A Silkworm Larvae Plasma Test for Detecting Peptidoglycan in Cerebrospinal Fluid Is Useful for the Diagnosis of Bacterial Meningitis

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1 A Silkworm Larvae Plasma Test for Detecting Peptidoglycan in Cerebrospinal Fluid Is Useful for the Diagnosis of Bacterial Meningitis Katsuya Inada*,1, Kiyomi Takahashi 1, Sadato Ichinohe 2, Hidetoshi Suda 4, Masakazu Tsuchiya 5, Junkichi Takahashi 5, Shuji Matsuura 5, Takeshi Kasai 3, Masao Yoshida 6, Shigeatsu Endo 3, and Shigehiro Sato 1 1 Department of Microbiology, 2 Department of Pediatrics, and 3 Department of Critical Care Medicine, School of Medicine, Iwate Medical University, Morioka, Iwate , Japan, 4 Hasuda-Issinkai Hospital, Hasuda, Saitama , Japan, 5 Wako Pure Chemical Industries, Ltd., Osaka, Osaka , Japan, and 6 Iwate Life Science Institute, Morioka, Iwate , Japan Received May 14, 2003; in revised form, June 25, Accepted July 9, 2003 Abstract: The silkworm larvae plasma (SLP) test has been established based on a cascade reaction triggered by either peptidoglycan or (1, 3)- -D-glucan to produce melanin. We applied this test to the diagnosis of bacterial meningitis. Cerebrospinal fluid (CSF) obtained from patients with bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, or fungi, showed positive reactions to the test. In contrast, CSF from patients with viral meningitis or noninfectious illnesses gave negative reactions. Therefore, this test seems to be useful for diagnosis of bacterial and fungal meningitis. When this test was used together with two types of limulus tests, an endotoxin-specific test, and a conventional test, meningitis was further characterized as gram-positive, gram-negative or fungal meningitis. The SLP test requires a computerized instrument for quantitative colorimetric measurement. A qualitative alternative of this test also can be accomplished by visually observing the darkening color. Thus, this method can be applied for simple and rapid diagnosis of meningitis. Key words: Bacterial meningitis, Diagnosis, Silkworm larvae plasma, Peptidoglycan Microbiol. Immunol., 47(10), , 2003 *Address correspondence to Dr. Katsuya Inada, Department of Microbiology, School of Medicine, Iwate Medical University, 19 1 Uchimaru, Morioka , Japan. Fax: kaind@mb.neweb.ne.jp Because bacterial meningitis is still a life-threatening disease, it should be diagnosed at an early stage for effective treatment with antibiotics (2). A limulus test to measure endotoxin (ET) content in cerebrospinal fluid (CSF) has been used to diagnose meningitis caused by gram-negative bacteria (6). We previously reported that the ET-specific colorimetric limulus test was useful for rapid diagnosis of gram-negative meningitis (3). The clearance of ET from CSF during treatment appeared to be useful in determining the timing for the discontinuation of antibiotic treatment. Recently, it has become possible to measure peptidoglycan (PG), the cell wall component of gram-positive and gram-negative bacteria, by a sophisticated test using the reagent from silkworm (Bombyx mori) larvae plasma (SLP) (11). The SLP test is based on melanin formation by SLP in the presence of PG. The prophenol-oxidase cascade reaction is preserved as a sclerosing, wound healing, and defense reaction of this insect against foreign matter entering the blood circulation (Fig. 1) (1). This cascade reaction is not PG-specific and is also reactive with (1,3)-β-D-glucan (GL), a cell wall component of fungi (1). In the present study, we applied this test to the detection of these cell wall components in CSF to diagnose bacterial and fungal meningitis. Furthermore, we were able to discriminate between meningitis caused by gram-negative bacteria, gram-positive bacteria, and fungi by using a combination of the SLP and limulus tests. Materials and Methods Objective cases and collection of CSF. Specimens obtained from 8 cases (0 32 years old; 3 females and 5 males) with bacterial meningitis, 11 cases with viral meningitis, and 10 cases with noninfectious illnesses Abbreviations: CSF, cerebrospinal fluid; ET, endotoxin; GL, (1, 3)-β-D-glucan; SLP, silkworm larvae plasma. 701

