Development and validation of a simple and rapid way to generate low volume of plasma to be used in point-of-care HIV virus load technologies

brazjinfectdis2020;24(1):30–33

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

Development

and

validation

of

a

simple

and

rapid

way

to

generate

low

volume

of

plasma

to

be

used

in

point-of-care

HIV

virus

load

technologies

Isabelle

Vasconcellos

b

_,

_Diana

_Mariani

a

_,

_Marcelo

_C.V.M.

_de

_Azevedo

b

_,

Orlando

C.

Ferreira

Jr.

a

_,

_Amilcar

_Tanuri

a,∗

a_{UniversidadeFederaldoRiodeJaneiro,DepartamentodeGenética-InstitutodeBiologia,RiodeJaneiro,RJ,Brazil}

b_{UniversidadeFederaldoestadodoRiodeJaneiro,HospitalUniversitárioGaffrèeeGuinle,LaboratóriodePesquisaemImunologiae}

AIDS,RiodeJaneiro,RJ,Brazil

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n

f

o

Articlehistory:

Received16July2019

Accepted27October2019

Availableonline21November2019

Keywords:

POCVL

HIVviralload

HIV Point-of-care Fingerstick-plasma mPIMA

a

b

s

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c

t

Anewpoint-of-care(POC)HIVvirusload(VL),mPIMAHIV-1/2VL,Abbott,USA,hasbeen

recentlydeveloped.ThisPOCVLrequiresnoskilledpersontorunandusesasmallplasma

volume(50␮L).However,obtaining50␮Lofplasmacanbeachallengeinlimitedresource

settings.Wevalidatedasimpleandeasymethodtoobtainenoughamountofplasmatoruna

POCVL.Thestudyutilized149specimensfrompatientsfailingantiretroviraltherapy.Atleast

250␮Lofwholebloodwascollectedinamicrotube/EDTAfromfingerstick(fs-plasma)and

immediatelycentrifuged.Parallelcollectionofvenousbloodtoobtainplasma(vp-plasma)

wasusedtocompareperformanceinaPOCVLassayandinmethodologyusedincentralized

laboratoriesAbbottM2000,Abbott,USA.Theprocedureforplasmacollectiontakesless

than10minandin94%ofthecasesonlyonefingerstickwassufficienttocollectatleast

250␮Lofblood.ThePearsoncorrelationcoefficientvalueforvp-plasmaversusfs-plasma

ranonmPIMAwas0.990.TheBland–Altman meandifference(md) forthiscomparison

werevirtuallyzero(md=−0.001)withlimitsofagreementbetween−0.225and0.223.In

addition,thePearsoncorrelationcoefficientvalueforfs-plasmainmPIMAversusvp-plasma

inAbbottM2000was0.948forvaluesabovethemPIMAlimitofquantification(LoQ;from800

to1,000,000copies/mL).Theseresultsvalidatethissimpleplasmaisolationmethodcapable

tobeimplementedinlowresourcecountrieswherePOCdecentralizationisdeeplyneeded.

©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:atanuri@biologia.ufrj.br(A.Tanuri).

