ww 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
High
degree
of
concordance
between
flow
cytometry
and
geno2pheno
methods
for
HIV-1
tropism
determination
in
proviral
DNA
Alex
José
Leite
Torres
a,∗,
Luis
Fernando
de
Macedo
Brígido
b,
Marcos
Herculano
Nunes
Abrahão
a,
Ana
Luiza
Dias
Angelo
a,
Gilcivaldo
de
Jesus
Ferreira
a,
Luana
Portes
Coelho
b,
João
Leandro
Ferreira
b,
Célia
Regina
Mayoral
Pedroso
Jorge
a,
Eduardo
Martins
Netto
a,
Carlos
Brites
aaLaboratóriodePesquisaemInfectologia,HospitalUniversitárioProfessorEdgardSantos,UniversidadeFederaldaBahia,
Salvador,BA,Brazil
bGrupodepesquisaparacaracterizac¸ãodeRetrovírusemhumanosdoInstitutoAdolfoLutz,SãoPaulo,SP,Brazil
a
r
t
i
c
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e
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n
f
o
Articlehistory: Received27June2014
Accepted16November2014
Availableonline18February2015
Keywords: HIV-1 Tropism Flowcytometry Geno2pheno
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Use of CCR5 antagonists requires previous viral tropism determination. The available
methodshavehighcost,aretime-consuming, orrequirehighlytrainedpersonnel,and
sophisticated equipment. We compared a flow cytometry-based tropism assay with
geno2phenomethodtodetermineHIV-1tropisminAIDSpatients,inBahia,Brazil.Wetested peripheralbloodmononuclearcellsof102AIDSpatientsunderantiretroviraltherapyby
usingacytometry-basedtropismassayandgeno2phenoassay.Cellularmembrane
recep-torswereidentifiedbyusingCXCR4,CCR5andCD4monoclonalantibodies,whiledetection ofcytoplasmicmRNAsforgagandpolHIVregionswasachievedbyusingalabeledprobe.
GenotypicidentificationofX4andR5tropicviruseswasattemptedbygeno2pheno
algo-rithm.Therewasahighdegreeofconcordancebetweencytometry-basedtropismassayand
geno2phenoalgorithmindeterminationofHIV-1tropism.Cytometry-basedtropismassay
demonstratedhighersensitivityandspecificityincomparisontogeno2pheno,whichwas
usedasagold-standard.Onesamplecouldnotbeamplifiedbygeno2phenomethod,butwas classifiedasduotropicbycytometry-basedtropismassay.Wedidnotfindanyassociation betweenCD4+countorplasmaHIV-1RNAviralloadandtropismresults.Theoverall
per-formancesofcytometry-basedtropismassayandgeno2phenoassaywerealmostidentical
indeterminationofHIV-1tropism.
©2015ElsevierEditoraLtda.Allrightsreserved.
∗ Correspondingauthor.
E-mailaddress:[email protected](A.J.L.Torres).
