New biomarkers for stage determination in Trypanosoma brucei rhodesiense sleeping sickness patients.

0.32 MB·English

by  Tiberti, Natalia

Tibertietal.ClinicalandTranslationalMedicine2013,2:1
http://www.clintransmed.com/content/2/1/1
RESEARCH Open Access
New biomarkers for stage determination in
Trypanosoma brucei rhodesiense sleeping
sickness patients
Natalia Tiberti1, Enock Matovu2, Alexandre Hainard1, John Charles Enyaru3, Veerle Lejon4, Xavier Robin1,
Natacha Turck1, Dieudonné Mumba Ngoyi5, Sanjeev Krishna6, Sylvie Bisser7,8, Bertrand Courtioux7,8,
Philippe Büscher4, Krister Kristensson9, Joseph Mathu Ndung'u10 and Jean-Charles Sanchez1*
Abstract
Accurate stagedetermination is crucial in the choice of treatment for patients suffering from sleeping sickness, also
known as human African trypanosomiasis (HAT).Current staging methods, based onthecounting of white blood
cells(WBC) and thedetectionof parasites inthe cerebrospinalfluid (CSF) have limited accuracy. We hypothesized
that immunemediators reliable for stagingT. b.gambiense HAT could also be used to stratify T. b.rhodesiense
patients, the less common form ofHAT.
A population comprising 85 T.b. rhodesiense patients, 14 stage 1(S1) and 71 stage 2 (S2) enrolled in Malawi and
Uganda, was investigated. The CSF levels ofIgM, MMP-9, CXCL13, CXCL10, ICAM-1, VCAM-1, neopterin and B2MG
were measuredand their staging performances evaluated using receiveroperating characteristic (ROC) analyses.
IgM, MMP-9 and CXCL13 were themost accurate markers for stage determination(partialAUC 88%, 86% and 85%,
respectively). The combinationin panels of threemoleculescomprising CXCL13-CXCL10-MMP-9 or
CXCL13-CXCL10-IgM significantly increased their staging ability to partial AUC 94% (pvalue < 0.01).
The present study highlighted new potential markers for stage determinationof T.b. rhodesiense patients. Further
investigations are needed to better evaluate these molecules,alone or in panels, as alternatives to WBC to make
reliable choice oftreatment.
Keywords: Sleepingsickness,Biomarkers,Trypanosomabruceirhodesiense,Cerebrospinalfluid,Stagedetermination
Background ultimately lead to coma and death, when untreated [3].
Human African trypanosomiasis (HAT), commonly The two forms of HAT differ in their clinical presenta-
known as sleeping sickness, is a neglected tropical dis- tions and geographic distribution. The gambiense form
ease caused by the Trypanosoma brucei parasite and iswidespreadinCentralandWesternAfrica andiscom-
transmitted to humans through the bite of the tsetse fly monlyconsidered tobeachronicinfection,whichslowly
[1]. Two morphologically identical subspecies of para- progresses from the first to the second stage. The rhode-
sites are responsible for the disease: Trypanosoma brucei siense form of sleeping sickness, that affects communi-
gambiense and T. b. rhodesiense [2]. In both cases, the ties in Eastern Africa, is a more aggressive illness, which
disease progresses from a haemolymphatic first stage rapidly progresses to the meningo-encephalitic stage [3]
(S1), to a meningo-encephalitic second stage (S2). The and accounts for less than 5% of all HATcases [4]. Con-
latter reflects invasion of the central nervous system trary to T. b. gambiense, for which a relatively safe drug
(CNS) by the parasites across the blood–brain barrier combination has recently been introduced for treatment
(BBB) with severe neurological complications, which can of S2 patients [4-6], treatment of S2 T. b. rhodesiense
patients still relies on melarsoprol [7-9]. Melarsoprol has
*Correspondence:[email protected] been reported to cause reactive encephalopathies in 8%
1DepartmentofHumanProteinSciences,UniversityofGeneva,Geneva, of T. b. rhodesiense treated patients, which are fatal in
Switzerland
57% of them [8]. As a drug to safely treat both stage 1
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©2013Tibertietal.;licenseeSpringer.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproduction
inanymedium,providedtheoriginalworkisproperlycited.
