Scandinavian Journal of Infectious Diseases
ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19
An Evaluation of Two Immunological Methods in the Diagnosis of Bacterial Meningitis: The Effect of Ultrasonic Treatment of the Cerebrospinal Fluid Øsystein Føsrre & Peter Gaustad To cite this article: Øsystein Føsrre & Peter Gaustad (1977) An Evaluation of Two Immunological Methods in the Diagnosis of Bacterial Meningitis: The Effect of Ultrasonic Treatment of the Cerebrospinal Fluid, Scandinavian Journal of Infectious Diseases, 9:4, 285-288, DOI: 10.3109/inf.1977.9.issue-4.05 To link to this article: http://dx.doi.org/10.3109/inf.1977.9.issue-4.05
Published online: 02 Jan 2015.
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=infd19 Download by: [RMIT University Library]
Date: 07 April 2016, At: 15:35
Scand J Infect Dis 9: 285-288, 1977
An Evaluation of Two Immunological Methods in the Diagnosis of Bacterial Meningitis: The Effect of Ultrasonic Treatment of the Cerebrospinal Fluid OYSTEIN FORRE and PETER GAUSTAD
Downloaded by [RMIT University Library] at 15:35 07 April 2016
From the Norwegian Defence Microbiological Laboratory, Oslo, Norway
ABSTRACT. Counterimmunoelectrophoresis (CIE) and fluorescent antibody (FA) technique were evaluated in the diagnosis of bacterial meningitis caused by Neisseria meningitidis (groups A, B, C), Streptococcuspneumoniae and Haemophilusinfluenzae type b. FA was positive in 80 % and CIE in 70% of the 59 cases of bacterial meningitis investigated. Ultrasonic treatment of the cerebrospinal fluid (CSF) increased the efficacy of CIE from 50 % to 70 %. By FA it was possible to demonstrate bacterial antigen in CSF up to 3 days after the beginning of antibacterial therapy. Bacterial antigens could be demonstrated by the two immunological methods from 2 to 24 days when stored at 20°C. At storage at 4°C the antigens were demonstrable for 2 to 13 weeks by FA and for 2 to 10 weeks by CIE.
INTRODUCTION During recent years the importance of rapid and adequate diagnosis and treatment of bacterial meningitis has increased, partly because of many epidemics in different parts of the world (3, 8), and partly because of increase in resistance to antibiotics of the etiological agents (2, 4, 5 , 10). Immunological methods may have advantages, and this study deals with an evaluation of fluorescent antibody (FA) technique and counterimmunoelectrophoresis (CIE) in the diagnosis of bacterial meningitis caused by Neisseria meningitidis (groups A, B, C), Streptococcus pneumoniae or Haemophilus influenzae type b. The results obtained by these techniques were compared with the results of gram staining and culture. Ultrasonic treatment of the cerebrospinal fluid (CSF) appeared to improve the efficiency of the CIE test. The efficacy of the methods during antibacterial therapy was also studied. Effects of storage at 4°C and at 20°C on the bacterial antigens were investigated. MATERIAL AND METHODS Patients and CSF specimens From September 1973 to April 1975, 199 samples of CSF from 185 patients from 0 to 84 years of age were examined.
The diagnostic procedures started 0 to 72 h after the lumbar puncture. The CSF was stored at room temperature (about 20°C). Lumbar puncture was in most cases carried out before treatment with antibiotics. In cases where the patients had received antibiotics before the puncture, informations about dosage and length of treatment were noted. In some cases CSF was examined during the antibiotic treatment.