http://dx.doi.org/10.1016/j.bjid.2014.11.007
Introduction
Toenterthehostcells,HIV-1requiresaninitialattachment
tocellsmembrane’s CD4receptor, whichleadstoan
inter-action between the viral envelope (gp120) and one of the
cellularcoreceptors: CCR5or CXCR4.1,2 Thisstep is
essen-tial to enable the fusion of viral envelope and host cell
membrane, and to allow the introduction ofviral genome
into cellcytoplasm.3 Itwas demonstratedthat CCR5-tropic
viruses predominateduringthe initial phasesof infection,
whereas CXCR4-binding viruses usually emerge in a later
phase,andare associatedwithafasterCD4+T-celldecline
and progressiontoAIDS.4 MostHIV variantsisolated from
drug-naive,chronicallyinfectedindividuals,useCCR5along
withCD4 toenter Tcells (R5 tropic viruses).On the other
hand, viruses able to use CXCR4 coreceptors (X4-tropic)
tend to emerge later in the course of HIV infection, in
comparison with R5 strains, and are recognized in nearly
half of patients with advanced disease stages. Coreceptor
switch mayoccur overtime inpatients underantiretroviral
therapy.5
CCR5antagonistsarearelativelynewclassofdrugsthat
block HIV entry into target cells. These drugs bind to a
hydrophobicpocket formed bythe transmembrane helices
ofCCR5, and induce conformationalchanges in the
extra-cellular loops (ECLs) ofthe receptor. Since they are active
onlyagainstR5viruses,itisnecessarytodeterminetheviral
tropism previously to the use of CCR5 antagonists in HIV
treatment.6–12
Differentgenotypicandphenotypictoolshavebeenused
todefineHIVtropism.13,14Theviraltropismhasbeen
deter-minedbyusingphenotypicmethods(likeMonogram’sTrofile),
by sequencing the V3 loop of HIV envelope, and by flow
cytometry-basedassays.15–17 Despite somerecent
improve-ments, genotypic algorithms are still restricted to referral
laboratories,andtheirpredictivevalueshavelimitations.18–20
Recently, a new algorithm (geno2pheno) showed a good
correlation with phenotypic methods.21 Another genotypic
approach is ultra-deep sequencing, but it is expensive,
requires sophisticatedequipment, and is notapplicable to
dailyclinicalpractice.22
Currently,themostusedtropismassaysare
phenotypic-(“Trofile”)orgenotyping-basedtests(geno2pheno).Bothtests
sharesomepracticallimitations,suchashighcost,prolonged
timetoobtain a confirmedresult,availability, considerable
proportion of “nonreportable” results, the requirement of
sampleswithadetectablelevelofHIVRNAcopies/ml,and
limited access in developing countries.23 Flow cytometry
tropismassay(FCTA) wasinitiallydevelopedasa
commer-cially available method (ViroTect Tropism Assay, Invirion
Diagnostics,USA),based onfluorescence-activated cell
sor-ting,withaturnaroundtimeof3h(InvirionDiagnostics,2007),
andasignificantlylowercost,incomparisonwiththeabove
describedmethods.
Inthepresentstudy,wecomparedtheperformanceofan
in-house flow cytometry-based assay, with geno2pheno
assay for determination of viral tropism in
periph-eral blood mononuclear cells (PBMC) of HIV-infected
patients.
Material
and
methods
We evaluated viral tropism of HIV strains in PBMCof 102
consecutiveHIV-1-infectedpatients,whoattendedtheAIDS
outpatient clinicofHospital Universitário Professor Edgard
Santos,FederalUniversityofBahia,Brazil.Patientsassignedto
routineevaluationofHIVviralload,andCD4/CD8+cellcount
wereinvitedtoenterthestudy.Theinstitutionalethicsreview
committeeapprovedthe study,andpatientswere recruited
aftersigningawritteninformedconsent.
Flowcytometrytropismassayprocedure
DeterminationofHIV-1tropismbyFCTAwasperformedby
mRNA in situ hybridization (FISH), combined with T cell
immunophenotyping to define expression of CCR5 and/or
CXCR4coreceptors.AlabeledprobedetectedepitopesofGag
andpolHIV-1regions.15
Briefly, we fixed and permeabilized PBMC with specific
buffers.AfterwashingwithPBS,amRNAprobelabeledwith
6-FAMfluorophoreandanintegrationbufferwereaddedto
cell suspension,andincubated for2hinwarmbathinthe
dark.Thesamplesweresubmittedtoanewwashingstep,and
monoclonalantibodiestoCD4(labeledwithAPC),toCXCR4
(PE),andtoCCR5(PerCP)wereadded,beforeacquisitionand
analysisbyBDCellQuestsoftwarefromFACSCalibur,withfour
fluorimetricparameters.