2 702 K. INADA ET AL Fig. 1. Cascade reactions in SLP. Fig. 2. Dilution effect of CSF for the reactivity in SLP. CSF was diluted with water, and 100 µl aliquots were incubated with 100 µl of SLP. Results with CSFs from 3 objective cases in the present study and from 5 encephalopathy cases (cases A D) with suspected postoperative bacterial infections are shown. The final concentration of the PG value was depicted in this figure. (9 with epilepsy, 1 Guillain-Barre syndrome case, and 1 ataxia) were examined. Meningitis was diagnosed by physical and laboratory findings; e.g., leukocyte counts, bacterial culture, isolation of virus, sugar and protein contents of CSF, blood leukocyte count, and the C-reactive protein titer. CSFs carefully were collected to avoid contamination of ET, GL, and PG, and were stored at 80 C until use. Blood-containing CSF was centrifuged at 3,000 rpm for 40 sec to remove blood cells prior to storage. The study was conducted with the informed

3 SLP TEST FOR DIAGNOSIS OF MENINGITIS 703 Fig. 3. Cascade reactions in horseshoe crab amebocyte lysate. In the chromogenic test, a synthetic chromogenic substrate, boc-leu-gly- Arg-paranitroaniline (pna), is added. The pna released from the substrate is detected by diazo-coupling reagents, and the amount of colored product is measured photometrically at 545 nm. consent of the patients or their family and the approval of the Ethical Committee of Iwate Medical University. Assay of PG and GL by the SLP test. The SLP reagent kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was used to detect PG and GL (11). Lyophilized substrate (3,4-dihydroxyphenylalanine; DOPA) was dissolved with Goods buffer, then added to a lyophilized SLP reagent. One-hundred µl of the CSF sample was mixed with 100 µl of the SLP-substrate mixture, then incubated at 30 C for 120 min in the computerized instrument (Toxinometer, Wako Pure Chemical Industries, Ltd.) (7). The transmittance ratio was set at 100% at the incubation. During the incubation, melanin-formation (dark color generation) occurred and the transmittance ratio decreased. The time (min; Ta) necessary to reach the threshold value (92/%) of the transmittance ratio was measured. The contents of PG and GL were calculated with a standard curve using a standard PG preparation derived from Micrococcus luteus. Because CSF from meningitis patients had an inhibitory activity for the SLP test, in a preliminary experiment, we attempted to eliminate the inhibitory activity by diluting CSF with water. We found that 8 16 fold-diluted CSF was appropriate for these measurements (Fig. 2). Therefore, 8 fold-diluted CSF was used in this study. Assay of ET and GL by limulus tests. Limulus tests are made based on the gel-clot formation due to ET or GL (Fig. 3). Among gel-clot methods for the limulus tests, two kinds of kits were used. HS-test Wako (Wako Pure Chemical Industries, Ltd.) is a conventional limulus gelation test for ET and GL. ES-test Wako (Wako Pure Table 1. Dilution effect of CSF for the recovery by LPS in the HS-test Dilution of CSF Final LPS conc. (pg/ml) :2 1:4 1:8 1:16 1:32 H 2O The CSF was diluted with water, and a 200 µl aliquot was added to the lyophilized reagent. Then 20 µl of LPS solution (E. coli O111:B4, W, Difco) was added to the reagent, and incubated at 37 C for 60 min. Gel-clot formation was observed. : positive result. Chemical Industries, Ltd.) is an ET-specific test in which an excess amount of GL (Curdlan) is added to prevent the GL-involved cascade (10). Limulus polyphemus amebocyte lysate is used in both these tests. Two-hundred µl of a CSF sample was added to a tube containing the lyophylized lysate, mixed thoroughly, and incubated for 60 min at 37 C. A positive reaction was determined by gel-clot formation. The sensitivity of ET detection is approximately 0.03 EU/ml. Colorimetric chromogenic quantitative limulus tests. The Toxicolor test (Seikagaku Corp., Tokyo) is a conventional limulus test that reacts with ET and GL. The Endospecy test (Seikagaku Corp.) is an ET-specific test due to the deletion of GL-sensitive factor G (8). The cutoff value of ET in CSF was set at 5 pg/ml. The value

