Bacterial meningitis (BM) remains a significant cause of childhood mortality and morbidity globally, often affecting children in developing, resource-poor countries [
Matrix metalloproteinases (MMPs) are a structurally related but genetically distinct group of proteolytic enzymes which play a central role in regulating tissue destruction, remodeling, and immune responses, including in BM [
The proinflammatory burden in the CSF during BM induces the production of reactive oxygen species, catalyzed by the enzyme myeloperoxidase (MPO), among others. Interestingly, in addition to targeting microbes, MPO is capable of both oxidatively activating latent pro-MMPs and inactivating TIMPs [
In a previous study, our group showed that CSF MMP-9 is strongly upregulated in BM and that increased MMP-9 levels on admission associate with severe disease and an increased risk of death [
To our knowledge, however, no previous studies have explored the relation between these inflammatory mediators in a clinical setting. By measuring the MMP-8, MMP-9, TIMP-1, and MPO levels in the CSF of children with BM, we addressed two questions in this study: First, how would these inflammatory mediators relate to each other in human subjects? Second, to what extent would the results reflect the outcomes of this severe disease?
This study was a retrospective analysis using the prospectively collected data from a large double-blind treatment trial on childhood BM in Latin America in 1996–2003 [
On arrival at the hospital, the patients’ clinical condition was graded using the age-adjusted Glasgow Coma Scale (GCS). Besides death, the disease outcomes were registered by defining as “severe neurological sequelae” all cases of blindness, quadriplegia, severe psychomotor retardation, or hydrocephalus requiring a shunt. “Any neurological sequelae” also comprised milder deficits such as ataxia and hemiparesis.
Concentrations of MMP-8 were determined with a time-resolved immunofluorometric assay (Medix Biochemica, Espoo, Finland). The monoclonal MMP-8-specific antibodies 8708 and 8706 were used as a catching antibody and a tracer antibody, respectively. The tracer antibody was labeled using a europium chelate. The assay buffer contained 20 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 5 mM CaCl2, 50
The levels of MPO and TIMP-1 were determined using commercially available ELISA kits. MPO ELISA (Immundiagnostik AG, Bensheim, Germany) and the Amersham Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Human Biotrak ELISA systems (Amersham Biosciences, GE Healthcare, Buckinghamshire, UK) were used according to the manufacturer’s instructions. The secondary antibody in each kit was conjugated with horseradish peroxidase, and tetramethylbenzidine was used as a substrate. The absorbance was measured at 450 nm using a Victor X4 Multilabel Reader (PerkinElmer Finland Oy, Turku, Finland). The interassay coefficient of variation was <3% for MPO and <12% for TIMP-1, while the corresponding detection limits were 0.294 ng/mL and 1.25 ng/mL [
The MMP-9 levels were assayed using zymography 11% sodium dodecyl sulphate-polyacrylamide gels, based on modification of the method of Lindberg et al. [
Normality of the measured variables was visually inspected. Associations with continuous patient characteristics, as well as interactions between the variables, were assessed using Spearman’s rank correlation, while the relation to categorical patient characteristics was analyzed using the Mann-Whitney
The predictive values of the studied variables were determined by binary logistic regression analysis. To facilitate the interpretation of the results, regression analyses were conducted using median-cut values of the variables. The odds ratios for death, death or severe neurological sequelae, and death or any neurological sequelae of all the studied molecules were adjusted for the level of consciousness on admission. This was done due to the pivotal impact of the child’s presenting status on the outcomes of BM [
Statistical analyses were conducted with IBM SPSS Statistics software, version 24 (IBM Corp., NY, US), except for the local regression procedure which was performed in R, version 3.4.2 (R Foundation for Statistical Computing, Vienna, Austria).
This series comprised 245 children with bacterial meningitis originating from the Dominican Republic (
Patient characteristics.
Characteristic | Result |
---|---|
Total number of patients | 245 |
Male sex (%) | 140/245 (57.1) |
Age, median months (IQR) | 8 (5–19) |
Duration of preadmission illness, median days (IQR) | 2 (1–3) |
GCS score on admission, median (IQR) | 13 (11–14) |
Etiology, number of cases (%) | |
|
122 (49.8) |
|
64 (26.1) |
|
10 (4.1) |
Other | 12 (4.9) |
Unknown | 37 (15.1) |
CSF test results on admission | |
Leukocyte count, median cells/ |
2,500 (900–8,050) |
Protein level, median mg/dL (IQR) | 158 (94–257) |
Glucose level, median mg/dL (IQR) | 15 (6–32) |
Disease outcomes, number of patients | |
Death (%) | 33/245 (13.5) |
Death or severe neurological sequelae (%) | 58/242 |
Death or any neurological sequelae (%) | 112/241 |
IQR: interquartile range.