HIV-1envgeneticsequencing
PBMCDNAwasextractedbyQIAGENprotocol.Amplification
ofpartialHIVenvelope(env)genebynestedPCRwasperformed
with2.5LofDNAinput.Reactionmixtureincluded10Mof
each primerED5,forwardpositionATGGGATCAAAGCCT
AAAGCCATG TG(6557→6582) and ED12,reverseposition
AGTGCTTCCTGCTGCTCCCAAGAACCCAAG(7811←7782)
withTaqPlatinumHighFidelity(Invitrogen)inafinalvolume
reactionof25L.Positiveandnegativecontrolswereincluded ineachrun.PCRconditionsconsistedof10cycles94◦C2min, 94◦C15s,55◦C30s,68◦C1min,25cycles94◦C15s,55◦C30s, 68◦C1minadd5s/cycle,and68◦C7min;2.5UlofPCR
prod-uctswereusedinasecondPCR(nested)withprimersED31,
forwardpositionCCTCAGCCATTACACAGGCCTGTCCAAAG
(6817→6845)andES8,reversepositionCACTTCTCCAATTGT
CCCTCA(7668←7648)withTaqDNAPolymerase(Invitrogen)
inafinalvolumereactionof25L.
PCR conditions consistedof 10 cycles 94◦C 2min, 94◦C
15s, 55◦C 30s, 72◦C 1min, 25 cycles 94◦C 15s, 55◦C 30s, 72◦C1min(increasingby5sineachsequentialelongation),
with afinal elongationperiodat72◦C 1min,add5s/cycle.
PCR reaction wasconsidered validwhen the PCRproducts
andpositivecontrolbandswerevisualizedaround700bpat
1.3%agarosegel withSYBRSafe DNAgel staining
(Invitro-gen, USA) along with controls. Nested PCR products were
quantified usinglowmassladder (Invitrogen,USA),diluted
accordingly(1:10)inwatertoallowaninputof5–10nanograms
ofDNAforrodamineincorporation.BigDyeKit(Ready
Reac-tionMix,AppliedBiosystems,FosterCity,CA)wasusedwith
CAATGTACAC3(6948→6969),ES7,forwardposition5CTG
TTAAATGGCAGTCTAGC3(7005→7021),ED33reverse
posi-tion5TTACAGTAGAAAAATTCCCCTC3(7360←7378)and
SQV3,reverseposition5GAAAAATTCCCTTCCACAATTAAA
3(7350←7370).Cyclesequencingconditionsconsistedof25 cycles96◦C10s,50◦C5s,60◦C4min,8◦Cinfinity.Aftercycle
sequencing, DNA precipitation (usinga solutionofsodium
acetateandethanol)wasfollowedbydenaturationusing
Hi-DiFormamide at94◦C 4minandresolvedatanautomated
sequencer Genetic Analyzer 3130 XL (Applied Biosystems).
SequenceswereeditedusingSequencer(Biocodes,USA)and
submittedtogeno2pheno evaluation(MaxPlanckInstitute,
Germany)usingclonaloption.
StatisticalparameterswereanalyzedbySPSSv.17software.
FCTAandgeno2phenoresultswerecomparedandevaluated
byPearsonChi-squaretest.Sensitivity,specificityandpositive
(PPV)andnegative(NPV)predictive valueswere also
calcu-latedbetweenthetwoassays.
Results
Atotalof102PBMCsamplesfromHIVinfectedpatientswere
evaluated.Medianagewas42years.ThemedianCD4+
lym-phocytecountwas489cells/mm3 (IQR:305–675), CD8+was
886cells/mm3(IQR:716–1229),andCD3+was1529cells/mm3
(IQR:1187–1946).ThemeanelapsedtimesinceHIVdiagnosis
wassixyears(range2–6years).Sixty-sixpatientspresented
withundetectableHIVviral load,while28 had aviralload
higherthan 1500copies/ml.Eight patientsdid nothave an
availableviralloadmeasurementinthepast12months.