Bacteriological methods Gram staining was performed in the conventional way by the local hospital or our laboratory. During this part of the work small gram-negative rods were tentatively registered as H. influenzae, gram-positive diplococci as S. pneumoniae and gram-negative diplococci as N. meningitidis. Culture was first done in the local hospital or laboratory, and subsequently in our laboratory. Blood agar and chocolate agar plates were used, and the cultures were incubated at 37°C for up to 2 days. The chocolate agar plates were incubated in an atmosphere of 5-10% C o t (candle jar). Immunological methods Counterimmunoelectrophoresis (CIE). CIE was carried out by a method described by Myhre (6). Each CSF sample was tested against the following specific antisera: N. meningitidis groups A, B, C antisera (provided by E. Holten, M.D., Rikshospitalet, Oslo), omnipotent S. pneumoniae antiserum (provided by Statens Seruminstitut, Copenhagen, Denmark), and H. influenzae type b antiserum (7). The slides were inspected for precipitation lines against a dark background and with oblique lighting Scand J Infect Dis 9
286
Q. F ~ r r and e P . Gaustad
Downloaded by [RMIT University Library] at 15:35 07 April 2016
Table I. Distribution according to etiological agent Agent
No. of patients
N. meningitidis Group A Group B Group C Unclassified S. pneumoniae H. influenzae type b Total
12 13 7 9 6 12 59
Antigen demonstrable Antigen demonstrable at 20°C (N= 13) at 4°C (N=33)
CIE FA
immediately after the run. In cases with negative CIE but positive clinical and/or other laboratory findings, the test was repeated after heating the CSF sample to 56°C for 30 min and ultrasonic treatment for 1 min (100 watt Ultrasonic Disintegrator, frequency 20 kc/s). Fluorescent antibody (FA) technique. Fluorescein isothiocyanate (F1TC)-conjugated IgG from the antisera were prepared according to Biegeleisen et al. (1). A Leitz Orthoplan microscope with an Osram HBC 200 light source with a 1.5 mm BG 12 exciter filter, a 495 interference filter and K 510 barrier filter was used. The immunofluorescence staining was carried out on 2 standard glass slides (26x76 mm) with open spaces for the staining prepared with “Spray PTFE plastiband” and glycerol. After drying and gentle heat-fixing, 1 drop of FITC-conjugated antiserum was placed on the CSF preparation, and the slides were placed at 37°C in a humid atmosphere for 20 min. The slides were rinsed in phosphate buffered saline (pH 7.2) for 10 min and in distilled water for further 10 min. Then the slides were dried in air at 37°C and a cover slip was mounted on each specimen with buffered glycerol (pH 8.5). One step inhibition test was used as positive control. A FA preparation was regarded as positive only when irnmunofluorescence was present in morphologically intact bacteria. Storage studies In some cases with demonstrated bacterial antigen the CSF was stored at 20°C or at 4°C to evaluate the storage effects on the bacteria. 13 samples stored at 20°C were
Table 11. Results with different techniques on 59 CSF samples
Gram stain Cultivation CIE CIE after ultrasound FA Scand J Infect Dis 9
Table 111. Bacterial antigen demonstrated by CIE and FA after storage at 4°C and 20°C
No. of samples examined
No. of positive samples
%
48 55 57
32 41 28
67 75 50
57 57
40 45
I0 80
positive
Mean
Range
Mean
Range
4
2-10 weeks 2-13 weeks
9 10
2-24 days 2-24 days
5
re-examined daily up to 26 day after lumbar puncture by CIE and FA, while 33 samples stored at 4°C were reexamined every 14th day as long as the bacterial antigens could be demonstrated.
