Extensive variation in drug-resistance mutational profile of Brazilian patients failing antiretroviral therapy in five large Brazilian cities

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w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Extensive

variation

in

drug-resistance

mutational

profile

of

Brazilian

patients

failing

antiretroviral

therapy

in

five

large

Brazilian

cities

Carlos

Brites

a,b,∗

,

Lauro

Pinto-Neto

c

,

Melissa

Medeiros

d

,

Estevão

Nunes

e

,

Eduardo

Sprinz

f

_,

_Mariana

_Carvalho

g

a_Fundac¸ão_Bahiana_de_Infectologia_(FBaI),_Salvador,_BA,_Brazil

b_Universidade_Federal_da_Bahia_(UFBA),_Laboratório_de_Pesquisa_em_Infectologia_(LAPI),_Salvador,_BA,_Brazil

c_Escola_de_Ciências_da_Saúde_da_Santa_Casa_de_Vitoria,_Vitória,_ES,_Brazil

d_Unichristus,_Fortaleza,_CE,_Brazil

e_Fundac¸ão_Oswaldo_Cruz_(FIOCRUZ),_Instituto_de_Infectologia_Evandro_Chagas,_Rio_de_Janeiro,_RJ,_Brazil

f_Hospital_de_Clínicas_de_Porto_Alegre,_Porto_Alegre,_RS,_Brazil

g_Universidade_de_Campinas_(UNICAMP),_Campinas,_SP,_Brazil

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Articlehistory:

Received14July2015 Accepted30March2016 Availableonline9June2016

Keywords: HIV Resistance Brazil Mutations

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Background:Development of drug-resistance mutations is the maincause of failure in

antiretroviraltherapy.InBrazil,thereisscarceinformationonresistancepatternforpatients failingantiretroviraltherapy.

Objectives: TodefinetheHIVmutationalprofileassociatedwithdrugresistanceinBrazilian

patientsfrom5largecities,afterfirst,secondorfurtherfailurestoantiretroviraltherapy.

Methods:Wereviewedgenotypingresultsof1520patientsfailingtherapyinfiveBrazilian

cities.Frequencyofmutations,meannumberofactivedrugs,viralsusceptibilitytoeach antiretroviralsdrug,andregionaldifferenceswereassessed.

Results:Meantimeofantiretroviralsusewas22.7±41.1months.Meanpre-genotypingviral

loadwas4.2±0.8log(2.1±2.0afterswitchingantiretrovirals).Meannumberofremaining activedrugswas9.4,9.0,and7.9after1st,2nd,and3rdfailure,respectively.Wedetected regionalvariationsindrugsusceptibility:whileBAandRSshowedthehighest(∼40%) resis-tanceleveltoATV/r,FPV/randLPV/r,intheremainingcitiesitwasaroundhalfofthisrate.We detected90%efavirenz/nevirapineresistanceinSP,only45%inRS,andlevelsbetween25% and30%intheothercities.RegardingNRTI,wefoundasimilarpattern,withRJpresenting thehighest,andCEthelowestsusceptibilityratesforallNRTI.Zidovudineresistancewas detectedinonly3%ofpatientsinRJ,against45–65%intheothercities.RJandRSshowed3% resistancetotenofovir,whileinCEitreached55%.DRV/r(89–97%)andetravirine(61–85%) werethemostactivedrugs,butagain,withawidevariationacrosscities.

∗ _{Corresponding}_author.

E-mailaddress:[email protected](C.Brites).

http://dx.doi.org/10.1016/j.bjid.2016.03.010

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Conclusions: TheresistancemutationalprofileofBrazilianpatientsfailingantiretroviral therapyisquitevariable,dependingonthecitywherepatientsweretested.Thisvariation likelyreflectsdistinctivechoiceofantiretroviralsdrugstoinitiatetherapy,adherenceto specificdrugs,orcirculatingHIV-1strains.Overall,etravirineandDRV/rremainasthemost activedrugs.