4 704 K. INADA ET AL Table 2. Differential diagnosis of bacterial and fungal meningitis by two limulus tests and the SLP test Gram-positive Gram-negative Fungal infection Viral infection infection infection SLP test Conventional limulus test Endotoxin-specific limulus test Table 3. Results of the SLP test and limulus tests in CSF from patients with bacterial meningitis No. Age/ Bacteria isolated from CSF SLP (pg/ml) Endospecy Toxicolor ES HS Note Sex( a) ) pg/ml pg/ml (gelation) (gelation) 1 10Y/M(2) Haemophilus influenzae 1, Y/F(1) H. influenzae 1, M/F(1) Escherichia coli Y/M(2) Gram-negative rod 1, Gram-negative rod was observed in a smear of CSF. 5 12Y/F(1) 1, Suspected gram-negative infection; CSF was pyogenic and the symptoms were reduced after cefotaxime treatment. 6 11Y/M(4) Staphylococcus aureus 15, Y/M(1) Streptococcus pneumoniae N.T. b) 8 1Y/M(1) 10, Fungemia; Candida albicans was isolated from blood. a) Day of illness. b) N.T.: not tested. (Toxicolor Endospecy) was regarded as the GL content. Eight-fold-diluted CSF was found to be adequate for these tests (Table 1), although Inagaki et al. (6) suggested that 1:2-fold diluted CSF was suitable for the colorimetric assays. The diagnostic criteria by these tests which we proposed. Table 2 shows our diagnostic criteria for bacterial and fungal meningitis based on the SLP test and two limulus tests. Results As shown in Tables 3 5, the SLP test showed positive results for bacterial meningitis due to gram-positive bacteria, gram-negative bacteria, and fungi, but not for viral meningitis nor noninfectious illnesses. In cases 1 3, as shown in Table 3, bacteria were isolated from CSF. In case 4, gram-negative bacteria were detected by a gram-stained smear of the CSF. Therefore, cases 1 4 were diagnosed as gram-negative meningitis. The chromogenic limulus tests (Endospecy and Toxicolor) showed positive results and both values were comparable. The two limulus gelation tests also showed positive reactions. As expected, the SLP test showed positive reactions. Thus, all the results of the SLP and limulus tests completely agreed with the diagnostic criteria. In case 2, the SLP test value gradually decreased with antibiotic treatment (Fig. 4). Case 5 was suspected to be meningitis, but bacteria were not isolated from CSF. However, CSF was pyogenic and the patient s symptoms became less severe after treatment with cefotaxime (Fig. 4). From the results of the limulus tests and the SLP test, this case was suspected to be gram-negative meningitis. In cases 6 and 7, patients were diagnosed as having gram-positive meningitis. Two chromogenic and two gelation methods of the limulus test showed negative reactions, but the SLP tests were positive. In case 6, a febrile response was found after a ventricle-peritoneal shunt installation and, therefore, complicated meningitis was suspected. The SLP test value was elevated in the process (Fig. 4). Case 8 was suspected to be bacterial meningitis, but no bacteria were isolated. The patient s underlying dis-

5 SLP TEST FOR DIAGNOSIS OF MENINGITIS 705 Table 4. Results of the SLP test and limulus tests in CSF from patients with aseptic meningitis No. Age/ Virus isolated from CSF SLP (Tg) Endospecy Toxicolor ES HS Note Sex( a) ) (pg/ml) pg/ml pg/ml (gelation) (gelation) 9 26Y/F(2) Echovirus N.D. N.D Y/F(1) Echovirus N.D. N.D Y/F(2) Echovirus N.D. N.D Y/F(2) N.D. b) 6.7 N.D. N.D Y/M(5) N.D. 6.7 N.D. N.D Y/F(6) N.D. 6.7 N.D. N.D Y/F(3) N.D. 6.7 N.D. N.D Y/M(3) N.D. 6.7 N.D. N.D. 17 3Y/M(2) N.D Y/M(1) N.D Y/M(1) N.D a) Day of illness. b) N.D.: not detected. Echovirus 30 was isolated from a throat swab. Significant level of anti-echovirus 30 antibody in serum was detected. Aseptic meningitis was diagnosed clinically and