platinum loop is a suitable alternate method requiring less time and equipment. However, both methods still require skill, time, and equipment not readily available in all office or clinic settings. Consequently, several miniaturized culture devices and chemical tests have been proposed as practical screening devices for bacteriuria. However, most of these procedures have met with rather limited acceptance. The findings of the present investigation confirm our previous preliminary observations 9 that the Limulus in-vitro endotoxin assay can be used successfully for the detection of significant bacteriuria. The results of the Limulus test can be available within l-'/ hr. of collection of a urine specimen. Although results of Gram stains can be obtained in a shorter period, more time is required of technical personnel for preparation and interpretation of a Gram-stained smear than is required for performance of a Limulus assay. Furthermore, performance of the Limulus test does not require the degree of subjective judgment for accurate quantitation of bacteria required by methods involving microscopy of urinary sediment. This is emphasized by the considerably greater numbers of both false-positive and false-negative results observed with the Gram stain. The sensitivity and specificity of the Limulus assay for endotoxin results in detection of nearly % of urines containing > Gram-negative bacteria per ml. A major deficiency of the Limulus assay for this purpose is that Grampositive bacteria and yeast are undetectable, due to their lack of endotoxin. Therefore, the predictive value of the Limulus assay for significant bacteriuria lies in the normal preponderance of Gram-negative uropathogens, which possess endotoxin. Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8

January 9 DETECTION OF BACTERIURIA The direct Gram stain of uncentrifuged urinary sediment appears to have a false-negative rate of approximately % irrespective of the group of organisms involved. The data in Table indicate that actually more incorrect responses regarding the basic identities of organisms seen in urinary sediment occurred with Gram-positive organisms. Therefore, both the Limulus assay and the direct Gram stain procedure seem better able to define bacteriuria accurately when Gram-negative organisms are involved. Neither the Limulus assay nor direct microscopy could be considered an acceptable method for detection of bacteriurias involving organisms per ml. However, the Limulus assay again defined significantly more urines in this group than was possible using the Gram stain. The percentage agreement of the Gram stain procedure with quantitative cultures reported here is lower than that of certain previous studies.,8 However, in the study by Hoeprich, 8 a Gram stain was considered predictive of bacteriuria of, organisms per ml. or more (and therefore positive) "if bacteria were seen in nearly every field." Even with these less stringent criteria, Hoeprich experienced a falsepositive rate of.%. Brupacher and associates considered a Gram-stained smear positive if "any bacteria were noted." The latter authors also considered a colony count of, bacteria per ml. to be significant. Therefore, the more stringent criteria for positivity (at least one organism per oil-immersion field and at least, organisms per ml.) used in the present investigation probably explain the lower predictive value of the Gram stain in our hands. Specimens in this study which produced growth of < organisms per ml. but which had either a positive Limulus test and a positive Gram stain or a positive Limulus test and a negative Gram stain Table. Sensitivity* and Specificity* of the Limulus Assay and the Direct Limulus Assay Sensitivity 8.% 9.% Specificity 9.% 9.8% False-positive rate.9%.% False-negative rate.8%.