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Bacterial antigen detection in cerebrospinal fluid of patients with meningitis
DIAGNMICROBIOLINFECTDIS 1985;3:373-379
373
ORIGINALARTICLES Bacterial Antigen Detection in Cerebrospinal Fluid of Patients with Meningitis D.J. Hoban, E. Witwicki, and G.W. Hammond This study compared the sensitivity and specificity of four test systems in detecting Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, and gram-negative organisms in cerebrospinal fluid (CSF), versus culture. The tests used on CSF from 155 patients with meningitis were the Phadebaet coagglutination (CoA) test, the Directigen latex agglutination (LA) test, counterimmunoelectrophoresis (CIE), and the Limulus amebocyte lysate (LAL) test. The sensitivity for patients with bacterial meningitis was 78% (18/23)for LA, 78% (25/32) for CoA, and 67% (18/27) for CIE for detection of H. infiuenzae type b; 71% (10/14) for CoA, 100% (6/6) for LA, and 50% (6/13) for CIE in detecting S_:.pneumoniae; and 33% (1/3) for LA and 50% (2/4) for CIE in detecting N...~.meningitidis. LAL had a sensitivity of 77% 137/48) in detecting CSF gram-negative endotoxin. The specificities of thase with bacterial meningitis for H__~,influenzae, S_:.pneumoniae, and N.._z.meningitidis tested by LA were, respectively. 100% (35/35), 96% (50/52), and 100% (54/54); for H._:.in//uenzae and S__:.pneumoniae using CoA 97% (62/64) and 96% (80/83); for/-/, influenzae, S.~.pneumoniae, and N_.:,meningitidis using CIE 67% (18/27), 50% (6/12), and 50% (2/4). The specificity of LAL was 86% (38144). The detection of bacterial antigen from CSF in patients with meningitis by commercial agglutination tests is mere sensitive than CIE and is highly specific.
INTRODUCTION
Several techniques are available to detect bacterial antigens in CSF, including coagglutination (Burdash and West, 1982; Carey, 1983; Collins and Kelly, 1983; Drow and Manning, 1983; Mayer et al., 1983, Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983), latex particle agglutination (Carey, 1983; Collins and Kelly, 1983; Drow and Manning, 1983; Ingram et al., 1983; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979), limulus amebocyte lysate assay (Ingram et al., 1983; Jorgensen and Lee, 1978), and counterimmunoelectrophoresis (Anhalt et al., 1978; Bortolussi et al., 1982; Carey, 1983; Collins and Kelly, 1983; Finch and Wilkinson, 1979; Olcen, 1978; Thirumoorthi and Dajani, 1979; Tompkins, 1983). The present study compared four methods including two commercial systems for their sensitivity and specificity in detection of Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and gram-negative organisms from culture-positive cerebrospinal fluid (CSF) of patients with meningitis. The Phadebact
From the Department of Clinical Microbiology (D.J.H. and E.W.), Health Sciences Centre, the Department of Medical Microbiology (D.J.H. and G.W.H.), University of Manitoba and the Cadham Provincial Laboratory (G.W.H.), Winnipeg, Manitoba, Canada. Address reprint requests to: Dr. D. Hoban, Assistant Director, Department of Clinical Microbiology, Health Sciences Centre, 700 William Avenue, Winnipeg, Manitoba, Canada, R3E OZ3. Received June 13, 1984; revised and accepted September 18, 1984. © 1985 ElsevierScience Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017
0732-8893/85/$03.30
374
D.J. Hoban et al.