RESULTS AND DISCUSSION
199 CSF samples from patients with suspected meningitis were examined. 59 samples (30%) were from patients with bacterial meningitis caused by N. meningitidis, S. pneumoniae or H. influenzae type b. The distribution according to etiological agents is given in Table I. In 8 CSF samples (4%) cultivation and gram staining yielded other bacteria or the clinical diagnosis was based on other criteria. Viruses caused the meningitis in 10% (21 samples). In 56% (1 11 samples) the suspected diagnosis of meningitis was not confirmed. Efficiency of the techniques before and after antibacterial treatment By means of FA and CIE, we were able to determine the etiological agent in 80% and 70% respectively of the 59 cases of bacterial meningitis investigated (Table 11). In N. meningitidis and H. influenzae meningitis, the serogroup or serotype could be determined. Gram staining and culture detected the agents in 67% and 75% of the 59 samples (Table 11). 12 samples which were negative after CIE examination turned out to be positive after ultrasonic treatment. In 3 cases several precipitation lines appeared after such treatment and no conclusive results could be obtained. The use of ultrasonic treatment of the CSF thus improved the diagnosis by CIE in cases of bacterial meningitis, since the percentages of positive specimens augmented from 50 to 70 %. Theoretically the use of ultrasonic treatment of the CSF may in some cases give false positive results or cross-reactions with other bacterial antigens (9), but this problem is apparently of little importance as only 3 instances
Downloaded by [RMIT University Library] at 15:35 07 April 2016
Diagnosis of bacterial meningitis
were noted in which no conclusive results could be obtained. In 13 culture-negative cases, FA and/or CIE demonstrated the causal agent. Cultivation detected the etiological organisms in 5 cases with negative FA and/or CIE. The present study indicates that the two immunological tests, CIE and FA, give approximately the same percentages of positive results as was obtained with routine culture. The time required is about 1 h for the immunological methods, while cultivation needs 24-48 h. However, CIE and FA cannot replace cultivation, since the evaluation of susceptibility and resistance to certain antibiotics of the bacteria causing the meningitis will probably be more and more important (1,4, 5, 10). In 8 of 16 samples microorganisms or antigens could be demonstrated in the CSF up to 3 days after the beginning of antibacterial therapy. Except in one sample where both FA and CIE were positive, only one of the techniques yielded a positive result. In 8 cases the examinations were negative (examination performed 3 days or more after therapy had been started). Culture, however, yielded a positive result only once after the onset of antibacterial therapy. FA was clearly the most effective test, since it could detect the causative agent in 6 samples up to 3 days of antibacterial treatment, while CIE demonstrated the bacterial antigens only in 2 samples. Since as little as one dose of antibiotic may inhibit bacterial growth, the immunological methods provide the only means of making a diagnosis in previously treated patients. Influence of conditions of storage on the antigens in CSF In 13 samples stored at 20°C the antigens of N. meningitidis (groups A, B, C), S. pneumoniae or H. influenzae type b could be demonstrated for 2 to 24 days, with mean values of 9 and 10 days for CIE and FA respectively. Stored at 4"C, the antigens in 33 samples were demonstrable by CIE for 2 to 10 weeks (mean 4 weeks) and by FA for 2 to 13 weeks (mean 5 weeks). Thus, the 2 immunological methods appeared equally efficient (Table 111). Cultivation is seldom positive in samples stored longer than 20 h at 20°C. The fact that the antigens may be demonstrated in CSF for weeks when stored at 4°C may have implications concerning the transport period to a laboratory and the temperature at which the transport should be carried out.
287
The limited number of samples stored at 20°C do not allow any conclusions concerning storage time of the different bacteria examined, but indicate that the capsular antigens in CSF may be demonstrable for several days. Stored at 4°C the capsular antigens of the various bacterial species and serogroups had great variations in type of demonstrable antigen in CSF, and more systematic studies are needed to elucidate this. We feel that CIE could be carried out in any microbiological laboratory because of the cheap equipment and because it is relatively simple to evaluate the results. In the case of FA, the situation is less certain. The microscopic equipment needed is expensive, and the interpretation requires considerable experience. It is therefore recommended that this test should be restricted to central laboratories or laboratories with special knowledge in the field. ACKNOWLEDGEMENTS We thank Tov Omland, M.D., Director of Norwegian Defence Microbiological Laboratory for valuable advice and encouragement, and we also thank Professor Helge Laake, M.D. and the technicians, Department of Infectious Diseases, Ullev%l Hospital, Oslo, Norway, who carefully have brought us CSF samples. We are grateful to Mrs Valeria Gacek for excellent technical assistance.