https://doi.org/10.1016/j.bjid.2019.10.010

1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC

brazj infect dis.2020;24(1):30–33

31

Introduction

HIVviralload(HIVVL)testingisanimportanttoolfor

monitor-ingsuccessfulanti-retroviraltreatment(ART).1_{The90–90–90}

HIVtargetslaunchedbyTheJointUnitedNationsProgramon

HIV/AIDS(UNAIDS)highlightedtheimportanceofviralload

testingandmadeanurgentcallforincreasedaccesstoviral

loadandotherHIVdiagnostictests.2,3

IndevelopedcountriesARVtreatmentismonitoredbyVL

testingusinghighthroughputstandardofcaretechnologiesin

centralizedlaboratories.Althoughcentralizedtestingisa

log-icalsolution,itposesaseriouschallengeincountrieswhere

roads are insufficient, many not paved and often blocked

byfloods.Alsochallengingaresamplecollection,coldchain

transportationtothecentrallaboratories,andresultreturnto

theclinicstoaidclinicaldecisionmaking.Infact,insome

set-tingsresultstakemorethanonemonthtoreturntohealth

professionalsandasubstantialamountofHIVVLtestsfailto

returntopatientfileshighlightingthelossofresourcesand

effort.4_{Forexample,inZimbabwe,useofcentralized}

labora-toriesforearlyinfantdiagnosisresultedindelaysofseveral

monthsforEIDdiagnosis and nearly halfofinfantstested

neverreceivedtheirresults.5

Point-of-care(POC)HIVvirusloadtechnologieshavebeen

recently developed allowing decentralized HIV VL test to

occur. POC assays are simpler and faster than

laboratory-based assays, requires minimal training, do not require

complex infrastructure and obviates the need for sample

transportation. However,theystill fallshortofexpectation

byrequiringinsomecases,largeplasmavolume(1mL)and

needscomputer analysis making it cumbersome for some

settings.AnewPOCHIVvirusloadtechnologybasedin

ther-mostablecartridge,mPIMAHIV-1/2VL(mPIMA),Abbott,USA,

hasbeenrecentlydeveloped.mPIMAHIV-1/2VLusesasmall

plasmavolume(50␮L)withaturnaroundtimeof1h,andhas

alimitofquantification(LoQ)of800copies/mLandadynamic

rangeextendingupto106_{copies/mL.Still,obtainingof50␮L}

ofplasmacan bechallengingin remotesitesor inlimited

resourcesettings.Inthiswork,ourgroupvalidatedasimple

andreliablewaytogenerate50␮Lofplasmafromdigital

punc-turebloodcollection,comparingtovenousbloodandtoM2000

AbbottHIVvirusloadassay,tobeusedinsettingslacking

reg-ularclinicallaboratoriescapableofdoingbloodcollectionby

venipunctureandregularcentrifugation.

Methods

The mPIMA was evaluated using 50␮L of plasma

speci-mensobtainedfrom wholebloodcollected byvenipuncture

(vp-plasma)ordigitalpuncture(fingerstick,fs-plasma)from

patientswithdetectable viralload.VL fromvp-plasmawas

firstmeasuredbytheAbbottM2000sqand149/160specimens

withVLfallingwithinthequantitativerangeofmPIMA(from

800to1×106_{Logcp/mL)weresubsequently runonmPIMA.}

Theremaining11specimenshadVLbelow800cp/mL(n=6)or

above106_{cp/mL(n=5)byAbbottM2000sq,andwereexcluded}

fromtheevaluation.

Basically, the mPIMA HIV-1/2 VL plasma test was run

followingoriginalinstructionsgeneratingplasmausingtwo

differentmethodologies. Thestandardprocedurewas done

through venipunctureusing anEDTA-Vacutainer tubes

fol-lowed by centrifugation at 600×g for 10min at room

temperature ina regular clinical centrifuge,and 50␮Lwas

collectedwitharegularadjustablevolumemicropipette.The

alternativeprocedurewasdevelopedcollecting250–500␮Lof

bloodinanEDTABDMicrotainertubewithBDMicrogard(BD

Cat#365974)usingafingerstickdonewithaBDHighFlow

lancet(BDCat#366594).Plasmawasseparatedbycentrifuging

themicrotubeatmaximumspeedfor5minusinga1.5–2.0mL

tubemicrofugeforsixtubes(USAScientific,CAT#2631-0006)

pluggedinacomputerUPSwhichgivestothemicrofugemore

than7hautonomy.Aftercentrifugation50␮Lplasmawas

col-lected usingafixvolumeplasticPasteurpipettes (Pastettes

50␮LDualBulb,CAT#LW4745-500,AlphaLaboratoriesLtd,UK)

andaddeddirectonmPIMAcartridgetorun(seeFig.1and

SupplementalVideo1formoredetails).

Forthisprocedurewehaveestablishedtwoparametersto

measurethefeasibilityofthemethod.Onewasthefrequency

inwhichtheminimalvolumeof250␮Lofbloodfrom

finger-prick collectionwould beobtained,andsecond,how many

digitalpuncturewouldbenecessarytoobtainsuchminimal

volume.

Results reported in copy number/mL (cp/mL) and were

log10transformedtologcp/mLforstatisticalanalysis.Analysis

includedagreementbetweenthetwoassaysassessedbythe

Bland–Altman (B&A)and linear correlationbetween assays

expressedbyPearsoncorrelationcoefficient. Analyseswere

performed withMicrosoft Excelsoftware. Theresultswere

alsocomparedtoAbbottM2000.

This study was ethically reviewed by Hospital

Graf-feeGuinleIRBunder#86604317.6.0000.5258.

Results

The149selectedspecimensthathavehadplasmaobtained

by twoselection methods(vp-plasma and fs-plasma)were

assayedbymPIMAforperformancecomparison.Inparallel,

both vp-plasma and fs-plasma were alsomeasured by the

Abbottm2000sqassay.

