Neopterin is a cerebrospinal fluid marker for treatment outcome evaluation in patients affected by Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers. CONCLUSIONS/SIGNIFICANCE: In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment

Baseline risk factors for treatment failure in late stage patients.

<p>TT: treatment; E: eflornithine treatment; M: melarsoprol treatment; M-E: combination of melarsoprol and eflornithine; M-N: combination of melarsoprol and nifurtimox.</p><p>n/N: n = number of patients experiencing treatment failure, N = total number of patients.</p><p>RR: risk ratio.</p><p>T−: absence of parasite in CSF; T+: presence of parasites in CSF.</p><p>NS−: absence of neurological sings, NS+: presence of neurological sings.</p><p>Age and WBC/µL categories were defined according to Mumba Ngoyi D. <i>et al.</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002088#pntd.0002088-MumbaNgoyi2" target="_blank">[8]</a>.</p

Performances of neopterin, CXCL13 and WBC in discriminating between S2 cured and S2 relapsed patient.

<p>3 M, 6 M, 12 M: 3, 6, 12 months after treatment.</p><p>Number of patients: 3 M cured n = 113, relapsed m = 105; 6 M cured n = 111, relapsed n = 64; 12 M cured n = 110, relapsed n = 18.</p><p>*pAUC was calculated between 90 and 100% of sensitivity.</p><p>SP% = specificity%; SE% = sensitivity%.</p><p>Cut-off corresponds to the best combination of SP and SE within the pAUC.</p><p>LR+: positive likelihood ratio, LR−: negative likelihood ratio.</p><p>TN: true negatives, number of cured patients correctly classified.</p><p>FN: false negatives, number of relapsing patients wrongly classified as cured.</p

Characteristics at baseline of the verification cohort.

*<p>n = 27, probable relapse.</p>†<p>No significant difference between S2 cured and S2 relapsed, Fisher's exact test.</p>‡<p>No significant difference between S2 cured and S2 relapsed, Mann-Whitney <i>U</i> test.</p>∥<p>Secondary case: patients already treated once for HAT.</p><p>P: pentamidine treatment; E: eflornithine treatment; M: melarsoprol treatment; M-E: combination of melarsoprol and eflornithine; M-N: combination of melarsoprol and nifurtimox. More details on treatment regimens are reported in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002088#pntd.0002088-MumbaNgoyi2" target="_blank">[8]</a>.</p

Kinetics of neopterin, CXCL13 and WBC during the follow-up.

<p>The variation in concentrations of the three markers in S1 cured patients, S2 cured patients and S2 relapsing patients are represented. Median concentrations at each time point are reported. Bars represent inter-quartile intervals. Numbers on the graphs represent the number of CSF samples assessed at each time point for each category of HAT patients. BT: before treatment; EoT: end of treatment; 3 M, 6 M, 12 M: 3, 6, 12 months after treatment. FU: follow-up.</p

Performance of the markers on the screening cohort.

<p>Number of patients: 39 S2 relapsed vs. 39 S2 cured.</p><p>SP%: specificity %; SE%: sensitivity%; 95%CI: 95% confidence interval.</p><p>The reported cut-off corresponds to the best combination of specificity and sensitivity obtained for each marker.</p