The expression rates in the CSF for MMP-8, TIMP-1, MPO, proform of MMP-9 (proMMP-9), and active form of MMP-9 (actMMP-9) were 99%, 94%, 84%, 93%, and 73%, respectively. The median concentrations were 453 ng/mL (interquartile range (IQR) 189–1,593 ng/mL) for MMP-8, 232 ng/mL (IQR 53–1,251) for TIMP-1, and 5,018 ng/mL (1,993–16,927) for MPO. The corresponding median densitometric values were 0.21 (IQR 0.00–0.78) for actMMP-9, 0.42 (IQR 0.14–1.46) for proMMP-9, and 0.64 (IQR 0.16–2.21) for total MMP-9 activity (Figure
Gelatin zymography analysis of meningitis cerebrospinal fluid (CSF). (a) Lanes 1-3 represent CSF with elevated levels of MMP-9, and lanes 4-6 represent CSF with low levels of MMP-9. (b) Lanes 1 and 2 represent western immunoblot analysis of MMP-9. Mobilities of 92 kDa pro MMP-9 (proMMP-9) and active MMP-9 (actMMP-9) species are indicated on the right.
While comparing the CSF MMP levels with the baseline patient characteristics, some differences between MMP-9 and MMP-8 were noted. First, total MMP-9 correlated with the patient’s presenting condition: the worse the GCS score, the higher the MMP-9 level (Table
Associations between MMP-8, MPO, TIMP-1, and MMP-9 and baseline patient characteristics
Characteristic | MMP-8 | MPO | TIMP-1 | Total MMP-9 |
---|---|---|---|---|
Age | ||||
Duration of preadmission illness in days | ||||
GCS on admission | ||||
CSF test results | ||||
CSF white cell count | ||||
CSF protein level | ||||
CSF glucose | ||||
MPO correlated, similarly to total MMP-9, negatively with the GCS score on admission but was also positively associated with the CSF white cell count and protein level. In contrast, TIMP-1 associated inversely with these CSF parameters. The relation of TIMP-1 to the GCS score on admission mimicked that of total MMP-9 and MPO (Table
Of all the examined variables, only MMP-9 distinguished between the different causative agents (
A correlation matrix for the studied variables is presented in Table
Correlation matrix for the studied molecules
Variable | MPO | MMP-8 | TIMP-1 | Total MMP-9 | Act% of MMP-9 | MMP8/TIMP-1 molar ratio |
---|---|---|---|---|---|---|
MPO | N/A | |||||
MMP-8 | N/A | |||||
TIMP-1 | N/A | |||||
Total MMP-9 | N/A | |||||
Act% of MMP-9 | N/A | |||||
MMP8/TIMP-1 molar ratio | N/A | |||||
The crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for death and for the composite outcomes of death and severe or any neurological sequelae are presented in Tables
Odds ratios for death
Variable | Unadjusted OR (95% CI) | Adjusted OR |
||
---|---|---|---|---|
MMP-8 | 5.3 (2.1–13.4) | <0.001 | 4.9 (1.8–12.9) | 0.001 |
MPO | 2.8 (1.2–6.3) | 0.02 | 2.0 (0.8–4.8) | 0.12 |
Total MMP-9 | 2.2 (1.0–4.8) | 0.04 | 1.4 (0.6–3.3) | 0.41 |
TIMP-1 | 0.7 (0.3–1.5) | 0.34 | 0.7 (0.3–1.6) | 0.37 |
MMP-8/TIMP-1 molar ratio | 3.3 (1.4–7.8) | 0.005 | 2.8 (1.2–7.0) | 0.02 |
Odds ratios for death or severe neurological sequelae
Variable | Unadjusted OR (95% CI) | Adjusted OR |
||
---|---|---|---|---|
MMP-8 | 2.5 (1.3–4.6) | 0.005 | 2.3 (1.2–4.6) | 0.02 |
MPO | 2.8 (1.4–5.2) | 0.002 | 2.2 (1.1–4.4) | 0.03 |
Total MMP-9 | 1.5 (0.8–2.7) | 0.21 | 0.9 (0.5–1.8) | 0.78 |
TIMP-1 | 0.8 (0.4–1.5) | 0.51 | 0.8 (0.4–1.5) | 0.46 |
MMP-8/TIMP-1 molar ratio | 1.5 (0.8–2.8) | 0.20 | 1.3 (0.6–2.5) | 0.50 |
Odds ratios for death or any neurological sequelae
Variable | Unadjusted OR (95% CI) | Adjusted OR |
||
---|---|---|---|---|
MMP-8 | 2.2 (1.3–3.8) | 0.003 | 2.2 (1.2–4.0) | 0.007 |
MPO | 1.8 (1.1–3.1) | 0.02 | 1.6 (0.9–2.8) | 0.12 |