We first looked whether pairs of assay and/or plasma

preparationmethodwerelinearlycorrelated.ThePearson

cor-relationcoefficientvalue(R)foreach comparisonattesteda

goodcorrelationbetweenpairs,asfollows:(a)vp-plasma

ver-susfs-plasma,bymPIMA(R=0.990)(seeFig.2);(b)vp-plasma

byAbbottm2000versusfs-plasmabymPIMA(R=0.948);and(c)

vp-plasma,Abbottm2000versusmPIMA(R=0.951)(seeFig.2A

andC).

Wealsoanalyzedlevelofagreementbetweenpairsofassay

and/orplasmapreparationmethodbyB&Aanalysis(Fig.3).

Mean difference (md) and limits ofagreement (LoA) were:

(a)vp-plasmaminusfs-plasma,bymPIMA(md=−0.001;LoA:

−0.225 to0.223); (b)vp-plasma byAbbott m2000minus

fs-plasmabymPIMA(md=−0.145;LoA:−0.655to0.365)and;(c)

vp-plasma,Abbott m2000minus mPIMA(md=−0.144;LoA:

32

braz j infectdis.2020;24(1):30–33

Fig.1–Alternativeproceduretogenerateplasmainsiteswithnolaboratoryinfrastructure.Eachframerepresents:1, fingerstick;2,collectingthebloodflow;3,bloodcollectedinmicrotube;4,minifugeassemblewithUPS;5,collectingplasma witha50␮Lfixedvolumepippet;6,loadingmPIMAcartridge;7,insertingthecartridgeinmPIMA;8,resultdisplayedin mPIMA;9,printingresult;10,fullmPIMAapparatussetup.

6,5 1,0 0,5 0,0 -0,5 -1,0 1,0 0,5 0,0 -0,5 -1,0 y = 0,9299x + 0,4336 Abbott VP (Log cp/mL) mPIMA VP (Log cp/mL) Ab bott VP - mPIMA VP (Log cp/mL) Ab bott VP - mPIMA FS (Log cp/mL) mPIMA FS (Log cp/mL)

Abbott VP + mPIMA VP/2 (Log cp/mL)

(Abbott VP + mPIMA FS)/2 (Log cp/mL) Abbott VP (Log cp/mL) y = 0,9036x + 0,544 R2 _{= 0,904} R2 = 0,8994 6,0 5,5 5,0 4,5 4,0 3,5 3,0 2,5 6,5 6,0 5,5 5,0 4,5 4,0 3,5 3,0 2,5 2,5 2,5 3,5 3,5 4,5 4,5 5,5 5,5 6,5 2,5 3,5 4,5 5,5 6,5 6,5 2,5 3,5 4,5 5,5 6,5

Fig.2–(A)and(C)showthelinearregressionand(B)and(D)Bland–Altmananalysis,respectively,fromVLresultsusingand vp-plasmaassayedbyAbbottm2000versusvp-plasmaandfs-plasmaassayedbymPIMA(n=149plasmaspecimens).In(A) and(C),acontinuouslineshowstheLRline.(B)and(D)showtheaverageVLobtainedbyAbbottvp-plasmaminusmPIMA vp-plasma(B)orminusmPIMAfs-plasma(D)versustheVLdifferencebetweenspecimens:dashedlineindicatesdemean differencebetweenVLoftwospecimensanddottedlinesthelimitsofagreementofthemeandifference.Alldatashown werelog10transformed.

Regardingtheparametersestablishedtoaccessfeasibility

offingerstickbloodcollection,wehaveobservedthatinonly

oneoccasion(0.7%)thecollectedbloodvolumewaslessthan

250␮L.Inthisparticularcase,therewasnoattempttoperform

asecond fingerstick.Excludingthiscase,139/148(94%)

col-lectionyieldedbloodvolumeequaltoorgreaterthan250␮L,

afteronesinglefingerstick.Forthelastnineindividuals,an

min-brazj infect dis.2020;24(1):30–33

33

B A 6,5 1,0 0,0 -1,0 6,0 5,5 5,0 4,5 4,0 3,5 3,0 2,5 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 6,5 mPIMA VP (Log cp/mL) mPIMA FS (Log cp/mL) Diff erence mPIMA VP-FS (Log cp/mL) mPIMA VP+FS) (Log cp/mL)

Fig.3–(A)and(B)showthelinearregressionandBland–Altmananalysis,respectively,fromVLresultsusingandVPandFS plasmacollectionmethodologies,generatedonthemPIMAtechnology(n=149plasmaspecimens).In(A),acontinuousline showstheLRanalysisanddashedlineindicatestheequalityline.(B)showstheaverageVLobtainedbyVPandFSversus thedifferencebetweenspecimensobtainedbyVPandFS:dashedlineindicatesdedifference(VP-FS)anddottedlinesthe limitsofagreement.Alldatashownwerelog10transformed.

imalvolume.Overall,the proceduretakeslessthan10min,

includingwhenaseconddigitalpunctureisneeded.