Initially, we used gate strategy Side Scatter versus CD4+
in dot plot FCTA analysis for selection of monocytes and
lymphocytes.Inthesequence,CD4+CCR5+andCD4+CXCR4+
positiveeventswereisolatedintwodifferentgates.Ineach
gateCCR5+wasplottedversusCXCR4+.Agatewasselectedfor
CCR5+CXCR4−cells,andanalyzedtodetectHIV-1probeversus
CD4+cellsinotherplot.Thesameprocedurewasperformed
toidentifyCCR5−CXCR4+cells,asshowninFigs.1and2.
Figs.1and2showtheresultsoftropismdeterminationfor
twodifferentpatients:onewithCCR5tropism(A1–A6)andthe
secondwithCXCR4tropism(B1–B6).Initially,SSCversusCD4+
dotplotwasusedasgatestrategiestomonocytesand
lympho-cytesselection.Inthefirstsample(A1–A6)weinitiallyselected
the CD4+CXCR4+cellspopulation(A1). Inthesequence,A2
determinestheaxispositiontothepositiveCXCR4+
popula-tion(firstquadrant),andA3demonstratesthatthereareno
cellsinthesecondquadrant(W1),whichmeans anegative
resultforX4tropism.WhenwelookattheCCR5+cells,A4
selectthe entireCD4+CCR5+cell population(R2). InA5we
havethedefinitionofaxispositiontotheCCR5+population
(firstquadrant),andinA6wecandetectHIV-infectedcellsin
the secondquadrant(W2),which giveusadefinitionofR5
tropism(Fig.3).
TodefineCXCR4tropism,inasecondpatient(B1–B6),the
analysiswasperformedinasimilarway.Theonlydifference
wasthatinB3wefoundcellsinw4(secondquadrant)which
indicatesapositiveresulttoCXCR4HIV-1tropism.Nocellsare
detectedinthesecondquadrant(w5)inB6.
HIV-1FCTAandgeno2phenotropismresultswerepaired
and defined 98% sensitivity, 96% specificity, and 98%
posi-tive92%negativepredictivevaluesofFCTA,incomparisonto
geno2phenomethod.Sixty-fivepatientspresentedR5tropism
accordingtobothmethods;onlyonepatienthaddiscordant
results.ThreepatientspresentsX4tropisminbothtestsand
28weredualtropicaccordingtothetwotestsandinfour sam-plesthetestshaddiscordantresults.Table1showstheoverall
performanceofbothtests.
TheFCTAperformancecomparedtogeno2phenowas
con-sistentregardlessofCD4+cellcountlevel.Table2displaysthe testresultsofviraltropismfordifferentCD4strata.
Discussion
Inthepresentstudy,useofFCTAorgeno2phenoalgorithm
fordeterminationofHIV-1tropisminpatientswithdifferent
strataofviralloadandCD4countprovidedpractically
iden-ticalresults.ThediscoveringofCCR5antagonistsanditsuse
Figure1–Negativecontrol.ThisfigureshowstheresultsofFCTAdeterminationforseronegativeindividuals.Inthefirst plotlymphocytesandmonocytespopulationsareidentifiedbyForwardScatterandCD4+gatestrategy.Insecondplot,only CD4+populationsareviewed,withnocelllabeledbyHIVprobe.
Figure2–CCR5HIVtropismresultmodel.TodefineCCR5(A)andCXCR4(B)tropism,wefirstidentifiedCD4+cells,
HIV-infectedcells(monocytesandlymphocytes)inR1,byusingHIVProbeversusCD4dotplot.Theselectedgatetoinfected cells,wasusedtodefineHIVtropism,byusingCD4+versusCCR5andCD4+versusCXCR4indotplotsforfinalanalysis.
forthetreatmentofHIV-1infectionhasledtodevelopmentof severalHIV-1tropismtests.Aphenotypictest(Trofile)wasthe firstassaycreatedforthatpurpose.Sincecoreceptortropismis arequisitetotheuseofsuchdrugs,theadditionofthesetests toclinicalpracticehasallowedhealthprofessionalstosafely
selectCCR5antagonistsasactivedrugstocompose
antiretro-viralregimens.24
Subsequently, other assays were developed: a Spanish
group created a different phenotypic assay (TROCAI), and
its results were comparedwith Trofile. TROCAI provided a
reportableresultinallpatientswithviralload>500HIV-1RNA
copies/mlofplasma,andin3/6patientswith<500HIVRNA
copies/ml.Patients respondingto maravirochad a 0–0.41%
proportionofX4-usingvirusinsupernatantofindicatorcells.
