Luminex multiplex bead array technology

The CSF mycobacterial load was inferred qualitatively by comparing patients with negative versus positive CSF culture, and semiquantitively from the GeneXpert Ct-values as described previously (Thuong et al., 2019 (link)), and inferred from CSF M. tuberculosis culture. CSF inflammatory cytokines in 178 Indonesian HIV-negative TBM patients were measured using a multiplex proximity extension assay (Olink) in two batches. Olink uses a multiplex assay that simultaneously recognize 96 target proteins through specific paired-antibodies which are coupled with unique oligonucleotides, for quantitative PCR measurement (Assarsson et al., 2014 (link)). For each protein, overlapping samples from two batches were fitted in a linear regression model, where the linear components were subsequently extracted, and used as correction factors for batches normalization. In 304 Vietnamese HIV-negative patients, 10 human cytokines were measured in CSF with Luminex multiplex bead array technology (Bio-Rad Laboratories, Hercules, CA) (Thuong et al., 2017 (link)). CSF total protein was used as proxy for blood–CSF barrier disruption as it showed a near-perfect correlation with the established marker CSF–serum albumin (r² = 0.98) (Svensson et al., 2020 (link)).

The CSF mycobacterial load was inferred qualitatively by comparing patients with negative versus positive CSF culture, and semiquantitively from the GeneXpert Ct-values as described previously,^{10 (link)} and inferred from CSF M. tuberculosis culture. CSF inflammatory cytokines in 178 Indonesian HIV-negative TBM patients were measured using a multiplex proximity extension assay (Olink) in two batches. Olink uses a multiplex assay that simultaneously recognize 96 target proteins through specific paired-antibodies which are coupled with unique oligonucleotides, for quantitative PCR measurement.^{11 (link)} For each protein, overlapping samples from two batches were fitted in a linear regression model, where the linear components were subsequently extracted, and used as correction factors for batches normalization. In 304 Vietnamese HIV-negative patients, 10 human cytokines were measured in CSF with Luminex multiplex bead array technology (Bio-Rad Laboratories, Hercules, CA).^{4 (link)} CSF total protein was used as proxy for blood-CSF barrier disruption as it showed a near-perfect correlation with the established marker CSF-serum albumin (r²=0.98).^{12 (link)}

Detailed clinical assessments were made at baseline and up to 9 months after randomization. Baseline disease severity was assessed by the modified British Medical Research Council (BMRC) grading system [14 ]. An HIV test was performed on all patients at enrollment. Concentrations of total and differential blood cell counts (including CD4⁺ T-cell counts in HIV-infected patients) and CSF leukocyte counts, lactate levels, glucose levels, and protein levels were measured by standard methods.
A panel of 10 human cytokines was measured in stored CSF specimens by Luminex multiplex bead array technology (Bio-Rad Laboratories, Hercules, CA). The panel consists of interleukin 1β (IL-1β), IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α). Assays were read on the Bio-Plex 200 platform, and the Bio-Plex Manager software 6.0 was used for bead acquisition and analysis. Additional details on the method for cytokine measurement is provided in the Supplementary Materials.