coagglutination test (CoA), the Directigen latex agglutination test (LA), counterimmunoelectrophoresis (CIE), and the limulus amebocyte lysate (LAL) test for detection of H. influenzae type b, S. pneumoniae, N. meningitidis, and gram-negative organisms in CSF were compared to culture results of these specimens. Specimens from patients with aseptic meningitis served as an additional specificity control. MATERIALS AND METHODS
Over an 18-month period, CSF specimens were collected from patients with suspected meningitis or central nervous system (CNS) shunt infections and pleocytosis (~>10 WBCs) and were evaluated. Ninety-nine CSF specimens were culture positive for bacteria (Table 1). Fifty-six CSF specimens that were culture-negative for bacteria (aseptic meningitis) were used as controls. These 56 CSF negative controls included the following diagnoses (with the number of CSF in each diagnostic group): viral meningitis (14), aseptic presumed viral meningitis (23), partially treated bacterial meningitis (4), Guillain-Barre syndrome (2), cryptococcal meningitis (3), suspected Mycobacterium tuberculosis meningitis (3), postmastoidectomy meningitis (1), postchemical meningitis (1), herpes simplex virus encephalitis (2), Salmonella gastroenteritis (1), and CNS lymphoma (2). The CSF that were culture positive for bacteria were analyzed using each method if specimen volume permitted. After culture and testing by the CIE test (if requested) at the time of specimen submission, all CSF specimens were frozen at -20°C and later tested in coded batches by CoA and LA without knowledge of the CSF culture results. Phadebact CoA (Pharmacia Diagnostic AB, Uppsala, Sweden) was performed and interpreted according to the manufacturer's instructions. A type b reagent and a reagent for types a and c through f were tested for H. influenzoe detection, and a third regent was used to detect S. pneumoniae. Directigen LA (Hynson, Wescott and Dunning, Baltimore, MD) was performed and interpreted as recommended by the manufacturer. The CSF was tested for H. influenzae type b, S. pneumoniae, and N. meningitidis types a and c. For the CIE test, antisera to H. influenzae type b was obtained from Wellcome Laboratories (Beckenham, England); antisera to S. pneumoniae came from Statens Serum Institute (Copenhagen, Denmark); and antisera to N. meningitidis came from Wellcome (poly A-D), Difco (Detroit, MI); (poly x, y, and z) and mono groups
TABLE 1. Bacteria Cultured from CSF Specimens
Organism Haemophilus influenzae
Streptococcus pneumoniae Neisseria meningitidis Streptococcus Group A Group B Alpha Staphylococcus aureus Coagulase( - ) Staphylococcus Escherichia coil Serratia marcescens Listeria monocytogenes Ktebsiella pneumoniae Acinetobacter lwoffi
Number positive 32 14 5
3 4 1 6 18 8
1 4
2 1
Bacterial Antigen Detection in Meningitis Patients
375
A,B,C,D,X,Y,Z and W135 were from Wellcome Laboratories. Counterimmunoelectrophoresis was performed as previously described (Anhalt et al., 1978). For the LAL test, reagents were purchased from Sigma Chemical Company (St. Louis, MO). Glass slides were heated for 4 hr at 150°C, and sterile pipette tips were used to dispense all reagents and CSF. Twenty microliters of LAL were added to 20 ~1 of CSF on a glass slide. The LAL and specimen were mixed and incubated at 37°C for I hr in a moist chamber. A positive result was determined when a solid gel formed that would not run when the slide was tilted. RESULTS The bacterial organisms isolated from the patients with bacterial meningitis and the number are shown in Table 1. Table 2 compares the sensitivity and specificity of CoA, LA, and CIE in detecting H. influenzae type b, S. pneumoniae, and N. meningitidis antigen in CSF compared to CSF culture results. For detection of H. influenzae type b, the sensitivity ranged from 67% (18/27) for CIE to 78% each for CoA (25/32) and LA (18/23). The specificity for each diagnostic test was similar: 100% for CIE (42/42) and LA (35/35), and 97% for CoA (62/64). The CoA H. influenzae type b reagent detected two streptococcus organisms; one was a group A and one was a group B. Both CoA and LA were 100% specific in patients with aseptic meningitis. These CSF specimens were not tested using CIE. In detecting S. pneumoniae in CSF, the sensitivity was higher for LA (100%-6/6) and CoA (71%--10/14), and lower for CIE (50%--6/12). For S. pneumoniae, the specificity was high: 100% (57/57) for CIE and 96% each for CoA (80/83) and LA (50/52). CoA was falsely positive for one N. meningitidis W135, one group A streptococcus, and one group B streptococcus. Latex agglutination was falsely positive for one H. influenzae type b and one N. meningitidis W135. The specificity of CoA and LA in aseptic CSF controls was 100%. Our number of CSF specimens that tested positive for N. meningitidis was too low to adequately evaluate sensitivity. Two missed isolates by LA (1 group B, 1 group W135) would not have been detectable within the test reagent pool available at the time of testing. The CIE demonstrated a sensitivity of 50% (2/4). The two missed isolates were a group B and a group A. The CoA test did not have available N. meningitidis reagents at the time. The specificity for both LA (54/54) and CIE (65/65) was 100%. The sensitivity and specificity of the LAL test for gram-negative endotoxin is shown in Table 3. The test is 77% (37/48) sensitive in detecting the presence of gramnegative organisms. The LAL test was falsely positive for six CSF samples that grew organisms other than gram negatives (1 group A streptococcus, 1 group B streptococcus, 1 S. pneumoniae, 1 L. monocytogenes, and 2 coagulase-negative staphylococcus). However, LAL produced false-positive results for two CSF specimens that were negative controls (one from a patient with aseptic meningitis and one from a patient with a stool culture positive for Salmonella). A summary of test results with the LAL, CoA, and LA for detection of H. influenzae type b, S. pneumoniae, and N. meningitidis when the CIE was negative on the identical specimens is shown in Table 4. Additional detection of both H. influenzae and S. pneumoniae were achieved with CoA and LA versus CIE. The LAL test detected two additional specimens positive for N. meningitidis. A cost analysis for materials used in each detection system is shown in Table 5. For routine diagnostic use CoA is the most cost effective. No objective time studies were carried out with each system, but subjective impressions indicate that both CoA
