AREVIR 2016 Detection of (drug-resistant) cytomegalovirus in immunosuppressed patients An overview Detlef Michel Institut für Virologie, Universitätsklinikum Ulm
HCMV - diagnostics genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
HCMV - diagnostic genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
HCMV-serology and transplantation • serology should be performed before transplantation on both donor and recipient • donor and recipient serostatus (D/R) are key predictors of infection risk and management • no role in the diagnosis of active HCMV disease after transplantation • interpretation of results can be difficult: – in D/R with recent transfusion of blood products – in children younger than 12 months, as passive transfer of antibody can lead to transient false-positive serologic results
HCMV - diagnostics genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
HCMV - diagnostics genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
HCMV monitoring after transplantation • qPCR is preferred for diagnosis, decisions regarding preemptive therapy, and monitoring response to therapy – due to the ability to harmonize and standardize these tests • commercial and in-house tests must be calibrated to the WHO international standard – results have to be reported as IU/ml • either plasma or whole blood is an acceptable specimen for qPCR – specimen type should not be changed when monitoring patients • if qPCR is not available, antigenemia is an acceptable alternative
pp65 antigenemia • does not require expensive equipment and is relatively easy to perform • lack of standardization, including subjective result interpretation − it is unlikely that better standardization of this assay will occur, because most laboratories have moved to molecular methods • assay performance diminishes when the absolute neutrophil count is less than 1000/mm 3 • blood specimens should be processed as fast as possible to avoid a decrease in test sensitivity − thus, transplant centers managing patients at distant sites whose blood samples are mailed into the laboratory may prefer to use qPCR rather than antigenemia
HCMV - diagnostics genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
HCMV - diagnostics genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
HCMV - diagnostics genotyping UL97 2-3 days Polymerase 4-7 days (UL54) serology antigenemia real-time PCR Quantiferon-CMV shell vial virus culture IgG, IgM, (pp65) DNA ELISA virämia isolate (interferon- γ ) (avidity) 5 to 6 h 2 to 4 h 1 to 3 days 1 to 6 weeks 6 h to 24 h 24 h phenotyping (after virus isolation 1-2 weeks) (ganciclovir, foscarnet, cidofovir)
Mechanism of action of anti-cytomegalovirus (HCMV) drugs mucosa cells valganciclovir ValGCV ganciclovir GCV P UL97 P P NA CDV P cellular cidofovir Kinases P infected cell P P P P P NA P NTP viral DNA- FOS Polymerase foscarnet viral DNA NA
Probability of viral resistance of HCMV In immunosppressed patients Long term therapy: HIV-infection (AIDS) 2% > 3 months 9% 9 months transplantation, general 10% lung transplantation, D+/R- 30% (>2 months) kidney-/liver transplantation <10%
Probability of viral resistance of HCMV In immunosppressed patients Long term therapy: HIV-infection (AIDS) 2% > 3 months 9% 9 months transplantation, general 10% lung transplantation, D+/R- 30% (>2 months) kidney-/liver transplantation <10% Our results after suspicion of viral resistance: (n ~ 1500 patients): 15% PCR/pp65 negative 50 % wild-type 35% GCV/FOS/CDV resistance
Impact of different mutations in UL97 on drug sensitivity Ratio IC50 *
From genotype to phenotype to consequences for therapy UL97 A478V T601M V466M C603S C603del A497T C592G C603W A594E A594V D605E M460V H520Q L592S L600I G598S C607F switch to FOS N597D is suggested E596G H469Y K599E A591V may permit GCV at higher doses maintain therapy
Distribution of UL97 mutations detected by genotyping (n = 235) E596D A594G A594T C607Y A594L C603F A594S L595F A594V different Deletions H520Q M460I C592G L595S M460V C603W
http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/mra/app/index.php?plugin=form
Requests to the MRA-webside (UL97 / UL54) ww.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/mra/app year MRA-HCMV 2010 2019 2011 2798 2012 4911 2013 5555 2014 5447 2015 6976 12.02.2016 661
„Evolution“ of resistant HCMV UL97 L595S 20% H411Y 20% UL97 D605E UL97 UL97 L595S 50% M460V 20% T409A 50% D605E Pol L595S 50% H411Y 50% T700A 20% D605E D605E Pol A809V 20% UL97 wt wt Pol Pol D605E n.d. wt May Jun Jul Aug Sep Okt Nov Dez Jan 21.6. 1.7. 26.8. 7.11. 6.12. 19.1. foscarvir ganciclovir cidofovir maribavir leflunomid
Cloning proofs accumulation of different Maribavir-resistant strains under therapy 409 411 MBV r direct sequencing from EDTA-blood T409M H411Y cloning clone 2 MBV r in E. coli V T V H R M Y MBV r
Observations during anti-HCMV therapy • mutations are distrubuted among different subpopulaions (strains) of the same clinical isolate • several strains/variants may „disappear“ under therapy (i.e. M460I), others maintain • during ganciclovir therapy pauses (under foscarnet or whithout) „repopulation“ with wildtype can occure • GCV-resistant variants have a selection advantage if therapy is continued Quellen: Schubert et al. 2013 BMC Infect Dis Drew and Liu 2012, Clin Transplant Strasfeld et al. 2010, J Infect Dis
Genotyping is expensive (?) Daily costs of anti-HCMV therapy (according prime costs) Ganciclovir (i.v.) 68,00 € (2x5mg/kg) Valganciclovir (p.o.) 101,00 € (2x900 mg) Foscarnet 382,00 € (2x90 mg/kg) Cidofovir 67,00 € (1x5 mg/kg/2 weeks; one vial 961,00 €) ------------------------------------------------------------------------------------------------- (Artesunate) (68,00 €) (2,4 mg/kg) (Leflunomid) (3,00 €) (20 mg )
Molecular aims of the HCMV therapy Ganciclovir Egress of the Cyclopropavir linear double strand Maribavir genome UL97 replication by „rolling circle“, circularisation concatemers UL54 (Polymerase) envelopment Ganciclovir egress Foscarnet Artesunate? Cidofovir Leflunomid? Maribavir Cyclopropavir Brincidofovir Letermovir Packaging: UL56 Import of DNA into UL56 UL89 capsid. Complex of UL89 UL56/UL89 proteins Cutting to genome lengths with capsid/DNA
Summary: HCMV and resistance • different diagnostic tools for the diagnosis of active HCMV infections are available • UL97 and UL54 genotyping is suitable for adjustment of therapy • different mutations in the same “patient virus population” are mostly distributed among different virus strains • in patients consecutively treated with different compounds “virus evolution” can be observed • new mutations require a continuously updated public database of the impact on the viral phenotype • new substances may require new genotypic (phenotypic?) assays (Letermovir, Maribavir, Artesunate, Leflunomid)
Ulm definition of phenotypic GCV resistance 90% perzentil 20 number of tested HCMV isolates 16 reduced resistent sensitive 12 sensitive median 8 3,6 µM 4 0 3 6 9 12 15 18 20 30 40 50 60 70 Ganciclovir HD (in µM) 50
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