“HIV Proviral DNA is an adequate genetic compartment for...
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[email protected] “HIV Proviral DNA is an adequate genetic compartment for detection of resistance mutations associated to integrase inhibitors (INIs) therapy in patient with undetected or low viral load” M Durán 1 , C. Palma 2 , L. Montecinos 2 , M. Ferrés 2 , A. Afani 1 , C. Pérez 2 , R. Tordecilla 1 , P Ferrer 1 1 Hospital Clínico Universidad de Chile, Laboratorio de Medicina Molecular, Santiago de Chile. 2 Pontificia Universidad Católica de Chile, Laboratorio de Infectología y Virología Molecular.
Transcript of “HIV Proviral DNA is an adequate genetic compartment for...
“HIV Proviral DNA is an adequate genetic compartment for detection of resistance
mutations associated to integraseinhibitors (INIs) therapy in patient with
undetected or low viral load”
M Durán1, C. Palma2, L. Montecinos2, M. Ferrés2, A. Afani1, C. Pérez2, R. Tordecilla1, P Ferrer1
1 Hospital Clínico Universidad de Chile, Laboratorio de Medicina Molecular, Santiago de Chile.
2 Pontificia Universidad Católica de Chile, Laboratorio de Infectología y Virología Molecular.
Integrase inhibitors (INIs) is afamily of drugs that block the transfer HIVproviral DNA into host´s genome.Resistance mutations have beenobserved in patients experiencing virologicfailure (VF) on Raltegravir (RAL),Elvitegravir (EVG) or Dolutegravir (DTG)treatment.
HIV RNA
Introduction
RT
We studied 47 patients with virologic failure (viral load>1,000 HIV RNA copies/mL) under antiretroviral treatmentincluded RAL, on simultaneously collected RNA andproviral DNA.
RNA genotyping used RT-PCR nested assay. For proviralDNA, reverse transcription was omitted.
PCR products were sequenced by Sanger’s method.Sequences obtained were analyzed using Recall®bioinformatics software.
Only the sequences approved were interpreted in twodatabases: Stanford and Geno2pheno.
The obtained mutations were compared for both nucleicacids and evaluate resistance to INIs..
GenotipyngOf Integrase
Geno2phenoStandford
Proviral DNA or viral RNA
Resistance Prediction on Databases Used
High LevelResistance
Low LevelResistance
Intermedia LevelResistance
Potential LowLevel ResistanceSusceptible
ResultsThe forty-one (87,2%) samples were concordant between RNAand proviral DNA for prediction of resistance to INIs.
Six samples (12.8%) were discordant. Five samples were RNA-Resitance/DNA-Susceptible and one was RNA-susceptible/DNA-Resistance.
For the samples resistance concordant RNA and Proviral DNAshowed similar number and kind of mutations associated to INIsresistance.
87%
13%8 R
33 S
Y143R
RNA Proviral DNA
T97A G140S Y143R Q148H N155H
H155NQ148H R263K
8
15
In the samples Resistance Concordant themutations detected in both compartments were:
T97A, G140S, Y143R, Q148H, and N155H.
In the six nonconcordant samples thefollowing mutations could not be detected byproviral DNA:
H155N (3 samples), Q148H and R263K andthese samples were susceptible for proviralDNA and resistant for RNA.
On the other hand, the mutation Y143Rcould not be detected by RNA and wassusceptible for RNA and resistant for proviralDNA.
Results
0
2
4
6
8
10
12
14
16
T66I L68V L74M T97A E138K G140A G140S Y143R S147G Q148H/R V151I N155H
% P
orce
ntaj
e
Percentage each mutations to INIs
More amplification andsequencing mistakes were foundworking with RNA (16 sequencesfailed) than with proviral DNA (8sequences failed).
Four samples (8,5%) could not begenotyped by RNA or proviralDNA and were considerednonreportable (indeterminate).
Results
16
84
FALLA VIROLÓGICA
2008
Infección
ConfirmaciónISP
Inicio TAR
NRTI
NNRTI
IP
RALFalla
Virológica
2009 2010 2011 2012 2013 2014 2015
Carga ViralCD4 2 (1-4) Genotipos
Transcriptasa Reversa / Proteasa
Genotipo Integrasa
DTG
?
?22 meses
EVG1995-2012
(1-90)
1997-2013
Nº esquemas: 4 (1-8)
398.550(1.200-2.700.000)
186 (9-868)
43,4 (28-69) años
93.6%
2007
N=47
?
Antecedentes Clínicos y epidemiológicos de los pacientes
Pac en TAR y VF
Rec
uent
o ba
sal d
e C
D4
Se encontraron diferencias en el recuento basal de CD4 P<0.05
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Comparación del Recuento de CD4 basales entre sensibles y Resistentes a INIs
INIs Resistentes
INIs Sensibles 255116
7
8
2
6 6
3
7
4
3
4 4 4 4
7
3
2 2 2
5
4
3
5
2 2
3
4
3
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Comparación del número de esquemas TAR entre Sensibles y Resistentes a INIs
TAR INIs Resistentes TAR INIs Sensibles
Núm
ero
de e
sque
mas
TAR
Se encontraron diferencias en el número de esquemas terapéuticos P<0.05
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Conclusions
Due to high level of concordant detected Proviral DNA offers a promisingapproach for resistances to INIs prediction in clinical practice, particularlyfor the assessment for treated patients with low or suppressed vireamia.
However due to discordant results It is not advisable to replace RNA. Werecommend use proviral DNA only in cases where RNA analysis is notpossible.
Mutations detected for RNA or proviral DNA may explain the virologicfailure due to INIs exposure.
Dr. Rolf Kaiser, AlemaniaAgosto 2012
Laboratorio de Medicina Molecular
Acknowledgment