376
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Bacterial Antigen Detection in Meningitis Patients
377
TABLE 3. The Sensitivity and Specificity of CSF Specimens from Patients with Meningitis evaluated by the L i m u l u s Amebocyte Lysate Test Sensitivity
Specificity
Gram-negative CSF isolates LAL
n
+
37
77%
-
11
--
Gram-positive CSF isolates
(%)
Other CNS infections
n
(%]
n
(%)
6
--
2 51
-96%
38
86%
TABLE 4. Improved Diagnosis of Bacterial Meningitis Detection of Bacterial Antigen in the CSF of the Culture Positive Specimens Negative by CIE
H. influenzae S. pneumoniae N. meningitidis
No. of CIE-negative specimens positive by an alternate test
No. negative by CIEa
CoA
LA
LAL
9 6 2
3/9 4/6 0/2
1/5 2/2 0/1
3/9 0/5 2/2
~Fhese isolates correspond to the isolates missed by CIE in Table 2.
TABLE 5. Cost Analysis for CSF Antigen Detection Test system Phadebact (CoA) CSF kit Directigen (LA) CIE
LAL
Antigens diluted S. pneumoniae, H. influenzae type b. Group B streptococcus, N. meningitidis types A, B, C, Y, and W135 S. pneumoniae, H. influenzae type b, and N. meningitidis types A, B, C S. pneumoniae, H. influenzae type b, N. meningitidis types A, B, C, D, and N. meningitidis types X, Y, Z, W135 Gram-negative endotoxin
aPrice indicated is for the cost of materials in Canadiandollars as of January 1, 1984.
Cost per CSF° $6.12
$10.36 $12.36
$0.50
:378
D.J. Hoban et al.
and LA take about the same amount of time to carry out, while CIE takes considerably longer. DISCUSSION
Latex agglutination has previously been used to detect H. influenzae type b (Bortolussi et al., 1982; Collins and Kelly, 1983; Drow and Manning, 1983; Ingrain et al., 1983; Thirumoorthi and Dajani, 1979), S. pneumoniae (Bortolussi et al., 1982; Ingrain et al., 1983; Thirumoorthi and Dajani, 1979), and N. meningitidis (Bortolussi et al., 1982; Ingrain et al., 1983; Thirumoorthi and Dajani, 1979). In these studies, the sensitivity has ranged from 10 to 70% for N. meningitidis, from 50 to 69% for S. pneumoniae, and from 81 to 100% for H. influenzae type b. In these studies, specificity ranged from 80 to 100%. Coagglutination has likewise been studied for the detection of H. influenzae type b (Collins and Kelly, 1983; Drow and Manning, 1983; Mayer et al., 1983; Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983), S. pneumoniae (Burdash and West, 1982; Mayer et al., 1983; Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983), and N. meningitidis (Olcen, 1978; Thirumoorthi and Dajani, 1979; Tompkins, 1983). The sensitivity ranged from 95 to 100%. Many studies have compared CIE to culture (Bortolussi et al., 1982; Collins and Kelly, 1983; Finch and Wilkinson, 1979; Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983). In these studies, the sensitivity ranged from 17 to 100% for N. meningitidis, from 0 to 93% for S. pneumoniae, and from 71 to 83% for H. influenzae type b. The specificity in each reported study was at or near 100%. Carey (1983) has recently summarized various methods for rapid diagnosis of bacterial meningitis by antigen detection. Little information is currently available comparing LAL to any of the above-mentioned techniques to detect gram-negative endotoxin in CSF. The sensitivity of LAL indicated that this technique is comparable to LA or CoA but that LAL test specificity was not as great. Jorgensen and Lee (1978) reported that LAL detected 92% of all patients with culture-proven gram-negative bacterial meningitis in assessing a series of reports. Our data with the commercial kits for specific bacterial antigens compared favorably to published results already discussed. Detection of H. influenzae type b was accomplished with similar sensitivity by CoA (Phadebact) and by LA (Directigen). And CIE was only slightly less sensitive. The specificity was very high for each method. In detecting S. pneumoniae in CSF, LA was the most sensitive followed by CoA and lastly by CIE. The specificity was also very high. The detection of N. meningitidis was difficult for all test systems. This is shown in published studies (Thirumoorthi and Dajani, 1979) and in our own data, although our number of positive specimens was very small. Poor sensitivity with the CIE reflects on the quality and titre of available antisera, especially group B. No single set of reagents currently detected the full range of common bacteria found in the CSF of patients with meningitis (H. influenzae, S. pneumoniae, and N. meningitidis types b and c). Therefore, the combination of a rapid agglutination procedure plus a method of detection of bacterial endotoxin may be optimal. Along with greater ease of performance, we noted that rapid slide tests such as LA or CoA and LAL may offer increased sensitivity versus CIE. Each system varied in cost per CSF based upon numbers and types of antigens to be tested. The availability of good commercial kits now offers laboratories the opportunity to accurately and rapidly detect bacterial antigen in CSF.
