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Paolo Grossi

Clinica di Malattie Infettive e Tropicali

Universit degli Studi dellInsubria

Ospedale di Circolo e Fondazione Macchi, Varese

Laboratory Tests to Monitor CMVInfection in Transplant Patients

IHMF Annual Meeting 2007

Dubrovnik, 10th

-11th

October 2007

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Cytomegalovirus (CMV)

Single most important pathogen affectingtransplant recipients

Direct effects: clinical syndromes such as

mononucleosis, pneumonia, colitis, and others Indirect: immunosuppression, super-

infections increased the risk of post-

transplant lymphoproliferative disease,diabetes, atherosclerosis and allograft injury

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CMV infection in transplant recipients

Primary infection

occurs when an allograft from a seropositiveindividual is transplanted into a seronegativerecipient; >90% of these individuals become ill.

Reactivation infection Reactivation infection occurs when endogenous,

latent infection is reactivated; ~15% ofthese become ill. Superinfection

both donor and recipient are seropositive, but thevirus that is reactivated is of donor origin; ~25% of

these people become ill.

In the absence of antiviral prophylaxis CMVinfection occurs 13 months posttransplant

In contrast, antiviral prophylaxis extends theincubation period

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Pre-tx HCMV serology in a cohort of Heartand Lung Transplant recipients

HCMV neg

10,6%

HCMV pos

89,4%

HCMV neg

13,4%

HCMV pos

86,6%

Heart n=649Heart n=649 Lung n=187Lung n=187

Median age=52Median age=52(range 8-71)(range 8-71)

Median age=47Median age=47(range 13-68)(range 13-68)

P.Grossi, unpublished dataP.Grossi, unpublished data

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HCMV associated clinical syndromes

Mononucleosis like syndromeGastrointestinal disease

Hepatitis - cholangitisNeurological diseaseRetinitisPneumoniaGraft rejection ?

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Modalities of CMV Prevention

Prophylactic Therapy

Preemptive TherapyPreemptive Therapy

Prophylaxis - Prevention of DiseaseGreek - Guard before, take Precautions

Preemption - Obtaining Something in AdvancePreemption - Obtaining Something in AdvanceIdentifying Patients with Subclinical CMV infection and treat them inIdentifying Patients with Subclinical CMV infection and treat them inAdvance, before they develop CMV disease.Advance, before they develop CMV disease.

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Treating CMV infection intransplant patients

Intravenous ganciclovir or the prodrugvalganciclovir are the drugs that are

commonly utilized to prevent or treatactive CMV disease

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Mechanisms of action of antiherpes drugs targeted at the viral DNA polymerase. Nucleosideanalogues are first activated by herpes simplex virus (HSV)- or varicella zoster virus (VZV)-encoded thymidine kinase (TK), or the cytomegalovirus unique long (UL)97-encoded kinase,followed by phosphorylation by cellular kinases. The inhibition of the viral DNA polymerase is

competitive with regard to the natural nucleoside triphosphates (dGTP, dTTP or dCTP), orpyrophosphate (PPi).

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CMV Prophylaxis in Solid Organ Transplant Recipients

Efficacy Comments

D+ or D-/R+ D+/R-

i.v. immunoglobulin Reduction of CMV

disease

No benefit with the

exception of Kidney

transplant

Very high cost

4-6 weeks

i.v. ganciclovir

Reduction of CMV

disease

No benefit Delayed onset

12 weeks

i.v. ganciclovir

Reduction of

CMV disease

High cost;

bacterial

superinfections;

12 weeksoral ganciclovir

Reduction ofCMV disease

Selection ofresistant strains.

12 weeks

oral valganciclovir

Reduction of

CMV disease

Delayed onset;Selection of

resistant strains ?

P.Grossi, 2007

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PV16000: Time to CMV Disease Up

To 6 Mo (EC, ITT)

Prophylaxis period

%

Patient s

withno

CM

VDisease

0

10

20

30

40

50

60

70

80

90

100

Time (days)

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

364 D+/R- SOT patientsInvestigator treatedInvestigator treated

CMVCMV

valganciclovirvalganciclovir

ganciclovirganciclovir

Paya C., et al AJT 2004;4:611-620

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Occurrence of late CMV disease at day 101365 inR+ and D+/R- patients by type of transplant

Limaye AP, et al. Lancet2000; 356: 64549

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HCMV resistance to antiviral drugs

F. Baldanti, G. Gerna. J Antimicrob Chemother2003;52:324

DNA polymerase codons mutated in ganciclovir and cidofovir-cross-resistant,foscarnet-resistant and multidrug-resistant HCMV strains are indicated by solid,dashed, and dotted vertical bars, respectively. UL97 codons mutated inganciclovir-resistant HCMV strains are shown by vertical solid bars.

