A combined CXCL10, CXCL8 and H-FABP panel for the staging of human African trypanosomiasis patients

Abstract

Background: Human African trypanosomiasis (HAT), also known as sleeping sickness, is a parasitic tropical disease. It progresses from the first, haemolymphatic stage to a neurological second stage due to invasion of parasites into the central nervous system (CNS). As treatment depends on the stage of disease, there is a critical need for tools that efficiently discriminate the two stages of HAT. We hypothesized that markers of brain damage discovered by proteomic strategies and inflammation-related proteins could individually or in combination indicate the CNS invasion by the parasite.

Methods: Cerebrospinal fluid (CSF) originated from parasitologically confirmed Trypanosoma brucei gambiense patients. Patients were staged on the basis of CSF white blood cell (WBC) count and presence of parasites in CSF. One hundred samples were analysed: 21 from stage 1 (no trypanosomes in CSF and <or=5 WBC/microL) and 79 from stage 2 (trypanosomes in CSF and/or >5 WBC/microL) patients. The concentration of H-FABP, GSTP-1 and S100beta in CSF was measured by ELISA. The levels of thirteen inflammation-related proteins (IL-1ra, IL-1beta, IL-6, IL-9, IL-10, G-CSF, VEGF, IFN-gamma, TNF-alpha, CCL2, CCL4, CXCL8 and CXCL10) were determined by bead suspension arrays.

Results: CXCL10 most accurately distinguished stage 1 and stage 2 patients, with a sensitivity of 84% and specificity of 100%. Rule Induction Like (RIL) analysis defined a panel characterized by CXCL10, CXCL8 and H-FABP that improved the detection of stage 2 patients to 97% sensitivity and 100% specificity.

Conclusion: This study highlights the value of CXCL10 as a single biomarker for staging T. b. gambiense-infected HAT patients. Further combination of CXCL10 with H-FABP and CXCL8 results in a panel that efficiently rules in stage 2 HAT patients. As these molecules could potentially be markers of other CNS infections and disorders, these results should be validated in a larger multi-centric cohort including other inflammatory diseases such as cerebral malaria and active tuberculosis.

Figure 1. Scatter plots correlating the level of GR3 molecules with the WBC count.

The horizontal dashed line corresponds to the cutoff value for the molecule that discriminates between S1 and S2 patients with a specificity of 100%. The left vertical dashed line corresponds to the WBC count cutoff value used for staging. The second vertical dashed line indicates the suggested cutoff value for staging. Patients between these lines (>5 and ≤20 WBC/µL) corresponded to potential intermediate stage patients. The diagonal line corresponds to the linear regression.

Figure 2. Box-plot of GR3 molecules and WBC classified according to the presence of the parasite in CSF.

Median and mean are represented as a solid line in the box and a diamond respectively. Whisks are defined as 5^th–95^th percentile without outliers. Half-width of the notch was calculated automatically by the software.

Figure 3. Box-plot of GR3 molecules and WBC classified according to the neurological signs.

Median and mean are represented as a solid line in the box and a diamond respectively. Whisks are defined as 5^th–95^th percentile without outliers. Half-width of the notch was calculated automatically by the software. Neurological signs of two patients were not reported (n = 98).