Acta Paediatr 81: 560-1. 1992
SHORT COMMUNICATION
Plasma endotoxin and glycolipid antibodies in children with meningitis ME A1 Awad, R Moosa, S Raziuddin and N Wardle King Saud College of Medicine. ABHA. Saudi Arabia
In spite of advances in antibiotic therapy, the mortality and morbidity of meningitis has not changed over the past 15 years (1). There is limited information in human studies about endotoxaemia during meningitis. Endotoxin has been measured in CSF in experimental Escherichia coli meningitis and it leads to permeability of the blood-brain barrier (2). Haemophilus injuenzae type-b lipo-oligosaccharide induces meningeal inflammation (3). Endotoxins will naturally affect cytokine production (4). Since bacteraemia or septicaemia precede meningitis, plasma endotoxaemia may occur. We measured plasma endotoxin as lipid-A by chromogenic substrate assay and IgG, IgM and IgA antiglycolipid A antibody levels in a series of paediatric patients with meningitis. There were 14 children with H. influenzae type-b meningitis, 1 1 with meningococcal meningitis and 10 with pneumococcal meningitis. Soon after the diagnosis was made, blood was obtained under sterile conditions for assay of: lipid-A of endotoxins (5) after removal of plasma inhibitors by perchloric acid extraction (6); interleukin- 1 by ELISA (Cistron kits); interleukin-2 receptor levels by sandwich ELISA (7); and serum antiglycolipid A antibody levels (8). None of the patients had immunoglobulin deficiency. For the lipid-A toxin assay, blood was collected in pyrogen-free tubes containing endotoxin-free heparin (Bayer) 50 pl of 50 u per ml. The assay was performed on batches of 30 samples together with standards of E. coli endotoxin 01 11.B4 using chromogenic substrate S2423 (Whitaker Bioproducts kit 56-640 U). In our assay, normal plasma samples gave minimal colouration at 8.4 (SD 8.5) pg/ml. Antibodies to the rough glycolipid A of endotoxins were measured by ELISA using a modification of the
method of Nardiello et al. (8). In our method, 100 pl of lipopolysaccharide (E. coli J5 and Salmonella Re 599, each 10 pg/ml in 0.05 M carbonate buffer, pH 9.6, were dispensed into each well of Dynatech microtitre plates. The ELISA colour reaction to antibody in 1/10 dilutions of serum samples was developed in standard fashion. IgG, IgM or IgA anti-human sera conjugated with alkaline phosphatase. Results of the four types of assay are given in Table 1. Plasma lipid-A endotoxin was highest during infection with the two gram-negative organisms but there was also endotoxaemia in the cases of pneumococcal meningitis. Values of plasma interleukin-1 and those of the soluble interleukin-2 receptor assay were high in all forms of infection. Antibodies to glycolipids of IgG and IgM type were high, even at the time of admission. At that stage IgA antibody values were minimal. From the antibody results it appears that the children must have had their infection for at least three days prior to admission. In that time some may have received an oral antibiotic but at an ineffective dose. The endotoxaemia has to be judged in cognisance of this (9). The other finding was endotoxaemia during pneumococcal meningitis. In another study from Abha (9,an adult with chronic renal failure who developed pneumococcal pneumonia with septicaemia had plasma endotoxin values of 40 pg/ml. Also, a man who had Streptococcus pyogenes septicaemia from a leg ulcer, and thereafter acute renal failure, had plasma endotoxin values of 40-60 pg/ml. Thus, severe gram-positive septicaemia can be complicated by a degree of endotoxaemia. This probably arises from increased permeability of the bowel mucosa and it may be accompanied by bacterial translocation.
Table 1. Mean fSD endotoxin values and glycolipid antibodies in children with meningitis.
