CMV in ICUCytomegalovirus (CMV) in Intensive Care Unit (ICU) patientsDavid Navarro, M.D., Ph.D.Microbiology Service, Unive...
PrimoinfecciónReactivacióntisular/mucosasEstocásticaInflamaciónStressViremiaLatencia/persistenciamultiorgánicaEnfermeda...
UL1-146TLR 1-14 TRS1IRL/SgpTLR11gpUL16gpUL18 gpUL27 gpUL33gpUL40 gpUL83gpUL111gpUL78 gpUL146US1-34gpUS2gpUS3gpUS6gpUS10gpU...
Innate immunityDC/MΦAdaptive immunityEarly control of CMV replicationDefinitive control of CMV replicationIFNs (α,β,λ)Anti...
CMV in ICUBarcelona, 2013
CMV in ICUClinical consequences of active CMV infection in ICU patients• Longer stay in ICU (hospital)• Longer time on me...
Pooled odds ratio for all-cause mortality: 1.93 (95% C.I. 1.29-2.88; P=0.001)•Clinical consequences of active CMV infectio...
~1,000 patients: 81% higher mortalityCMV in ICU
Limaye et al., JAMA, 2008CMV in ICUplasma CMV DNA load (Q r-t PCR)Barcelona, 2013
Randomized-controlled clinical trial of antiviral therapy•CMV in critically ill patients: pathogen or bystander?CMV in ICU...
CMV in ICU• Real-time PCR for CMV monitoring• Blood + Lower respiratory tract are screened• Monitoring at least once a wee...
CMV in ICUPlasmaCMV DNAemiaQ r-t PCR•Severe sepsis/septic shock•Mechanical ventilation at entryUnderlying diseaseLimaye et...
•Weekly virological monitoring in plasma and tracheal aspirates by Q-RT-PCR (L.O.D.=10copies/mL)No patient received antiv...
TA TA+PL PL (no TA specimens available)Active CMV infection•Dissociated episodesQ-RT-PCR(~10 copies/mL)CMV in ICUChilet et...
CMV in ICUCMV DNA detected earlier in the LRT (11 days/16.5 days in plasma)Longer time to resolution in the LRT (28 days...
TA TA+PL PL /leukocytes(no TA specimens available in 9 patients)Active CMV infectionDissociated episodesQualitative PCR(No...
•Direct effect: Cytopathogenicity: High level virus replication10-40% of patients with ALI/ARDS have histopathological evi...
•Experimental mCMV model (sepsis/ileo-cecal puncture)Pro-inflammatory status (SIRS)CMV reactivation in LRTTNF-α/IL-1β/LPS ...
CMV in ICUPrevious CMV infection increases pulmonary TNF-α response in sepsisCook and Trgovcich, 2011•Experimental mCMV m...
CMV in ICU•Stronger rationale for the strategy of antiviral prophylaxis over pre-emptive therapy•Exerts greater benefitial...
CMV mRNA inLung tissue(Incidence)Lung fibrosisGomori stain(collagen)(Severity)Ganciclovir treatment3 weeks following infec...
CMV in ICU•Efficacy of Ganciclovir/Valganciclovir for Prevention of Cytomegalovirus Reactivationin Acute Injury of the Lun...
•Serum IL-6 level (predicts clinical outcome in ICU patients with ALI/ARDS)•Change between baseline and 14 days post-rando...
“Pre-emptive antiviral therapy of activeCMV infection in ICU patients with severesepsis, septic shock and mechanicallyvent...
•Primary Outcome Assessment:Clearance of active CMV infection in LRT and blood (plasma)The following virological parameter...
•>18 years old• No canonic immunosuppression• CMV-seropositive• Severe sepsis/septic shock•Mechanical ventilation within 4...
IV Ganciclovir/Valganciclovir:First 5 days : IV ganciclovir: 10 mg/kg daily,given as 5 mg/kg every 12 hours (adjustedfor r...
• Clinical outcomes at 7, 14, 21 , 28, 60 , 90 days post-randomizationOrgan system failure at 14 and 28 daysDuration of me...