REFERENCES 1. Biegeleisen, J. Z., Jr, Mitchell, M. S., Marcus, B. B., Rhoden, D. L. & Blumberg, R. W.: Immuno-
fluorescence techniques for demonstrating bacterial pathogens associated with cerebrospinal meningitis. I. Clinical evaluation of conjugates on smears prepared directly from cerebrospinal fluid sediments. J Lab Clin Med 65: 976, 1965. 2. Holten, E., Vaage, L. & Jyssum, K.: Sulphonamideresistant meningococci in Norwegian naval recruits. Scand J Infect Dis 2: 11 1, 1970. 3. Marget, W. & Lang, W.: Die MeningokokkenMeningitis in Sao Paulo, Brasilien. Infection 2: 171, 1973. 4. Khan, W., Ross, S., Rodriguez, W., Controni, G. &
Saz, A. K.: Haemophilus influenzae type b resistant to ampicillin. JAMA 229: 298, 1974. 5. Millar, J. W., Siess, E. E., Feldman, H. A., Silverman, C. & Frank, P.: In vivo and in vitro resistance to sulfadiazine in strains of Neisseria meningitidis. JAMA 186: 139, 1963. 6. Myhre, E. B.: Rapid diagnosis of bacterial meningitis. Scand J Infect Dis 6: 237, 1974. 7. Omland, T.: Serological studies on Haemophilus influenzae and related species. Acta Pathol Microbiol Scand 57: 279, 1963. Scand J Infect Dis 9
288
0.Ferre and P . Gaustad
Downloaded by [RMIT University Library] at 15:35 07 April 2016
8. Sivonen, A., Renkonen, 0. V., Weckstrom, P. & Makela, P. H.: Group A meningococcal epidemic in Finland. Bacteriological aspects. Abstract ScottishScandinavian Conference on Infectious Diseases, p. 79, 1974. 9. Robbins, J. B., Myerowitz, R. L., Whisnant, J. K., Argaman, M., Schneerson, R., Handzel, Z. T. & Gotschlich, E.: Enteric bacteria cross-reactive with Neisseria meningitidis groups A and C and Diplococcus pneumoniae type I and 111. Infect Immun 6: 651, 1972. 10. Tomeh, M. O., Starr, S. E. & McGowan, J. E.: Ampicillin-resistant Haemophilus influenzae type b infection. JAMA 229: 295, 1974. Address f o r reprints:
P . Gaustad, M.D., Kaptein W . Wilhelmsen og Frues Bakteriologiske Institutt, Rikshospitalet, Oslo 1, Norway
Scand J Infect Dis 9
ISSN: 0036-5548 (Print) 1651-1980 (Online) Journal homepage: http://www.tandfonline.com/loi/infd19
An Evaluation of Two Immunological Methods in the Diagnosis of Bacterial Meningitis: The Effect of Ultrasonic Treatment of the Cerebrospinal Fluid Øsystein Føsrre & Peter Gaustad To cite this article: Øsystein Føsrre & Peter Gaustad (1977) An Evaluation of Two Immunological Methods in the Diagnosis of Bacterial Meningitis: The Effect of Ultrasonic Treatment of the Cerebrospinal Fluid, Scandinavian Journal of Infectious Diseases, 9:4, 285-288, DOI: 10.3109/inf.1977.9.issue-4.05 To link to this article: http://dx.doi.org/10.3109/inf.1977.9.issue-4.05
Published online: 02 Jan 2015.
Submit your article to this journal
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=infd19 Download by: [RMIT University Library]
Date: 07 April 2016, At: 15:35
Scand J Infect Dis 9: 285-288, 1977
An Evaluation of Two Immunological Methods in the Diagnosis of Bacterial Meningitis: The Effect of Ultrasonic Treatment of the Cerebrospinal Fluid OYSTEIN FORRE and PETER GAUSTAD
Downloaded by [RMIT University Library] at 15:35 07 April 2016
From the Norwegian Defence Microbiological Laboratory, Oslo, Norway
ABSTRACT. Counterimmunoelectrophoresis (CIE) and fluorescent antibody (FA) technique were evaluated in the diagnosis of bacterial meningitis caused by Neisseria meningitidis (groups A, B, C), Streptococcuspneumoniae and Haemophilusinfluenzae type b. FA was positive in 80 % and CIE in 70% of the 59 cases of bacterial meningitis investigated. Ultrasonic treatment of the cerebrospinal fluid (CSF) increased the efficacy of CIE from 50 % to 70 %. By FA it was possible to demonstrate bacterial antigen in CSF up to 3 days after the beginning of antibacterial therapy. Bacterial antigens could be demonstrated by the two immunological methods from 2 to 24 days when stored at 20°C. At storage at 4°C the antigens were demonstrable for 2 to 13 weeks by FA and for 2 to 10 weeks by CIE.
