Transcript Diapositiva 1 - Universidad de Sevilla

Métodos genotípicos de
resistencia a antivirales
JOSÉ C. PALOMARES
Catedrático Microbiología
Jefe de Sección Microbiología Diagnóstica Molecular
. H. U. Valme
Agentes Antiretrovirales (VIH)
Nucleoside RTIs
• Zidovudine
• Didanosine
• Zalcitabine
• Stavudine
• Lamivudine
• Abacavir
• Emtricitabine
• Tenofovir
Nonnucleos(t)ide RTIs
Protease Inhibitors
• Nevirapine
• Delavirdine
• Efavirenz
• Etravirine
• Rilpivirina
• Saquinavir
• Ritonavir
• Indinavir
• Nelfinavir
• Amprenavir
• Lopinavir/r
• Atazanavir
• Fosamprenavir
• Tipranavir
• Darunavir
Integrase Inhibitors
Boosters
• Ritonavir
• Cobicistat
• Raltegravir
• Dolutegravir*
• Elvitegravir
Fusion Inhibitor
• Enfuvirtide (T-20)
CCR5 Antagonist
• Maraviroc
• Vicriviroc
Agentes Antivirales: Virus Hepatitis
VHB
•Lamivudine
•Tenofovir
•Entecavir
•Adefobir
•Telbivudina
VHC
Clásicos
•Interferón
•Ribavirina
Antivirales Acción Directa (DAAs)
Inhibidores Proteasa
•Boceprevir
•Telaprevir
•Simeprevir
•Asunaprevir
•Faldaprevir
Inhibidores Replicasa NS5A
•Daclatasvir
•Ledipasvir
Inhibidores Polimerasa NS5B
• Sofosbuvir
• Deleobuvir
INCIDENCIA DE RESISTENCIA POR CLASES
Detección Fenotípica de Resistencia

Phenotypic susceptibility tests measure viral replication in cell culture in the
presence of serialARV dilutions. Plotting the inhibition of viral replication at
increasing ARV concentrations creates a sigmoidal dose-response curve
that is usually summarized by the ARV concentration that inhibits viral
replication by 50% (IC ). The IC of an ARV cannot be translated directly into
the in vivo activity of the ARV because the virus inoculum and cells used in a
phenotypic assay often do not reflect “in vivo”conditions.
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Rather, phenotypic susceptibility testing determines the relative antiviral
activity of an ARV against a tested HIV-1 isolate versus against a wild-type
control virus. Therefore, drug susceptibility results are reported as levels of
fold-resistance, which are calculated by dividing the IC of the investigated
virus by the IC of a control virus. Plasma HIV-1 RNA levels >1000 copies
per mL are generally required for phenotypic susceptibility testing.
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Detección Genotípica de Resistencia
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Genotypic resistance testing relies on detecting known drugresistance mutations in the enzymatic targets of antiviral therapy:
protease, reverse transcriptase, and, if specially requested, integrase
and glycoprotein (gp)41.
The standard approach to genotypic resistance testing is direct
polymerase chain reaction (PCR) dideoxynucleotide (Sanger)
sequencing.
Detección Genotípica de Resistencia

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Genotypic testing produces a nucleotide sequence usually encompassing
the complete 297 nucleotides (or 99 amino acids) of HIV-1 protease, and the
50 polymerase coding region of HIV-1 reverse transcriptase, usually
encompassing amino acid positions 40–240, the part of reverse
transcriptase containing the vast majority of NRTI- and NNRTI-resistance
mutations. Integrase sequencing is usually ordered as a separate test.
The sensitivity of genotypic resistance tests ranges from 100 to 1000 plasma
HIV-1 RNA copies per mL, depending upon the assay used.At low plasma
HIV-1 RNA levels, genotypic resistance testing is likely to be sequencing
only a small number of circulating virus variants.
Detección Genotípica de Resistencia