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and stage 2 patients is yet to be identified, and as S2 (NEUROTRYP study [13]) and Uganda (FINDTRYP
treatment is associated with severe side effects and tox- study), in regions endemic for T. b. rhodesiense HAT.
icity [8], stage determination remains a key step in the The studies were approved by the Ministry of Health
management of patients suffering from T. b. rhodesiense and Population, Lilongwe, Malawi and by the Uganda
HAT. NationalCouncil forScienceandTechnology(UNCST).
Staging is based on the examination of the cerebro- All patients signed a written informed consent before
spinal fluid (CSF) by microscopy. According to WHO, inclusion into the study. Children (< 18 years old) or
patients having ≤ 5 white blood cells (WBC) per micro- patients with altered mental status were only included in
liter of CSF and absence of parasites are considered to the studies after written consent of a parent or a guard-
be in the first stage of the disease, while patients having ian. All enrolled patients had the possibility to withdraw
more than 5 WBC/μL and/or presence of parasites in at any moment. Details on sample collection, inclusion
the CSF are considered as S2 [10]. These methods suffer and exclusion criteria of the two cohorts are reported in
from limited specificity and reproducibility of the count- Additionalfile1:Table1.
ing of WBC and lack of sensitivity in finding of parasites CSFsampleswerecollectedbylumbarpunctureandthe
in CSF [11,12] (Dieudonné Mumba Ngoyi, personal number of WBC counted. The presence of parasites was
communication). determinedusingeitherthemodifiedsinglecentrifugation
The discovery of surrogate markers to complement or (Malawi) [24] or double centrifugation (Uganda) [25]
replace the counting of WBC in the staging of HAT is methods. CSF samples were stored in liquid nitrogen at
highly desired [11,13,14]. Many studies have focused on the site of collection, followed by storage at −80°C.
thestaginginT.b.gambienseHAT[13,15-20],whileless Patients were diagnosed, staged and treated for HAT
attention has been paid to T. b. rhodesiense, with a pau- according to the guidelines of the national sleeping sick-
city of data on staging markers [13,21,22]. Some pro- nesscontrolprogramofthecountryofsamplecollection.
and anti-inflammatory factors have been shown to be Inthepresentstudy,patients’stagewasassignedaccord-
associated with the late stage of T. b. rhodesiense sleep- ing to WHO recommendations [10], i.e., stage 1 when
ing sickness, including IL-10, IL-6, CXCL10 and neop- CSF WBC ≤ 5/μL and absence of parasite in CSF, stage 2
terin[13,21,22]. when CSF WBC > 5/μL and/or parasites detected in the
The aim of the present study was to investigate eight CSF. Patients were excluded when information to classify
immune-related factors, shown to be powerful markers themaccordingtothesecriteriawasnotavailable.
for stratificationof T. b. gambienseHAT patients [13,15-
20,23], as staging markers for T. b. rhodesiense sleeping Immunoassays
sickness. The levels of the markers were measured in pre-
treatment CSF using commercially available immunoas-
Methods says (ELISA or multiplex bead suspension assay) follow-
Patients ing manufacturers’ instructions as reported elsewhere
Eighty five patients (14 stage 1 and 71 stage 2) with evi- [23]. These included IgM (ICL, OR, USA), B2MG
dence of parasites in blood, lymph or CSF were investi- (Calbiotech, CA, USA), neopterin (BRAHMS, Germany),
gated in the present study (Table 1). Patients were CXCL10 (Bio-Rad, CA, USA), VCAM-1, ICAM-1,
enrolled by active or passive case finding in Malawi CXCL13andMMP-9(R&DSystems,UK).
Table1Pre-treatmentcharacteristicsoftheinvestigated Statistics
patients
Statistical analyses were performed using IBM SPSS Sta-
Stage1(n=14) Stage2(n=71) tistics version 20.0.0 (IBM, NY, USA). Comparisons be-
Demography tween groups were performed using the Mann–Whitney
Sex,F(n)* 7 32 U test, setting the level of significance at 0.05. Correla-
Age,years[mean±SD]† 37.1[±19.3] 36.9[±15.8] tions between molecules and the number of WBC were
assessed through the Spearman correlation rho coeffi-
Geographicalorigin
cient. ROC analyses were performed using pROC pack-
Malawi,n 3 27
age for S+ version 8.1 (TIBCO, Software Inc.) [26]. All
Uganda,n 11 44 testsweretwo-tailed.
