Review of Viral LoadReview of Viral LoadTechnologiesTechnologies
Susan A. Fiscus, Ph.D.Susan A. Fiscus, Ph.D.University of North CarolinaUniversity of North Carolina
at Chapel Hillat Chapel Hill
Model for HIV Assays in Resource-Model for HIV Assays in Resource-Poor SettingsPoor Settings
ReferenceReferenceCenterCenter
Provincial orProvincial ordistrict leveldistrict level
Primary care orPrimary care orrural settingrural setting
Viral loadViral load ExpensiveExpensive Complex technologyComplex technology Gold standardGold standard
P24/Reverse transcriptase?P24/Reverse transcriptase? Lower costLower cost Less complexLess complex
technologytechnology
Ship samples (DBS orShip samples (DBS orfixatives)fixatives)
Least resourceLeast resourceintensiveintensive
Least complexLeast complex
Commercially Available ViralCommercially Available ViralLoad Load –– HIV RNA HIV RNA
*Roche Monitor, 1.5 *Roche Monitor, 1.5 –– RT-PCR RT-PCR **bioMerieuxbioMerieux NucliSensNucliSens--
isothermal NASBAisothermal NASBA *Bayer Versant - *Bayer Versant - bDNAbDNA bioMerieuxbioMerieux EasyQEasyQ –– molecular molecular
beaconbeacon PrimagenPrimagen Retina Rainbow Retina Rainbow ––
molecular beaconmolecular beacon
* * FDA approvedFDA approved
Pros and Cons of HIV RNAPros and Cons of HIV RNAAssaysAssays
AdvantagesAdvantages•• High ThroughputHigh Throughput•• Well validatedWell validated•• 3 are FDA approved3 are FDA approved•• Clinician familiarityClinician familiarity•• Most subtypesMost subtypes•• Manufacturers QAManufacturers QA
reagentsreagents•• Work with DBSWork with DBS•• Possible reducedPossible reduced
price through largeprice through largevolume purchasevolume purchase
DisadvantagesDisadvantages•• ExpensiveExpensive
equipmentequipment•• ExpensiveExpensive
reagentsreagents•• TechnologicallyTechnologically
complexcomplex•• EquipmentEquipment
maintenancemaintenance
Other AssaysOther Assays Real time PCRReal time PCR P24 antigenP24 antigen CavidiCavidi RT RT FlowFlow Point of Care Point of Care ––
•• DipstickDipstick•• NanoparticlesNanoparticles•• Chip technologyChip technology•• Shipping specimensShipping specimens
Pros and Cons of Real TimePros and Cons of Real TimePCR AssaysPCR Assays
AdvantagesAdvantages•• ReagentsReagents
inexpensiveinexpensivecompared tocompared tocommerciallycommerciallyavailable kitsavailable kits
•• Can be very sensitiveCan be very sensitive(Palmer)(Palmer)
•• Can be very specificCan be very specific((RouziouxRouzioux))
•• Assays for additionalAssays for additionalpathogens can bepathogens can bedevelopeddeveloped
DisadvantagesDisadvantages•• Very expensive equipmentVery expensive equipment•• Home brew assays, soHome brew assays, so
variability in reagents and novariability in reagents and nomanufacturermanufacturer’’s QAs QA
•• May need different primersMay need different primersand probes for differentand probes for differentsubtypessubtypes
•• Royalty charge for use of Royalty charge for use of TaqTaq•• ReproducibilityReproducibility•• Technologically complexTechnologically complex•• Prone to contaminationProne to contamination•• Clinical validation yet to beClinical validation yet to be
donedone
HD24 Ag AssayHD24 Ag AssaySpecimen preparationSpecimen preparation
Dilute 50 Dilute 50 ulul of plasma with 250 of plasma with 250ulul of detergent-containing of detergent-containing lysislysisbufferbuffer
Mix and incubate 10 min at roomMix and incubate 10 min at roomtemptemp
Heat for 5 min at 100C in dryHeat for 5 min at 100C in dryheat blockheat block
