w w w . e l s e v ie r . c o m / l o c a t e / b j i d
The
Brazilian
Journal
of
INFECTIOUS
DISEASES
Original
article
In
vitro
basal
T-cell
proliferation
among
asymptomatic
Human
T
cell
Leukemia
Virus
type
1
patients
co-infected
with
hepatitis
C
and/or
Human
Immunodeficiency
Virus
type
1
Tatiane
Assone
a,b,
Tatiana
M.
Kanashiro
a,
Maira
P.M.
Baldassin
a,
Arthur
Paiva
a,b,
Michel
E.
Haziot
c,
Jerusa
Smid
c,
Augusto
Penalva
de
Oliveira
c,
Luiz
Augusto
M.
Fonseca
d,
Philip
J.
Norris
e,f,
Jorge
Casseb
a,b,∗aUniversidadedeSãoPaulo,FaculdadedeMedicina,DepartamentodeDermatologia,LaboratóriodeInvestigac¸ãoemDermatologiae
Imunodeficiências,SãoPaulo,SP,Brazil
bInstitutodeMedicinaTropicaldeSãoPaulo,SãoPaulo,SP,Brazil
cInstitutodeDoenc¸asInfecciosas“EmilioRibas”(IIER),SãoPaulo,SP,Brazil
dUniversidadedeSãoPaulo,FaculdadedeMedicina,DepartamentodeMedicinaPreventiva,SãoPaulo,SP,Brazil
eBloodSystemsResearchInstitute,SanFrancisco,California,USA
fUniversityofCalifornia,SanFrancisco,California,USA
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received13November2017 Accepted2February2018 Availableonline28February2018
Keywords: HCV HTLV-1 HIV-1 T-cellproliferation
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s
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c
t
Background:Infection withHumanTcellLeukemiaVirustype1canbeassociatedwith
myelopathy/tropicalspasticparaparesis(HAM/TSP)andotherinflammatorydiseases. Lym-phocytes from about half of Human T cell Leukemia Virus type 1-infected subjects spontaneouslyproliferateinvitro,andhowthisphenomenonrelatestosymptomatic dis-easeandviralburdenispoorlyunderstood.
Objective:ToevaluateT-cellproliferationinvitroamongpatientsco-infectedwithHumanT
cellLeukemiaVirustype1/HepatitisCVirus/HumanImmunodeficiencyVirustype1.
Materialand methods:From610 Human T cell Leukemia Virus-infected patients of the
Human T cell Leukemia Virus outpatient clinic from Institute of Infectious Diseases “EmilioRibas” inSãoPaulo,273agreedtoparticipate:72hadHAM/TSP(excludedfrom this analysis) and 201 were asymptomatic, a classification performed during a reg-ular neurological appointment. We selected the subgroup made up only by the 201 asymptomatic subjects to avoid bias by the clinical status as a confounder effect, who had laboratory results of Human T cell Leukemia Virus type 1 proviral load and T-cell proliferation assay in our database. They were further grouped accord-ing to their serological status in four categories: 121 Human T cell Leukemia Virus type 1 asymptomatic mono-infected carriers; 32 Human T cell Leukemia Virus type 1/HepatitisCVirus,29Human TcellLeukemiaVirustype 1/HumanImmunodeficiency
∗ Correspondingauthor.
E-mailaddress:[email protected](J.Casseb). https://doi.org/10.1016/j.bjid.2018.02.002
1413-8670/©2018SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Virus type 1, and 19 Human T cell Leukemia Virustype 1/Human Immunodeficiency Virus type 1/Hepatitis C Virus co-infected patients. Clinical data were obtained from medical records and interviews. DNA Human T cell Leukemia Virus type 1 provi-ral load (PVL) and T-cell proliferation (LPA) assay were performed for all samples.
Results: Fromatotalof273subjectswithHumanTcellLeukemiaVirustype1,80presented
co-infections:29 hadHumanImmunodeficiencyVirustype1,32 hadHepatitisCVirus, and19hadHumanImmunodeficiencyVirustype1andHepatitisCVirus.Comparingthe groupsbasedontheirserologicalstatus,independentlyofbeingasymptomaticcarriers,we observedasignificantincreaseofPVL(p<0.001)andLPA(p=0.001).However,whengroups werestratifiedaccordingtotheirclinicalandserologicalstatus,therewasnosignificant increaseinHumanTcellLeukemiaVirustype1PVLandLPA.