Discussion

The results generated in this study shown that there

are no meaningful VL differences between plasma

preparation methods when measurements were done by

mPIMA.Whenthefs-plasmaviralloadinmPIMAiscompared

withAbbottM2000,themethodologyuseduntilnowin

cen-tralizedlaboratoriesinBrazil,wehadanexcellentcorrelation

givingaPearson correlationcoefficient valueforfs-plasma

in mPIMA versus vp-plasma in Abbott M2000 of 0.948 for

values above the mPIMA limit of quantification (from 800

to 1,000,000cp/mL). This value for the Pearson correlation

coefficientissimilartotheonefoundbyCeffaaetal.(2016)

whenthey compared Abbott M2000with another

method-ology(CepheidGeneXpert).Itisimportanttohighlightthat

AbbottM2000andothermethodologieslikeGeneXpertneed

around1mLofplasma.6 _{AbbottM2000assay use0.6mLof}

plasmatorun,evenwhenadaptationsaremade,suchasfor

DBS.7

Inaddition,thecollectionof50␮Lofplasmabyfingerstick

wasfeasibleandeasytoimplementinsiteswithoutlaboratory

infrastructure.Ofnote,VLmeandifferencebetweenplasma

preparationmethodswasvirtuallyzero(md=−0.001)on

sam-plesanalyzedbymPIMA.

Thisperformanceofplasma obtainedbyfingerstick

(fs-plasma)willmakePOCHIVVLtechnologyavailabletofollow

upand monitorpatients underHAART insettings without

laboratoryinfrastructure,like mobileclinicsor standalone

outpatientclinics.TheuseofaUPSlinkedmicrofugemakes

theplasmaseparationfeasibletobedonewithouttheneedof

regularpowersource.UsingPOCVLassaysplustheproposed

plasmaseparation methodology could substantially reduce

waitingtimeforviralloadresultsanddrasticallyshortenthe

timetoinitiationofART.Surely,thiswillimpactonHIV

trans-missioninlocalcommunitiesandhelpcountriesachieving

UNAIDS90/90/90target.

Funding

ThisworkwassupportedbyAlereInc.#FUJB234/2016.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Appendix

A.

Supplementary

data

Supplementary data associated with this article can be

found, in the online version, at https://doi.org/10.1016/j.

bjid.2019.10.010.

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c

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s

1.WorldHealthOrganization.Consolidatedguidelinesontheuse ofantiretroviraldrugsfortreatingandpreventingHIV infection:ecommendationsforapublichealthapproach;2015.

2.UNAIDS.90–90–90anambitioustreatmenttargettohelpend theAIDSepidemic;2014.

3.UNAIDS.LandmarkHIVdiagnosticaccessprogramwillsave $150mandhelpachievenewglobalgoalsonHIV;2014.

4.SaitoS,DuongYT,MetzM,etal.ReturningHIV-1viralload resultstoparticipant-selectedhealthfacilitiesinnational population-basedHIVimpactassessment(PHIA)household surveysinthreesub-SaharanAfricanCountries,2015to2016.J IntAIDSSoc.2017;20Suppl.7:19–25.

5.FrankSC,CohnJ,DunningL,etal.Clinicaleffectand

cost-effectivenessofincorporationofpoint-of-careassaysinto earlyinfantHIVdiagnosisprogrammesinZimbabwe:a modellingstudy.LancetHIV.2019;6:182–90.

6.CeffaaS,LuhangabR,AndreotticM,etal.Comparisonofthe CepheidGeneXpertandAbbottM2000HIV-1realtime molecularassaysformonitoringHIV-1viralloadand detectingHIV-1infection.JVirolMethods.2016;229:35–9.

7.ZehC,NdiegeK,InzauleS,etal.Evaluationoftheperformance

ofAbbottm2000andRocheCOBASAmpliprep/COBASTaqman

assaysforHIV-1viralloaddeterminationusingdried

bloodspotsanddriedplasmaspotsinKenya.PLoSONE.

2017;12:e0179316,