Introduction

Resistancetoantiretrovirals(ARV)isausualfindingin HIV-infected patients failing antiretroviral therapy (ART). The mutationalpatternafterinitialfailureisquitepredictable,but insubsequentARVregimensitmaybecomeverycomplex,and frequentlylimittheavailabletreatmentoptions.1_In_addition, theuseofdifferentalgorithmsforinterpretationofmutational profilesobtainedbygenotypictestsmightprovidedivergent resultsregardingsensitivityofHIVtoARVdrugstobeusedin salvagetherapyregimens.

The Brazilian Ministry of Health (BMOH) provides free universal access to ARV drugs since the beginning of the epidemic.Thecurrentofficialrecommendation forpatients failing therapy is tochoose salvage regimens according to HIV-1 antiretroviral drug sensitivity, assessedby genotypic resistancetests.2_However,_although_resistance_tests_are sup-posedtobereadilyavailable,manylogisticalproblemshave impairedthisstrategy,duetolongturnaroundtimeofresults insomeareasofthecountry,andtothefactthatmany physi-ciansdecidetoswitchtherapywithoutapreviousresistance test.

Inaddition,thegenotypiccharacteristicsofHIVinpatients failingARTisstillunclear,sincetheavailableinformationis restrictedto small,specificgroups ofpatients,from differ-entsites.InBrazil,circulationofdifferentviralsubtypeshas beenreported,withvariableprevalenceaccordingtodifferent regions.3–6_There_is_scarce_information_on_regional_differences regardingmutationalprofile,availabilityofremainingactive drugs,andthevariabilityofsusceptibilityratesfordifferent ARVdrugs,accordingtotheuseofdifferentalgorithms.

Inthelastyears,theroutinetoolforgenotypic interpreta-tionwasalocallydevelopedalgorithm(RENAGENO),inwhich interpretationofresultsarereleasedalongwiththatprovided byTruGeneplatform (Siemens HealthcareDiagnostics, Inc, USA).7 _In_last_decade_BMOH_trained_around₄₀₀_doctors_for interpretinggenotypicreports,andtoprovidesuggestionsfor thenextARVregimentobeusedbyphysicians.Thisstrategy madeeasiertheselectionofappropriateARVdrugsinsalvage therapyregimens.

Inthepresentworkweevaluatedalargenumberof resis-tancetests, infivelargeBraziliancities. Thisallowedusto definethe frequencyofmutationsafterfirstorsubsequent failures,aswellasthedifferencesbetweendrugsusceptibility ratesacrosstheselocations,andmeannumberofremaining activedrugsforpatientsateachsite.

Methods

Wereviewedreportsofresistancetestsperformedfrom2010 to2013infivelargeBraziliancities, fromdifferent regions. Allavailablereportsfrompatientstestedinreferencecenters forHIVcareinPortoAlegre(RS,Southregion),Campinas(SP, Southeastregion),RiodeJaneiro(RJ,Southeastregion),Vitoria (ES,Southeastregion),Salvador (BA,Northeastregion), and Fortaleza(CE,Northeastregion)werereviewed.

Frequencyofdetecteddrug-resistancemutations(DRM), previoususeofantiretroviraldrugs,andpatients’ characteris-ticswereevaluated,andthemeannumberoffullyactivedrugs wascalculatedforeachcityandfortheoverallstudy popula-tion.Fullyactivedrugswereattributedaweightequaltoone. Drugspartiallyactive(“intermediateresistance”)weregivena 0.5weight,whileforcompleteresistancetheattributedweight waszero.Thesusceptibilityforeachdrugwascomparedby usingtheBrazilianalgorithmofinterpretation(RENAGENO), accordingtopatient’sorigin.7

Statisticalanalyses

We used SPSS (StatisticalPackage for SocialSciences) ver-sion 17.0 toperform all statisticalcalculations. Descriptive analyses(frequencies,mean,standarddeviations)were per-formed,andcomparisonsoffrequenciesbetweengroupswere assessedbychi-squaretest.

Results

A total of 1512genotypic tests was reviewedin the study period,butonly1481hadenoughinformationtobeincluded intheanalysis.Mosttestswereperformedinpatientsfrom Salvador(552),followedbyVitória(246),Fortaleza(219),Rio deJaneiro(152),PortoAlegre(112),andCampinas(102tests).