Conclusion: No significant increase of basal T-cell proliferation among Human T cell
LeukemiaVirustype1co-infectedwasobserved.Thisinteractionmaybeimplicatedin liverdamage,worseningtheprognosisofco-infectedpatientsor,onthecontrary,inducing ahigherspontaneousclearanceofHepatitisCVirusinfectioninHumanTcellLeukemia Virustype1co-infectedpatients.
©2018SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Introduction
HumanT-celllymphotropicvirustype1(HTLV-1)isaretrovirus etiologically linked to adult T-cell leukemia/lymphoma,1,2 HTLV-1-associated myelopathy/tropical spastic parapare-sis (HAM/TSP), and other inflammatory diseases.3 This virus is endemic in Japan, Caribbean Basin, and some countries in Latin America,4,5 with 5–10 million peo-ple infected worldwide.6,7 In Brazil, the highest preva-lence of HTLV-1 is found in the North and North-east, particularly in the cities of Belem, São Luiz, and Salvador.8
Inareasendemicforretroviruses,the higherprobability ofoccurrenceofco-infections(HIVandHTLV-1,forexample), withhepatitisCvirus (HCV)may changesome characteris-ticsofthedisease,suchasanalteredresponsetotreatment,9 andespeciallyinthepathogenesisofliverdisease.9 Indeed, cell mediated immunity involved in the development and progressionofliver diseaseassociatedwiththe interaction betweenHCVandHTLV-1maycontributetochangesinthe naturalhistoryofthediseasecausedbytheseviruses,suchas thedevelopmentofhepatocellularcarcinomainco-infected subjects.10
In previous findings, HAM/TSP progression was associ-ated with T-cell activation in the spinal cord, leading to aninflammatoryprocess anddemyelinization.11 Apossible causeforhigherimmuneactivationcouldbethepresenceof higherDNAproviralloads(PVL)amongHAM/TSPpatients.12 Alternatively,thosefindingscouldbeduetodurationof dis-ease, sincewe studieda broadrange ofTSP/HAM cases.13 In case ofHTLV-1/HIV-1co-infection, down-regulation of T-cell proliferation, usually present with HIV infection, may notoccur,afinding thatcould berelatedtothe lower sur-vival rate of such patients.14 Based on the consequences ofco-infectiononHAM/TSPdevelopment,weexaminedthe
possibilityofanassociationamongasymptomatic HTLV-1-co-infectedsubjects,increaseofHTLV-1DNAproviralload,and T-cellproliferationinalargecohortofHTLV-1-infected sub-jectsinSaoPaulocity,Brazil.
Material
and
methods
TheHTLVoutpatientclinicfrom InstituteofInfectious Dis-eases“EmilioRibas”(IIER)hasbeenfollowingacohortof610 HTLV-1-infectedpatients for19 years,startinginJuly1997. For the purpose ofthis study, we recruited a total of 201 HTLV-1-infectedsubjectswhowere olderthan 18yearsand remainedinactivefollow-upfromJune2011toMay2012,and wereclinicallyasymptomatic.TheEthicalReviewBoardofthe IIERapprovedthe protocol (Number13/2011),and asigned informedconsentwasobtainedfromallparticipantspriorto studyinclusion.
All201volunteerswereasymptomaticaccordingto neuro-logical evaluationand wereselectedif laboratoryresultsof HTLV-1 proviral loadand T-cellproliferation were available andretrievableinthepatient’srecord.Eligiblepatientswere classified accordingto theirserologicalstatus infour cate-gories:121HTLV-1asymptomaticmonoinfectedcarriers; 32 HTLV-1/HCV,29HTLV-1/HIV-1,and19HTLV-1/HIV-1/HCV co-infectedpatients.
Blood samples were collected in acid-citrate-dextrose solution, and PBMCwere separatedbyFicoll density gradi-entcentrifugation(Pharmacia,Uppsala,Sweden).Cellswere washed with saline solution; cell number was adjusted to 2×106cells and then stored at −80◦C. DNA was
extracted using a commercial kit (Illustra Tissue and Cells GenomicPrep Mini Spin kit, Easton Turnpike, Fair-field, CA) according to manufacturer’s instructions. After this procedure the DNA was stored at −80◦C for later
QuantificationofHTLV-1proviralload
The HTLV-1 proviral load was quantified by real-time PCR using primers and probes targeting the pol gene: SK110 (5-CCCTACAATCCAACCAGCTCAG-3), and SK111 (5-GTGGTGAAGCTGCCATCGGGTTTT-3).
