Paolo Grossi

1
Laboratory Tests to Monitor CMV Infection in
Transplant Patients
Clinica di Malattie Infettive e
Tropicali Università degli Studi dellInsubria
Ospedale di Circolo e Fondazione Macchi, Varese

• Paolo Grossi

IHMF Annual Meeting 2007 Dubrovnik, 10th-11th
October 2007
2
Cytomegalovirus (CMV)

• Single most important pathogen affecting
  transplant 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

3
CMV infection in transplant recipients

• Primary infection
• occurs when an allograft from a seropositive
  individual is transplanted into a seronegative
  recipient gt90 of these individuals become ill.
• Reactivation infection
• Reactivation infection occurs when endogenous,
  latent infection is reactivated 15 of these
  become ill.
• Superinfection
• both donor and recipient are seropositive, but
  the virus that is reactivated is of donor origin
  25 of these people become ill.
• In the absence of antiviral prophylaxis CMV
  infection occurs 13 months posttransplant
• In contrast, antiviral prophylaxis extends the
  incubation period

4
Pre-tx HCMV serology in a cohort of Heart and
Lung Transplant recipients
Lung n187
Heart n649
Median age47 (range 13-68)
Median age52 (range 8-71)
P.Grossi, unpublished data
5
HCMV associated clinical syndromes
Mononucleosis like syndrome
Gastrointestinal disease
Hepatitis - cholangitis
Neurological disease
Retinitis
Pneumonia
Graft rejection ?
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Modalities of CMV Prevention
Prophylactic Therapy
Prophylaxis - Prevention of Disease Greek -
Guard before, take Precautions
Preemptive Therapy
Preemption - Obtaining Something in Advance
Identifying Patients with Subclinical CMV
infection and treat them in Advance, before they
develop CMV disease.

7
Treating CMV infection in transplant patients

• Intravenous ganciclovir or the prodrug
  valganciclovir are the drugs that are commonly
  utilized to prevent or treat active CMV disease

8
Mechanisms of action of antiherpes drugs targeted
at the viral DNA polymerase. Nucleoside analogues
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), or
pyrophosphate (PPi).
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CMV Prophylaxis in Solid Organ Transplant
Recipients
P.Grossi, 2007
10
PV16000 Time to CMV Disease Up To 6 Mo (EC, ITT)
100
90
80
70
364 D/R- SOT patients
60
Investigator treated CMV
Patients with no CMV Disease
50
40
valganciclovirganciclovir
30
20
Prophylaxis period
10
0
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
Time (days)
Paya C., et al AJT 20044611-620
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Late CMV Disease Definition

• CMV disease occurring gt3 monthspost-SOT
• May be primary infection (D/R-) or recurrence
  (R)
• May present with atypical symptoms
• Diagnosis can be missed
• Patient may not be followed by primary center or
  may not be followed as closely

12
Occurrence of late CMV disease at day 101365 in
R and D/R- patients by type of transplant
Limaye AP, et al. Lancet 2000 356 64549
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HCMV resistance to antiviral drugs
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 in
ganciclovir-resistant HCMV strains are shown by
vertical solid bars.
F. Baldanti, G. Gerna. J Antimicrob Chemother
200352324330
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Antiviral prophylaxis and CMV-specific immune
reconstitution

• Emerging data suggest that potent antiviral
  agents (e.g., ganciclovir) may inhibit the
  development of long-term protective immunity
  against CMV in transplant recipients.
• Ganciclovir affects cellular DNA synthesis and
  has an overall inhibitory 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 of late onset
CMV disease occurred in ganciclovir recipients.
In all patients, the presence of a CTL response
to CMV conferred protection from subsequent CMV
disease (P .005), and these protective CTL
responses are shown to be specific for structural
virion proteins similar to the responses in
immunocompetent CMV seropositive individuals.
These data confirm the importance of CMV-specific
T-cell responses and suggest that a delay in
recovery of these responses as a result of
ganciclovir prophylaxis may contribute to the
occurrence of late CMV disease.
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Antiviral prophylaxis and CMV-specific immune
reconstitution

• Long-term control of viral replication is
  critically dependent on an adequate 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 after infection 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 generation and expansion of CD8
  T-cell responses

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How Do We Deal With Late-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 withholding antiviral
  agents until they would be maximally effective
• An accurate detection method to identify patients
  at risk for disease is an essential component of
  this strategy

