657
Absence of Intrathecal Synthesis of Some Interferon-a Subtypes in Bacterial Meningitis J. Raymond, C. Benichou, D. de Boissieu, K. Mensah, M. Bergeret, and P. Lebon
Laboratoire de Bacteriologie et Virologie and Departement de Pediatrie. Hiipita! St.- Vincent-de-Paul et Universite Paris V. Paris. France
Sixty-five cerebrospinal fluid (CSF) samples from 54 infants and children with bacterial meningitis were analyzed for the presence of interferon (IFN)-a with a biologic assay. Of the 65 samples, 3 were positive (2-4 IU/mL) and only 1 of 56 collected early was at 4 IU/mL. These results suggest that some subtypes of IFN-a already reported as present in viral infections of the central nervous system are not detected in bacterial meningitis by our IFN assay. This difference may be helpful in differentiating bacterial from viral infections and also in evaluating the quality of the virologic investigation; moreover, the rarity of IFN-1' in CSF in bacterial meningitis needs further investigation to understand its role in the pathogenesis of the disease.
Materials and Methods Patients and clinical specimens. This study was done prospectively over 4 years from January 1987 to March 1991 in children from I day to 10 years old with clinically and biologically diagnosed meningitis. Cultures of CSF for bacteria and viruses and analyses of soluble antigens (Wellcome Diagnostics, Dartford, UK) were systematically done by standard methods. Laboratory and clinical results were compared and routine biochemical analysis of the CSF was done.
Received 7 February 1992; revised 2 April 1992. Reprints or correspondence: Dr. Pierre Lebon. Laboratoire de Bacteriologie-Virologie, Hopital St.-Vincent-de-Paul, 82, Ave. Denfert Rochereau, 75674 Paris Cedex 14, France. The Journal of Infectious Diseases 1992;166:657-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0036$01.00
Sixty-five CSF samples and 15 sera from 54 patients with proved bacterial meningitis were examined for IFN-a; 56 CSF samples were collected between day 0 and 2 after admission. Of the 65 CSF samples, 23 were assayed for IFN-'Y. The causative organism, identified by bacterial culture or positivity of soluble antigens, was Hemophilus influenzae in 27 patients, Streptococcus pneumoniae in 8, Streptococcus agalactiae in 4, Neisseria meningitidis in 5, Escherichia coli in 3, Enterobacter cloacae in I, and Listeria monocytogenes in 4 (4 CSF samples were provided by P. Berche, University of Paris). CSF IFN-'Y was measured in 16 patients with meningitis due to H. influenzae (9), S. pneumoniae (2), N. meningitidis (3), E. coli ( I), and E. cloacae ( I) (table I). Five sera and 5 CSF samples from newborns with enteroviral meningitis were included as positive controls. Four BI and one B5 coxsackieviruses were isolated from the CSF samples in which IFN-a and -1' were titered (table 2). IFN assays. IFN-a was measured in a biologic assay as previously described [10, II], using Madin-Darby bovine kidney (MDBK) cells and vesicular stomatitis virus as the challenge virus. An aliquot (50 ~L) of sera or CSF was mixed with I00 ~L ofcell suspension (25,000 cells/Oi l mL) and incubated for 24 h at 37°C. The cells were then washed and challenged with vesicular stomatitis virus at an MOl of 0.1. After an 18-h incubation, the titer was estimated as the reciprocal of the highest dilution that protected 50%of the cell population. This test can detect as little as I IV /ml, of IFN-a2 subtype. IFN-'Ywas measured by solid-phase RIA (Centocor, Malvern, PA). Briefly, polystyrene beads coated with anti-human IFN-'Y monoclonal antibodies were incubated with specimens and controls. Unbound material was removed, and beads were incubated again with these monoclonal antibodies labeled with 1251. After washing, the bound radioactivity was determined by counting the beads in a well-type gamma counter. IFN-')' concentrations were determined from a standard curve. In this test the monoclonal antibodies are specific for the epitopes of biologic activity of human IFN [12] and can detect titers as low as 0.20IV/mL. IFN-a and -1' titers were expressed in international reference units (IV) after comparison with the international reference samples titrated under the same conditions. Characterization ofIFN. Positive CSF samples were neutral-
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 26, 2015
Conditions for interferon (IFN)-a, -11, and --y induction differ depending on the nature of the cells, the inducers, and the mechanism ofIFN gene activation [1-3]. The induction ofIFN also has been demonstrated in vivo during viral, bacterial, or parasitic infections [4, 5]. However, many studies have not identified the type of endogenous IFN found in serum or cerebrospinal fluid (CSF) of patients [4]. Few studies of encephalitis and meningitis have demonstrated the intrathecal origin ofIFN synthesis when comparing the level in CSF to that in serum collected at the same time. Since IFN-a can be induced in vitro with bacteria [6, 7], we have verified that they can also be produced in vivo in bacterial meningitis. We measured the IFN-a levels in CSF samples collected at the acute phase of bacterial meningitis using a biologic assay very sensitive to some subtypes ofIFNa but not to IFN-'Y. We used the same cells as those used in previous studies of viral infections [8-10]. IFN-'Y was also studied in some cases with an immunoassay that does not detect IFN-a. Five cases of viral meningitis were included as positive controls.