Table1showsthemaincharacteristicsofpatientssubmitted toHIV-1genotyping.

Mostpatients(675subjects–43.1%)werefailingtheir sec-ond ARV regimen, 431 (37.8%) were failing their first ARV treatment, and the remaining375 (15%)had already failed threeormoreregimens.ThemostfrequentlydetectedDRM (>10%)areshowninTables2and3.Thesusceptibilityrates ofHIV-1strainstoeachARVdruginthefivedifferent loca-tionsareshowninFigure1a–c.Itshouldbepointedoutthe clear difference betweenthe susceptibility rates to reverse

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Table1–CharacteristicsofpatientssubmittedtoHIV-1genotypinginfivedifferentcitiesinBrazilfrom2010to2013. Overall BA SP RS CE RJ ES Numberoftests 1383 552 102 112 219 152 246 Age 41.1±11.0 41.8±9.3 43.8±10.2 44.3±8.8 42.0±9.8 39.0±10.6 37.2±15.3 Pre-genotyping VL(log) 4.2±0.8 4.2±0.9 4.1±0.7 4.3±1.2 4.1±0.6 4.2±0.7 4.3±0.7 Pre-genotyping CD4count (cells/mm3₎ 299±251 313±260 282±253 245±198 285±253 267±239 263±247

Allresultsareexpressedasmean±standarddeviation.

Table2–Frequency(%)ofmostcommondrug-resistancemutationstoHIV-1reversetranscriptaseinhibitors,according tothepatient’sorigin.

Mutation Allsamples BA SP RS CE RJ ES

41L 21.2 31.0 19.4 36.6 20.4 10.5 0.0 67N 22.9 27.5 17.5 30.9 20.0 9.9 21.5 70R 18.2 23.2 6.8 22.0 15.6 9.9 17.9 184V 70.9 81.2 54.4 74.0 65.6 55.9 68.1 210W 15.7 20.5 9.7 21.1 11.6 4.6 15.9 215F 11.3 13.6 10.7 18.7 13.2 5.3 4.4 215Y 16.5 26.1 0.0 17.9 19.2 0.0 8.8 103N 40.0 44.0 50.5 28.5 38.0 30.3 40.6 108I 10.2 11.8 12.6 4.1 11.6 5.9 10.0

Table3–Mostfrequent(>10%)drug-resistancemutationsinHIV-1protease,accordingtooriginofsample.

Mutation Allsamples BA SP RS CE RJ ES

10I 17.7 19.2 19.4 27.6 10.0 11.8 20.3 10V 10.6 13.0 13.6 6.5 8.4 4.6 11.6 20R 15.0 14.3 11.7 26.0 13.6 10.5 16.7 36I 47.0 47.3 49.5 53.7 46.8 40.1 46.2 46I 15.3 19.2 8.7 26.8 8.4 9.9 13.9 54V 12.6 13.9 13.6 30.9 5.2 5.3 12.0 60E 11.0 9.2 8.7 0.0 9.2 21.7 16.3 62V 24.4 23.7 26.2 0.0 24.8 37.5 28.7 71V 14.1 20.8 12.6 28.5 12.0 5.9 0.0 90M 12.6 15.9 11.7 23.6 8.4 2.0 10.8 93L 23.3 21.0 29.1 3.3 22.4 24.3 36.3

transcriptaseinhibitors (zidovudine– AZT,tenofovir– TDF, didanosine–ddI,stavudine–d4T,nevirapine–NVP,efavirenz –EFV,andetravirine–ETV)amongtestsoriginatedfrom differ-entsites(p<0.001forallcomparisons).Forinstance,genotypic testsofpatientsfromSPshowedthehighestrateofresistance toEFVandNVP(92%),whileinRSalmost2/3(58%)oftests detectedsusceptibilitytothesenon-nucleosidereverse trans-criptase inhibitors (NNRTI, p<0.001). The remaining three sitessharedasimilarprofile,withdetectedsusceptibilityfor NNRTIinaround1/3ofsamples.Weobservedthesame pat-ternforalldrugs,withalargevariabilityinsusceptibilityrates fordifferentcities,especiallyforsomeARV:inCEwedetected resistancetoETRin52%ofsamples,whileinRSonly8%of testsshowedresistancetothisdrug(p<0.001).