CMV in ICU•Only 1/3 of CMV-seropositive ICU patients will develop active CMV infection•2/3 of CMV-seropositive ICU patient...
•CMV-related risk factorsLatent CMV burden may determinethe risk of CMV reactivation• CMV-specific IgG levels?• Peripheral...
CMV in ICU•Host-related risk factors•Genomic risk profile?•Immune risk profile?Evaluation at the time of ICU admissionBarc...
IL 10 (2068) Intron - C/GCCR5 (-2086) 5’ UTR - A/GAA substitutionCCL2 (MCP1) (1543) 3´-UTR - C/TIL-10CCR5MCP-1CMV in ICU•G...
CMV in ICUGeners numberGenotypeActive CMV Infection (%)P valueNo YesIL10 C/C 14 (73,7) 5 (26,3) 0,081rs1878672 C/G 15 (45,...
CMV in ICUGeners numberGenotypePLASMA (IU/mL)P valueTA (IU/mL)CMV DNA peak*P valueCMV DNA peak*CCR5 A/A 398 (91-12649) 0,2...
CMV in ICU•Host-dependant risk factors•Immune risk profile?•CMV pp65 and IE-1 IFN-γ-producing CD8+and CD4+T cells protect ...
pepmix pp65 and IE-1(1 µg/mL/peptide)+ CD28/CD49d moAbsBrefeldin (at 2 h.)6 h.Lysis/FixationPermeabilizationStaining (CD3+...
CMV in ICUActive CMV InfectionT-cell subsetcells/µlNo YesNoYesIFNγ CD8+ T cells IFNγ CD4+ T cells•First immunological eval...
CMV in ICU•First immunological evaluation at the time of ICU admission (median 2days)Ongoing episode (n=5)During follow-up...
CMV in ICU•LPS (bacterial sepsis) induces a contraction of mCMV-specificmemory CD8+ T cellsTotal CD8+T cellsmCMV-specific ...
CMV in ICUTLR4 -/- Knock-out mice•Contraction of mCMV-specific CD8+ T cells after heterologousantigenic stimulation (LPS) ...
CMV in ICU•Functional NK-cell deficit in ICU patients developing an episode of active CMV infection•Immunological analyses...
CMV in ICUChiche et al., 2012•Patients developing an episode of active CMV infection display an impaired functional NK-cel...
CMV in ICUChiche et al., 2012•Immunological data on specimensobtained the week right before detection of active CMV infect...
CMV in ICU•Functional NK-cell deficit mediated by IL-10 and IL-15•Patients with an episode of active CMV infection display...
CMV in ICUvon Müller L et al. J Infect Dis. 2007;196:1288-1295Active CMV infection (pp65 AG)No active CMV infection (pp65 ...
CMV in ICUvon Müller L et al. J Infect Dis.2007;196:1288-1295Active CMV infection (pp65 AG)No active CMV infection (pp65 A...
CMV in ICU0,00,10,20,30,40,50,6cells/µl 0,00,20,40,60,81,01,21,41,61,82,06,012,018,024,030,036,0cells/μlBaseline•The magni...
•IL-10 levels in LRT >blood (may impair CMV-specific T-cell functionality in the LRT)•IL-10 levels correlate (P=0.001) wit...
CMV in ICU•Conclusions•Active CMV infection is a frequent event in ICU patients withoutcanonical immunosuppression•Screeni...
CMV in ICUMarifina ChiletDayana BravoM. Aª ClariElisa CostaBeatriz MuñozEstela GiménezIsabel CorralesGerardo AguilarJavier...