CSFexaminations Toassessthestagingabilityofeachmarkerandtocom-
Trypanosomepositive,n 0 64 pare their performances at high specificity, corrected par-
WBC/μL(median,range) 3[2–5] 21[4–1140] tial areas under the ROC curves (pAUC) were calculated
between 90 and 100% of specificity (SP) [26]. A cut-off
*Fisher’sexacttest,nosignificantdifferences.
†Mann–WhitneyUtest,nosignificantdifferences. correspondingto100%specificitywasalsocomputed.
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To evaluate the power ofthemarkersin predicting the Stagingabilityofthemarkers
presence of trypanosomes in CSF, patients were classi- ROC analyses were performed to further assess the abil-
fied based on the absence (n=21) or presence (n=64) of ity of the markers to discriminate between S1 and S2
parasites in their CSF. Complete AUC were then com- patientsintermsofspecificity andsensitivity.
puted as well as the cut-off corresponding to the best To highlight markers able to correctly rule out the
combinationofspecificity (SP) andsensitivity (SE). highest number of S1 patients, partial AUC between 90
Panels of markers were obtained using an in-house and 100% of specificity were calculated together with a
software, PanelomiX, based on a method of optimization cut-off in marker concentration at 100% of SP (Figure 1,
of cut-off values by iterative combination of biomarkers Additionalfile1:Table2).
and thresholds (rule-induction-like method) [18]. Highly IgM, MMP-9 and CXCL13 had a pAUC higher than
specific combinations of three molecules were com- 80%, but for 100% of specificity IgM showed higher sen-
puted. Those showing the highest pAUC for discrimin- sitivity (SE 77%, 76.6-87.3 95% CI), compared to MMP-9
ation between stage 1 and stage 2 patients were kept. (72% SE, 60.6-81-7 95% CI) and CXCL13 (69% SE,
Statistical comparison between the pAUC of the panels 57.8-80.395%CI).
and those of the individual markers was obtained A second group of markers with a pAUC between 70-
through the Bootstrap test for two correlated ROC 80% comprised VCAM-1, B2MG, ICAM-1 and neop-
curves. terin. CXCL10 was highlighted as the less accurate mar-
ker, with a pAUC of only 55% and 3% sensitivity for
Results 100%specificity.
Concentrationofmarkersinpatients’CSF To further assess the ability of the eight molecules to
The CSF levels of IgM, B2MG, MMP-9, CXCL13, identify patients with advanced S2 HAT, patients were
CXCL10, ICAM-1, VCAM-1 and neopterin were mea- classified based on the absence (T-, n=21) or the pres-
sured on a population of 85 patients, comprising 14 ence (T+, n=64) of parasites in the CSF. The total area
stage 1and71stage 2(Table1). under the ROC curve was considered. All markers, ex-
All molecules showed a higher CSF concentration in cept neopterin and CXCL10, discriminated between
S2 patients compared to S1. IgM, MMP-9 and CXCL13 T- and T+ patients with AUC > 80% and with perfor-
showed the highest fold increase (S2/S1 concentration mances comparable to those of WBC (95% CI around
ratio of 68, 12 and 187, respectively). However, the com- AUC overlapping)(Table3).
parison using the Mann–Whitney U test, highlighted Interestingly,whenthespecificityandsensitivitycorre-
significant differences between the two stages for all spondingtothebest cut-offweretaken into account,the
markers (Table 2). These differences were confirmed three best markers, i.e. IgM, MMP-9 and CXCL13,
when patients from Uganda (S1 n=11; S2 n=44) were turned out to be more specific (SP > 85%) than WBC,
considered separately, while only MMP-9, IgM and which inturnwasmoresensitive(SE>95%) (Table3).