Cool to room temperatureCool to room temperature
Up24 Antigen Assay
YYYYY
YYYYY*****
Y Capture antibodyAntigen 2 hr with shaking RT
Y* Detector antibody-biotin 1 hr, 37
TTTTStreptavidin-HRP 15 min, 37
**** T* Biotinyl-Tyramide 15 min, RT
Streptavidin-HRP 15 min, RT
OPD 30 min, RT
Heat Dissociated p24 AntigenHeat Dissociated p24 Antigen Assay works very well to diagnoseAssay works very well to diagnose
infants (infants (SutthentSutthent, 2003; Sherman, 2004;, 2003; Sherman, 2004;NouhinNouhin, Bangkok; , Bangkok; DeBaetsDeBaets, 2005;, 2005;RespessRespess CROI) CROI)
New buffer described by Dr. J.New buffer described by Dr. J.SchupbachSchupbach (JAIDS, 2003) increases (JAIDS, 2003) increasessensitivity of the assay (Jennings, ICAAC,sensitivity of the assay (Jennings, ICAAC,2003; Fiscus, CROI 2004)2003; Fiscus, CROI 2004)
In general studies using the kit bufferIn general studies using the kit bufferhave performed less favorably (have performed less favorably (BonardBonard,,2003; 2003; PradoPrado, 2004) compared to those, 2004) compared to thoseusing the using the SchupbachSchupbach buffer ( buffer (RibasRibas, 2003;, 2003;SchupbachSchupbach, 2003; Stevens, in press), 2003; Stevens, in press)
HDp24 for Infant DiagnosisHDp24 for Infant Diagnosis
multiplemultiple100%100%92.3%92.3%150150DeBaetsDeBaets(DRC)(DRC)
A/EA/E99.2%99.2%91.1%91.1%167167NouhinNouhin(Cambodia)(Cambodia)
BB98.7%98.7%95.8%95.8%749749RespessRespess(US)(US)
multiplemultiple100%100%100%100%8787DeBaetsDeBaets(DRC)(DRC)
CC98.5%98.5%98%98%203203ShermanSherman(So Africa)(So Africa)
A/E, BA/E, B100%100%100%100%142142SutthentSutthent(Thailand)(Thailand)
SubtypeSubtypeSpecSpecSensSensNNAuthorAuthor
Performance of p24 and Up24Performance of p24 and Up24for Detection of Acute HIVfor Detection of Acute HIV
(0.11, 0.59)(0.11, 0.59)0.250.25LR-LR-
(67.0, 492.3)(67.0, 492.3)181.7181.7LR+ Up24LR+ Up24
(58.5, 447.8)(58.5, 447.8)161.8161.8LR+ p24LR+ p24
(0.988, 0.999)(0.988, 0.999)0.9950.995SpecificitySpecificity
(0.604, 0.966)(0.604, 0.966)0.8420.842Sensitivity Up24Sensitivity Up24
(0.476, 0.927)(0.476, 0.927)0.7500.750Sensitivity p24Sensitivity p24
(95% CI)(95% CI)EstimateEstimate
Patient MonitoringPatient Monitoring
0
1
2
3
4
5
6
11/1
3/19
98
1/13
/199
9
3/13
/199
9
5/13
/199
9
7/13
/199
9
9/13
/199
9
11/1
3/19
99
1/13
/200
0
3/13
/200
0
5/13
/200
0
7/13
/200
0
9/13
/200
0
11/1
3/20
00
1/13
/200
1
3/13
/200
1
Dates
Log1
0 va
lues
vl
p24
Pros and Cons of HeatPros and Cons of HeatDissociated p24 AntigenDissociated p24 Antigen
AdvantagesAdvantages•• Equipment generallyEquipment generally
availableavailable•• Less technologicallyLess technologically
complexcomplex•• High through putHigh through put•• Less prone toLess prone to
contaminationcontamination•• Excellent for infantExcellent for infant
diagnosisdiagnosis•• Appears to work wellAppears to work well
in diagnosing acutein diagnosing acuteHIV infectionHIV infection
•• Very reproducibleVery reproducible
DisadvantagesDisadvantages•• DoesnDoesn’’t measure t measure virionvirion--
associated molecule, soassociated molecule, sosometimes get differentsometimes get differentresults than RNAresults than RNA
•• Works best with non-kitWorks best with non-kitbuffer, so has similar QAbuffer, so has similar QAproblems to other problems to other ““home-home-brewbrew”” assays assays
•• Usually not as sensitive asUsually not as sensitive asmost of the other assaysmost of the other assays
•• Need more data on otherNeed more data on othersubtypes and clinical validationsubtypes and clinical validation