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Cytomegalovirus (CMV) in Intensive Care Unit (ICU) patients (David Navarro, PhD)

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La infección activa (IA) por el CMV es frecuente en el paciente crítico sin inmunosupresión canónica (0-55% de los pacientes seropositivos para el CMV). Esta es comúnmente el resultado de la reactivación viral en el tracto respiratorio inferior, sin que pueda descartarse la reinfección como origen, en algunos casos. La IA por el CMV se ha asociado consistentemente con una mayor mortalidad, especialmente relacionada con el desarrollo de ALI/ARDS, una estancia en UCI más prolongada, una mayor duración del período de ventilación mecánica y un riesgo mayor de “superinfección” bacteriana y fúngica. No existen, sin embargo, vínculos incontestables de causalidad. Solo un ensayo clínico controlado puede precisar el papel del CMV como agente patogénico en este grupo de pacientes. En este seminario se tratarán los siguientes temas: (i) posible patogenia de la infección por el CMV en el paciente crítico; (ii) ensayos clínicos de tratamiento antiviral en marcha; (iii) factores biológicos (genómicos e inmunológicos) de riesgo para el desarrollo de IA en estos pacientes.

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Cytomegalovirus (CMV) in Intensive Care Unit (ICU) patients (David Navarro, PhD)

  1. 1. CMV in ICUCytomegalovirus (CMV) in Intensive Care Unit (ICU) patientsDavid Navarro, M.D., Ph.D.Microbiology Service, University Clinic Hospital, Valencia, SpainDepartment of Microbiology, School of Medicine, University of Valencia, SpainBarcelona, 2013
  2. 2. PrimoinfecciónReactivacióntisular/mucosasEstocásticaInflamaciónStressViremiaLatencia/persistenciamultiorgánicaEnfermedad orgánica/tisularDéficit Th-1PMM (CD33+) Monocitos EndotelioTNF-αCatecolaminasDCsMθTúbulo renal Células acinaresInfección crónicaReinfecciónCMV in ICUBarcelona, 2013
  3. 3. UL1-146TLR 1-14 TRS1IRL/SgpTLR11gpUL16gpUL18 gpUL27 gpUL33gpUL40 gpUL83gpUL111gpUL78 gpUL146US1-34gpUS2gpUS3gpUS6gpUS10gpUS11RegulaciónnegativapresentaciónHLA I/IIHomólogoreceptor FcIII.Inhibe ADCCBloqueaactivaciónvía NKG2DBloqueaactivaciónde NKLiga LIR1Bloqueaactivaciónde NKHomólogosreceptores α-quimioquinasQuimioquinahomóloga IL-8HomólogoIL-10.Inhibe Th1gpUS28Inmunosupresor y pro-inflamatorioInhibe presentaciónde IE-1Homólogosreceptoresde β-quimioquinas yfractalinaM θ (M1/M2)IL-10IL-18vIL-10IL-1IL-6TNF-αNOiNOSCOX-2InmunosupresiónInflamaciónDCsBloqueopresentaciónHLA I/IICMV in ICUBarcelona, 2013
  4. 4. Innate immunityDC/MΦAdaptive immunityEarly control of CMV replicationDefinitive control of CMV replicationIFNs (α,β,λ)Antiviral cytokinesNK cellsTCD8+TCD4+TregsB cellsComplexHierarchicalRedundantNeutralizingADCCCMV in ICUBarcelona, 2013
  5. 5. CMV in ICUBarcelona, 2013
  6. 6. CMV in ICUClinical consequences of active CMV infection in ICU patients• Longer stay in ICU (hospital)• Longer time on mechanic ventilation• Higher incidence of bacterial and fungal “superinfection”•Higher mortality (raw)/ALI/ARDSDomart et al., Chest, 1990Cook et al., Am J Surg, 1998Heininger et al., Crit Care Med, 2001Cook et al., Crit Care Med, 2003Von Müller et al., Emerg Infect Dis, 2004Jaber et al., Chest, 2005Ziemann et al., Crit Care Med, 2008Limaye et al., JAMA, 2008Chiche et al., Crit Care Med, 2009Heininger et al., Crit Care, 2011Coisel et al., PLOS one, 2012Barcelona, 2013