Hence,itwasusedathresholdof0.5%tocategorizeTROCAI
resultsasR5(<0.5%)ordual/mixed(>0.5%).Theconcordance
betweenTROCAIandTrofile(ES)was22/24(91.6%).In
compar-isonwiththegenotypicmethoditreached22/26(84.6%).23
Another testwasdevelopedtodetermine HIV-1tropism
bygenotyping,whichusedduplicatePCRamplificationofthe
regionencodingV3loop.Theevaluationofitsperformance
was attempted by comparing with results of the TROFILE
assay.ThesensitivityofV3genotypinginpredicting
CXCR4-usingvirusesincreasedfrom56.8%(withsinglePCR)to68.2%,
withduplicatePCR(p=0.13).Theconcordancebetweenthe
duplicatePCRapproachandTrofilewasof76%forHIV-1
sub-typeBand83%fornon-BHIV-1subtypes.25
Results genotypic assays are interpreted by using
spe-cific algorithmsfor definitionof viral tropismof aspecific
sample. In a study that evaluated the performance of
genotypicalgorithmsforpredictingHIVtropism,PSSMX4X5
Figure3– CXCR4HIVtropismresultmodel.
CI,76.7–86.4%).Geno2pheno assay(false-positive rate,20%)
showed the highest sensitivity to predict CXCR4 (76.7%;
95% CI, 61.8–87.5%), with a specificity of 73.6% (95% CI,
68.7–77.2%).Althoughnosignificant differenceinthe
accu-racyofthe bioinformaticstoolswasdetected:1%and 2.5%
withGeno2pheno,respectively,whenassessingHIVtropism
insamplesfrom drug-naïvepatients and insamples
origi-natedfromtreatment-experiencedpatients.21
InastudyconductedbyVilchezet al.,16 acommercially
availableFCTAkit(ViroTectTropismAssay,Invirion,USA)was
comparedwithTROFILE.Inatotalof288samples,FCTA
pro-vided lessinconclusive results(6%)than TROFILE (13%).In
addition,itwasmorelikelythanstandardTROFILE,todetect
CXCR4virusesinthatpopulation.Althougharecent
enhance-mentofTROFILEincreasedthesensitivityofthetesttodetect X4viruses,thestudyresultsindicatethatFCTAmaybea
reli-ableoptionforHIV-1tropismdetermination.
Geno2pheno methodology forHIV-1 tropism
determina-tion becameafasterandcheaperalternativetophenotypic
tests.Theavailableevidenceindicate thatgeno2pheno
pro-vides an efficient way to evaluate viral tropism, and it is
thetestrecommendedbytheEuropeanAIDSClinicalSociety
(EACS)guidelines.25
Accordingtosomestudies,CD4countcan bean
impor-tant predictive factor indetermining viraltropism. Among
thealgorithms,PSSMX4R5showedthehighestconcordance
ratewithESTROFILE.ThebestcorrelationwithTROFILEwas
obtainedinpatientspresentingwithCD4+cellcountbetween
200and 500cells/mm3.Incontrast,theweakercorrelations
were found in patients with CD4+ cell count lower than
200cells/mm3.Inourwork,therewasnovariationintest
per-formanceacrossdifferentstrataCD4cellcounts.Ofnote,there
wasahigherlikelihoodofidentifyingR5virusesinpatients
Table1–HIV-1tropismclassificationaccordingtotheassayemployed.