The internal HTLV-1 TaqMan probe (5
-FAMCTTTACTGACAAACCCGACCTACCCATGGATAMRA-3) was selected using the Oligo (version 4, National Biosciences, Plymouth, MI, USA). For quantifica-tion of the human albumin gene, the primers Alb-S (5-GCTGTCATCTCTTGTGGGCTGT-3) and Alb-AS (5 -AAACTCATGGGAGCTGCTGGTT-3) and albumin TaqMan probe (5-FAMCCTGTCATGCCCACACAAATCTCTCCTAMRA-3) wereusedasdescribedpreviously.15,16Basedonthemedian of asymptomatic individuals, 200copies/104 PBMCs of PVL
wasthevalueusedasacutofftodiscriminatefromHAM/TSP subjects.
T-cellproliferation(LPA)assayusingperipheralblood mononuclearcellcultures(PBMC)
T-cellproliferationassaywasperformedasdescribedindetail elsewhere.17 Briefly, 10mL of peripheral heparinized blood wascollectedfromeverypatientandcontrol,andPBMCswere isolatedusingFicoll-Hypaque (Pharmacia,NewJersey,USA) gradient,washedtwotimesinsterilesalineandresuspended inRPMI1640(Difco,NY,USA).PBMCsfrompatientsand con-trols,2×106cells/mLinRPMIwith10%fetalcalfserumwere
incubatedat37◦Cand5%CO2forthreedayswithPHAand
OKT3,and sixdayswithPWMandCMAintriplicatein 96-wellplates(Costar,Cambridge,MA).Cellswerepulsedwith tritiatedthymidine(0.5Ci/mL,AmershamInt.,England)18h beforeharvesting ina semi-automaticcell harvester(Flow Laboratories,United Kingdom)and counted ina -counter (Beckman,USA).Themeancountsperminute(CPM)ofthe triplicatesampleswerecalculatedandtheresultsexpressed asthedifference betweenthe cpmofstimulated and non-stimulatedcultures.Thestimulationindexwasmeasuredby theratiobetweenspontaneous/stimulatedresults.17
Statisticalanalysis
Student’st-testwasusedforparametriccontinuousvariable, andthechi-squaretestforproportions.Possibledifferencesin patientcharacteristicsorlaboratoryvaluesamongthegroups wereevaluatedwithMann–Whitney’stestandKruskal–Wallis test.Logistic regressionanalysis wasperformed toidentify independentvariablesassociatedwithHTLV-1co-infection.
Results
Studysubjectscharacteristics
Thecharacteristicsofthe participantsaccordingtodisease statusarepresentedinTable1.PresenceofHIV-1co-infection (p=0.001)andHIV-1/HCV(p=0.017)wasmorefrequentinthe asymptomaticgroup.Theasymptomaticgroupconsistedof 201subjectsinfectedwithHTLV-1with80havingco-infections
Table1–Characteristicsof273HTLV-1-infectedsubjects.
Variable Asymptomatic 201(73.63%) HAM/TSP 72(26.37%) p-Value Age(years) 0.003 18–50 85(42.28) 17(23.61) >50 113(56.22) 55(76.39) Gender 0.016 Male 101(50.24) 25(34.72) Female 100(49.76) 47(65.28) Serologicalstatus HIV-1 29(19.86) 2(2.78) 0.001 HCV 32(22.86) 13(18.31) 0.446 HIV-1/HCV 19(13.01) 2(2.82) 0.017
DNAproviralloadHTLV-1 <0.001 <200 156(77.62) 35(48.61) >200 45(22.38) 37(51.39) T-cellproliferation 0.001 <2000 88(43.78) 17(23.61) >2000 113(56.22) 55(76.39) LymphocytesTCD4+ 0.059 ≤500 32(20.12) 6(8.70) >500 127(79.88) 63(91.30) LymphocytesTCD8+ 0.4939 ≤500 42(31.11) 21(32.31) >500 93(68.89) 44(67.69)
PVL,absoluteHTLV-1proviralload;LPA,T-cellproliferationassay.