B2MG could significantly discriminate between Mala-
wian S1 (n=3) and S2 (n=27) patients (Additional Combinationofmarkersintopanels
Figure 1). Furthermore,allmarkerssignificantlycorrelated To evaluate whether a combination of markers could in-
(Spearman correlation) with the number of WBC counted crease the staging ability, panels of three molecules cor-
in the CSF, the current staging method, with MMP-9, responding to 100% specificity were calculated. Two
CXCL13 and CXCL10 having a rho coefficient > 0.5 different combinations showing the same staging perfor-
(Table2). mances (pAUC 94%, 89.9-97.3 95% CI; SE 87.3%, 78.9-
Table2Concentrationofmarkersinearly(S1)andlate(S2)stageT.b.rhodesiensepatients
Marker [S1],median [S2],median [S2]/[S1] pvalue* Spearmanrho†
IgM[μg/mL] 0.96 65.4 68.1 <0.0001 0.491
MMP-9[pg/mL] 108.6 1309.9 12.1 <0.0001 0.554
CXCL13[pg/mL] 8.2 1531.2 186.7 <0.0001 0.529
VCAM-1[ng/mL] 22.6 67.3 3.0 <0.0001 0.372
B2MG[ng/mL] 964 3447 3.6 <0.0001 0.426
ICAM-1[ng/mL] 1.99 9.6 4.8 <0.0001 0.457
Neopterin[nmol/L] 41.2 112.9 2.7 0.001 0.360
CXCL10[ng/mL] 8.9 41.9 4.7 0.005 0.508
*Mann–WhitneyUtest.
†CorrelationbetweenthenumberofCSFWBCandCSFlevelsforeachmarker.Correlationwassignificantat0.01level.
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Figure1ROCcurvesrepresentingthestagingabilitiesoftheeightmarkers.DarkgrayareasrepresentthecorrectedpAUCbetween90and
100%ofspecificityobtainedforeachmarker.LightgrayzonesrepresentapAUCof100%.Thevalueofthecut-offcorrespondingto100%
specificitycomprisedwithinthepAUCisreportedoneachgraphtogetherwiththecorrespondingsensitivity%(SE%).Additionalresultsare
reportedinAdditionalfile1:Table2.
Table3AbilityofmarkerstoclassifyT.b.rhodesiensepatientsaccordingtothepresenceofparasitesinCSF
Marker pvalue* AUC%(95%CI) Cut-off SP%(95%CI) SE%(95%CI)
IgM[μg/mL] <0.0001 84.5(73.9-95.2) 17.8 85.7(66.7-100) 81.3(71.9-90.6)
MMP-9[pg/mL] <0.0001 85.2(74.3-96) 499.8 90.5(76.2-100) 76.6(65.6-85.9)
CXCL13[pg/mL] <0.0001 80.4(69.1-91.8) 200.3 90.5(76.2-100) 73.4(62.5-84.4)
VCAM-1[ng/mL] <0.0001 83.2(73.7-92.7) 43.9 76.2(57.1-90.5) 78.1(67.2-87.5)
B2MG[ng/mL] <0.0001 82.0(71.3-92.7) 1462 66.7(47.6-85.7) 85.9(76.6-93.8)
ICAM-1[ng/mL] <0.0001 83.3(73–93.6) 4.7 81.0(61.9-95.2) 73.4(62.5-84.4)
Neopterin[nmol/L] <0.0001 75.5(63.9-87.1) 69.4 81.0(61.9-95.2) 65.6(54.7-76.6)
CXCL10[ng/mL] 0.002 73.1(59.3-86.8) 7.5 47.6(28.6-66.7) 92.2(84.4-98.4)
WBC(Cells/μL) <0.0001 87.4(77.4-97.4) 6.5 71.4(52.4-90.5) 95.3(89.1-100)
PatientswithoutparasitesdetectedinCSF,n=21;patientswithparasitedetectedinCSF,n=64.
*Mann–WhitneyUtest.
SP%=specificity%;SE%=sensitivity%;95%CI=95%confidenceinterval.