Normal children (n = 30) Lipid-A (pg/ml) Interleukin-1 (pg/ml) Sol. interleukin-2 receptor (units/ml) Antiglycolipid IgG (OD units) Antiglycolipid IgM (ODunits) Antiglycolipid IgA (OD units)
8.4f8.5 70+30 260 f40 0.205f0.210 0.262 f0.220 0.045+ 0.030
Haemophilus influenzae
Neisseria meningitidis
(n = 14)
(n=II)
Streptococcus pneumoniae (n= 10)
183f44 940f6 10 1040 f220 0.540 f0.140 I .365 f 0.220 0. I50f 0.140
175f53 780 f 370 1060 f 280 0.490f0.110 1.250f 0.220 0.061 f 0.040
74 f 25 430 f 2 10 940f 230 0.460 0.140 0.86ofO.110 0.068 f0.030
ACTA PEDIATR 81 (1992)
Plasma endotoxin and glycolipid antibodies in children with meningitis
References I . Saez-Llorens X,Ramilo 0, Mustafa MM, Mertsola J, McCracken JH. The molecular pathophysiology of bacterial meningitis: current concepts and applications. J Pediatr 1990116:671-6 2. Tauber MG, Shibl AF, Hackbarth CJ, Larrick JW, Sande MA. Antibiotic therapy, endotoxin concentrations in CSF and brain edema in experimental E. coli meningitis in rabbits. J Infect Dis 1987;156:456-62 3. Hansen EJ, McCracken GH, Syrogiannopoulos G. Haemophilus influenzae type b lipo-oligosaccharide induces meningeal inflammation. Pediatr Infect Dis 1987;156:1250-1 4. Mustafa MM, Lebel MH, Ramilo 0, et al. Correlation of interleukin 1 beta and cachectin concentrations in CSF and outcome from bacterial meningitis. J Pediatr 1989;115:208-13 5. Vijaykumar E, Raduddin S, Wardle EN. Plasma endotoxin in patients with trauma, sepsis and severe haemorrhage. Clin Intensive Care 1991;2:4-9
56 1
6. Obayashi T. Addition of perchlonc acid to blood samples for colorimetric Limulus test. J Clin Lab Med 1984;104:321-30 7. Rubin LA, Kurman CC, Fritz ME, et al. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro. J Immunol 1985;135:3127-77 8. Nardiello S, Pizella T, Russo M, Galanti B. Serodiagnosis of typhoid fever by ELISA determination. J Clin Microbiol l984;20:7 18-2 I 9. Shenep JL, Flynn PM, Barrett FF, Stidham GL, Westenkirchner DF. Serial quantitation of endotoxaemia and bacteraemia during therapy for gram-negative bacterial sepsis. J Infect Dis 1987; 157:565-8 Received May 23, 1991. Accepted Nov. 6, 1991 EN Wardle. Greenways. 21 Common Road, North Leigh, Nr. Oxford, OX8 6RD. U K
SHORT COMMUNICATION
Plasma endotoxin and glycolipid antibodies in children with meningitis ME A1 Awad, R Moosa, S Raziuddin and N Wardle King Saud College of Medicine. ABHA. Saudi Arabia
In spite of advances in antibiotic therapy, the mortality and morbidity of meningitis has not changed over the past 15 years (1). There is limited information in human studies about endotoxaemia during meningitis. Endotoxin has been measured in CSF in experimental Escherichia coli meningitis and it leads to permeability of the blood-brain barrier (2). Haemophilus injuenzae type-b lipo-oligosaccharide induces meningeal inflammation (3). Endotoxins will naturally affect cytokine production (4). Since bacteraemia or septicaemia precede meningitis, plasma endotoxaemia may occur. We measured plasma endotoxin as lipid-A by chromogenic substrate assay and IgG, IgM and IgA antiglycolipid A antibody levels in a series of paediatric patients with meningitis. There were 14 children with H. influenzae type-b meningitis, 1 1 with meningococcal meningitis and 10 with pneumococcal meningitis. Soon after the diagnosis was made, blood was obtained under sterile conditions for assay of: lipid-A of endotoxins (5) after removal of plasma inhibitors by perchloric acid extraction (6); interleukin- 1 by ELISA (Cistron kits); interleukin-2 receptor levels by sandwich ELISA (7); and serum antiglycolipid A antibody levels (8). None of the patients had immunoglobulin deficiency. For the lipid-A toxin assay, blood was collected in pyrogen-free tubes containing endotoxin-free heparin (Bayer) 50 pl of 50 u per ml. The assay was performed on batches of 30 samples together with standards of E. coli endotoxin 01 11.B4 using chromogenic substrate S2423 (Whitaker Bioproducts kit 56-640 U). In our assay, normal plasma samples gave minimal colouration at 8.4 (SD 8.5) pg/ml. Antibodies to the rough glycolipid A of endotoxins were measured by ELISA using a modification of the