INTRODUCTION During recent years the importance of rapid and adequate diagnosis and treatment of bacterial meningitis has increased, partly because of many epidemics in different parts of the world (3, 8), and partly because of increase in resistance to antibiotics of the etiological agents (2, 4, 5 , 10). Immunological methods may have advantages, and this study deals with an evaluation of fluorescent antibody (FA) technique and counterimmunoelectrophoresis (CIE) in the diagnosis of bacterial meningitis caused by Neisseria meningitidis (groups A, B, C), Streptococcus pneumoniae or Haemophilus influenzae type b. The results obtained by these techniques were compared with the results of gram staining and culture. Ultrasonic treatment of the cerebrospinal fluid (CSF) appeared to improve the efficiency of the CIE test. The efficacy of the methods during antibacterial therapy was also studied. Effects of storage at 4°C and at 20°C on the bacterial antigens were investigated. MATERIAL AND METHODS Patients and CSF specimens From September 1973 to April 1975, 199 samples of CSF from 185 patients from 0 to 84 years of age were examined.
The diagnostic procedures started 0 to 72 h after the lumbar puncture. The CSF was stored at room temperature (about 20°C). Lumbar puncture was in most cases carried out before treatment with antibiotics. In cases where the patients had received antibiotics before the puncture, informations about dosage and length of treatment were noted. In some cases CSF was examined during the antibiotic treatment.
Bacteriological methods Gram staining was performed in the conventional way by the local hospital or our laboratory. During this part of the work small gram-negative rods were tentatively registered as H. influenzae, gram-positive diplococci as S. pneumoniae and gram-negative diplococci as N. meningitidis. Culture was first done in the local hospital or laboratory, and subsequently in our laboratory. Blood agar and chocolate agar plates were used, and the cultures were incubated at 37°C for up to 2 days. The chocolate agar plates were incubated in an atmosphere of 5-10% C o t (candle jar). Immunological methods Counterimmunoelectrophoresis (CIE). CIE was carried out by a method described by Myhre (6). Each CSF sample was tested against the following specific antisera: N. meningitidis groups A, B, C antisera (provided by E. Holten, M.D., Rikshospitalet, Oslo), omnipotent S. pneumoniae antiserum (provided by Statens Seruminstitut, Copenhagen, Denmark), and H. influenzae type b antiserum (7). The slides were inspected for precipitation lines against a dark background and with oblique lighting Scand J Infect Dis 9
286
Q. F ~ r r and e P . Gaustad
Downloaded by [RMIT University Library] at 15:35 07 April 2016
Table I. Distribution according to etiological agent Agent
No. of patients
N. meningitidis Group A Group B Group C Unclassified S. pneumoniae H. influenzae type b Total
12 13 7 9 6 12 59
Antigen demonstrable Antigen demonstrable at 20°C (N= 13) at 4°C (N=33)
CIE FA
immediately after the run. In cases with negative CIE but positive clinical and/or other laboratory findings, the test was repeated after heating the CSF sample to 56°C for 30 min and ultrasonic treatment for 1 min (100 watt Ultrasonic Disintegrator, frequency 20 kc/s). Fluorescent antibody (FA) technique. Fluorescein isothiocyanate (F1TC)-conjugated IgG from the antisera were prepared according to Biegeleisen et al. (1). A Leitz Orthoplan microscope with an Osram HBC 200 light source with a 1.5 mm BG 12 exciter filter, a 495 interference filter and K 510 barrier filter was used. The immunofluorescence staining was carried out on 2 standard glass slides (26x76 mm) with open spaces for the staining prepared with “Spray PTFE plastiband” and glycerol. After drying and gentle heat-fixing, 1 drop of FITC-conjugated antiserum was placed on the CSF preparation, and the slides were placed at 37°C in a humid atmosphere for 20 min. The slides were rinsed in phosphate buffered saline (pH 7.2) for 10 min and in distilled water for further 10 min. Then the slides were dried in air at 37°C and a cover slip was mounted on each specimen with buffered glycerol (pH 8.5). One step inhibition test was used as positive control. A FA preparation was regarded as positive only when irnmunofluorescence was present in morphologically intact bacteria. Storage studies In some cases with demonstrated bacterial antigen the CSF was stored at 20°C or at 4°C to evaluate the storage effects on the bacteria. 13 samples stored at 20°C were
Table 11. Results with different techniques on 59 CSF samples
Gram stain Cultivation CIE CIE after ultrasound FA Scand J Infect Dis 9
Table 111. Bacterial antigen demonstrated by CIE and FA after storage at 4°C and 20°C
No. of samples examined
No. of positive samples
%
48 55 57
32 41 28
67 75 50
57 57
40 45
I0 80
positive
Mean
Range
Mean
Range
4
2-10 weeks 2-13 weeks
9 10
2-24 days 2-24 days
5
re-examined daily up to 26 day after lumbar puncture by CIE and FA, while 33 samples stored at 4°C were reexamined every 14th day as long as the bacterial antigens could be demonstrated.