  7. 7. Pooled odds ratio for all-cause mortality: 1.93 (95% C.I. 1.29-2.88; P=0.001)•Clinical consequences of active CMV infection in ICU patientsCMV in ICUBarcelona, 2013
  8. 8. ~1,000 patients: 81% higher mortalityCMV in ICU
  9. 9. Limaye et al., JAMA, 2008CMV in ICUplasma CMV DNA load (Q r-t PCR)Barcelona, 2013
  10. 10. Randomized-controlled clinical trial of antiviral therapy•CMV in critically ill patients: pathogen or bystander?CMV in ICU•Prospective or retrospective observational studies do not prove causalityBarcelona, 2013
  11. 11. CMV in ICU• Real-time PCR for CMV monitoring• Blood + Lower respiratory tract are screened• Monitoring at least once a week• CMV Surveillance for > 2 weeks• CMV-seropositive (reactivation/reinfection)• Burn and Surgical/Trauma ICUs• Poor clinical scores at entry0-55%Barcelona, 2013
  12. 12. CMV in ICUPlasmaCMV DNAemiaQ r-t PCR•Severe sepsis/septic shock•Mechanical ventilation at entryUnderlying diseaseLimaye et al., JAMA, 2008Major risk factors55%38%25%18%CMV DNAemia >1,000 copies/mL45%20%15%5%Barcelona, 2013
  13. 13. •Weekly virological monitoring in plasma and tracheal aspirates by Q-RT-PCR (L.O.D.=10copies/mL)No patient received antiviral therapy53 patients undergoing mechanical ventilation65% severe sepsis or septic shock•Simultaneous screening of the LRT and the blood compartment for thepresence of CMV DNA is imperative for an optimal diagnosis of active CMVinfection in ICU patientsCMV in ICUBarcelona, 2013
  14. 14. TA TA+PL PL (no TA specimens available)Active CMV infection•Dissociated episodesQ-RT-PCR(~10 copies/mL)CMV in ICUChilet et al., J Med Virol, 2010Barcelona, 2013
  15. 15. CMV in ICUCMV DNA detected earlier in the LRT (11 days/16.5 days in plasma)Longer time to resolution in the LRT (28 days/20 days in plasma)Chilet et al., J Med Virol, 2010Barcelona, 2013
  16. 16. TA TA+PL PL /leukocytes(no TA specimens available in 9 patients)Active CMV infectionDissociated episodesQualitative PCR(No defined L.O.D.)Heininger et al., Critical Care, 2011CMV in ICUBarcelona, 2013
  17. 17. •Direct effect: Cytopathogenicity: High level virus replication10-40% of patients with ALI/ARDS have histopathological evidencesof CMV pneumonitisPapazian et al., Anesthesiology, 1998Papazian et al., Crit Care Med, 2007Chiche et al., Crir Care Med, 2009•“Indirect effects”: Low level persistent virus replication•CMV may increase lung inflammation (pro-inflammatory)•CMV may increase the incidence of bacterial and fungal “superinfection”•CMV may trigger immunopathogenic effects (CTLs) causing lung injury•How may CMV injure immune competent hosts?CMV in ICUBarcelona, 2013
  18. 18. •Experimental mCMV model (sepsis/ileo-cecal puncture)Pro-inflammatory status (SIRS)CMV reactivation in LRTTNF-α/IL-1β/LPS (systemic and LRT)Pulmonar fibrosis(ALI/ARDS)TNF-α/IL-1β/TGF-βInfected alveolar macrophagesActivated CMV-specific CD8+/CD4+T cellsViremiaCook et al., 2002Cook et al., 2006Cook et al., 2007Forster et al., 2010•Pathogenesis of active CMV infection in ICU patientsCMV in ICUBarcelona, 2013
  19. 19. CMV in ICUPrevious CMV infection increases pulmonary TNF-α response in sepsisCook and Trgovcich, 2011•Experimental mCMV model of sepsis induced by ileo-cecal punctureBarcelona, 2013