method of Nardiello et al. (8). In our method, 100 pl of lipopolysaccharide (E. coli J5 and Salmonella Re 599, each 10 pg/ml in 0.05 M carbonate buffer, pH 9.6, were dispensed into each well of Dynatech microtitre plates. The ELISA colour reaction to antibody in 1/10 dilutions of serum samples was developed in standard fashion. IgG, IgM or IgA anti-human sera conjugated with alkaline phosphatase. Results of the four types of assay are given in Table 1. Plasma lipid-A endotoxin was highest during infection with the two gram-negative organisms but there was also endotoxaemia in the cases of pneumococcal meningitis. Values of plasma interleukin-1 and those of the soluble interleukin-2 receptor assay were high in all forms of infection. Antibodies to glycolipids of IgG and IgM type were high, even at the time of admission. At that stage IgA antibody values were minimal. From the antibody results it appears that the children must have had their infection for at least three days prior to admission. In that time some may have received an oral antibiotic but at an ineffective dose. The endotoxaemia has to be judged in cognisance of this (9). The other finding was endotoxaemia during pneumococcal meningitis. In another study from Abha (9,an adult with chronic renal failure who developed pneumococcal pneumonia with septicaemia had plasma endotoxin values of 40 pg/ml. Also, a man who had Streptococcus pyogenes septicaemia from a leg ulcer, and thereafter acute renal failure, had plasma endotoxin values of 40-60 pg/ml. Thus, severe gram-positive septicaemia can be complicated by a degree of endotoxaemia. This probably arises from increased permeability of the bowel mucosa and it may be accompanied by bacterial translocation.
Table 1. Mean fSD endotoxin values and glycolipid antibodies in children with meningitis.
Normal children (n = 30) Lipid-A (pg/ml) Interleukin-1 (pg/ml) Sol. interleukin-2 receptor (units/ml) Antiglycolipid IgG (OD units) Antiglycolipid IgM (ODunits) Antiglycolipid IgA (OD units)
8.4f8.5 70+30 260 f40 0.205f0.210 0.262 f0.220 0.045+ 0.030
Haemophilus influenzae
Neisseria meningitidis
(n = 14)
(n=II)
Streptococcus pneumoniae (n= 10)
183f44 940f6 10 1040 f220 0.540 f0.140 I .365 f 0.220 0. I50f 0.140
175f53 780 f 370 1060 f 280 0.490f0.110 1.250f 0.220 0.061 f 0.040
74 f 25 430 f 2 10 940f 230 0.460 0.140 0.86ofO.110 0.068 f0.030
ACTA PEDIATR 81 (1992)
Plasma endotoxin and glycolipid antibodies in children with meningitis
References I . Saez-Llorens X,Ramilo 0, Mustafa MM, Mertsola J, McCracken JH. The molecular pathophysiology of bacterial meningitis: current concepts and applications. J Pediatr 1990116:671-6 2. Tauber MG, Shibl AF, Hackbarth CJ, Larrick JW, Sande MA. Antibiotic therapy, endotoxin concentrations in CSF and brain edema in experimental E. coli meningitis in rabbits. J Infect Dis 1987;156:456-62 3. Hansen EJ, McCracken GH, Syrogiannopoulos G. Haemophilus influenzae type b lipo-oligosaccharide induces meningeal inflammation. Pediatr Infect Dis 1987;156:1250-1 4. Mustafa MM, Lebel MH, Ramilo 0, et al. Correlation of interleukin 1 beta and cachectin concentrations in CSF and outcome from bacterial meningitis. J Pediatr 1989;115:208-13 5. Vijaykumar E, Raduddin S, Wardle EN. Plasma endotoxin in patients with trauma, sepsis and severe haemorrhage. Clin Intensive Care 1991;2:4-9
56 1
6. Obayashi T. Addition of perchlonc acid to blood samples for colorimetric Limulus test. J Clin Lab Med 1984;104:321-30 7. Rubin LA, Kurman CC, Fritz ME, et al. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro. J Immunol 1985;135:3127-77 8. Nardiello S, Pizella T, Russo M, Galanti B. Serodiagnosis of typhoid fever by ELISA determination. J Clin Microbiol l984;20:7 18-2 I 9. Shenep JL, Flynn PM, Barrett FF, Stidham GL, Westenkirchner DF. Serial quantitation of endotoxaemia and bacteraemia during therapy for gram-negative bacterial sepsis. J Infect Dis 1987; 157:565-8 Received May 23, 1991. Accepted Nov. 6, 1991 EN Wardle. Greenways. 21 Common Road, North Leigh, Nr. Oxford, OX8 6RD. U K