Bacterial A n t i g e n Detection in Meningitis Patients
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The assistance of Mrs. Young, Mrs. N. McFarlane and Mrs. R. Malazdrewicz is greatly appreciated. This study was supported in part by a grant from the National Health and Research Development Project, Canada. REFERENCES Anhalt JP, Kenny GE, Rytel MW (1978) Cumitech 8 Detection of microbial antigens by counterimmunoelectrophoresis. T.L. Gavan Coordinating ed. Washington, DC: American Society for Microbiology. Bortolussi R, Wort AJ, Casey S (1978) The latex agglutination test versus counterimmunoelectrophoresis for rapid diagnosis of bacterial meningitis. Con Med Assoc ] 15:489~t93. Burdash NM, West ME (1982) Identification of Streptococcus pneumoniae by the Phadebact coagglutination test. J Clin Microbiol 15:391-394. Carey RB (1983) Rapid diagnosis of bacterial meningitis by antigen detection. Clin Microbiol Newsletter 5:117-120. Collins JK, Kelly MT (1983) Comparison of Phadebact coagglutination, Bactagen latex agglutination and counterimmunoelectrophoresis for detection of Haemaphilus influenzae type b antigens in cerebrospinal fluid. J Clin Microbiol 17:1005-1008. Drow, DL, Manning DD (1983) Comparison of coagglutination, latex agglutination and enzymelinked immunosorbent assay for detection of Haemophilus in~luenzae type b infection. Diagn Microbiol Infect Dis 1:317-322. Finch CA, Wilkinson HW (1979) Practical considerations in using counterimmunoelectrophoresis to identify the principal causative agents of bacterial meningitis. J Clin Microbiol 10:519-524. Ingram DL, Pearson AW, Occhiuti AR (1983) Detection of bacterial antigens in body fluids with the Wellcogen Haemophilus influenzae b, Streptococcus pneumoniae, and Neisseria meningitidis (ACYW135) latex agglutination tests. J Clin Microbiol 18:1119-1121. Jorgensen JH, Lee JC (1978) Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay. J Clin Microbiol 7:12-17. Kelsey MC, Lipscomb AP, Mowles JM (1982) Limulus amebocyte lysate endotoxin test: An aid to the diagnosis in the septic neonate. J Infect 4:69-72, Mayer ME, Geiseler PJ, Harris B (1983) Coagglutination for detection and serotyping of bacterial antigens: Usefulness in acute pneumonias. Diag Microbiol Infect Dis 1:277-285. Olcen P (1978) Serological methods for rapid diagnosis of Hoemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in cerebrospinal fluid: A comparison of coagglutination, immunofluorescence and immunoelect~ophoresis. Scand J Infect Dis 10:283-289. Suksanong M, Dajani AS (1977) Detection of Haemophilus influenzae, type b antigens in body fluids, using specific antibody-coated Staphylococci. J Clin Microbiol 5:81-85. Thirumoorthi MC, Dajani AS (1979) comparison of Staphylococcal coagglutination, latex agglutination, and counterimmunoelectrophoresis for bacterial antigen detection. J Clin Microbiol 9:28-32. Tompkins DS (1983) Comparison of Phadebact co-agglutination tests with counterimmunoelectrophoresis for the detection of bacterial antigens in cerebrospinal fluid. J Clin Pathol 36:819-822.