(66.12%):29HIV-1(36.25%),32HCV(40.0%)and19HIV-1and HCV (23.75%) co-infected patients. There were moremales thanfemales(p<0.001)[Table2].Therewasnoagedifference between co-infected subjects monoinfected asymptomatic subjects.
SpontaneousT-cellproliferation(LPA),HTLV-1DNA proviralload(PVL),CD8+TandCD4+Tcellscount
HTLV-1 affects the immunological response and exerts an influenceinTcellproliferationandTcellcounts,turningthe hostvulnerabletothedevelopmentofHAM/TSP.14Hence,the HTLV-1DNAproviralloadhasbeenimplicatedasan impor-tantfactorinthedevelopmentofHAM/TSP.18Asymptomatic patientsalsoshowanincreaseinthosefactors,albeitmild, withoutacorrespondingchangeintheirclinicalstatus.19We soughttounderstandhowthespontaneousT-cell prolifera-tion,HTLV-1DNAproviralload,TCD8+andTCD4+cellcounts
wereassessedinasymptomaticco-infectedsubjects. ThePVL,expressedasthecopynumberofHTLV-1 provi-ralDNAper104PBMC,wassignificantlyhigherinHAM/TSP
patientsthaninasymptomaticsubjects(p<0.001),incontrast tolymphocyteT-cellproliferation(LPA)assays,sinceHAM/TSP patients’ lymphocytes proliferated significantly more than asymptomatic patients, as shown by their respective CPM (p=0.001).
Therearesomelimitationstothisstudy,sincewehave col-lectedsecondarydataregardingTCD4+(for42asymptomatic patientsandforthreeHAM/TSPcarriers)andTCD8+(for66 asymptomaticpatientsandforsevenHAM/TSPsubjects)from theclinicalcharts(Tables1and3).
Nodifferencesbetweenmonoinfectedandco-infected sub-jectsastoT-cellproliferationandHTLV-1DNAproviralloads
Table2–Demographiccharacteristicsof201asymptomaticHTLV-1-monoandco-infectedsubjects.
Variables HTLV-1
Mono-infectedsubjects
Presenceofco-infectionwithHTLV-1 p-Value
HIV-1 HCV HIV-1/HCV N 121 29 32 19 Age(years) 18–50 60(49.59) 10(34.48) 10(31.25) 6(31.58) NS ≥51 61(50.41) 19(65.52) 22(68.75) 13(68.42) Sex <0.0001 Male 39(32.23) 22(75.86) 24(75.00) 16(84.21) Female 82(67.77) 7(24.14) 8(25.00) 3(15.79)
Table3–Proviralloads,lymphoproliferationandTlymphocytescellcountsamong201monoandco-infected HTLV-1-infectedsubjects.
Variables HTLV-1
Mono-infectedsubjects
Presenceofco-infectionwithHTLV-1
HIV-1 HCV HIV-1/HCV
N 121 29 32 19
DNAproviralloadHTLV-1
<200 95(78.51) 22(75.86) 24(75.00) 15(78.94) >200 26(21.49) 7(24.14) 8(25.00) 4(21.05) T-cellproliferation <1000 54(44.63) 12(41.38) 13(40.62) 9(47.37) >1000 67(55.37) 17(58.62) 19(59.38) 10(52.63) LymphocytesTCD4+count ≤500 – 12(48.00) 10(38.47) 10(58.82) >500 91(100) 13(52.00) 16(61.53) 7(41.18) LymphocytesTCD8+count ≤500 39(42.86) 1(6.67) 2(10.53) – >500 52(57.14) 14(93.33) 17(89.47) 10(100)
PVL,HTLV-1DNAproviralload;LPA,basallymphocyteproliferationassay.
were observed(Table3). Onthe same Table, it isapparent thatTCD8+andTCD4+cellcountsofmonoandco-infected
patients were different, with the former presentinghigher countsofbothtypesofcells,but ahighnumberofmissing valuesforbothvariablesprecludedastatisticalanalysistobe done.