Concise Communications
658
JID 1992; 166 (September)
Table 1. Interferon (IFN)-a and -'Y from patients with bacterial meningitis. IFN-')'
IFN-a
No. positive/ no. tested
No. positive/ no. tested Bacteria Haemophilus influenzae Streptococcus pneumoniae Streptococcus agalactiae Neisseria meningitidis Escherichia coli Enterobacter cloacae Listeria monocytogenes
NOTE.
No. of cases
CSF
Serum
No. of cases
27 8 4 5 3 3 4
1/32 2/10 0/4 0/5 0/5 0/5 0/4
2/8 0/4 NO 0/3 NO NO ND
9 2 0 3 I I 0
3/13 0/3
1/2
0/3 0/2 0/2
2/2
IFN-a. As shown in table I, IFN-a was not found in 62 (95%) of65 CSF specimens. Of the 3 positive cases, S. pneumoniae was isolated in 2. In the first, the IFN-a titer was 4 IU ImL at day 0 and became
Absence of Intrathecal Synthesis of Some Interferon-a Subtypes in Bacterial Meningitis J. Raymond, C. Benichou, D. de Boissieu, K. Mensah, M. Bergeret, and P. Lebon
Laboratoire de Bacteriologie et Virologie and Departement de Pediatrie. Hiipita! St.- Vincent-de-Paul et Universite Paris V. Paris. France
Sixty-five cerebrospinal fluid (CSF) samples from 54 infants and children with bacterial meningitis were analyzed for the presence of interferon (IFN)-a with a biologic assay. Of the 65 samples, 3 were positive (2-4 IU/mL) and only 1 of 56 collected early was at 4 IU/mL. These results suggest that some subtypes of IFN-a already reported as present in viral infections of the central nervous system are not detected in bacterial meningitis by our IFN assay. This difference may be helpful in differentiating bacterial from viral infections and also in evaluating the quality of the virologic investigation; moreover, the rarity of IFN-1' in CSF in bacterial meningitis needs further investigation to understand its role in the pathogenesis of the disease.
Materials and Methods Patients and clinical specimens. This study was done prospectively over 4 years from January 1987 to March 1991 in children from I day to 10 years old with clinically and biologically diagnosed meningitis. Cultures of CSF for bacteria and viruses and analyses of soluble antigens (Wellcome Diagnostics, Dartford, UK) were systematically done by standard methods. Laboratory and clinical results were compared and routine biochemical analysis of the CSF was done.