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ORIGINALARTICLES Bacterial Antigen Detection in Cerebrospinal Fluid of Patients with Meningitis D.J. Hoban, E. Witwicki, and G.W. Hammond This study compared the sensitivity and specificity of four test systems in detecting Haemophilus influenzae type b, Neisseria meningitidis, Streptococcus pneumoniae, and gram-negative organisms in cerebrospinal fluid (CSF), versus culture. The tests used on CSF from 155 patients with meningitis were the Phadebaet coagglutination (CoA) test, the Directigen latex agglutination (LA) test, counterimmunoelectrophoresis (CIE), and the Limulus amebocyte lysate (LAL) test. The sensitivity for patients with bacterial meningitis was 78% (18/23)for LA, 78% (25/32) for CoA, and 67% (18/27) for CIE for detection of H. infiuenzae type b; 71% (10/14) for CoA, 100% (6/6) for LA, and 50% (6/13) for CIE in detecting S_:.pneumoniae; and 33% (1/3) for LA and 50% (2/4) for CIE in detecting N...~.meningitidis. LAL had a sensitivity of 77% 137/48) in detecting CSF gram-negative endotoxin. The specificities of thase with bacterial meningitis for H__~,influenzae, S_:.pneumoniae, and N.._z.meningitidis tested by LA were, respectively. 100% (35/35), 96% (50/52), and 100% (54/54); for H._:.in//uenzae and S__:.pneumoniae using CoA 97% (62/64) and 96% (80/83); for/-/, influenzae, S.~.pneumoniae, and N_.:,meningitidis using CIE 67% (18/27), 50% (6/12), and 50% (2/4). The specificity of LAL was 86% (38144). The detection of bacterial antigen from CSF in patients with meningitis by commercial agglutination tests is mere sensitive than CIE and is highly specific.
INTRODUCTION
Several techniques are available to detect bacterial antigens in CSF, including coagglutination (Burdash and West, 1982; Carey, 1983; Collins and Kelly, 1983; Drow and Manning, 1983; Mayer et al., 1983, Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983), latex particle agglutination (Carey, 1983; Collins and Kelly, 1983; Drow and Manning, 1983; Ingram et al., 1983; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979), limulus amebocyte lysate assay (Ingram et al., 1983; Jorgensen and Lee, 1978), and counterimmunoelectrophoresis (Anhalt et al., 1978; Bortolussi et al., 1982; Carey, 1983; Collins and Kelly, 1983; Finch and Wilkinson, 1979; Olcen, 1978; Thirumoorthi and Dajani, 1979; Tompkins, 1983). The present study compared four methods including two commercial systems for their sensitivity and specificity in detection of Haemophilus influenzae type b, Streptococcus pneumoniae, Neisseria meningitidis, and gram-negative organisms from culture-positive cerebrospinal fluid (CSF) of patients with meningitis. The Phadebact
From the Department of Clinical Microbiology (D.J.H. and E.W.), Health Sciences Centre, the Department of Medical Microbiology (D.J.H. and G.W.H.), University of Manitoba and the Cadham Provincial Laboratory (G.W.H.), Winnipeg, Manitoba, Canada. Address reprint requests to: Dr. D. Hoban, Assistant Director, Department of Clinical Microbiology, Health Sciences Centre, 700 William Avenue, Winnipeg, Manitoba, Canada, R3E OZ3. Received June 13, 1984; revised and accepted September 18, 1984. © 1985 ElsevierScience Publishing Co., Inc. 52 Vanderbilt Avenue, New York, NY 10017
0732-8893/85/$03.30
374
D.J. Hoban et al.
