Value of lipocalin 2 as a potential biomarker for bacterial meningitis

Abstract

Objectives: Central nervous system (CNS) infections are common causes of morbidity and mortality worldwide. We aimed to discover protein biomarkers that could rapidly and accurately identify the likely cause of the infections, essential for clinical management and improving outcome.

Methods: We applied liquid chromatography tandem mass spectrometry on 45 cerebrospinal fluid (CSF) samples from a cohort of adults with and without CNS infections to discover potential diagnostic biomarkers. We then validated the diagnostic performance of a selected biomarker candidate in an independent cohort of 364 consecutively treated adults with CNS infections admitted to a referral hospital in Vietnam.

Results: In the discovery cohort, we identified lipocalin 2 (LCN2) as a potential biomarker of bacterial meningitis (BM) other than tuberculous meningitis. The analysis of the validation cohort showed that LCN2 could discriminate BM from other CNS infections (including tuberculous meningitis, cryptococcal meningitis and virus/antibody-mediated encephalitis), with sensitivity of 0.88 (95% confident interval (CI), 0.77-0.94), specificity of 0.91 (95% CI, 0.88-0.94) and diagnostic odds ratio of 73.8 (95% CI, 31.8-171.4). LCN2 outperformed other CSF markers (leukocytes, glucose, protein and lactate) commonly used in routine care worldwide. The combination of LCN2, CSF leukocytes, glucose, protein and lactate resulted in the highest diagnostic performance for BM (area under the receiver operating characteristics curve, 0.96; 95% CI, 0.93-0.99). Data are available via ProteomeXchange with identifier PXD020510.

Conclusions: LCN2 is a sensitive and specific biomarker for discriminating BM from a broad spectrum of other CNS infections. A prospective study is needed to assess the diagnostic utility of LCN2 in the diagnosis and management of CNS infections.

Keywords: Biomarkers; Central nervous system infections; Lipocalin 2; Mass spectrometry; Meningitis.

Fig. 1

Overview of protein marker discovery phases and origin of clinical samples used for the analysis. Patients of the discovery cohort include TBM (n = 20), BM (n = 10), encephalitis (n = 10) and five patients with non-CNS infection. Of the ten patients with cBM, seven were infected with Streptococcus suis and three with S. pneumoniae and of the patients with encephalitis, HSV was the cause in 5, DENV in 3, JEV in 1 and MuV in 1. ^#Including: S. suis (n = 20), S. pneumoniae (n = 6), Escherichia coli (n = 5), Neisseria meningitidis (n = 2), Burkholderia pseudomallei (n = 1), Enterococcus faecalis (n = 1), Enterococcus gallinarum (n = 1), Streptococcus agalactiae (n = 1), Staphylococcus aureus (n = 1), Streptococcus gallolyticus subsp. gallolyticus (formerly known as S. bovis type I) (n = 1) and Gram staining positive only (n = 5). ∗Including: HSV (n = 11), varicella zoster virus (n = 7), DENV (n = 5), JEV (n = 2), DENV/JEV (n = 1), MuV (n = 1), measles virus (n = 1) and influenza A virus (n = 1). $Including: cryptococcal meningitis (n = 14), anti-NMDAR encephalitis (n = 17), eosinophilic meningitis (n = 10) and neurotoxoplasmosis (n = 2). BM, bacterial meningitis; cBM, confirmed bacterial meningitis; cEN, confirmed encephalitis; cTBM, confirmed tuberculous meningitis; DENV, dengue virus; EN, encephalitis; HSV = herpes simplex virus; JEV, Japanese encephalitis virus; MuV, mumps virus; NI, non-CNS infections; NMDAR, N-methyl-d-aspartate receptor; sBM, clinically suspected bacterial meningitis; sEN, clinically suspected encephalitis; sTBM, clinically suspected tuberculous meningitis; TBM, tuberculous meningitis.

Fig. 2

Results of LC-MS/MS and LCN2 ELISA analysis of discovery cohort. (A) Heat map showing clinical entities clustering based on protein/peptide profiles obtained from label-free quantitative MS analysis of 45 patients of the discovery phase. Columns represent clinical entities; rows, individual proteins; and black line, relative position of LCN2. (B) Dot plots demonstrating the difference in CSF LCN2 levels between BM and non-BM groups obtained from quantitative ELISA analysis, (C) AUROC based on LCN2 levels measured by MS (blue line) and quantitative ELISA (red line) analysis. Colors indicate differentially expressed proteins: red indicates upregulated; white, unchanged; and blue, downregulated. AUROC, area under the receiver operating characteristics curve; BM, bacterial meningitis; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; LC-MS/MS, liquid chromatography tandem mass spectrometry; LCN2, lipocalin 2; MS, mass spectrometry; non-BM, nonbacterial meningitis (encephalitis, tuberculous meningitis or non–central nervous system infections).

Fig. 3

Results of LCN2 ELISA and AUROC analysis of validation cohort. (A) LCN2 concentrations in patients with meningitis, tuberculous meningitis, encephalitis and others (cryptococcal meningitis, anti-NMDAR encephalitis, eosinophilic meningitis, neurotoxoplasmosis and non-CNS infections). (B) AUROC showing diagnostic values of LCN2 in discriminating bacterial meningitis from other CNS infection entities. Others indicates patients with other CNS infections (cryptococcal meningitis, anti-NMDAR encephalitis, neurotoxoplasmosis or eosinophilic meningitis) or non-CNS infections. AUROC, area under the receiver operating characteristics curve; BM, bacterial meningitis; CNS, central nervous system; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; LCN2, lipocalin 2; NMDAR, N-methyl-d-aspartate receptor; non-BM, nonbacterial meningitis.

Fig. 4

Diagnostic values of LCN2 in predicting bacterial meningitis in comparison and in combination with existing CSF parameters. (A) AUROC showing that LCN2 is better than the existing CSF parameters in distinguishing between bacterial meningitis with other CNS infections (tuberculous meningitis, encephalitis, anti-NMDAR encephalitis, cryptococcal meningitis, neurotoxoplasmosis, eosinophilic meningitis or non-CNS infections). (B) AUROC values of subgroup analyses. (C) AUROC showing that LCN2 significantly improves the discriminatory ability of the diagnostic model for bacterial meningitis using the remaining CNS infections groups as controls. (D) AUROC values of subgroup analyses. AUROC, area under the receiver operating characteristics curve; CNS, central nervous system; CSF, cerebrospinal fluid; LCN2, lipocalin 2; NMDAR, N-methyl-d-aspartate receptor; WCC, white blood cell (leukocyte) count.