Key words: Limulus amoebocyte lysate assay, Limulus lysate micro-technique procedure, Chromogenic substrate assay, Rapid detection of bacteriuria

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Key words: Limulus amoebocyte lysate assay, Limulus lysate micro-technique procedure, Chromogenic substrate assay, Rapid detection of bacteriuria

Fig. 1 Preparation of chromogenic substrate LAL assay on urine specimens

Fig. 2 Standard curve of LAL assay by using E. coli (O111-B4) standard endotoxin Each point represents the means }the standard deviation. Table 1 Results of quantitative urine culture (228 samples) ND.*; not detected(<2 ~103CFU/ml)

Fig. 3 Comparison of the results of LAL assay on urine specimens including E. coli (ATCC 25922) according to various dilution rates and incubation times LAL assay was performed on undiluted urine specimens (N) and diluted to 1: 10 ( ), 1: 100 ( œ) and 1: 1000 ( ) with endotoxin free water. Fig. 4 Results of the absorbance values in LAL assay on diluted 58urine specimens (ND group)

Fig. 5 Results of the absorbance assay on diluted urine specimens group) values in LAL (GPC, YLO Fig. 6 Results of the absorbance values in LAL assay on diluted urine specimens (GNB group) Fig. 7 Comparison of the absorbance values in LAL assay on specimens of four urine groups (GNB, GPC, YLO and ND groups) (): number of urine isolated 105CFU/ml of bacteria p values compared GNB group with each GPC,YLO and ND group

Table 2 Comparison of the absorbance values in LAL assay between GNB group and other 3 groups Table 3 Correlation of the results of LAL assay with four groups a): GNB, GPC and YLO were the groups of a): GNB, Gram-negative bacteria ( 105CFU/ml) GPB, Gram-positive cocci ( 105CFU/ml) YLO, Yeast-like organism( 105CFU/ml) ND, Not detected(<2 ~103CFU/ml) b): N, Number of urine specimens c): Av, Average of absorbance values; SD, Standard deviation d): p values compared GNB group with each GPC, YLO and ND group urine contained 105 CFU/ml of each kind of bacteria in urine. ND was the group of urine not detected by the quantitative urine culture (<2 ~103CFU/ml) b): above 0.15 with OD values at 410nm Table 4 Results of LAL assay on urine specimens compared with quantitative culture a): Total No. of GPC, YLO and ND group

Detection of endotoxin in antibiotic solutions with limulus amobocyte lysate. Antimicrob. Agents Chemother., 23: 649-652, 1983. 4) Iwanaga, S., Morita, T., Harada, T., Naka- mura, S., Kimura, T. & Sakakibara, S.: Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostatis.. 7: 183 13: 158-162, 1981. 485-488, 1983. 1) Levin, J. & Bang, F. B.: The role of endotoxin in the extracellular coagulation of limulus blood. Bull. Johns Hopkins Hosp., 115: 265 2) Siegel, S. E. & Nachum, R.: Use of the limulus lysate assay (LAL) for the detection and quantitation of endotoxin. In Prespective in toxinology (Bernheimer, A. W. ed.) p.72-81, John Willy & Sons, New York, 1977. 3) Case, M. J., Ryther, S. S., Novitsky, T. J.: 6) Jorgensen, J. H. & Jones, P. M.: Comparative evaluation of the limulus assay and the direct Gram stain for detection of significant bacteriuria,. Am. J. CLin. Pathol., 63: 142-148, 1975. 7) Jorgensen, J. H., Carvajal, H. F., Chipps, B. E. & Smith, R. F.: Rapid detection of Gramnegative bacteriuria by use of the limuls endotoxin assay. Appl. Microbiol., 26: 38-42, 1973. 8) Jorgensen, J. H. & Alexander, G. A.: Rapid ditection of significant bacteriuria by use of an automated limulus amoebocyte lysate assay. J. Clin. microbiol., 16: 587-589, 1982. 9) Nachum, R. & Shanbrom, E.: Rapid detection of Gram-negative bacteriuria by limulus amoebocyte lysate assay. J. Clin. Microbiol., 10) Nachum, R. & Berzofsky, R. N.: Chromogenic limulus amoebocyte assay for rapid detection of Gram-negative bacteriuria. J. Clin. Microbiol., 21: 759-763, 1985. 11) Prior, R. B. & Spagna, V. A.: Rapid evaluation of gonococcal and nongonococcal urethritis in men with limulus amoebocyte lysate and a chromogenic substrate. J. Clin. Microbiol., 17: 12) Kakinuma, A., Asano, T., Torii, H. & Sugino, Y.: Gelation of limulus amoebocyte lysate by an antitumor (1 3)-ƒÀ-D-glucan. Biochem. Biophys. Res. Communs., 101: 434-439, 1981. 14) Obayashi, T., Tamura, H., Tanaka, S., Ohki, M., Takahashi, S., Arai, M., Masuda, M. & Kawai, T.: A new endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical aplications. Clinical Chimica Acta, 149: 55-65, 1985. 15) Friedman, R. B., Kwean, H. C. & Szczecinski, M.: Imporved sensitivity of chromogenic substrate assays for urokinase and plasmin. Thrombosis Reserch, 12: 37-46, 1977. 16) Kass, E. H.: Asymptomatic infections of the

urinary tract. Trans. Assoc. Am. Physicians, 69: 56-64, 1956. 17) Prior, R. B. & Spagna, V. A.: Application of a limulus test device in rapid evaluation of gonoconcal and nongonococcal urethritis in males. J. Clin. Microbiol., 14: 256-260, 1981. Evaluation of Screening of Bacteriuria -Rapid Detection of Gram-Negative Bacteriuria by Using the chromogenic Limulus Lysate Microassay- Takahiro NARITA, Kazuyuki SUGAHARA, Keizo YAMAGUCHI & Toshiaki USUI Department of Clinical Laboratory, School of Medicine Nagasaki University Hospital The use of Limulus Amoebocyte Lysate (LAL) assay in determining bacteriuria due to gramnegative organisms had been studied extensively. Many authors and workers have published papers concerning this study. And Iwanaga et al., in 1978, introduced the chromogenic substrate LAL assay. In this study, bacteriuria due to gram-negative bacteria (GNB) was detected by using chromogenic substrate LAL assay. On the basis of this method, micro-technique procedure was applied using small amount of urine specimens and solution composed of LAL and chromogenic substrate. We used 100- fold diluted urine on LAL assay, mixed and incubated at 37 Ž for 30 minutes and examined macroscopically by using spectrophotometer (microplate reader). Out of 228 urine samples tested, 31 out of the 32 samples (96.9%) were positive which correlated well with urine samples which containing more than 105 CFU/ml of gram-negative bacteria. Out of 8 urine samples which contained more than 104 CFU/ml up to 105 CFU/ml of GNB, 6 (75.0%) were positive. Out of 11 urine samples which contained less than 104 of GNB, 4 (36.4%) were positive. The most common organisms isolated from urinary tract infections are gram-negative organisms. So the LAL assay is a rapid, simple and reliable method in determining of clinically significant gram-negative bacteriuria.

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