  20. 20. CMV in ICU•Stronger rationale for the strategy of antiviral prophylaxis over pre-emptive therapy•Exerts greater benefitial results on CMV-induced indirect effects in the SOT settingHodson et al. Lancet, 2005Reischig et al. Am J Transplant ,2008Kliem et al., Am J Transplant, 2008•Data obtained in the murine model of sepsis-induced mCMV reactivationForster et al., Antiviral Res, 2010Only a randomized-controlled clinical trial of antiviral therapy may reveal thepathogenetic role, if any, of CMV in ICU patients•When should antivirals be administered? Prophylactic vs. Pre-emptiveBarcelona, 2013
  21. 21. CMV mRNA inLung tissue(Incidence)Lung fibrosisGomori stain(collagen)(Severity)Ganciclovir treatment3 weeks following infection Forster et al., 20107 days (Pre-emptive)7 days (Pre-emptive)Ganciclovir treatment•Experimental mCMV model (sepsis/ileo-cecal puncture)CMV in ICUCMV in ICUProphylaxisProphylaxisProphylaxisProphylaxisProphylaxisProphylaxisBarcelona, 2013
  22. 22. CMV in ICU•Efficacy of Ganciclovir/Valganciclovir for Prevention of Cytomegalovirus Reactivationin Acute Injury of the Lung•Funding: U.S.National Heart, Lung, & Blood Institute•Fred Hutchinson Cancer Research Center, Seattle, WA•Michael Boeckh, MD, Vaccine and Infectious Disease DivisionMain inclusion criteria•CMV IgG seropositive•Intubated and requiring mechanical positive pressure ventilation•Acute Lung Injury/ARDSMain exclusion criteria•Known or suspected immunosuppression•Hospitalized for > 96 hoursIntervention•Ganciclovir 5mg/kg iv, or valganciclovir 900 mg orally or placebo at entryMulticenter randomized placebo-controlled double-blind trialBarcelona, 2013
  23. 23. •Serum IL-6 level (predicts clinical outcome in ICU patients with ALI/ARDS)•Change between baseline and 14 days post-randomization in placebo & ganciclovir groups•Primary endpoint•Secondary endpoints•Incidence of CMV reactivation at 28 days•Additional inflammatory cytokines•Clinical outcomes•Length of stay•Incidence of CMV disease•SafetyCMV in ICUBarcelona, 2013
  24. 24. “Pre-emptive antiviral therapy of activeCMV infection in ICU patients with severesepsis, septic shock and mechanicallyventilated”Número EudraCT: 2011-002603-15Clinical trial code: GANCMV-2011FIS de Investigación Clínica Independiente 2010.Código: EC10-318Randomized, unicentric, parallel group, double-blind placebo controlled phase II clinical‑ ‑trialCMV in ICUBarcelona, 2013
  25. 25. •Primary Outcome Assessment:Clearance of active CMV infection in LRT and blood (plasma)The following virological parameters will be comparedbetween the groups:Area under the curve (CMV DNA load in TA and plasma)Peak viral load (in each compartment)Time to CMV DNA load clearance• Secondary outcome measurement: IL-6 in TAand plasma (weekly monitorization).CMV in ICUBarcelona, 2013
  26. 26. •>18 years old• No canonic immunosuppression• CMV-seropositive• Severe sepsis/septic shock•Mechanical ventilation within 48 h. of admissionInclusion criteriaPre-emptive therapy•Initiation: CMV DNA load >10 copies/mL in TA/plasma (or both)•Discontinuation: Negative PCR in TA (BAS) and plasma(treatment up to 14 days)CMV surveillance•Twice/week in TA and plasma•Bronchoaspirates will be screened (once/week) if mechanicalventilaton is discontinuedCMV in ICUBarcelona, 2013
  27. 27. IV Ganciclovir/Valganciclovir:First 5 days : IV ganciclovir: 10 mg/kg daily,given as 5 mg/kg every 12 hours (adjustedfor renal function). After first 5 days (fora maximun of 14 days), either IVganciclovir 5 mg/kg QD or POvalganciclovir 900 mg po QD (bothadjusted for renal function).Placebo For the first 5 days, dosing of intravenousplacebo is daily, given every 12 hours.After first 5 days (for at least 14 days),either IV placebo QD or PO placebo QD. Aminimum interval of 6 hours is requiredbetween the first and second dose. Theplacebo is an IV solution or a PO tabletthat does not contain any activemedications.CMV in ICUBarcelona, 2013
  28. 28. • Clinical outcomes at 7, 14, 21 , 28, 60 , 90 days post-randomizationOrgan system failure at 14 and 28 daysDuration of mechanical ventilationLung injury scoreBacteremia and/or fungemiaMortalityComposite of survival status, ventilation status, and IL-6 levels• Length of stay• CMV disease (biopsy-proven)• SafetyTime to neutropenia ( <500/µL)Use of G-CSFTime to renal insufficiency (creatinine clearance < 60, < 30 ml/min)Time to thrombocytopenia (platelet count < 50,000, < 20,000/µL)CMV in ICUBarcelona, 2013
  29. 29. CMV in ICU•Only 1/3 of CMV-seropositive ICU patients will develop active CMV infection•2/3 of CMV-seropositive ICU patients would be treated unnecessarilyIt is unlikely that clinical studies will help to narrow the group athighest risk for CMV reactivationLooking for biological risk factors•“Further studies must be conducted to identify subsets of patients whoare most at risk to develop active CMV infection”Barcelona, 2013
  30. 30. •CMV-related risk factorsLatent CMV burden may determinethe risk of CMV reactivation• CMV-specific IgG levels?• Peripheral CMV-specific CD8+T-cell levels?Evaluation at the time of ICU admissionInference byCMV in ICUCook and Trgovcich, Antiviral Res, 2012Is CMV DNA load in saliva predictive of the developmentof LRT/systemic CMV reactivation?Salivary glands are a majorsite of chronic virus replicationmCMV murine modelBarcelona, 2013
  31. 31. CMV in ICU•Host-related risk factors•Genomic risk profile?•Immune risk profile?Evaluation at the time of ICU admissionBarcelona, 2013
  32. 32. IL 10 (2068) Intron - C/GCCR5 (-2086) 5’ UTR - A/GAA substitutionCCL2 (MCP1) (1543) 3´-UTR - C/TIL-10CCR5MCP-1CMV in ICU•Genomic risk profile?•Pairwise analysis•Testing for a Dominant/Co-dominant/Recessive modelBravo et al., manuscript in preparation, 2013Barcelona, 2013
  33. 33. CMV in ICUGeners numberGenotypeActive CMV Infection (%)P valueNo YesIL10 C/C 14 (73,7) 5 (26,3) 0,081rs1878672 C/G 15 (45,5) 18 (54,5)C/C 14 (73,7) 5 (26,3) 0,134G/G 13 (50) 13 (50)C/G 15 (45,5) 18 (54,5) 0,796G/G 13 (50) 13 (50)G/G-C/G 28 (47,5) 31 (52,5) 0,064C/C 14 (73,7) 5 (26,3)C/C-C/G 29 (55,8) 23 (44,2) 0,64G/G 13 (50) 13 (50)CCR5 A/A 16 (66,7) 8 (33,3) 0,201rs1800023 A/G 20 (48,8) 21 (51,2)A/A 16 (66,7) 8 (33,3) 0,30G/G 6 (46,2) 7 (53,8)A/G 20 (48,8) 21 (51,2) 1,0G/G 6 (46,2) 7 (53,8)G/G-A/G 26 (48,1) 28 (51,9) 0,148A/A 16 (66,7) 8 (33,3)A/A-A/G 36 (55,4) 29 (44,6) 0,56G/G 6 (46,2) 7 (53,8)MCP1 C/C 30 (61,2) 19 (38,8) 0,105rs13900 C/T 12 (41,4) 17 (58,6)Bravo et al., manuscript in preparation, 2013RecessivemodelG risk alleleBarcelona, 2013
  34. 34. CMV in ICUGeners numberGenotypePLASMA (IU/mL)P valueTA (IU/mL)CMV DNA peak*P valueCMV DNA peak*CCR5 A/A 398 (91-12649) 0,24 14542 (91-6024200) 0,03rs1800023 A/G 249 (91-7585) 781 (91-77624)A/A 398 (91-12649) 0,76 14542 (91-6024200) 0,19G/G 331 (91-1995) 1183 (218-19952)A/G 249 (91-7585) 0,54 781 (91-77624) 0,49G/G 331 (91-1995) 1183 (218-19952)G/G-A/G 292 (91-7585) 0,29 1047 (91-77624) 0,03A/A 398 (91-12649) 14542 (91-6024200)A/A-A/G 331 (91-12649) 0,78 1303 (91-6024200) 0,98G/G 331 (91-1995) 1183 (218-19952)IL-10 C/C 812 (245-1737) 0,44 1288 (319-3388) 0,54rs1878672 C/G 331 (91-12649) 2575 (91-6024200)C/C 812 (245-1737) 0,15 1288 (319-3388) 0,63G/G 91 (91-1995) 333 (91-41686)C/G 331 (91-12649) 0,22 2575 (91-6024200) 0,24G/G 91 (91-1995) 333 (91-41686)G/G-C/G 331 (91-12649) 0,17 1250 (91-6024200) 0,91C/C 812 (245-1737) 1288 (319-3388)C/C-C/G 331 (91-12649) 0,094 1656 (91-6024200) 0,25G/G 91 (91-1995) 333 (91-41686)MCP1 C/C 331 (91-3890) 0,77 1641 (91-1122018) 0,86rs13900 C/T 331 (91-12649) 1183 (91-6024200)Bravo et al., manuscript in preparation, 2013•Recessivemodel(A risk allele)Barcelona, 2013
  35. 35. CMV in ICU•Host-dependant risk factors•Immune risk profile?•CMV pp65 and IE-1 IFN-γ-producing CD8+and CD4+T cells protect againstthe development of active CMV infection in theAllo-SCT settingSolano et al., Haematologica, 2008Tormo et al., Bone Marrow Transplant, 2010Tormo et al., Bone Marrow Transplant, 2011Barcelona, 2013
  36. 36. pepmix pp65 and IE-1(1 µg/mL/peptide)+ CD28/CD49d moAbsBrefeldin (at 2 h.)6 h.Lysis/FixationPermeabilizationStaining (CD3+/CD4+orCD8+/CD69+/IFNγ)BD FastimmuneSolano et al., Haematologica, 2008CMV in ICUFlow cytometry for ICSBarcelona, 2013
  37. 37. CMV in ICUActive CMV InfectionT-cell subsetcells/µlNo YesNoYesIFNγ CD8+ T cells IFNγ CD4+ T cells•First immunological evaluation at the time ICU admission (median 2 days)•Virological monitoring (TA+PLASMA) by real-time PCRClari et al. J Med Virol, 2013•Immune risk profile? •32 patients•80% Septic shockBarcelona, 2013
  38. 38. CMV in ICU•First immunological evaluation at the time of ICU admission (median 2days)Ongoing episode (n=5)During follow-up (n=5)Ongoing episode (n=3)During follow-up (n=2)Ongoing episode (n=1)During follow-up (n=1)•Patients with active CMV infection (n=17)Clari et al. J Med Virol, 2013•Patients without active CMV infection (n=15)Peak CMV DNA load:2,830 copies/mL in TA and100 copies/mL in plasmaPeak CMV DNA load: 230copies/mL in TA and 100copies/mL in plasma•Enumeration of CMV p65 and IE-1-specific IFN-γ T cells at ICU admission may predict the risk of active CMV infectionand allow the inference of the level of CMV replication in the LRT within the episodesBarcelona, 2013
  39. 39. CMV in ICU•LPS (bacterial sepsis) induces a contraction of mCMV-specificmemory CD8+ T cellsTotal CD8+T cellsmCMV-specific CD8+T cellsmCMV murine modelBarcelona, 2013
  40. 40. CMV in ICUTLR4 -/- Knock-out mice•Contraction of mCMV-specific CD8+ T cells after heterologousantigenic stimulation (LPS) is driven by TLR-4Barcelona, 2013
  41. 41. CMV in ICU•Functional NK-cell deficit in ICU patients developing an episode of active CMV infection•Immunological analyses(at admission/days 7,14,21, 28,35)15 cases and 15 controlsPBMCsK562 cells (Direct)P815 cells (ADCC)ICSIFN-γCD107b•Virological monitoring: pp65 AGBarcelona, 2013
  42. 42. CMV in ICUChiche et al., 2012•Patients developing an episode of active CMV infection display an impaired functional NK-cell activity(IFN-γ production) the week before diagnosis of active CMV infection but not at ICU admissionADCCADCCDirectDirectBarcelona, 2013
  43. 43. CMV in ICUChiche et al., 2012•Immunological data on specimensobtained the week right before detection of active CMV infection (pp65 AG)•Expression of activating and inhibitory NK-cell receptors was comparable in cases and controlsBarcelona, 2013
  44. 44. CMV in ICU•Functional NK-cell deficit mediated by IL-10 and IL-15•Patients with an episode of active CMV infection displayed higher plasma IL-10/IL-15 levels and lower plasma TGFβ2 levels than patients not developing itChiche et al., 2012Barcelona, 2013
  45. 45. CMV in ICUvon Müller L et al. J Infect Dis. 2007;196:1288-1295Active CMV infection (pp65 AG)No active CMV infection (pp65 AG)•Cytotoxic NK-cell activity is depressed in cases and controlsBarcelona, 2013
  46. 46. CMV in ICUvon Müller L et al. J Infect Dis.2007;196:1288-1295Active CMV infection (pp65 AG)No active CMV infection (pp65 AG)•An expansion of CMV-specific CD4+and CD8+T cells occurs in patients with active CMV infection•Can we predict the outcome of active CMV infection in ICUpatients by measuring CMV-specific T-cell responses?Chilet et al., 2010Blanquer et al., 2011Barcelona, 2013
  47. 47. CMV in ICU0,00,10,20,30,40,50,6cells/µl 0,00,20,40,60,81,01,21,41,61,82,06,012,018,024,030,036,0cells/μlBaseline•The magnitude of expansion of both T-cell subsets in patients with decreasing CMV DNA loads was notsignificantly different from that seen in patients with increasing CMV DNA loads (P=0.72).Baseline Peak levelIncreasing CMV DNA loadsin plasma and/or LRTDecreasing CMV DNA loadsin plasma and LRTClari et al. J Med Virol, 2013pp65 and IE-1-specific CD8+T cells pp65 and IE-1-specific CD8+T cellsPeak level•Monitoring of peripheral blood CMV-specific T-cell responses may no reflect the actual replicative state in the lungsBarcelona, 2013
  48. 48. •IL-10 levels in LRT >blood (may impair CMV-specific T-cell functionality in the LRT)•IL-10 levels correlate (P=0.001) with CMV DNA loads in LRTCMV in ICU•Monitoring of peripheral blood CMV-specific T-cell responses may no reflect the actual replicative state in the lungsBarcelona, 2013
  49. 49. CMV in ICU•Conclusions•Active CMV infection is a frequent event in ICU patients withoutcanonical immunosuppression•Screening of the LRT for the presence of CMV DNA is imperative for an optimal diagnosis ofactive CMV infection•Whether CMV is a truly pathogen in ICU patients or a mere by-standerremains to be elucidated•Further studies must be conducted to identify subsets of patients who are mostat risk to develop active CMV infection•There might be a genotypic and an immunological risk profile for the developmentof active CMV infection• Monitoring of peripheral blood CMV-specific T cells may not reflect the actualstatus of CMV replication in the LRTBarcelona, 2013
  50. 50. CMV in ICUMarifina ChiletDayana BravoM. Aª ClariElisa CostaBeatriz MuñozEstela GiménezIsabel CorralesGerardo AguilarJavier BeldaJosé BlanquerJosé CarbonellLiliana HenaoMicrobiology ICUBarcelona, 2013

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