by laboratory findings of CSF. Table 5. Results of the SLP test and limulus tests in CSF from patients with noninfectious illnesses No. Age/ Illnesses SLP (pg/ml) Endospecy Toxicolor ES HS Sex pg/ml pg/ml (gelation) (gelation) 20 8Y/M Epilepsy suspected Y/M Epilepsy suspected Y/M Epilepsy suspected Y/M Ataxia Y/M Guillain-Barre syndrome M/F Epilepsy suspected Y/M Epilepsy suspected Y/M Epilepsy Y/M Epilepsy Y/M Epilepsy suspected ease was Hirschsprung Disease complicated with sepsis. Candida albicans was isolated from the blood. Two nonspecific limulus tests, Toxicolor and the HS-test, were found to be positive. SLP test was also positive. However, this was not the case for the endotoxin-specific test. Consequently, this case was considered to be meningitis due to fungi. As shown in Fig. 4, the SLP value remained high for a significant period of time which is characteristic of a fungal infection. Cases 9 19 (Table 4) were diagnosed as aseptic meningitis. The limulus tests and SLP test showed negative results. Cases (Table 5) were noninfectious illnesses, and no positive results were observed by any test. Discussion A limulus test has been established for the detection of ET using horseshoe crab amebocyte lysate. An additional pathway (GL-involved pathway) was found in the lysate (Fig. 3), and three types of limulus tests, a conventional, ET-specific, and GL-specific tests were developed in Japan. For these tests, gelation, turbidimetric and colorimetric methods are available. Here we used conventional and ET-specific tests in gelation and colorimetric assay methods. The limulus test has been used as a supporting tool for the diagnosis of gramnegative meningitis (3, 6). This test also can be used for the diagnosis of fungal meningitis. Since the SLP test for measuring PG and GL has been developed, we attempted to establish a new diag-

6 706 K. INADA ET AL Fig. 4. Kinetics of SLP values in 5 cases of bacterial meningitis. nostic tool for bacterial and fungal meningitis by detecting bacterial components in CSF using both limulus and SLP tests. Then we made a diagnostic chart to differentiate three forms (or types) of meningitis using these tests (Table 2). The negation of bacterial or fungal meningitis suggests viral meningitis or noninfectious illnesses. An inhibitory activity for the SLP test is thought to be present in CSF due to the interfering activity for the limulus test which also is present in plasma and CSF. Interfering proteins for the limulus test are present in plasma, and thus plasma pretreatment methods have been developed; e.g. the new PCA method (4) and the dilution (1:10 with detergent solution) and heating (80 C, 5 min) method (5). The protein content of CSF is less than that in plasma, and only a 1:2 dilution of CSF with distilled water was reported to be sufficient for the limulus tests (6). However, Suda et al. suggested that CSF should be treated by the same method as that used for plasma (9). In limulus gelation tests carried out in the present study (Table 2), it was also suggested that the CSF must be diluted by more than 1:2. We have found in a preliminary experiment that patient CSF has an inhibitory activity for the SLP test. Therefore, we ascertained which dilution rate for CSF was adequate for the SLP test. Figure 2 indicates that CSF has remarkable inhibitory activity, and thus a 1:8 dilution is more appropriate than a lesser dilution rate for the SLP test. We diluted CSF with water only to reduce the inhibitory activity in CSF because this procedure is simple and quick, and such a pretreatment method has less chance of contamination. The present study demonstrates that the SLP test is useful for the diagnosis of bacterial meningitis. In the bacterial meningitis cases, the CSF PG levels were significantly elevated (Table 3). As for case 5, although bacteria were not detected in the CSF, it can be diagnosed as gram-negative bacterial meningitis based on the result of the Endospecy test and confirmed by both the limulus gelation tests and