CavidiCavidi ExaVirExaVir Assay (RT) Assay (RT)
Newer version of assay muchNewer version of assay muchmore sensitive (Jennings,more sensitive (Jennings,unpublished; unpublished; GreengrassGreengrass, in, inpress; press; ShaoShao, Bangkok), Bangkok)
Being evaluated as anBeing evaluated as analternative to VL testingalternative to VL testing(Stevens, in press; (Stevens, in press; GreengrassGreengrass,,in press)in press)
Phenotype assay Phenotype assay –– Simon, Simon,Bangkok; Bangkok; NtsalaNtsala, Bangkok, Bangkok
RT Viral Load Assay: Virion separation procedure
RT Viral Load Assay: RT Viral Load Assay: RT ActivityRT ActivityDeterminationDetermination
VirologicVirologic Results Results In Malawi we have tested 126 samplesIn Malawi we have tested 126 samples
from 40 subjectsfrom 40 subjects Correlation coefficient for log10 HIV RNACorrelation coefficient for log10 HIV RNA
and log10 RT is 0.89and log10 RT is 0.89
050
100
1stQtr
3rdQtr
EastWestNorth2
34
56
logr
na
2 3 4 5 6lo g R T
Patient 862538
0
20 000
40 000
60 000
80 000
100 000
Dec
-99
Mar
-00
Jun-
00
Sep-0
0
Dec
-00
Mar
-01
Jun-
01
Sep-0
1
Dec
-01
Mar
-02
Jun-
02
HIV
RN
A c
op
ies/
ml
HIV RNA RT (converted)
Patient 803461
0
20 000
40 000
60 000
80 000
100 000
Sep-9
9
Dec
-99
Mar
-00
Jun-
00
Sep-0
0
Dec
-00
Mar
-01
Jun-
01
Sep-0
1
Dec
-01
Mar
-02
Jun-
02
HIV
RN
A c
opie
s/m
l
HIV RNA RT (converted)
Patient 862874
0
20 000
40 000
60 000
80 000
100 000
May
-98
Aug-9
8
Nov-9
8
Feb-9
9
May
-99
Aug-9
9
Nov-9
9
Feb-0
0
May
-00
Aug-0
0
Nov-0
0
HIV
RN
A c
opie
s/m
l
HIV RNA RT (converted)
Patient 862842
0
20 000
40 000
60 000
80 000
100 000
Sep-0
0
Dec
-00
Mar
-01
Jun-
01
Sep-0
1
Dec
-01
Mar
-02
Jun-
02
Sep-0
2
HIV
RN
A c
opie
s/m
l
HIV RNA RT (converted)
Comparison of viral load monitoring using CavidiExaVir, Bayer bDNA and Roche Amplicor in HIV patientson ARVs from the Australian cohort, Melbourne,Australia
Australian cohort (Greengrass et al, In Press)
Pros and Cons of the Pros and Cons of the ExaVirExaVirAssayAssay
AdvantagesAdvantages•• Should work on allShould work on all
subtypessubtypes•• InexpensiveInexpensive
equipmentequipment•• Sensitive to aboutSensitive to about
500 cp/ml500 cp/ml•• Phenotype fromPhenotype from
same RT prepsame RT prep•• Less prone toLess prone to
contaminationcontaminationthan PCR assaysthan PCR assays
DisadvantagesDisadvantages•• Long assay (3 days)Long assay (3 days)•• Phenotype assayPhenotype assay
only for only for NNRTIsNNRTIs and andT analog T analog NRTIsNRTIs
•• Nearly as expensiveNearly as expensiveas RNA assays if youas RNA assays if youcan get a largecan get a largevolume discountvolume discount
Point of Care TestsPoint of Care Tests
Dipstick Dipstick –– Helen Lee Helen Lee NanoparticlesNanoparticles for p24 Ag detection for p24 Ag detection
(Kim, (Kim, WolinskyWolinsky)) NielNiel Constantine Constantine Chip Technology Chip Technology –– Bill Rodriguez, Bill Rodriguez,
othersothers Shipping specimensShipping specimens
•• Dried blood spotsDried blood spots•• Sample tanker Sample tanker –– stabilizes dried plasma- stabilizes dried plasma-
Rob LloydRob Lloyd
Obstacles to ImplementationObstacles to Implementation InfrastructureInfrastructure Guidelines and consensus protocolsGuidelines and consensus protocols Lack of trained laboratory personnelLack of trained laboratory personnel Training toolsTraining tools Monitoring and evaluation toolsMonitoring and evaluation tools Supply managementSupply management Data managementData management Quality assurance (ProficiencyQuality assurance (Proficiency