RESULTS AND DISCUSSION
199 CSF samples from patients with suspected meningitis were examined. 59 samples (30%) were from patients with bacterial meningitis caused by N. meningitidis, S. pneumoniae or H. influenzae type b. The distribution according to etiological agents is given in Table I. In 8 CSF samples (4%) cultivation and gram staining yielded other bacteria or the clinical diagnosis was based on other criteria. Viruses caused the meningitis in 10% (21 samples). In 56% (1 11 samples) the suspected diagnosis of meningitis was not confirmed. Efficiency of the techniques before and after antibacterial treatment By means of FA and CIE, we were able to determine the etiological agent in 80% and 70% respectively of the 59 cases of bacterial meningitis investigated (Table 11). In N. meningitidis and H. influenzae meningitis, the serogroup or serotype could be determined. Gram staining and culture detected the agents in 67% and 75% of the 59 samples (Table 11). 12 samples which were negative after CIE examination turned out to be positive after ultrasonic treatment. In 3 cases several precipitation lines appeared after such treatment and no conclusive results could be obtained. The use of ultrasonic treatment of the CSF thus improved the diagnosis by CIE in cases of bacterial meningitis, since the percentages of positive specimens augmented from 50 to 70 %. Theoretically the use of ultrasonic treatment of the CSF may in some cases give false positive results or cross-reactions with other bacterial antigens (9), but this problem is apparently of little importance as only 3 instances
Downloaded by [RMIT University Library] at 15:35 07 April 2016
Diagnosis of bacterial meningitis
were noted in which no conclusive results could be obtained. In 13 culture-negative cases, FA and/or CIE demonstrated the causal agent. Cultivation detected the etiological organisms in 5 cases with negative FA and/or CIE. The present study indicates that the two immunological tests, CIE and FA, give approximately the same percentages of positive results as was obtained with routine culture. The time required is about 1 h for the immunological methods, while cultivation needs 24-48 h. However, CIE and FA cannot replace cultivation, since the evaluation of susceptibility and resistance to certain antibiotics of the bacteria causing the meningitis will probably be more and more important (1,4, 5, 10). In 8 of 16 samples microorganisms or antigens could be demonstrated in the CSF up to 3 days after the beginning of antibacterial therapy. Except in one sample where both FA and CIE were positive, only one of the techniques yielded a positive result. In 8 cases the examinations were negative (examination performed 3 days or more after therapy had been started). Culture, however, yielded a positive result only once after the onset of antibacterial therapy. FA was clearly the most effective test, since it could detect the causative agent in 6 samples up to 3 days of antibacterial treatment, while CIE demonstrated the bacterial antigens only in 2 samples. Since as little as one dose of antibiotic may inhibit bacterial growth, the immunological methods provide the only means of making a diagnosis in previously treated patients. Influence of conditions of storage on the antigens in CSF In 13 samples stored at 20°C the antigens of N. meningitidis (groups A, B, C), S. pneumoniae or H. influenzae type b could be demonstrated for 2 to 24 days, with mean values of 9 and 10 days for CIE and FA respectively. Stored at 4"C, the antigens in 33 samples were demonstrable by CIE for 2 to 10 weeks (mean 4 weeks) and by FA for 2 to 13 weeks (mean 5 weeks). Thus, the 2 immunological methods appeared equally efficient (Table 111). Cultivation is seldom positive in samples stored longer than 20 h at 20°C. The fact that the antigens may be demonstrated in CSF for weeks when stored at 4°C may have implications concerning the transport period to a laboratory and the temperature at which the transport should be carried out.