Received 7 February 1992; revised 2 April 1992. Reprints or correspondence: Dr. Pierre Lebon. Laboratoire de Bacteriologie-Virologie, Hopital St.-Vincent-de-Paul, 82, Ave. Denfert Rochereau, 75674 Paris Cedex 14, France. The Journal of Infectious Diseases 1992;166:657-9 © 1992 by The University of Chicago. All rights reserved. 0022-1899/92/6603-0036$01.00
Sixty-five CSF samples and 15 sera from 54 patients with proved bacterial meningitis were examined for IFN-a; 56 CSF samples were collected between day 0 and 2 after admission. Of the 65 CSF samples, 23 were assayed for IFN-'Y. The causative organism, identified by bacterial culture or positivity of soluble antigens, was Hemophilus influenzae in 27 patients, Streptococcus pneumoniae in 8, Streptococcus agalactiae in 4, Neisseria meningitidis in 5, Escherichia coli in 3, Enterobacter cloacae in I, and Listeria monocytogenes in 4 (4 CSF samples were provided by P. Berche, University of Paris). CSF IFN-'Y was measured in 16 patients with meningitis due to H. influenzae (9), S. pneumoniae (2), N. meningitidis (3), E. coli ( I), and E. cloacae ( I) (table I). Five sera and 5 CSF samples from newborns with enteroviral meningitis were included as positive controls. Four BI and one B5 coxsackieviruses were isolated from the CSF samples in which IFN-a and -1' were titered (table 2). IFN assays. IFN-a was measured in a biologic assay as previously described [10, II], using Madin-Darby bovine kidney (MDBK) cells and vesicular stomatitis virus as the challenge virus. An aliquot (50 ~L) of sera or CSF was mixed with I00 ~L ofcell suspension (25,000 cells/Oi l mL) and incubated for 24 h at 37°C. The cells were then washed and challenged with vesicular stomatitis virus at an MOl of 0.1. After an 18-h incubation, the titer was estimated as the reciprocal of the highest dilution that protected 50%of the cell population. This test can detect as little as I IV /ml, of IFN-a2 subtype. IFN-'Ywas measured by solid-phase RIA (Centocor, Malvern, PA). Briefly, polystyrene beads coated with anti-human IFN-'Y monoclonal antibodies were incubated with specimens and controls. Unbound material was removed, and beads were incubated again with these monoclonal antibodies labeled with 1251. After washing, the bound radioactivity was determined by counting the beads in a well-type gamma counter. IFN-')' concentrations were determined from a standard curve. In this test the monoclonal antibodies are specific for the epitopes of biologic activity of human IFN [12] and can detect titers as low as 0.20IV/mL. IFN-a and -1' titers were expressed in international reference units (IV) after comparison with the international reference samples titrated under the same conditions. Characterization ofIFN. Positive CSF samples were neutral-
Downloaded from http://jid.oxfordjournals.org/ at Monash University on August 26, 2015
Conditions for interferon (IFN)-a, -11, and --y induction differ depending on the nature of the cells, the inducers, and the mechanism ofIFN gene activation [1-3]. The induction ofIFN also has been demonstrated in vivo during viral, bacterial, or parasitic infections [4, 5]. However, many studies have not identified the type of endogenous IFN found in serum or cerebrospinal fluid (CSF) of patients [4]. Few studies of encephalitis and meningitis have demonstrated the intrathecal origin ofIFN synthesis when comparing the level in CSF to that in serum collected at the same time. Since IFN-a can be induced in vitro with bacteria [6, 7], we have verified that they can also be produced in vivo in bacterial meningitis. We measured the IFN-a levels in CSF samples collected at the acute phase of bacterial meningitis using a biologic assay very sensitive to some subtypes ofIFNa but not to IFN-'Y. We used the same cells as those used in previous studies of viral infections [8-10]. IFN-'Y was also studied in some cases with an immunoassay that does not detect IFN-a. Five cases of viral meningitis were included as positive controls.
Concise Communications
658
JID 1992; 166 (September)
Table 1. Interferon (IFN)-a and -'Y from patients with bacterial meningitis. IFN-')'
IFN-a
No. positive/ no. tested
No. positive/ no. tested Bacteria Haemophilus influenzae Streptococcus pneumoniae Streptococcus agalactiae Neisseria meningitidis Escherichia coli Enterobacter cloacae Listeria monocytogenes
NOTE.
No. of cases
CSF
Serum
No. of cases
27 8 4 5 3 3 4
1/32 2/10 0/4 0/5 0/5 0/5 0/4
2/8 0/4 NO 0/3 NO NO ND
9 2 0 3 I I 0
3/13 0/3
1/2
0/3 0/2 0/2
2/2
IFN-a. As shown in table I, IFN-a was not found in 62 (95%) of65 CSF specimens. Of the 3 positive cases, S. pneumoniae was isolated in 2. In the first, the IFN-a titer was 4 IU ImL at day 0 and became