Total MMP-9 | 1.8 (1.1–3.0) | 0.03 | 1.2 (0.7–2.2) | 0.47 |
TIMP-1 | 1.4 (0.8–2.3) | 0.24 | 1.2 (0.7–2.2) | 0.45 |
MMP-8/TIMP-1 molar ratio | 1.2 (0.7–2.0) | 0.49 | 1.1 (0.6–2.0) | 0.70 |
The risk of death estimated for different concentrations of MMP-8 using local regression. The vertical dashed line represents the median MMP-8 concentration, while the area shaded in grey shows the 95% confidence interval.
When neurological deficits were included in the calculations, the results remained similar. For death or severe neurological sequelae, the adjusted OR for median-cut MMP-8 was 2.3 (95% CI 1.2–4.6); for death or any neurological sequelae, the equivalent OR was 2.2 (95% CI 1.2–4.0) (Tables
A CSF MPO concentration above the median doubled the odds of death or severe neurological sequelae (OR 2.2, 95% CI 1.1–4.4). However, it did not increase the odds of death or any neurological sequelae. Adjusted median-cut values for CSF MMP-9, TIMP-1, or the molar ratio of MMP-8 and TIMP-1 did not predict worse composite outcomes (Tables
Our results suggest that elevated MMP-8 levels in the CSF of children with BM predict poor disease outcomes, especially an increased risk of death. Furthermore, we demonstrated that the actions of MPO, MMP-8, and MMP-9 during the proinflammatory burst of BM run parallel to each other, counterbalanced by TIMP-1.
Prior data on these issues are sparse. A few studies have registered upregulation of MMP-8 in CSF [
Previous data suggest that MPO can oxidatively modify the function of MMP-8, MMP-9, and TIMP-1 [
The comparison of the MMP concentrations with the other CSF parameters revealed that MMP-8 related to the CSF white cell count, while MMP-9 did not. The contradictory results of previous studies suggest that CSF pleocytosis is merely one of several factors affecting the amount of MMPs in the CSF [
MMP-8 and MMP-9 differed in terms of correlation with the patient’s presenting condition. Consistent with our previous study [
We acknowledge limitations in our study. Due to increased vaccination against
In conclusion, CSF MMP-8 presents as an attractive prognostic marker for BM in children. Our results, however, warrant a prospective study with a control group to validate these findings and to further elucidate the potential of this molecule, for example, in the differential diagnosis of meningitis. Rapid quantitative point-of-care tests for MMP-8 in salivary/body fluids are already available [
The datasets analyzed during this study are not publicly available due to patient-related confidentiality. However, these data are available from the corresponding author on reasonable request.
Prof. Timo Sorsa is an inventor of US patents 5652223, 5736341, 5866432, and 6143476. All the other authors report no conflict of interest.
The authors thank all the participating patients and their families in Latin America for providing these unique data for research. Furthermore, we thank Mitja Lääperi for statistical assistance. This work was supported by Finska Läkaresällskapet; Stiftelsen Dorothea Olivia, Karl Walter och Jarl Walter Perkléns Minne; Päivikki ja Sakari Sohlbergin Säätiö; Lastentautien Tutkimussäätiö; a King Khalid University scholarship, administered through the Saudi Arabian Culture Mission in Germany; the Finnish Medical Foundation; the Helsinki University Hospital Research Foundation under grants (TYH 2016251, TYH 2017251, TYH 2018229, Y1014SLO17, and Y1014SLO18); and Karolinska Institutet, Stockholm, Sweden.