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94.4 95% CI) were obtained. Both panels comprised behavior of the 8 molecules was observed in T. b. gam-
CXCL10 (cut-off 2.24 ng/mL) and CXCL13 (cut-off 23.3 biense patients, which may reflect the differences in
pg/mL) in combination with either MMP-9 (cut-off immunopathogenesis [29] and clinical presentation
499.8 pg/mL) or IgM (cut-off 17.8 μg/mL) (Table 4). [3,30] of the two forms of HAT. It has already been pro-
Both panels were considered positive when at least two posed, for example, that different activation pathways of
out ofthreemoleculeswereabovetheircut-offs. macrophages and astrocytes may take place in the two
When compared to the individual molecules, the two forms of HAT [22]. Such differences may be responsible
panels were significantly more accurate for stage deter- of the less accurate staging ability of neopterin on T. b.
mination (Bootstrap test for two correlated ROC curves, rhodesiense patients, compared to its very high staging
p<0.05). These combinations enabledthe correctclassifi- poweronT.b.gambiensepatients.
cation of all S1 patients (100% SP) and 62 out of 71 S2 The role of IgM, the best individual marker in the
patients(87% SE). present study, in disease progression has been exten-
sively studied. An increased CSF concentration of IgM
Discussion ofintrathecaloriginwasshownto bea good indicator of
Stage determination in T. b. rhodesiense sleeping sick- brain involvement in HAT [15], leading to the develop-
ness patientsis acriticalstep inensuring thatthe appro- ment of a rapid latex agglutination test (Latex/IgM) for
priate treatment is used [14]. An imperfect gold stage determination in the field [31]. However, when
standard for staging and the lack of a safe S2 drug high- assessed under field conditions, this assay did not repre-
light the need for new tools for staging this form of dis- sent an advantage compared to counting of WBC [31].
ease [27,28]. In the present study we investigated, on a Furthermore, when used for evaluation of the outcome
small population of patients suffering from T. b. rhode- aftertreatment,IgMlevelswerenotanoptimalindicator
siense HAT, a number of molecules (MMP-9, CXCL10, of recovery due to their slow normalization [32]. Studies
CXCL13, IgM, neopterin, ICAM-1, VCAM-1 and in animal models have shown that HAT meningo-
B2MG) known to be over-expressed in the CSF of late encephalitis is characterized by an increased number of
stage T. b. gambiense patients [23]. Since melarsoprol is leukocytes in the CNS [33]. CXCL13, also known as
still the only treatment for S2 rhodesiense patients, we BCA-1, is a chemokine mainly produced by dendritic
evaluated their staging ability as highly specific markers, cells [34], which specifically attracts B and T lympho-
to try to limit unnecessary exposure of patients to this cytes to the site of inflammation [35]. Its over-
toxic drug. IgM, MMP-9 and CXCL13 were shown to be expression in CSF has been associated with increased
the most accurate discriminators between early and late WBC and intrathecal production of immunoglobulins in
stagedisease(pAUC≥85%)andshowedthesameaccur- many pathological conditions [36,37], including late
acy as WBC in distinguishing between patients having stage T. b. gambiense HAT [19]. On the other hand,
parasites in their CSF from those without. Furthermore, MMP-9 (matrix-metalloproteinase 9), an enzyme invol-
combination of the molecules into panels of three mar- ved in tissue homeostasis and remodeling [38,39], has
kers (IgM-CXCL13-CXCL10 or MMP-9-CXCL13- been extensively studied in a number of pathologies af-
CXCL10) significantly increased the staging accuracy, fecting the CNS [39-42], in addition to T. b. gambiense
leading to the correct classification of all S1 patients and HAT [17]. Due to its ability to degrade β-dystroglycan,
62out of71S2patients. this protein has been proposed to be involved in the pas-
All the markers investigated here are known to be sage of leukocytes through the glia limitans to reach the
involved in the immune response elicited by the pres- brainparenchyma[43].However,thetemporalrelationship
ence of the parasite in the host. Interestingly, a different between the events leading to CNS invasion and the
Table4PanelsofmarkersforstagingT.b.rhodesiensepatientsobtainedthroughacombinationof3molecules
Panel Markers Cut-off pAUC%(95%CI)* SE%(95%CI)* pvalue†
1 CXCL10[ng/mL] 2.2 94(89.9-97.3) 87.3(78.9-94.4) 0.0001
CXCL13[pg/mL] 23.3 0.01
MMP-9[pg/mL] 499.8 0.01
2 CXCL10[ng/mL] 2.2 94(89.9-97.3) 87.3(78.9-94.4) 0.0001
CXCL13[pg/mL] 23.3 0.01
IgM[μg/mL] 17.8 0.02
*pAUCandSE%werecalculatedfor100%SP.