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Antiviral prophylaxis and CMV-specificimmune reconstitution

Emerging data suggest that potent antiviral agents (e.g.,ganciclovir) may inhibit the development of long-termprotective immunity against CMV in transplant recipients.

Ganciclovir affects cellular DNA synthesis and has an overallinhibitory effect onT-cell proliferation

Long-term ganciclovir prophylaxis was associated with

delayed IgG seroconversion

inhibition of antibody maturation

impaired immunoglobulin class switching from IgM to IgG

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Between day 40 and day 90. recovery of deficient CD8+ and CD4+ CMV-specifii T-cell responses occurred in the majority of individuals that

received placebo, but in a minority of ganciclovir recipients. Two cases oflate onset CMV disease occurred in ganciclovir recipients. In all patients,the presence of a CTL response to CMV conferred protection fromsubsequent CMV disease (P= .005), and these protective CTL responsesare shown to be specific for structural virion proteins similar to the

responses in immunocompetent CMV seropositive individuals. These dataconfirm the importance of CMV-specific T-cell responses and suggestthat a delay in recovery of these responses as a result of ganciclovirprophylaxis may contribute to the occurrence oflate CMV disease.

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Antiviral prophylaxis and CMV-specificimmune reconstitution

Long-term control of viral replication is critically dependent on anadequate development of CMV-specific T-helper cell and CD8+cytotoxic T-cell responses

In renal transplant recipients undergoing primary CMV infection,CMV-specific CD4+ T cells appear in the circulation ~7 days afterinfection or detection of CMV-DNA.

This is followed by the generation of specific antibodies and CMV-specific CD8+ T cells that confer protection against CMV disease.

CD4+ helper T cells play a pivotal role in facilitating the generationand expansion of CD8+ T-cell responses

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How Do We Deal WithLate-Onset Disease?

OPTIONS Do nothing accept the risk of late onset

disease and treat as it arises

Prolong prophylaxis Is more better? Not necessarily push disease further, high

number needed to treat (NNT)

Use better prophylaxis? Careful virologic monitoring of high-risk

patients after completing prophylaxis

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Preemptive therapy

Based on the principle of withholdingantiviral agents until they would be

maximally effective An accurate detection method to

identify patients at risk for disease

is an essential component of thisstrategy

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Viremia

18 h cocolture

p72

HCMV p72 staining

peripheralblood

PBLisolation

shell vialpreparation

Quantification of PBL carrying infectious virus

(immunofluorescenceor immunoperoxidase)

Antigenemia

peripheralblood

PBLseparation

cytospinpreparation HCMV pp65 staining

Quantification of pp65-positive PBL

(immunofluorescenceor immunoperoxidase)

DNAemia

peripheral bloodDNA extraction

Polymerase Chain Reaction

revelation (gel electrophoresisand densitometric analysis)

PBL isolation

plasma separation

Quantification of HCMV DNA in PBL, plasma or whole blood

whole blood

internal standard

external standard

1 2 3 4 5 6 87

sample

ds DNA (amplification product)

RNAemia

Peripheralblood

RNAextraction

Nucleic AcidSequence BasedAmplification (NASBA)

chemiluminescent hybridization

Detection (or quantification) of HCMV mRNAs (IE or Late)

RT-PCR

gel electrophoresis

ssRNA(amplification product)

mRNA

cDNA

ds DNA

Courtesy of Prof. G.Gerna, IRCCS S.Matteo, Pavia, Italy

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Grossi 1996

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

weeks after transplantation

Timing of blood testing for HCMV detection

Virological monitoring was performed by shellvial assay and tube culture (viremia) and CMVdirect pp65 antigen detection andquantification (antigenemia) following theschedule shown below.

Virological monitoring

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Preemptive Antiviral TherapyStarting Criteria

CMV seronegative first positive antigenemia

CMV seropositive antigenemia >=100/200,000

Steroid boluses orATG/OKT3 therapy forrejection

any positive antigenemia

P.Grossi, et al Transplantation 1995;59:847 851.

In all recipients antiviral therapy is continued to antigenemia clearance.Recurrent episodes of active infection are treated with additional coursesof ganciclovir starting with antigenemia values 100/200,000.

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HCMV Infection in 303 Thoracic OrganTransplant Recipients

(n=52)24.6%

(n=108)51.2%

(n=44)20.9%

(n=7)3.3%

(n=44)47.8%

(n=23)25.0%

(n=22)23.9% (n=3)3.3%

Preemptive therapy Infection not eligible

No infection Protocol violation

Heart (n=211)D+/R- = 22, 10.4% Lung (n=92)D+/R- = 13, 14.1%

P.Grossi, et al. J Heart and Lung Transplant 1998;17:50

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Incidence of CMV infection and disease inheart (n=211) and lung (n=92) transplant

recipients treated preemptively.