coagglutination test (CoA), the Directigen latex agglutination test (LA), counterimmunoelectrophoresis (CIE), and the limulus amebocyte lysate (LAL) test for detection of H. influenzae type b, S. pneumoniae, N. meningitidis, and gram-negative organisms in CSF were compared to culture results of these specimens. Specimens from patients with aseptic meningitis served as an additional specificity control. MATERIALS AND METHODS
Over an 18-month period, CSF specimens were collected from patients with suspected meningitis or central nervous system (CNS) shunt infections and pleocytosis (~>10 WBCs) and were evaluated. Ninety-nine CSF specimens were culture positive for bacteria (Table 1). Fifty-six CSF specimens that were culture-negative for bacteria (aseptic meningitis) were used as controls. These 56 CSF negative controls included the following diagnoses (with the number of CSF in each diagnostic group): viral meningitis (14), aseptic presumed viral meningitis (23), partially treated bacterial meningitis (4), Guillain-Barre syndrome (2), cryptococcal meningitis (3), suspected Mycobacterium tuberculosis meningitis (3), postmastoidectomy meningitis (1), postchemical meningitis (1), herpes simplex virus encephalitis (2), Salmonella gastroenteritis (1), and CNS lymphoma (2). The CSF that were culture positive for bacteria were analyzed using each method if specimen volume permitted. After culture and testing by the CIE test (if requested) at the time of specimen submission, all CSF specimens were frozen at -20°C and later tested in coded batches by CoA and LA without knowledge of the CSF culture results. Phadebact CoA (Pharmacia Diagnostic AB, Uppsala, Sweden) was performed and interpreted according to the manufacturer's instructions. A type b reagent and a reagent for types a and c through f were tested for H. influenzoe detection, and a third regent was used to detect S. pneumoniae. Directigen LA (Hynson, Wescott and Dunning, Baltimore, MD) was performed and interpreted as recommended by the manufacturer. The CSF was tested for H. influenzae type b, S. pneumoniae, and N. meningitidis types a and c. For the CIE test, antisera to H. influenzae type b was obtained from Wellcome Laboratories (Beckenham, England); antisera to S. pneumoniae came from Statens Serum Institute (Copenhagen, Denmark); and antisera to N. meningitidis came from Wellcome (poly A-D), Difco (Detroit, MI); (poly x, y, and z) and mono groups
TABLE 1. Bacteria Cultured from CSF Specimens
Organism Haemophilus influenzae
Streptococcus pneumoniae Neisseria meningitidis Streptococcus Group A Group B Alpha Staphylococcus aureus Coagulase( - ) Staphylococcus Escherichia coil Serratia marcescens Listeria monocytogenes Ktebsiella pneumoniae Acinetobacter lwoffi
Number positive 32 14 5
3 4 1 6 18 8
1 4
2 1
Bacterial Antigen Detection in Meningitis Patients
375
A,B,C,D,X,Y,Z and W135 were from Wellcome Laboratories. Counterimmunoelectrophoresis was performed as previously described (Anhalt et al., 1978). For the LAL test, reagents were purchased from Sigma Chemical Company (St. Louis, MO). Glass slides were heated for 4 hr at 150°C, and sterile pipette tips were used to dispense all reagents and CSF. Twenty microliters of LAL were added to 20 ~1 of CSF on a glass slide. The LAL and specimen were mixed and incubated at 37°C for I hr in a moist chamber. A positive result was determined when a solid gel formed that would not run when the slide was tilted. RESULTS The bacterial organisms isolated from the patients with bacterial meningitis and the number are shown in Table 1. Table 2 compares the sensitivity and specificity of CoA, LA, and CIE in detecting H. influenzae type b, S. pneumoniae, and N. meningitidis antigen in CSF compared to CSF culture results. For detection of H. influenzae type b, the sensitivity ranged from 67% (18/27) for CIE to 78% each for CoA (25/32) and LA (18/23). The specificity for each diagnostic test was similar: 100% for CIE (42/42) and LA (35/35), and 97% for CoA (62/64). The CoA H. influenzae type b reagent detected two streptococcus organisms; one was a group A and one was a group B. Both CoA and LA were 100% specific in patients with aseptic meningitis. These CSF specimens were not tested using CIE. In detecting S. pneumoniae in CSF, the sensitivity was higher for LA (100%-6/6) and CoA (71%--10/14), and lower for CIE (50%--6/12). For S. pneumoniae, the specificity was high: 100% (57/57) for CIE and 96% each for CoA (80/83) and LA (50/52). CoA was falsely positive for one N. meningitidis W135, one group A streptococcus, and one group B streptococcus. Latex agglutination was falsely positive for one H. influenzae type b and one N. meningitidis W135. The specificity of CoA and LA in aseptic CSF controls was 100%. Our number of CSF specimens that tested positive for N. meningitidis was too low to adequately evaluate sensitivity. Two missed isolates by LA (1 group B, 1 group W135) would not have been detectable within the test reagent pool available at the time of testing. The CIE demonstrated a sensitivity of 50% (2/4). The two missed isolates were a group B and a group A. The CoA test did not have available N. meningitidis reagents at the time. The specificity for both LA (54/54) and CIE (65/65) was 100%. The sensitivity and specificity of the LAL test for gram-negative endotoxin is shown in Table 3. The test is 77% (37/48) sensitive in detecting the presence of gramnegative organisms. The LAL test was falsely positive for six CSF samples that grew organisms other than gram negatives (1 group A streptococcus, 1 group B streptococcus, 1 S. pneumoniae, 1 L. monocytogenes, and 2 coagulase-negative staphylococcus). However, LAL produced false-positive results for two CSF specimens that were negative controls (one from a patient with aseptic meningitis and one from a patient with a stool culture positive for Salmonella). A summary of test results with the LAL, CoA, and LA for detection of H. influenzae type b, S. pneumoniae, and N. meningitidis when the CIE was negative on the identical specimens is shown in Table 4. Additional detection of both H. influenzae and S. pneumoniae were achieved with CoA and LA versus CIE. The LAL test detected two additional specimens positive for N. meningitidis. A cost analysis for materials used in each detection system is shown in Table 5. For routine diagnostic use CoA is the most cost effective. No objective time studies were carried out with each system, but subjective impressions indicate that both CoA