†ComparisonbetweenthepAUC(90-100%SP)ofthepanelandthoseofthemarkersindividuallyconsideredobtainedthroughtheBootstraptestfortwo
correlatedROCcurves.
Bothpanelsarepositivewhen≥2moleculesareabovetheircut-off.
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appearanceof varioussignsandsymptomsofnervoussys- highly toxic stage 2 drug, respectively. However, it
temdysfunctionneedstobeinvestigatedfurther. should be emphasized that a new staging biomarker for
The markers investigated in the present study were rhodesiense HAT would be combined with the detection
combined into panels in order to increase their accuracy of parasites in CSF, which would increase the sensitivity,
in stage determination. The utility of this approach to and with clinical evaluation of the neurological status of
achieve a better diagnostic accuracy has already been thepatients.
shown [18,44]. Using this method, we highlighted highly Theabsenceofinformationonneurologicalsignsexhib-
specific combinations comprising CXCL13, CXCL10 and itedbypatientsinthepresentstudypreventedanefficient
MMP-9 or IgM. Interestingly, CXCL10 was present in assessment of the association between the levels of the
both panels. This molecule was not efficient in staging markers andthesigns ofCNS involvement.Thisaspectis
T. b. rhodesiense patients when considered individually. particularly important in the light of a recent publication
However, when combined to CXCL13 and MMP-9 or on T. b. rhodesiense HAT reporting the poor association
IgM,ithelpedinreachingasignificantlyincreasedstaging between disease progression, the levels of a number of
accuracy. This chemokine, which specifically attracts T cytokinesandpatients’neurologicalmanifestations[51].
lymphocytestothesiteofinflammation[45],wasreported The 8 markers investigated here behaved differently
to be produced by activated astrocytes in trypanosome- when assessed on T. b. gambiense or T. b. rhodesiense
infected mice [46]. The activation of astrocytes and ma- samples, underlining the differences between the two
crophages are early events in stage 2 infection [47-49], forms of disease, and suggesting that potentially new
suggesting that CXCL10 may represent an early indicator rhodesiense-specificmarkerscouldbediscovered.
ofCNSinvolvementinHAT. Despite the high staging accuracy shown by the com-
Interestingly, the markers did not show the same sta- binations of markers described in the present study (i.e.
ging performances when assessed on patients classified CXCL10-CXCL13-MMP-9 and CXCL10-CXCL13-IgM),
according to their geographic origin (i.e. Malawi or their translation into a rapid field diagnostic test could
Uganda). Although the low number of Malawian S1 be difficult, due to a potential increase in the costs of
patients (n=3) certainly represents a bias and may be re- production, suggesting that deeper investigations should
sponsible for the differences observed, this result could be performed. The individual staging power of the mole-
reflect the variable clinical presentation of rhodesiense cules should be assessed on a larger cohort of T. b. rho-
disease observed in different foci [27]. This may suggest desiense HAT patients, including CSF samples collected
that potentially different markers will be needed to stage duringthepost-therapeuticfollow-up,andthepossibility
T. b. rhodesiense patients according to their geographical of their translation into a point-of-care test for stage de-
origin andtheparasitestrain. termination in the field should be evaluated. Further-
The present study has a number of limitations that more, their study in animal models, as already done for
should be considered. First, the data presented resulted IL-10 [52], could help in the further characterization of
from analyses on a small number of patients. This is a therole ofthesemarkersindiseaseprogression.
common problem associated to the investigation of this
form of HAT. Collecting samples from T. b. rhodesiense Conclusions
patients is considerably difficult, not only due to the The results presented in this work on T. b. rhodesiense
lower incidence of this disease compared to the gam- sleeping sickness highlight the potential utility of IgM,
biense form, but also as a consequence of a less effective MMP-9 and CXCL13, alone or combined with CXCL10
active screening, since the CATT test can only detect T. in staging patients. We believe that this work has paved
b. gambiense cases [50]. To further evaluate the staging the way for further investigations on the role of these
properties of the markers, larger cohorts of patients markers in detecting the meningo-encephalitic stage of
should be investigated. Moreover, due to the reported T. b. rhodesiense HAT, and therefore making a more ac-
differences between T. b. rhodesiense HAT among foci, curate choice oftreatment.
the results presented here should be validated in a more
controlled set of patients (i.e. in which the same para-
Additional file
sitologicalexaminationswereperformed).