1994 1995 1996 1997 1998 19990%

20%

40%

60%

80%

100%

No infection Asymptomatic Symptomatic

P.Grossi, et al. J Heart and Lung Transplant 1998;17:50

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Meta-Analysis: The Efficacy of Strategies ToPrevent Organ Disease by Cytomegalovirus in

Solid Organ Transplant Recipients

Kalil A.C., et al Ann Intern Med. 2005;143:870-880

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Recurrence of HCMV infection in HCMV D+/R-Thoracic Organ Transplant Recipients

Early

symptomatictherapy (n=21)

Preemptive

therapy (n=21)

p-value

# of patients with

recurrence%

8 (38) 17 (80.9) 0.011

# of episodes 12 25 0.0196

# of treated

episodes (%)

8 ( 66.6) 18 (72) n.s.

# of symptomatic

recurrence (%)

0 2 (8.6)

P.Grossi, unpublished data

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Incidence of CMV Infection and TreatmentsAccording to the Treatment Arm

Antigenemia

(n=42)

Nasba

(n=40)

p-value

HCMV infection 39 (92.9%) 30 (75.0%) 0.03

Treated

preemptively

15 (38.5%) 25 (83.3%) 0.02

N. of pts with

diseases

0 0

G.Gerna, P.Grossi, et al. TRANSPLANTATION 2003;75:10121019.

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Peak DNAemia levels asdetermined by

quantitative PCR (QPCR)in solid organ transplantrecipients either at thetime when antigenemia-

based preemptivetreatment was decided(treated), or followingselection from serial

results in the absenceof treatment(untreated).

Lilleri D., et al. Journal of Medical Virology 2004;73:412418

Correlation of HCS (A) and Amplicor-whole blood (B) with the in-

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G.Gerna, et al. J. Med. Virol. , 2004;73:412419.

Correlation of HCS (A) and Amplicor-whole blood (B) with the in-house QPCR. Retrospectively determined QPCR DNAemia cutoffvalues were interpolated on regression curves to determine thecorresponding DNAemia cutoff values (dotted lines) of the two

commercial assays tested for both SOTR and HSCTR.

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Quantification of HCMV-specificCD4+ and CD8+ T cells

A new methodological approach in order to provide amore comprehensive evaluation of the T-cell immuneresponse in transplant recipients was recently

reported This method is not limited by HLA-restriction,allows simultaneous expression of different viralproteins on the DC membrane, along with

simultaneous quantification and functional evaluationof both HCMV-specific CD4+ and CD8+ T cells byCFC.

G.Gerna, et al. AJT 2006; 6: 23562364

Vi l i d i l i f ll f h

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Virologic and immunologic follow-up of two hearttransplant recipients. G.Gerna, et al. AJT2006;6:23562364

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di i l i i i

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Studies in organ transplant recipients usingvalganciclovir as prophylaxis or preemptive therapy

Singh N. Rev. Med. Virol. 2006; 16: 281287.

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Strippoli GF, et al. 2006 The Cochrane Collaboration.

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Prophylaxis vs Pre-Emptive TherapyProphylaxis Pre-emptive

Patientss selection All (100%) Targeted to high riskpatients ~30%

Duration of administration 90-100 days 14-21 days

Cost of surveillance testing None 500-1000 US$/ptDrug acquisition cost 6294 US$/pt 1762 US$/pt

Logistic Easy Difficult

Potential for toxicity High LowLate onset disease ++ -

Resistance High Low

Results from a large randomized trial are needed!

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Conclusionso CMV infection continue to be a significant cause

of morbidity in transplant recipientso Studies have demonstrated the limitation of

antiviral prophylaxis

o Pre-emptive therapy based on virologicalmonitoring has been proven to reduce the riskof viral disease in immunocompromised hosts

o Monitoring of CMV DNAemia in solid organ

transplant recipients seems to represent animprovement with respect to pp65 antigenemiasince it better reflects virus replication in vivoand is more standardizable and automatable.

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Conclusions

osingle cutoff may be safely used for initiation of pre-emptive antiviral therapy both in primary andreactivated CMV infection

o

CMV DNA quantification on whole blood is the electiveassay for monitoring viral load, since directlycorrelating with viral replication and clinical symptoms;However, standardization of the different methods is

mandatory.

imultaneous monitoring of HCMV infection and HCMV-specific T-cell immunity predicts T-cell mediated

t l f HCMV i f ti