FCTAaclassification Geno2phenoclassification
CCR5b Dualinfection CXCR4c Total
n (%) n (%) n (%)
CCR5 65 (100) 1 (3.4) 0 (0) 66
DualInfection 0 (0) 28 (96.6) 4 (57.1) 32
CXCRd 0 (0) 0 (0) 3 (42.9) 3
Total 65 29 7
TheoverallconcordanceratebetweenbothtestsforidentifyingR5viruswas100%(95%CI:94.4–100%).Thirty-fivepatientswereclassifiedas non-R5(32asduotropic/mixedvirusesandthreeasX4)bygeno2pheno.Allbutonesamplewereequallyclassifiedasnon-R5byFCTA:one samplewasclassifiedasnon-R5bygeno2phenoandasR5byFCTA.Theseresultscorrespondto97.2%specificity(95%CI:85.8–99.5%)ofFCTA usinggeno2phenoasgoldstandard.Oneadditionalsamplecouldnotbeamplifiedingeno2phenoandwasclassifiedasduotropicbyFCTA.
a FCTA,flowcytometrytropismassay. b Co-receptorC-Cmotif5.
c Co-receptorC-Xmotif4. dShadeareaisthespecificityzone.
Sensitivity:100%;specificity:97.2%.
Table2–HIV-1tropismaccordingtoCD4+cellcount stratification,andtothetypeofassayused.
RangeCD4counts Geno2pheno Total CCR5 Non-CCR5 n(%) n(%) n(%) FCTA (0–249) CCR5 9(100) 0(0) 9(60) Non-CCR5 0(0) 8(100) 8(40) (250–499) CCR5 23(100) 1(7.1) 24(62.9) Non-CCR5 0(0) 11(92.9) 11(37.1) (500–749) CCR5 18(100) 0(0) 18(69.2) Non-CCR5 0(0) 8(100) 8(30.8) (>750) CCR5 15(100) 0(0) 15(75) Non-CCR5 0(0) 7(100) 7(25)
knownthatX4virusesemergelateinthecourseofHIV-1 infec-tion,andourresultsareconsistentwithsuchcontention.
In our study, FCTA presented a performance to define
viraltropismcomparabletothatfoundbyusinggeno2pheno methodology.FCTAhastheadvantageofbeinglesstime con-suming,andhasalowercostthangenotypictests.Itisalso technicallysimpler,andrequireslesssophisticated laborato-riesthangenotypic–basedmethods.Brazilhasalarge(over90 sites),alreadyin-placenetworkofreferrallaboratories capa-bleofperformingflow-cytometryassaysformanagementof HIV-infectedpatientsinpublichealthsettings.Ontheother hand,onlyoftheselaboratoriesareabletoperformgenotypic tropismtests,andareresponsibleforreceivingsamplesfrom allBrazilianstates.
Maraviroc,theonlyCCR5antagonistapprovedtoclinical use,wasrecentlymadeavailablefortreatingBrazilian HIV-infectedpatientswithpreviousfailuretootherdrugs/classes. Itisexpectedasignificantincreaseinthenumberoftropism testsreferredtoreferencelaboratories,andthereisaconcern ontheircapability totimelyrespondtosuchanincreasing demand. A simpler, faster, tropism test would be of help insuch situation. Itmakes FCTAa potentially useful tool, withsignificantlylowercosts,andshorterturnaroundtime. Itcould bewidelyused, duetothe alreadyexisting condi-tions.Moreover,our resultsdemonstrated thatFCTAhas a
high concordancerate withgeno2pheno algorithmin addi-tiontonotrequirenucleicacidamplification.Otheradvantage isitscapacitytodefineviraltropismeveninpatientsunder suppressivetherapy,onceitusesproviralDNA,anddoesnot dependondetectableHIV-1RNAinplasma.
Inconclusion,FCTAshowedanexcellentcorrelationwith genotypic assay and could representa cheaper, faster and easiertool.Itsimplementationcouldprovidelargeraccessof BrazilianpatientstotreatmentwithCCR5antagonists.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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