Acomparisonofmonoandco-infectedgroupsofpatients, independentlyofbeingasymptomaticcarriers,showeda sig-nificantincrease inPVL (p=0.006)and LPA(p=0.01) (Fig. 1). However,whenpatientswerestratifiedbasedontheirclinical andserologicalstatus,nosignificantincreaseofHTLV-1PVL and T-cellproliferation compared toHTLV-1 asymptomatic monoinfected subjects with HAM/TSP could be observed (Fig. 2A–D). Fig. 2Ashows a significant difference between HTLV-1 asymptomatic monoinfected subjects compared to HTLV-1/HIV-1asymptomaticcarriers.
Discussion
Previousreportsshowedcontradictoryresultsregarding HTLV-1-HCVco-infection.20–22IfHTLV-1couldworsentheprognosis of HCV disease, as postulated by some authors, it would havebeen possibletoseethosepooreroutcomesinalarge cohort.Inreality,weobservedanincreaseinthenumberof
co-infectedsubjectsinourcohortovertheyears,makingclear that co-infections haveno impact onthe clinical outcome ofHTLV-1-infectedpatients.Neither T-cellproliferation nor DNAHTLV-1proviralloadwereincreasedamongHCV/HTLV-1 subjectscomparedtopatientswithHTLV-1/HIV-1co-infection orHTLV-1/HIV-1/HCV.Itseemsthat,evenwithHIVinfection under control, some proteins of this virus cause a down-regulationofT-cellproliferation.Onthelastyears,fewnew casesofHAM/TSPhavebeenreportedamong HIV-1/HTLV-1co-infectedsubjects,despitetheoccurrenceof10newcaseson thefirstyearsofintroductionofHAART.22,23
It should be pointed out the higher occurrence of co-infected cases in males. One could infer that males are more exposed to HCV and HIV infections, as intravenous drugusewasmorecommoninmalesinthe1990s.24 These co-infectionscouldaffectimmunologicalparametersof HTLV-1-infectedpatients.
AcommonfindinginHTLV/HIVco-infectionisanincreased CD4T-cellcountwithoutanyadditionalimmunological ben-efit to the patient,19,25–28 with the possibility of causing an undue delay in the start of antiretroviral treatment in co-infected patients.29 It is possible that earlier diagnosis, associatedwith bettercontrol ofHIVinfection with newer antiretroviral regimens may, have contributed to reduce the occurrence of HAM/TSP among HIV/HTLV co-infected
10000 **
A
B
CPM 1000 10 1 0.1 HTL V-1 only HTL V-1 only HIV -1 HIV -1 HCV HCV/HIV -1 HCV/HIV -1 HCV PVL (per 10 4 PBMC) 100 100000 10000 1000 100 10Fig.1–HTLV-1Proviralloadandlymphoproliferationamongco-infectedpatients.(A)PVL,HTLV-1proviralload;B,basal T-cellproliferation;(B)CPM,countsperminute;thisfiguredescribesallHTLV-1monoinfectedsubjectscomparedtothose withco-infection,**ThePVLhadasignificantassociationwhencomparedtoHTLV-1-monoinfectedgroupand
HCV/HIV-1/HTLV-1co-infectedsubjects(A).TherewasnodifferenceinthelevelofspontaneousLPAamongmonoinfectedor viralco-infectedgroups(B).
10000 1000 10 1 100 100000 10000 1000 100 10 PVL (per 10 4 PBMC) CPM * * * * * PVL LPA **
B
A
HTL V-1 AS HTL V-1 AS HTL V-1 HAM /TSP HTL V-1 HAM/ TSP HCV AS HCV AS HCV HAM/TSP HC V HAM/TSPFig.2–HTLV-1proviralloadandlymphoproliferationamongHCVco-infectedasymptomaticsubjectsandHAM/TSP patients.Thisfiguredepictsthesubjectswhenstratifiedbasedontheirclinicalco-infectionstatus.A****showsan associationonPVLamongasymptomaticvsHAM/TSP.WeobservedasignificantincreaseinHTLV-1PVLinHTLV-1 monoinfectedHAM/TSPsubjectscomparedtoHTLV-1monoinfectedasymptomaticsubjects.ThehigherspontaneousLPA forHTLV-1.B*LPAwasassociatedamongasymptomaticvsHAM/TSP,andwecanobservedinB**thatLPAwasassociated whencomparedasymptomaticHTLV-1vsHCVwithHAM/TSPsubjects.(A)PVL,HTLV-1proviralload;B,basalT-cell proliferation;(B)CPM,countsperminute.