Another drawback could be represented by the choice
Additionalfile1:Table1.Detailsofthestudiesfromwhichsamples
of selecting highly specific markers, with the conse- wereobtained.Table2.Detailedcalculationfortheevaluationofthe
quence of compromising the sensitivity. Management of stagingabilityoftheeightmarkers.Figure1.Comparisonofthelevelsof
themarkersbetweenstage1(S1)andstage2(S2)T.b.rhodesiense
T. b. rhodesiense patients is far from being optimal, thus
patientsclassifiedaccordingtothecountryofsamplecollection.Foreach
both choices of high specificity or sensitivity would be country,differencesbetweenS1andS2wereassessedusingtheMann-
associated either toariskofmissingthe diagnosis oflate WhineyUtest.*correspondstoapvalue<0.05;**correspondstoap
value<0.001;***correspondstoapvalue<0.0001.
stage patients, or to the exposure of S1 patients to a
Tibertietal.ClinicalandTranslationalMedicine2013,2:1 Page7of8
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Abbreviations 8. BurriC:ChemotherapyagainsthumanAfricantrypanosomiasis:istherea
HAT:HumanAfricantrypanosomiasis;WBC:Whitebloodcells; roadtosuccess?Parasitology2010,137:1987–1994.
CSF:Cerebrospinalfluid;ROC:Receiveroperatingcharacteristic;AUC:Area 9. KuepferI,SchmidC,AllanM,EdieluA,HaaryEP,KakemboA,KibonaS,Blum
undertheROCcurve;pAUC:PartialAUC;S1:Stage1;S2:Stage2;CNS:Central J,BurriC:Safetyandefficacyofthe10-daymelarsoprolscheduleforthe
nervoussystem;WHO:Worldhealthorganisation;NECT:Nifurtimox- treatmentofsecondstagerhodesiensesleepingsickness.PLoSNeglTrop
eflornithinecombinationtherapy;SP:Specificity;SE:Sensitivity; Dis2012,6:e1695.
CI:Confidenceinterval.. 10. WHO:ControlandsurveillanceofAfricantrypanosomiasis.ReportofaWHO
ExpertCommittee.WorldHealthOrganTechRepSer1998,881(I-VI):1–114.
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Competinginterests
JosephNdung’uisanemployeeoftheFoundationforInnovativeNew stageornottostage?TransRSocTropMedHyg2008,102:306–307.
Diagnostics(FIND).VeerleLejon,PhilippeBüscherandSanjeevKrishnawere 12. SimarroPP,JanninJ,CattandP:EliminatinghumanAfrican
consultantsforFINDatthetimeofthestudy.Allotherauthorsdeclarethat trypanosomiasis:wheredowestandandwhatcomesnext?PLoSMed
theyhavenocompetinginterests. 2008,5:e55.
13. AminDN,NgoyiDM,NhkwachiGM,PalombaM,RottenbergM,BuscherP,
KristenssonK,MasochaW:Identificationofstagebiomarkersforhuman
Authors’contributions Africantrypanosomiasis.AmJTropMedHyg2010,82:983–990.
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1DepartmentofHumanProteinSciences,UniversityofGeneva,Geneva, H-FABPpanelforthestagingofhumanAfricantrypanosomiasis
Switzerland.2DepartmentofBiotechnicalandDiagnosticsSciences,College patients.PLoSNeglTropDis2009,3:e459.
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Kinshasa,D.R.Congo.6DivisionofCellularandMolecularMedicine,St. immunoglobulinscomparedtofielddiagnosisofcentralnervoussystem
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