% * Based on calculations described in text. were further investigated by review of the medical charts of the patients comprising these two groups. Current antimicrobial chemotherapy was a probable explanation for negative urine cultures from five of these patients. An additional eight patients with positive Limulus assays were diabetic and might be expected to have an increased incidence of bacteriuria; however, urine cultures failed to confirm this supposition. No apparent explanation was found for the positive Limulus tests of urine from the remaining patients. Another important factor in the evaluation of any potential screening test is the cost of performing the procedure. The cost of a Limulus assay as performed in this study would be approximately ^! per specimen. While this seems reasonably inexpensive, it cannot compare to the almost negligible cost of preparing a Gram-stained smear. The lack of predictive value for bacteriuria obtained by visualization of leukocytes in unsedimented urine is in agreement with findings of the previous study of Pryles and Eliot. The data in Table emphasize that bacteriuria may be present in the absence of pyuria as defined, and pyuria may be present without bacteriuria. Therefore, the visualization of significant numbers of leukocytes in urinary sediment may be suggestive of urinary tract infection, but pyuria alone cannot be considered indicative of infection. In conclusion, both the Limulus assay Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8

8 JORGENSEN AND JONES A J. C.P. Vol. and examination of a Gram-stained smear of urinary sediment can be used for detection of significant bacteriuria with reasonable success. The Limulus assay shows both greater sensitivity and greater specificity than the Gram stain procedure. Furthermore, the Limulus assay seems considerably less susceptible to subjective errors of interpretation than methods involving microscopy. Since the presence of antimicrobial agents in urine does not affect either the Limulus test or the Gram stain, either test could be used to assess the effectiveness of treatment of previously diagnosed urinary tract infections. A disadvantage of both methods is that with few exceptions, information regarding the identity of organisms or the presence of mixed infections is not obtained. Acknowledgments. The technologists and technicians of the Microbial Pathology Laboratory of the Bexar County Hospital assisted in the performance of this investigation. References. Brody LM, Webster C, Kark RM: Identification of elements of urinary sediment with phase contrast microscopy. JAMA :-8, 98. Brupacher R, Domingue G: Experiences with a screening test for bacteriuria. Am J Clin Pathol 9:-, 9. Bulger RJ, Kirby MM: Simple tests for significant bacteriuria. Arch Intern Med : -, 9. Cooper JF, Hochstein HD, Seligmann EB Jr: The Limulus test for endotoxin (pyrogen) in radiopharmaceuticals and biologicals. Bull Parenter Drug Assoc :-, 9. Craig WA, Kunin CM: Quantitative urine culture method using a plastic "paddle" containing dual media. Appl Microbiol :99-9, 9. Craig WA, Kunin CM, De Groot J: Evaluation of new urinary tract infection screening devices. Appl Microbiol :9-, 9. Greenberg ND, Stamler J, Zackler J, et al: Detection of urinary tract infections in pregnant women. Public Health Rep 8:8-8, 9 8. Hoeprich PD: Culture of the urine. J Lab Clin Med :899-9, 9 9. Jorgensen JH, Carvajal HF, Chipps BE, et al: Rapid detection of gram negative bacteriuria by use of the Limulus endotoxin assay. Appl Microbiol :8-, 9. JorgensenJH, Smith RF: Preparation, sensitivity and specificity of Limulus lysate for endotoxin assay. Appl Microbiol :-8, 9. Kass EH: Asymptomatic infections of the urinary tract. Trans Assoc Am Physicians 9:-, 9. Neter E: Evaluation of the tetrazolium test for the diagnosis of significant bacteriuria. JAMA 9:9, 9. Pryles CV: The diagnosis of urinary tract infection. Pediatrics :-, 9. Pryles CV, Eliot CR: Pyuria and bacteriuria in infants and children. Am J Dis Child :8-, 9. Rojas-Corona R, Skarnes R, Tamakuma S, et al: The Limulus coagulation test for endotoxin: A comparison with other assay methods. Proc Soc Exp Biol Med :99-, 99. Smith LG, Schmidt J: Evaluation of three screening tests for patients with significant bacteriuria. JAMA 8:-, 9. Smith LG, Thayer WR, Malta EM, et al: Relationship of the Griess nitrite test to bacterial culture in the diagnosis of urinary tract infection. Ann Intern Med :-, 9 8. Trippodo NC, Jorgensen JH, Priano LL, et al: Cerebrospinal fluid levels of endotoxin during entotoxemia. Proc Soc Exp Biol Med : 9-9, 9 9. Wilson JMG.Jungner G: Principles and Practice of Screening for Disease. World Health Organization, Public Health 98 Papers, Geneva, Downloaded from https://academic.oup.com/ajcp/article-abstract////8 on April 8