376
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Bacterial Antigen Detection in Meningitis Patients
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TABLE 3. The Sensitivity and Specificity of CSF Specimens from Patients with Meningitis evaluated by the L i m u l u s Amebocyte Lysate Test Sensitivity
Specificity
Gram-negative CSF isolates LAL
n
+
37
77%
-
11
--
Gram-positive CSF isolates
(%)
Other CNS infections
n
(%]
n
(%)
6
--
2 51
-96%
38
86%
TABLE 4. Improved Diagnosis of Bacterial Meningitis Detection of Bacterial Antigen in the CSF of the Culture Positive Specimens Negative by CIE
H. influenzae S. pneumoniae N. meningitidis
No. of CIE-negative specimens positive by an alternate test
No. negative by CIEa
CoA
LA
LAL
9 6 2
3/9 4/6 0/2
1/5 2/2 0/1
3/9 0/5 2/2
~Fhese isolates correspond to the isolates missed by CIE in Table 2.
TABLE 5. Cost Analysis for CSF Antigen Detection Test system Phadebact (CoA) CSF kit Directigen (LA) CIE
LAL
Antigens diluted S. pneumoniae, H. influenzae type b. Group B streptococcus, N. meningitidis types A, B, C, Y, and W135 S. pneumoniae, H. influenzae type b, and N. meningitidis types A, B, C S. pneumoniae, H. influenzae type b, N. meningitidis types A, B, C, D, and N. meningitidis types X, Y, Z, W135 Gram-negative endotoxin
aPrice indicated is for the cost of materials in Canadiandollars as of January 1, 1984.
Cost per CSF° $6.12
$10.36 $12.36
$0.50
:378
D.J. Hoban et al.
and LA take about the same amount of time to carry out, while CIE takes considerably longer. DISCUSSION
Latex agglutination has previously been used to detect H. influenzae type b (Bortolussi et al., 1982; Collins and Kelly, 1983; Drow and Manning, 1983; Ingrain et al., 1983; Thirumoorthi and Dajani, 1979), S. pneumoniae (Bortolussi et al., 1982; Ingrain et al., 1983; Thirumoorthi and Dajani, 1979), and N. meningitidis (Bortolussi et al., 1982; Ingrain et al., 1983; Thirumoorthi and Dajani, 1979). In these studies, the sensitivity has ranged from 10 to 70% for N. meningitidis, from 50 to 69% for S. pneumoniae, and from 81 to 100% for H. influenzae type b. In these studies, specificity ranged from 80 to 100%. Coagglutination has likewise been studied for the detection of H. influenzae type b (Collins and Kelly, 1983; Drow and Manning, 1983; Mayer et al., 1983; Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983), S. pneumoniae (Burdash and West, 1982; Mayer et al., 1983; Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983), and N. meningitidis (Olcen, 1978; Thirumoorthi and Dajani, 1979; Tompkins, 1983). The sensitivity ranged from 95 to 100%. Many studies have compared CIE to culture (Bortolussi et al., 1982; Collins and Kelly, 1983; Finch and Wilkinson, 1979; Olcen, 1978; Suksanong and Dajani, 1977; Thirumoorthi and Dajani, 1979; Tompkins, 1983). In these studies, the sensitivity ranged from 17 to 100% for N. meningitidis, from 0 to 93% for S. pneumoniae, and from 71 to 83% for H. influenzae type b. The specificity in each reported study was at or near 100%. Carey (1983) has recently summarized various methods for rapid diagnosis of bacterial meningitis by antigen detection. Little information is currently available comparing LAL to any of the above-mentioned techniques to detect gram-negative endotoxin in CSF. The sensitivity of LAL indicated that this technique is comparable to LA or CoA but that LAL test specificity was not as great. Jorgensen and Lee (1978) reported that LAL detected 92% of all patients with culture-proven gram-negative bacterial meningitis in assessing a series of reports. Our data with the commercial kits for specific bacterial antigens compared favorably to published results already discussed. Detection of H. influenzae type b was accomplished with similar sensitivity by CoA (Phadebact) and by LA (Directigen). And CIE was only slightly less sensitive. The specificity was very high for each method. In detecting S. pneumoniae in CSF, LA was the most sensitive followed by CoA and lastly by CIE. The specificity was also very high. The detection of N. meningitidis was difficult for all test systems. This is shown in published studies (Thirumoorthi and Dajani, 1979) and in our own data, although our number of positive specimens was very small. Poor sensitivity with the CIE reflects on the quality and titre of available antisera, especially group B. No single set of reagents currently detected the full range of common bacteria found in the CSF of patients with meningitis (H. influenzae, S. pneumoniae, and N. meningitidis types b and c). Therefore, the combination of a rapid agglutination procedure plus a method of detection of bacterial endotoxin may be optimal. Along with greater ease of performance, we noted that rapid slide tests such as LA or CoA and LAL may offer increased sensitivity versus CIE. Each system varied in cost per CSF based upon numbers and types of antigens to be tested. The availability of good commercial kits now offers laboratories the opportunity to accurately and rapidly detect bacterial antigen in CSF.