The nucleotide sequence is then translated to its amino acid sequence. The
amino acid sequence is then compared either with the sequence of a wildtype subtype B laboratory strain or to a consensus wild-type subtype B
amino acid sequence. The differences between a sequenced clinical virus
and the reference wild-type sequence generates a list of mutations.
Mutations are reported using a shorthand in which each mutation is denoted
by the one-letter code for the wild-type reference amino acid, followed by the
amino acid position, followed by the one-letter code for the amino acid
mutation found in the sequence.
Mutations in the Envelope Gene
Associated With Resistance to Entry
Inhibitors
Enfuvirtide
Maraviroc
Maraviroc activity is limited to patients with only CCR5 (R5) -using virus detectable; CXCR4 (X4) -CCR5 mixed tropic viruses and X4using viruses do not respond to maraviroc treatment. Some cases of virologic failure during maraviroc therapy are associated with
outgrowth of X4 virus that pre-exists as a minority population below the level of assay
detection. Mutations in the HIV-1 gp120 molecule that allow the virus to bind to R5 receptors in the presence of drug have been
described in viruses from some patients whose virus remained R5 at the time of virologic failure. A number of such mutations have
been identified, and the phenotypic manifestation of this drug resistance is a reduction in the maximal percentage inhibition (MPI)
rather than the increase in the 50% inhibitory concentration (IC50; defined by fold increase) that is characteristic of resistance to other
classes of antiretrovirals. The resistance profile for maraviroc is too complex to be depicted on the figures. The frequency and rate at
which maraviroc resistance mutations emerge are not yet know
Mutations Selected by nRTIs
Abacavir
Didanosine
Emtricitabine
Lamivudine
Stavudine
Tenofovir
Zidovudine
Mutations Selected by NNRTIs
Efavirenz
Etravirine
Nevirapine
Mutations in the Integrase Gene
Associated With Resistance to
Integrase Inhibitors
Raltegravir
Raltegravir failure was associated with integrase mutations in 2 distinct genetic pathways defined by 2 or more
mutations including: (1) a signature (major) mutation at either Q148H/K/R or N155H; and (2) 1 or more minor mutations
unique to each pathway.
Minor mutations described in the Q148H/K/R pathway include L74M + E138A, E138K, or G140S.
The most common mutation pattern in this pathway is Q148H + G140S; this Q148H + G140S pattern exhibits the greatest
loss of drug susceptibility.
Mutations described in the N155H pathway include this primary mutation plus either L74M, E92Q, T97A, E92Q + T97A,
Y143H, G163K/R, V151I, or D232N (Hazuda et al, Antivir Ther, 2007).
Mutations Selected by PIs
Atazanavir
+/-ritonavir
Darunavir/
ritonavir
Fosamprenavir/
ritonavir
Indinavir/
ritonavir
Lopinavir/
ritonavir
Mutations Selected by PIs (cont)
Nelfinavir
Saquinavir/
ritonavir
Tipranavir
/ritonavir
Limitaciones de estudios
genotípicos y fenotípicos
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Necesaria una carga viral 1,000 copias/mL
Sensibilidad (no detecta poblaciones minoritarias,
precisa prevalencia de 20%+)
Calidad (ensayo, laboratorio)
Problemas con amplificación por PCR
(contaminación)
Coste
Detección de variantes
minoritarias
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Varias técnicas que permiten detección de variantes
con prevalencia incluso menor del 1%:
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RT-PCR alelo específica
Oligonucleotido ligation assay (OLA) Detectan mutaciones puntuales
LigAmp
Secuenciación de genoma único (SGS) o secuenciación clonal
Ultra-deep sequencing
Multi-genome sequencing (MGS)
SUPERVIVENCIA EN RALACION A RESPUESTA
VIROLOGICA TRAS CAMBIO TRATAMIENTO POR
GENOTIPO
PERSPECTIVAS
Existen variados mecanismos de resistencia frente a ARVs.
Múltiples mutaciones generan diferentes efectos sobre la
sensibilidad a los ARVs.
Las resistencias se adquieren al infectarse o se seleccionan con
el tratamiento.
Una vez establecida la resistencia, evoluciona, se diversifica y se
hace irreversible.
Tecnologías de
Secuenciación
General
Sanger
Secuenciación 2ª generación:
454
Illumina
SOLiD
Ion Torrent
Secuenciación 3ª generación:
PacBio
Nanopore
Secuenciación Sanger
Traditional DNA sequencing method
Ideal for small sequencing projects
Read length around 600-700 bp
Around 5-10$ per reaction
384 reactions in parallel at most
Applied Biosystems is the main
technological provider
Secuenciación Sanger
Secuenciación Sanger
Secuencia y calidad
Phred score = - 10 log (prob error)
Secuenciación 2ª
generación (NGS)
454
First NGS platform
Pirosequencing based chemistry
Long reads (400-1500 bp)
Most expensive cost per base
Ideal for de novo sequencing projects
Owned by Roche
½, ¼ and 1/8 run can be ordered
>1 million reads
GS FLX+ and GS Junior
454
454
Illumina
Previously known as Solexa
Reversible terminators based
sequencing technique
Short reads (75 or 250bp depending on
the version)
Lowest cost per base
Ideal for resequencing projects
Highest throughput
Runs divided in 8 lines
up to 3000 million reads
Can sequence both ends of the
molecules (paired ends)
HiSeq2500 and MiSeq
Illumina
SOLiD
Ligation based sequencing chemistry
Short reads (35 - 75bp depending on the
version)
Only for resequencing projects
It does not produce nucleotide
sequences, but colors
115 or 320 million reads
SOLiD
SOLiD
Really?
Ion Torrent
Around 60-80 M reads.
200 pb length.
Sequences based on H+ production
Error rates lower than other 2nd
generation
Error pattern similar to 454, with
homopolymer problem.
Very cheap per run.
Belongs to Life technologies (Applied
Biosystems)
Sanger vs NGS
Sanger
NGS
Num. sequences per
reaction
1 clone
Millions of molecules
Max. parallelization
384
Several millions
Sequence quality
High
Low
Sequence length
600-800 bp
35-1000 (depends on the
platform)
Throughtput
Low
High
Sanger vs NGS
Comparación
Cost per raw Megabase of DNA
Secuenciación 3ª
generación
PacBio
3rd generation platform (single
molecule)
Polymerase based chemistry (SMRT)
Longest NGS reads (more than 1000bp)
Very high error rate
Ideal for de novo sequencing projects
45000 reads
PacBio
3rd generation, single molecule detection. No amplification step required.
Nucleotides labeled on the phosphate removed during the polymerization.
Sequencing based on the time required by the polymerase to incorporate a
nucleotide (Polymerase requires milliseconds versus microseconds for the
stochastic diffusion)
Nanopore
Sequencing technologies
3rd Generation
2nd Generation
Sanger
Modified from Michael Stromberg
Bioinformatic challenges
Huge data files handling.
Beefy computers required.
Software still being developed or
missing.
Ad-hoc software required during the
analysis.
Existing software tailored to
experienced bioinformaticians.
EDSAC by Computer Laboratory Cambridge
Dollar for dollar rule proposed