Theresistanceratestoproteaseinhibitors(PI)also signifi-cantlyvariedaccordingtooriginofpatients.Patientstested in BA or RS showed the highest (30–50%) resistance rates to boosted atazanavir (ATV/r), fosamprenavir (FPV/r), and lopinavir (LPV/r) whilethose livingin the remainingcities hadlevelsofresistancetothisclassbelow20%,asshownin

Figure1c(p<0.001).Ofnote,resistancetoboosted-darunavir

(DRV/r)wasdetectedinlessthan5%ofsamples,exceptinRS, wherewefound14%ofsamplespresentingresistancetothat PI.DRV/rwastheonlyARVdrugthatpreservedsusceptibility regardlessofpatient’sorigin.

Themostfrequentdrug-resistancemutationsafterfirst, second,and thirdfailures areshown inFigure 2. The over-allmeannumberofactivedrugswas8.8±3.2,9.2±2.7,and 7.9±3.9forpatientsfailingfirst,second,orthird/moreARV regimens,respectively.Thisnumberwassimilarforthe differ-entcities,andrangedfromeight(BA)to10.5(RJ).Somespecific mutationslike65Ralsoshowedagreatvariation,withan over-allmeanprevalenceof4.4%,butrangingfrom2.4%inRS,to 6.2%inRJ(p<0.01).

ThesusceptibilitypatternforallARVdrugsdecreasedover subsequentfailures,exceptforETRandDRV/rwhichkeptthe samelevelofactivityagainstHIV-1acrossinitialorsubsequent ARVregimensfailures,assummarizedinFigure3.

ThefrequencyofHIV-1subtypeswasalsovariableacross cities. Asexpected,moststrainswere classifiedassubtype B.Thelowest(55.4%)andhighest(78.6%)subtypeBfrequency wasobservedinESandBA,respectively.SubtypeCwasmainly

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Tenofovir Abacavir Estavudine Didanosine Lamivudine Zidovudine Bahia São Paulo Ceará Rio de Janeiro

Espirito Santo R.Gde do SUL .00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 RS SP RJ ES CE BA

Efavirenz Nevirapine Etravirine

.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 RS SP RJ ES CE BA Atazanavir/ritonavir Fosamprenavir/ritonavir

Lopinavir/ritonavir

Darunavir/ritonavir Tipranavir/ritonavir

Fig.1–(a)SusceptibilityofHIV-1(%)toNRTIdrugsafter1st treatmentfailureinfivecitiesofdifferentBrazilianstates. (b)SusceptibilityofHIV-1toNNRTIafter1sttreatment failure,accordingtopatient’sorigin.(C)Susceptibility(%)of HIV-1toPIsafter1sttreatmentfailure,accordingto patient’sorigin.

detectedin RS(31.7%),with othersites presentingamuch lower frequency (0.4–3.8%). Subtype F was less frequently detectedinRS(3.2%),withthehighestfrequenciesobservedin CE(9.6%)andES(11.5%).RecombinantBFstrainsweremostly detectedinBAand SP(4.7%each),but absentinCE. Other multiplerecombinantformswerealsodetectedinall sites, rangingfrom3.5%inRSto29.9%inES(p<0.01forcomparisons betweensites).

Discussion

Wedetectedahighvariabilityinsusceptibilityratestomost ARVdrugs,accordingtopatient’sorigin.TheonlyARVdrug withastablesusceptibilityrate, regardlessoflocation,was DRV/r.Themostmarkeddifferenceswerefoundingenotypic tests fromRS, RJ,and CE, whichshowed adistinct pattern of susceptibility to NRTI when compared to BA or ES. We observedthatsusceptibilityratestothesedrugswere signifi-cantlyhigherinRJincomparisontootherlocations,whilefor ddIandTDFtheratesinRJweredifferentfromBA,CE,andES, althoughsimilartothatfoundinRS.