patients.Ithasbeen notedthatthe CD8Tcellscountwas increasedinallco-infectedpatients,bothwithHIVandHCV. Thisupregulationmaybeduetothepresenceofanincreased viralload ortoimmunedysfunctioncausedbyother, non-identifiedfactors.30
Nevertheless,duringtheacutephaseofHCVinfection,a potentT-cellresponsewouldbeanimportantfactorforviral clearance.24,31 However,inthechronicphasetheactivation ofCTLs leadsto progressiveliver damage.Thus, anti-HCV responsecouldbeimpairedinHTLV-1-infectedpatients,the lysis ofHCV-infectedhepatocyteswould beminor,causing adelayinthe decisiontoperformliverbiopsyand/or initi-atinganti-HCVtreatment.9Thehigherproliferativecapacity couldhavedecreasedthepotentialforliverdamage.Infact, HCV/HTLV-1 co-infected patients may have less immune mediatedliverinjury,butotherfactorsassociatedwithHTLV-1 can influence disease progression.32,33 Furthermore, HTLV-1/HCVco-infected patientsshowedless hepatic injury,and similardatashownintheMiyazaki CohortStudyindicated apossiblenegativeinteractionbetweenHTLV-1andHCVwith
respect to abnormallevels of ALT, with the prevalenceof elevatedALTlevelsbeinglowerinco-infectedsubjectsthan in subjects withHCV alone.34 In Bahia, one study showed thatHIV/HTLV-1andHIV/HCVco-infectionscouldworsenthe clinicalrelatedoutcomes,butvirologicandimmunologic out-comesweresimilar.Liverfunctiontestsweremoreimpairedin patientswithmoresevereimmunosuppression.35This obser-vationisprobablyduetoanineffectiveantiviraltherapyfor bothdiseasesinthepast.
AsforHTLV/HIV co-infection,clinical stageatdiagnosis should alsobecarefullyconsidered intheinterpretationof clinicalstudiesinvolvingHTLV/HIV/HCVco-infections,since ahigherCD4countduetoHTLV-1infectionmayleadtobias inthe stratificationofpatients.Moreover,chronichepatitis Chasaprolongedcourseandtheclinicalfollow-upmaynot belong enoughtoallowfordefinitiveconclusions. Current studieshavereportedhigherspontaneousclearanceofHCV infection in patients with triple infection (HTLV/HIV/HCV) than inthosewithHIV/HCV orHCV only.24 Infact, HTLV-1 maymodulatetheimmuneresponseinHTLV/HIVco-infected
patientsbyincreasingthe productionofgamma-interferon and other pro-inflammatory cytokines related to Th1 type response.Thismechanismcouldbringaboutanenhancement oftheimmuneresponseagainstHCV,providingconditions forincreasedspontaneousclearanceofHCV,whichdepends stronglyontheendogenousproductionofinterferons.
Inconclusion,weobservedanonsignificant increaseof basalT-cellproliferationamongHTLV-1co-infectedpatients. However,HIV-1co-infectionmayinducedown-regulationof T-cellproliferation capacity.Thisinteraction may be impli-catedinliverdamage,worseningtheprognosisofco-infected patientsor,onthecontrary,ahigherspontaneousclearance ofHCVinfectioninHTLV-1co-infectedpatients.Itispossible thatadecreasedT-cellproliferationcanbeafeasiblestrategy toavoidHAM/TSPdevelopmentortoobtainanimprovement ofitssymptoms,asseenwiththeuseofcorticosteroids. Fur-therstudiesincludingmorepatientsandlongerobservation periodsareneededtoclarifythismatter.
Authors’
contributions
TAperformedthestatisticalanalysisandwrotethemaintext; TMandMPMBperformedthelabassays;AP,MH,JS,APOselect the volunteers and discussed the main text; LAMand PJN revisedthestatisticalanalysisanddiscussionoftext;JC con-ceivedandreadthefinalversionofthismanuscript.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
To all participants who contributed to this study. Sup-port: CNPq: 234058/2014-5; FAPESP: 2012/23397-0; FAPESP: 2014/22827-7.
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