the SLP test. In case 8, Candida albicans was isolated from the blood, and fungal meningitis was indeed suspected from the results of both the SLP test and limulus tests. In diagnosed or suspected cases of viral meningitis, both the SLP test and limulus tests were negative (Table 4). In noninfectious illness, all tests showed negative results. These data clearly indicate that the SLP test is useful to differentially diagnose bacterial and viral meningitis, and additional limulus tests demonstrated reliability for further differentiating gram-positive, gram-negative, and fungal meningitis. However, more cases will be needed in order to set a cut-off value in the SLP test to discriminate bacterial and fungal meningitis from viral meningitis. In the present study, the quantitative SLP test was performed with a computerized instrument. However, we also can visually observe the generation of a dark color

7 SLP TEST FOR DIAGNOSIS OF MENINGITIS 707 in the reaction mixture both during and at the end of incubation. Consequently, the results obtained by the limulus gelation tests, judged by visual observation, were comparable with the results obtained by the colorimetric quantitative limulus test. Therefore, although these tests could not identify the specific species of bacteria the qualitative SLP test and limulus tests are suggested to be useful for rapid diagnosis of meningitis. A part of this study was supported by the Grants-in-Aid for Advanced Medical Science Research by the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors are grateful to Ms. Nagiko Chiba for excellent technical assistance, and to Ms. V.L. Braymer for preparation of this manuscript. References 1) Ashida, M., and Yamazaki, H.I Biochemistry of the phenoloxidase system in insects with special reference to its activation, p In Onishi, E., and Ishizaki, H. (eds), Molting and metamorphosis, Japan Sci. Soc. Press, Tokyo. 2) Gray, L.D., and Fedorko, D.P Laboratory diagnosis of bacterial meningitis. Clin. Microbiol. Rev. 5: ) Ichinohe, S., Inada, K., Nemoto, T., Murata, A., Ichinohe, N., Fujiwara, T., and Yoshida, M Usefulness of endotoxinspecific limulus tests for the measurement of endotoxin in cerebrospinal fluid in diagnosis of bacterial meningitis. Kansenshougaku Zasshi 69: (in Japanese). 4) Inada, K., Endo, S., Takahashi, K., Suzuki, M., Narita, T., Yoshida, M., Suda, H., Komuro, T., and Yoshida, M Establishment of a new perchloric acid treatment to allow determination of the total endotoxin content in human plasma by the limulus test and clinical application. Microbiol. Immunol. 35: ) Inada, K., Yamashita, H., Yoshida, M., Harada, K., Tsuchiya, M., and Matsuura, S Evaluation of detergent-dilution and heating method for measurement of endotoxin in plasma. Kiso-to-Rinsho 26: (in Japanese). 6) Inagaki, M., Nakajima, M., Ando, Y., Takashima, S., Takeshita, K., Tanaka, S., Tamura, H., and Obayashi, T Measurement of endotoxin in cerebrospinal fluid from neonates and infants using a new endotoxin-specific chromogenic test. Clin. Chim. Acta 175: ) Oishi, H., Takaoka, Y., Hatayama, T., Matsuo, T., and Sakata, Y Automated limulus amebocyte lysate (LAL) test for endotoxin analysis using a new Toxinometer ET-201. J. Parenter. Sci. Technol. 39: ) Obayashi, T., Tamura, H., Tanaka, S., Ohki, S., Takahashi, S., Arai, M., Masuda, M., and Kawai, T A new chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical applications. Clin. Chim. Acta 149: ) Suda, H., Inada, K., and Yoshida, M Usefulness of a new perchloric acid treatment to measure endotoxin in cerebrospinal fluid by the limulus test. Acta Paediatr. Jpn. 35: ) Tsuchiya, M., Takaoka, A., Tokioka, N., and Matsuura, S Development of an endotoxin-specific Limulus amebocyte lysate test blocking β-glucan-mediated pathway by carboxymethylated curdlan and its application. Jpn. J. Bacteriol. 45: ) Tsuchiya, M., Asahi, N, Suzuoka, F., Ashida, M., and Matsuura, S Detection of peptidoglycan and β-glucan with silkworm larvae plasma test. FEMS Immunol. Med. Microbiol. 15: