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

CMV in ICU
Cytomegalovirus (CMV) in Intensive Care Unit (ICU) patients
David Navarro, M.D., Ph.D.
Microbiology Service, University Clinic Hospital, Valencia, Spain
Department of Microbiology, School of Medicine, University of Valencia, Spain
Barcelona, 2013

Primoinfección
Reactivación
tisular/mucosas
Estocástica
Inflamación
Stress
Viremia
Latencia/persistencia
multiorgánica
Enfermedad orgánica/tisular
Déficit Th-1
PMM (CD33+) Monocitos Endotelio
TNF-α
Catecolaminas
DCs
Mθ
Túbulo renal Células acinares
Infección crónica
Reinfección
CMV in ICU
Barcelona, 2013

UL1-146TLR 1-14 TRS1IRL/S
gpTLR11
gpUL16
gpUL18 gpUL27 gpUL33
gpUL40 gpUL83
gpUL111gpUL78 gpUL146
US1-34
gpUS2
gpUS3
gpUS6
gpUS10
gpUS11
Regulación
negativa
presentación
HLA I/II
Homólogo
receptor FcIII.
Inhibe ADCC
Bloquea
activación
vía NKG2D
Bloquea
activación
de NK
Liga LIR1
Bloquea
activación
de NK
Homólogos
receptores α-
quimioquinas
Quimioquina
homóloga IL-8Homólogo
IL-10.
Inhibe Th1
gpUS28
Inmunosupresor y pro-inflamatorio
Inhibe presentación
de IE-1
Homólogos
receptores
de β-quimioquinas y
fractalina
M θ (M1/M2)
IL-10
IL-18
vIL-10
IL-1
IL-6
TNF-α
NO
iNOS
COX-2
Inmunosupresión
Inflamación
DCs
Bloqueo
presentación
HLA I/II
CMV in ICU
Barcelona, 2013

Innate immunity
DC/MΦ
Adaptive immunity
Early control of CMV replication
Definitive control of CMV replication
IFNs (α,β,λ)
Antiviral cytokines
NK cells
TCD8+
TCD4+
Tregs
B cells
Complex
Hierarchical
Redundant
Neutralizing
ADCC
CMV in ICU
Barcelona, 2013

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/ARDS
Domart et al., Chest, 1990
Cook et al., Am J Surg, 1998
Heininger et al., Crit Care Med, 2001
Cook et al., Crit Care Med, 2003
Von Müller et al., Emerg Infect Dis, 2004
Jaber et al., Chest, 2005
Ziemann et al., Crit Care Med, 2008
Limaye et al., JAMA, 2008
Chiche et al., Crit Care Med, 2009
Heininger et al., Crit Care, 2011
Coisel et al., PLOS one, 2012
Barcelona, 2013

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 patients
CMV in ICU
Barcelona, 2013

~1,000 patients: 81% higher mortality
CMV in ICU

CMV in ICU
plasma 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
•Prospective or retrospective observational studies do not prove causality
Barcelona, 2013

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 entry
0-55%
Barcelona, 2013

CMV in ICU
Plasma
CMV DNAemia
Q r-t PCR
•Severe sepsis/septic shock
•Mechanical ventilation at entry
Underlying disease
Major risk factors
55%
38%
25%
18%
CMV DNAemia >1,000 copies/mL
45%
20%
15%
5%
Barcelona, 2013

•Weekly virological monitoring in plasma and tracheal aspirates by Q-RT-PCR (L.O.D.=10
copies/mL)
No patient received antiviral therapy
53 patients undergoing mechanical ventilation
65% severe sepsis or septic shock
•Simultaneous screening of the LRT and the blood compartment for the
presence of CMV DNA is imperative for an optimal diagnosis of active CMV
infection in ICU patients
CMV in ICU
Barcelona, 2013

TA TA+PL PL (no TA specimens available)
Active CMV infection
•Dissociated episodes
Q-RT-PCR
(~10 copies/mL)
CMV in ICU
Chilet et al., J Med Virol, 2010
Barcelona, 2013

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, 2010
Barcelona, 2013

TA TA+PL PL /leukocytes
(no TA specimens available in 9 patients)
Active CMV infection
Dissociated episodes
Qualitative PCR
(No defined L.O.D.)
Heininger et al., Critical Care, 2011
CMV in ICU
Barcelona, 2013

•Direct effect: Cytopathogenicity: High level virus replication
10-40% of patients with ALI/ARDS have histopathological evidences
of CMV pneumonitis
Papazian et al., Anesthesiology, 1998
Papazian et al., Crit Care Med, 2007
Chiche 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 ICU
Barcelona, 2013

•Experimental mCMV model (sepsis/ileo-cecal puncture)
Pro-inflammatory status (SIRS)
CMV reactivation in LRT
TNF-α/IL-1β/LPS (systemic and LRT)
Pulmonar fibrosis
(ALI/ARDS)
TNF-α/IL-1β/
TGF-β
Infected alveolar macrophages
Activated CMV-specific CD8+/CD4+T cells
Viremia
Cook et al., 2002
Cook et al., 2006
Cook et al., 2007
Forster et al., 2010
•Pathogenesis of active CMV infection in ICU patients
CMV in ICU
Barcelona, 2013

CMV in ICU
Previous CMV infection increases pulmonary TNF-α response in sepsis
Cook and Trgovcich, 2011
•Experimental mCMV model of sepsis induced by ileo-cecal puncture
Barcelona, 2013

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 setting
Hodson et al. Lancet, 2005
Reischig et al. Am J Transplant ,2008
Kliem et al., Am J Transplant, 2008
•Data obtained in the murine model of sepsis-induced mCMV reactivation
Forster et al., Antiviral Res, 2010
Only a randomized-controlled clinical trial of antiviral therapy may reveal the
pathogenetic role, if any, of CMV in ICU patients
•When should antivirals be administered? Prophylactic vs. Pre-emptive
Barcelona, 2013

CMV mRNA in
Lung tissue
(Incidence)
Lung fibrosis
Gomori stain
(collagen)
(Severity)
Ganciclovir treatment
3 weeks following infection Forster et al., 2010
7 days (Pre-emptive)
7 days (Pre-emptive)
Ganciclovir treatment
•Experimental mCMV model (sepsis/ileo-cecal puncture)
CMV in ICUCMV in ICU
Prophylaxis
Prophylaxis
Prophylaxis
Prophylaxis
Prophylaxis
Prophylaxis
Barcelona, 2013

CMV in ICU
•Efficacy of Ganciclovir/Valganciclovir for Prevention of Cytomegalovirus Reactivation
in 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 Division
Main inclusion criteria
•CMV IgG seropositive
•Intubated and requiring mechanical positive pressure ventilation
•Acute Lung Injury/ARDS
Main exclusion criteria
•Known or suspected immunosuppression
•Hospitalized for > 96 hours
Intervention
•Ganciclovir 5mg/kg iv, or valganciclovir 900 mg orally or placebo at entry
Multicenter randomized placebo-controlled double-blind trial
Barcelona, 2013

•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
•Safety
CMV in ICU
Barcelona, 2013

“Pre-emptive antiviral therapy of active
CMV infection in ICU patients with severe
sepsis, septic shock and mechanically
ventilated”
Número EudraCT: 2011-002603-15
Clinical trial code: GANCMV-2011
FIS de Investigación Clínica Independiente 2010.
Código: EC10-318
Randomized, unicentric, parallel group, double-blind placebo controlled phase II clinical‑ ‑
trial
CMV in ICU
Barcelona, 2013

•Primary Outcome Assessment:
Clearance of active CMV infection in LRT and blood (plasma)
The following virological parameters will be compared
between 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 TA
and plasma (weekly monitorization).
CMV in ICU
Barcelona, 2013

•>18 years old
• No canonic immunosuppression
• CMV-seropositive
• Severe sepsis/septic shock
•Mechanical ventilation within 48 h. of admission
Inclusion criteria
Pre-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 mechanical
ventilaton is discontinued
CMV in ICU
Barcelona, 2013

IV Ganciclovir/
Valganciclovir:
First 5 days : IV ganciclovir: 10 mg/kg daily,
given as 5 mg/kg every 12 hours (adjusted
for renal function). After first 5 days (for
a maximun of 14 days), either IV
ganciclovir 5 mg/kg QD or PO
valganciclovir 900 mg po QD (both
adjusted for renal function).
Placebo For the first 5 days, dosing of intravenous
placebo is daily, given every 12 hours.
After first 5 days (for at least 14 days),
either IV placebo QD or PO placebo QD. A
minimum interval of 6 hours is required
between the first and second dose. The
placebo is an IV solution or a PO tablet
that does not contain any active
medications.
CMV in ICU
Barcelona, 2013

• Clinical outcomes at 7, 14, 21 , 28, 60 , 90 days post-
randomization
Organ system failure at 14 and 28 days
Duration of mechanical ventilation
Lung injury score
Bacteremia and/or fungemia
Mortality
Composite of survival status, ventilation status, and IL-6 levels
• Length of stay
• CMV disease (biopsy-proven)
• Safety
Time to neutropenia ( <500/µL)
Use of G-CSF
Time to renal insufficiency (creatinine clearance < 60, < 30 ml/min)
Time to thrombocytopenia (platelet count < 50,000, < 20,000/µL)
CMV in ICU
Barcelona, 2013

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 at
highest risk for CMV reactivation
Looking for biological risk factors
•“Further studies must be conducted to identify subsets of patients who
are most at risk to develop active CMV infection”
Barcelona, 2013

•CMV-related risk factors
Latent CMV burden may determine
the risk of CMV reactivation
• CMV-specific IgG levels?
• Peripheral CMV-specific CD8+
T-cell levels?
Evaluation at the time of ICU admission
Inference by
CMV in ICU
Cook and Trgovcich, Antiviral Res, 2012
Is CMV DNA load in saliva predictive of the development
of LRT/systemic CMV reactivation?
Salivary glands are a major
site of chronic virus replication
mCMV murine model
Barcelona, 2013

CMV in ICU
•Host-related risk factors
•Genomic risk profile?
•Immune risk profile?
Evaluation at the time of ICU admission
Barcelona, 2013

IL 10 (2068) Intron - C/G
CCR5 (-2086) 5’ UTR - A/G
AA substitution
CCL2 (MCP1) (1543) 3´-UTR - C/T
IL-10
CCR5
MCP-1
CMV in ICU
•Genomic risk profile?
•Pairwise analysis
•Testing for a Dominant/Co-dominant/Recessive model
Bravo et al., manuscript in preparation, 2013
Barcelona, 2013

CMV in ICU
Gene
rs number
Genotype
Active CMV Infection (%)
P value
No Yes
IL10 C/C 14 (73,7) 5 (26,3) 0,081
rs1878672 C/G 15 (45,5) 18 (54,5)
C/C 14 (73,7) 5 (26,3) 0,134
G/G 13 (50) 13 (50)
C/G 15 (45,5) 18 (54,5) 0,796
G/G 13 (50) 13 (50)
G/G-C/G 28 (47,5) 31 (52,5) 0,064
C/C 14 (73,7) 5 (26,3)
C/C-C/G 29 (55,8) 23 (44,2) 0,64
G/G 13 (50) 13 (50)
CCR5 A/A 16 (66,7) 8 (33,3) 0,201
rs1800023 A/G 20 (48,8) 21 (51,2)
A/A 16 (66,7) 8 (33,3) 0,30
G/G 6 (46,2) 7 (53,8)
A/G 20 (48,8) 21 (51,2) 1,0
G/G 6 (46,2) 7 (53,8)
G/G-A/G 26 (48,1) 28 (51,9) 0,148
A/A 16 (66,7) 8 (33,3)
A/A-A/G 36 (55,4) 29 (44,6) 0,56
G/G 6 (46,2) 7 (53,8)
MCP1 C/C 30 (61,2) 19 (38,8) 0,105
rs13900 C/T 12 (41,4) 17 (58,6)
Recessive
model
G risk allele
Barcelona, 2013

CMV in ICU
Gene
rs number
Genotype
PLASMA (IU/mL)
P value
TA (IU/mL)
CMV DNA peak*
P value
CMV DNA peak*
CCR5 A/A 398 (91-12649) 0,24 14542 (91-6024200) 0,03
rs1800023 A/G 249 (91-7585) 781 (91-77624)
A/A 398 (91-12649) 0,76 14542 (91-6024200) 0,19
G/G 331 (91-1995) 1183 (218-19952)
A/G 249 (91-7585) 0,54 781 (91-77624) 0,49
G/G 331 (91-1995) 1183 (218-19952)
G/G-A/G 292 (91-7585) 0,29 1047 (91-77624) 0,03
A/A 398 (91-12649) 14542 (91-6024200)
A/A-A/G 331 (91-12649) 0,78 1303 (91-6024200) 0,98
G/G 331 (91-1995) 1183 (218-19952)
IL-10 C/C 812 (245-1737) 0,44 1288 (319-3388) 0,54
rs1878672 C/G 331 (91-12649) 2575 (91-6024200)
C/C 812 (245-1737) 0,15 1288 (319-3388) 0,63
G/G 91 (91-1995) 333 (91-41686)
C/G 331 (91-12649) 0,22 2575 (91-6024200) 0,24
G/G 91 (91-1995) 333 (91-41686)
G/G-C/G 331 (91-12649) 0,17 1250 (91-6024200) 0,91
C/C 812 (245-1737) 1288 (319-3388)
C/C-C/G 331 (91-12649) 0,094 1656 (91-6024200) 0,25
G/G 91 (91-1995) 333 (91-41686)
MCP1 C/C 331 (91-3890) 0,77 1641 (91-1122018) 0,86
rs13900 C/T 331 (91-12649) 1183 (91-6024200)
•Recessive
model
(A risk allele)
Barcelona, 2013

CMV in ICU
•Host-dependant risk factors
•Immune risk profile?
•CMV pp65 and IE-1 IFN-γ-producing CD8+
and CD4+
T cells protect against
the development of active CMV infection in the
Allo-SCT setting
Solano et al., Haematologica, 2008
Tormo et al., Bone Marrow Transplant, 2010
Tormo et al., Bone Marrow Transplant, 2011
Barcelona, 2013

pepmix pp65 and IE-1
(1 µg/mL/peptide)
+ CD28/CD49d moAbs
Brefeldin (at 2 h.)
6 h.
Lysis/Fixation
Permeabilization
Staining (CD3+
/CD4+
or
CD8+
/CD69+
/IFNγ)
BD Fastimmune
Solano et al., Haematologica, 2008
CMV in ICU
Flow cytometry for ICS
Barcelona, 2013

CMV in ICU
Active CMV Infection
T-cell subset
cells/µl
No YesNoYes
IFNγ 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 PCR
Clari et al. J Med Virol, 2013
•Immune risk profile? •32 patients
•80% Septic shock
Barcelona, 2013

CMV in ICU
•First immunological evaluation at the time of ICU admission (median 2days)
Ongoing episode (n=5)
During follow-up (n=5)
•Patients with active CMV infection (n=17)
•Patients without active CMV infection (n=15)
Peak CMV DNA load:
2,830 copies/mL in TA and
100 copies/mL in plasma
Peak CMV DNA load: 230
copies/mL in TA and 100
copies/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 infection
and allow the inference of the level of CMV replication in the LRT within the episodes
Barcelona, 2013

CMV in ICU
•LPS (bacterial sepsis) induces a contraction of mCMV-specific
memory CD8+ T cells
Total CD8+
T cells
mCMV-specific CD8+
T cells
mCMV murine model
Barcelona, 2013

CMV in ICU
TLR4 -/- Knock-out mice
•Contraction of mCMV-specific CD8+ T cells after heterologous
antigenic stimulation (LPS) is driven by TLR-4
Barcelona, 2013

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 controls
PBMCs
K562 cells (Direct)
P815 cells (ADCC)
ICS
IFN-γ
CD107b
•Virological monitoring: pp65 AG
Barcelona, 2013

CMV in ICU
Chiche 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 admission
ADCC
ADCC
Direct
Direct
Barcelona, 2013

CMV in ICU
Chiche et al., 2012
•Immunological data on specimens
obtained the week right before detection of active CMV infection (pp65 AG)
•Expression of activating and inhibitory NK-cell receptors was comparable in cases and controls
Barcelona, 2013

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 it
Chiche et al., 2012
Barcelona, 2013

CMV in ICU
von Müller L et al. J Infect Dis. 2007;196:1288-1295
Active CMV infection (pp65 AG)
No active CMV infection (pp65 AG)
•Cytotoxic NK-cell activity is depressed in cases and controls
Barcelona, 2013

CMV in ICU
von Müller L et al. J Infect Dis.
2007;196:1288-1295
Active 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 ICU
patients by measuring CMV-specific T-cell responses?
Chilet et al., 2010
Blanquer et al., 2011
Barcelona, 2013

CMV in ICU
0,0
0,1
0,2
0,3
0,4
0,5
0,6
cells/µl 0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
6,0
12,0
18,0
24,0
30,0
36,0
cells/μl
Baseline
•The magnitude of expansion of both T-cell subsets in patients with decreasing CMV DNA loads was not
significantly different from that seen in patients with increasing CMV DNA loads (P=0.72).
Baseline Peak level
Increasing CMV DNA loads
in plasma and/or LRT
Decreasing CMV DNA loads
in plasma and LRT
pp65 and IE-1-specific CD8+
T cells pp65 and IE-1-specific CD8+
T cells
Peak level
•Monitoring of peripheral blood CMV-specific T-cell responses may no reflect the actual replicative state in the lungs
Barcelona, 2013

•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 LRT
CMV in ICU
•Monitoring of peripheral blood CMV-specific T-cell responses may no reflect the actual replicative state in the lungs
Barcelona, 2013

CMV in ICU
•Conclusions
•Active CMV infection is a frequent event in ICU patients without
canonical immunosuppression
•Screening of the LRT for the presence of CMV DNA is imperative for an optimal diagnosis of
active CMV infection
•Whether CMV is a truly pathogen in ICU patients or a mere by-stander
remains to be elucidated
•Further studies must be conducted to identify subsets of patients who are most
at risk to develop active CMV infection
•There might be a genotypic and an immunological risk profile for the development
of active CMV infection
• Monitoring of peripheral blood CMV-specific T cells may not reflect the actual
status of CMV replication in the LRT
Barcelona, 2013

CMV in ICU
Marifina Chilet
Dayana Bravo
M. Aª Clari
Elisa Costa
Beatriz Muñoz
Estela Giménez
Isabel Corrales
Gerardo Aguilar
Javier Belda
José Blanquer
José Carbonell
Liliana Henao
Microbiology ICU
Barcelona, 2013

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

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