testing)testing)
ConclusionsConclusions Commercially available viral loadCommercially available viral load
assays are becoming less expensive,assays are becoming less expensive,but are still technologically complexbut are still technologically complexand best suited for large referenceand best suited for large referencelabslabs
Real time PCR assays, though lessReal time PCR assays, though lessexpensive for reagents, suffer fromexpensive for reagents, suffer fromhigh equipment costs and lack of QAhigh equipment costs and lack of QAof reagentsof reagents
HD P24 antigen seems very suitableHD P24 antigen seems very suitablefor infant diagnosis, and much lessfor infant diagnosis, and much lessexpensive than NATexpensive than NAT
Conclusions (2)Conclusions (2) Alternative assays for viral loadAlternative assays for viral load
(p24, RT, etc) may be useful in(p24, RT, etc) may be useful inprovincial labs, but:provincial labs, but:•• Are in a state of fluxAre in a state of flux•• P24 may not strictly correlate withP24 may not strictly correlate with
HIV RNA VLHIV RNA VL•• P24 assay gives best results withP24 assay gives best results with
an external an external lysislysis buffer buffer•• P24 and RT assays need moreP24 and RT assays need more
clinical validation, especially withclinical validation, especially withthe latest versionsthe latest versions
Conclusions (3)Conclusions (3) Primary care or rural settings for thePrimary care or rural settings for the
moment will have to ship samples tomoment will have to ship samples toa reference laboratorya reference laboratory
Point of care testing may be availablePoint of care testing may be availablein the next few years, but results willin the next few years, but results willhave to carefully QAhave to carefully QA’’d and costs mayd and costs maymake it better to ship samples to amake it better to ship samples to areference lab with high throughput,reference lab with high throughput,QA, and negotiated kit pricesQA, and negotiated kit prices
Conclusions (4)Conclusions (4) There are many obstacles toThere are many obstacles to
introducing laboratory assays inintroducing laboratory assays ingeneral to labs in resource poorgeneral to labs in resource poorsettingssettings
Many groups large and small areMany groups large and small aretrying to tackle the problems, manytrying to tackle the problems, manyof which are country specificof which are country specific
Collaboration by the large groups forCollaboration by the large groups fora global strategy, and by all playersa global strategy, and by all playersin a given country for the local plan,in a given country for the local plan,will reduce confusion and avoidwill reduce confusion and avoidwaste of time and effort.waste of time and effort.
Are VL Assays Necessary forAre VL Assays Necessary forStarting and Monitoring ART?Starting and Monitoring ART? Starting ART Starting ART –– Probably not Probably not
necessary necessary –– CD4 is probably adequate CD4 is probably adequate Monitoring ART for an individualMonitoring ART for an individual
•• Early to assess efficacyEarly to assess efficacy•• Later to determine failure and timeLater to determine failure and time
to switchto switch•• But itBut it’’s hard to make comparisonss hard to make comparisons
unless you have the baseline valueunless you have the baseline value Important for public healthImportant for public health
considerationsconsiderations