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The assistance of Mrs. Young, Mrs. N. McFarlane and Mrs. R. Malazdrewicz is greatly appreciated. This study was supported in part by a grant from the National Health and Research Development Project, Canada. REFERENCES Anhalt JP, Kenny GE, Rytel MW (1978) Cumitech 8 Detection of microbial antigens by counterimmunoelectrophoresis. T.L. Gavan Coordinating ed. Washington, DC: American Society for Microbiology. Bortolussi R, Wort AJ, Casey S (1978) The latex agglutination test versus counterimmunoelectrophoresis for rapid diagnosis of bacterial meningitis. Con Med Assoc ] 15:489~t93. Burdash NM, West ME (1982) Identification of Streptococcus pneumoniae by the Phadebact coagglutination test. J Clin Microbiol 15:391-394. Carey RB (1983) Rapid diagnosis of bacterial meningitis by antigen detection. Clin Microbiol Newsletter 5:117-120. Collins JK, Kelly MT (1983) Comparison of Phadebact coagglutination, Bactagen latex agglutination and counterimmunoelectrophoresis for detection of Haemaphilus influenzae type b antigens in cerebrospinal fluid. J Clin Microbiol 17:1005-1008. Drow, DL, Manning DD (1983) Comparison of coagglutination, latex agglutination and enzymelinked immunosorbent assay for detection of Haemophilus in~luenzae type b infection. Diagn Microbiol Infect Dis 1:317-322. Finch CA, Wilkinson HW (1979) Practical considerations in using counterimmunoelectrophoresis to identify the principal causative agents of bacterial meningitis. J Clin Microbiol 10:519-524. Ingram DL, Pearson AW, Occhiuti AR (1983) Detection of bacterial antigens in body fluids with the Wellcogen Haemophilus influenzae b, Streptococcus pneumoniae, and Neisseria meningitidis (ACYW135) latex agglutination tests. J Clin Microbiol 18:1119-1121. Jorgensen JH, Lee JC (1978) Rapid diagnosis of gram-negative bacterial meningitis by the Limulus endotoxin assay. J Clin Microbiol 7:12-17. Kelsey MC, Lipscomb AP, Mowles JM (1982) Limulus amebocyte lysate endotoxin test: An aid to the diagnosis in the septic neonate. J Infect 4:69-72, Mayer ME, Geiseler PJ, Harris B (1983) Coagglutination for detection and serotyping of bacterial antigens: Usefulness in acute pneumonias. Diag Microbiol Infect Dis 1:277-285. Olcen P (1978) Serological methods for rapid diagnosis of Hoemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in cerebrospinal fluid: A comparison of coagglutination, immunofluorescence and immunoelect~ophoresis. Scand J Infect Dis 10:283-289. Suksanong M, Dajani AS (1977) Detection of Haemophilus influenzae, type b antigens in body fluids, using specific antibody-coated Staphylococci. J Clin Microbiol 5:81-85. Thirumoorthi MC, Dajani AS (1979) comparison of Staphylococcal coagglutination, latex agglutination, and counterimmunoelectrophoresis for bacterial antigen detection. J Clin Microbiol 9:28-32. Tompkins DS (1983) Comparison of Phadebact co-agglutination tests with counterimmunoelectrophoresis for the detection of bacterial antigens in cerebrospinal fluid. J Clin Pathol 36:819-822.