Noteworthy,wedetectedahighsusceptibilityrate(97%)for AZTinRJ,incontrasttoCE,whereonly55%ofsampleswere susceptibletoAZT.ThismayreflectthepreferentialuseofAZT orTDFasfirstlinetherapyfortheremainingsites,wherewe observedacontrastingsusceptibilitybetweenTDFandAZT, butitwasnotdetectedinRJ,wherebothdrugstestedactive foralmost100%ofsamples.Apotentialexplanationforthis factcouldrelyondistinctadherenceratestofirstlinetherapy indifferentsites.

For NNRTIdrugsthe patternwasalsodifferent,withRS showinganalmosttwo-foldhigher(>50%)susceptibilityrates to EFV and NVP, in comparison with other sites (25–35%,

p<0.001 for comparison with other sites). In contrast, SP showedthelowestsusceptibilityrates(around10%)forthese drugs.EvenforETR,adrugnotusedasfirst-linetherapy,we detectedsignificantdifferencesbetweensites,withlower sus-ceptibilityratesfoundinCE(48%),andhigherratesinRS(92%) and RJ(79%).ForPI,wefoundamorehomogenouspicture, withCE,ES,RJ,andSPshowingsimilar(around80%) suscep-tibilityratesforalldrugsinthisclass.However,inRSandBA wefoundmuchlowersusceptibilityratesforATV/r,LPV/r,and FPV/r.

Severalpreviousstudieshaveshownasimilarlevelofdrug resistanceindifferentBrazilianregions;howeverthesample sizewereusuallysmallerthanours,andmostanalyseswere restrictedtoaspecificsetting.8–13_Even_in_such_populations_we cannoticedifferentprevalenceofmutationslike103N.Toledo etal.evaluatedasampleof467genotypictestsinParanastate, anddetecteda46%prevalenceof103Nmutation,whileWestin etal.,inMinasGeraisstatefoundalowerprevalencevalue (32%).10,5_In_Northeast_Brazil,_Cavalcanti_et_al._reported_58.7% resistancetoNNRTI,with103Nand190Aasthemostprevalent drugresistancemutationsinthisclass.13

Wedidnotget informationon mostusedfirst-lineARV regimensineachcity,buttheGuidelinesoftheBMOH recom-mendstouseasinitialARTregimenstwoNRTIdrugsplusEFV orNVP.2_Our_results_suggest_that_compliance_with_these rec-ommendationsaredistinctindifferentsettings.Thedistinct

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225H 108I 103N 215Y 215F 214F 211K 210W 184V 118I 70R 67N 41L

1st Failure 2nd Failure ≥3rd Failure

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10I _10V _13V _15V _20R _33F _35D 36I _41K 46I 50L 54V 60E 62V 63P 71V 77I 82A 89M 90M

1st.Failure 2nd. Failure 3rd.Failure

Fig.2–(a)Mostfrequent(>10%)mutationstoRTIdrugsat1,2or≥₃_treatment_failure._(B)_Most_frequent_mutations_(%)_to_PI drugsaccordingtothenumberofprevioustreatmentfailures.

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ZidovudineLamivudine Didanosine Stavudine Abacavir Tenofovir EfavirenzNevirapine Etravirine Atazanavir/ritonavir

Fosamprenavir/ritonavir

Lopinavir/ritonavirDarunavir/ritonavirTipranavir/ritonavir

10 20 30 40 50 60 70 80 90 100

1a. Failure 2nd. Failure ≥3 failures

Fig.3–MeansusceptibilityratestoARVdrugsaccordingtothenumberofpreviousfailurestoART.

susceptibility rates detected suggest that in some settings (BAandRS)proteaseinhibitorsweremorefrequentlyusedas thethirddrugeitherinfirst-lineorsalvageregimens,while inothers NNRTI were the drug ofchoicefor starting ther-apy.Otheralternativeexplanationisawiderprevioususeof unboostedPI(nelfinavir,indinavir,oratazanavir),since muta-tionslike90Mand36I,and71Vareassociatedwithuseofthese drugs.