287
The limited number of samples stored at 20°C do not allow any conclusions concerning storage time of the different bacteria examined, but indicate that the capsular antigens in CSF may be demonstrable for several days. Stored at 4°C the capsular antigens of the various bacterial species and serogroups had great variations in type of demonstrable antigen in CSF, and more systematic studies are needed to elucidate this. We feel that CIE could be carried out in any microbiological laboratory because of the cheap equipment and because it is relatively simple to evaluate the results. In the case of FA, the situation is less certain. The microscopic equipment needed is expensive, and the interpretation requires considerable experience. It is therefore recommended that this test should be restricted to central laboratories or laboratories with special knowledge in the field. ACKNOWLEDGEMENTS We thank Tov Omland, M.D., Director of Norwegian Defence Microbiological Laboratory for valuable advice and encouragement, and we also thank Professor Helge Laake, M.D. and the technicians, Department of Infectious Diseases, Ullev%l Hospital, Oslo, Norway, who carefully have brought us CSF samples. We are grateful to Mrs Valeria Gacek for excellent technical assistance.
REFERENCES 1. Biegeleisen, J. Z., Jr, Mitchell, M. S., Marcus, B. B., Rhoden, D. L. & Blumberg, R. W.: Immuno-
fluorescence techniques for demonstrating bacterial pathogens associated with cerebrospinal meningitis. I. Clinical evaluation of conjugates on smears prepared directly from cerebrospinal fluid sediments. J Lab Clin Med 65: 976, 1965. 2. Holten, E., Vaage, L. & Jyssum, K.: Sulphonamideresistant meningococci in Norwegian naval recruits. Scand J Infect Dis 2: 11 1, 1970. 3. Marget, W. & Lang, W.: Die MeningokokkenMeningitis in Sao Paulo, Brasilien. Infection 2: 171, 1973. 4. Khan, W., Ross, S., Rodriguez, W., Controni, G. &
Saz, A. K.: Haemophilus influenzae type b resistant to ampicillin. JAMA 229: 298, 1974. 5. Millar, J. W., Siess, E. E., Feldman, H. A., Silverman, C. & Frank, P.: In vivo and in vitro resistance to sulfadiazine in strains of Neisseria meningitidis. JAMA 186: 139, 1963. 6. Myhre, E. B.: Rapid diagnosis of bacterial meningitis. Scand J Infect Dis 6: 237, 1974. 7. Omland, T.: Serological studies on Haemophilus influenzae and related species. Acta Pathol Microbiol Scand 57: 279, 1963. Scand J Infect Dis 9
288
0.Ferre and P . Gaustad
Downloaded by [RMIT University Library] at 15:35 07 April 2016
8. Sivonen, A., Renkonen, 0. V., Weckstrom, P. & Makela, P. H.: Group A meningococcal epidemic in Finland. Bacteriological aspects. Abstract ScottishScandinavian Conference on Infectious Diseases, p. 79, 1974. 9. Robbins, J. B., Myerowitz, R. L., Whisnant, J. K., Argaman, M., Schneerson, R., Handzel, Z. T. & Gotschlich, E.: Enteric bacteria cross-reactive with Neisseria meningitidis groups A and C and Diplococcus pneumoniae type I and 111. Infect Immun 6: 651, 1972. 10. Tomeh, M. O., Starr, S. E. & McGowan, J. E.: Ampicillin-resistant Haemophilus influenzae type b infection. JAMA 229: 295, 1974. Address f o r reprints:
P . Gaustad, M.D., Kaptein W . Wilhelmsen og Frues Bakteriologiske Institutt, Rikshospitalet, Oslo 1, Norway
Scand J Infect Dis 9