Thispossibilityissupportedbythefindingofaninverse relationship between susceptibility rates for PI, compared toNNRTI drugsinthe mostdivergent scenario: thelowest susceptibilityratesforPI(especiallyATV/randFPV/r)andthe highestratesforNNRTIobservedinRS.However,thiswasnot

trueforSP,wherewedetectedthelowestsusceptibilityrates forNNRTI,but nodifferenceforPI. ForETR,the trendwas similar,exceptforCE,wherewedetected48%susceptibility, againsthigherrates(62%-92%)intheremainingsites. Inter-estingly,allITRNshowedbetteractivityinRJ,incomparison to other cities, but NNRTI susceptibility rates were similar acrosssites.

WeknowthatprevioususeofNVPorEFVdoesnotimply incross-resistancetoETR.14,15_However,_if_patients_remain_on failingregimenscontainingthesedrugsforlongenoughtime, itisexpectedthatviralvariantswithamorecomplexDRM profilewilleventuallybeselected,whichcouldleadto cross-resistancetoETR.Again,thelackofinformationonhowlong

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thesepatientsremainedonfailingregimenslimitsthereach ofsuchconclusions.

Oneinterestingpointisthefindingthatpatientsstill pre-serveahighGSS,evenconsideringthatmosthadprevious treatmentfailuretomorethanoneARVregimen.Therefore mostfailingpatientscanbesuccessfullytreated,withahigh likelihoodtoachievesustainedviralsuppression.Thisisin accordancetorecentpublicationsbyBrazilianauthors show-ingthatevenheavilypre-treatedpatients areabletoreach virologicalsuppressionafteroneyearofasalvage regimen includingnewARVdrugs/classes.16–19_On_the_other_hand,_low resistanceratesafterfailingregimenssuggeststhatadherence totherapyisamajorcomponentoftreatmentfailureinthese centers,anditdeservesacarefulevaluationbyphysiciansand healthauthorities.

The observed frequency of subtypes also demonstrates thedifferentepidemiologicalscenariointhedistinctregions. While most HIV strains belonged to subtype B, a clear divergence regarding subtype C was observed, which was basicallyrestrictedtoRS,butwasalsopresentinSP,BA,and ES,asalreadydetectedinpreviousstudies.20,21 _Noteworthy, ES showed the lowest frequency of subtypeB (55.4%), but the highestproportionof other recombinants (29.9%), sug-gesting a distinct dynamic ofHIV-1 diversity in that area. Whethersubtypes variationcouldexplainsomedifferences inmutationalprofilesobservedindifferentcities,itisstilla controversialissue.22–26

AlthoughmutationsassociatedwithHIV-1subtypeC(like V106N)mayconferresistancetoEFVandNVPtheyhaveno impactonETVactivity.27_In_addition,_in_the_present_study_it wasnotafrequentfinding.Somerecentdataindicatethat subtypeC-associatedmutationsconferringresistancetoETV arerare,andmostHIV-1strainsaresusceptibletothesedrugs afterfailuretofirstgenerationNNRTI.28_The_higher suscepti-bilityratetoETVdetectedinRS(thesitepresentingthehigher frequencyofsubtypeC)underscorestheabsenceofan asso-ciationbetweenHIV-1subtypeandsusceptibilityratetoETV.

Our study has some clearlimitations: we did not have accesstoinformationontreatmentoutcomesaftertherapy modification,whichprecludedconfirmationthattheobserved susceptibilityratesheldtrueinclinicalpractice.Inaddition, informationonadherencetotherapywasnotavailable,which couldexplainsomeofstudyfindings.

However,thisstudyhasalargesamplesize,andincludes patientsfromdifferentBrazilianregions,providing informa-tiononthemostfrequentDRM,andsusceptibilityratesfor patients indifferent phases of treatmentof HIV infection. In addition, it underscores the need for virological moni-toring todetect drugfailure, and touse genotypictests to guidethechoiceofsalvagetherapyregimens,asalready sug-gestedinotherstudiesondrugresistanceinresource-limited settings.29_{Understanding}_the_factors_associated_with_failing treatment,andthedistinctresistanceprofileinearlyorlater treatmentfailuresisanessentialsteptofacethispersistent challengeinHIVtherapy.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisworkwassupportedbyanInvestigator’sInitiativeGrant, fundedbyJanssen-CilagFarmacêuticaLtda.

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