19
Courtesy of Prof. G.Gerna, IRCCS S.Matteo, Pavia,
Italy
20
Virological monitoring
Virological monitoring was performed by shell
vial assay and tube culture (viremia) and CMV
direct pp65 antigen detection and
quantification (antigenemia) following the
schedule shown below.
Timing of blood testing for HCMV detection
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
Grossi 1996
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Preemptive Antiviral Therapy
Starting Criteria
CMV seronegative
first positive antigenemia
CMV seropositive
antigenemia gt100/200,000
Steroid boluses or
ATG/OKT3 therapy for
any positive antigenemia
rejection

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

P.Grossi, et al Transplantation 199559847-851.
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HCMV Infection in 303 Thoracic Organ Transplant
Recipients
Heart (n211) D/R- 22, 10.4
Lung (n92) D/R- 13, 14.1
(n108)
51.2
(n23)
25.0
(n44)
47.8
(n52)
24.6
(n22)
(n7)
(n3)
(n44)
23.9
3.3
3.3
20.9
Preemptive therapy
Infection not eligible
No infection
Protocol violation
P.Grossi, et al. J Heart and Lung Transplant
19981750
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Incidence of CMV infection and disease in heart
(n211) and lung (n92) transplant recipients
treated preemptively.
100
80
60
40
20
0
1994
1995
1996
1997
1998
1999
No infection
Asymptomatic
Symptomatic
P.Grossi, et al. J Heart and Lung Transplant
19981750
24
Meta-Analysis The Efficacy of Strategies To
Prevent Organ Disease by Cytomegalovirus in Solid
Organ Transplant Recipients
Kalil A.C., et al Ann Intern Med. 2005143870-880
25
Recurrence of HCMV infection in HCMV D/R-
Thoracic Organ Transplant Recipients
P.Grossi, unpublished data
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Incidence of CMV Infection and Treatments
According to the Treatment Arm
G.Gerna, P.Grossi, et al. TRANSPLANTATION
20037510121019.
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Peak DNAemia levels as determined by quantitative
PCR (QPCR) in solid organ transplantrecipients
either at the time when antigenemia-based
preemptive treatment was decided (treated), or
following selection from serial results in the
absence of treatment (untreated).
Lilleri D., et al. Journal of Medical Virology
200473412418
28
Correlation of HCS (A) and Amplicor-whole blood
(B) with the in-house QPCR. Retrospectively
determined QPCR DNAemia cutoff values were
interpolated on regression curves to determine
the corresponding DNAemia cutoff values (dotted
lines) of the two commercial assays tested for
both SOTR and HSCTR.
G.Gerna, et al. J. Med. Virol. , 200473412419.
29
Quantification of HCMV-specific CD4 and CD8 T
cells

• A new methodological approach in order to provide
  a more comprehensive evaluation of the T-cell
  immune response in transplant recipients was
  recently reported
• This method is not limited by HLA-restriction,
  allows simultaneous expression of different viral
  proteins on the DC membrane, along with
  simultaneous quantification and functional
  evaluation of both HCMV-specific CD4 and CD8 T
  cells by CFC.

G.Gerna, et al. AJT 2006 6 23562364
30
Virologic and immunologic follow-up of two heart
transplant recipients. G.Gerna, et al. AJT
2006623562364
31
(No Transcript)
32
Studies in organ transplant recipients using
valganciclovir as prophylaxis or preemptive
therapy
Singh N. Rev. Med. Virol. 2006 16 281287.
33
Strippoli GF, et al. 2006 The Cochrane
Collaboration.
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Prophylaxis vs Pre-Emptive Therapy
Results from a large randomized trial are needed!
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Conclusions

• CMV infection continue to be a significant cause
  of morbidity in transplant recipients
• Studies have demonstrated the limitation of
  antiviral prophylaxis
• Pre-emptive therapy based on virological
  monitoring has been proven to reduce the risk of
  viral disease in immunocompromised hosts
• Monitoring of CMV DNAemia in solid organ
  transplant recipients seems to represent an
  improvement with respect to pp65 antigenemia
  since it better reflects virus replication in
  vivo and is more standardizable and automatable.

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Conclusions

• A single cutoff may be safely used for initiation
  of pre-emptive antiviral therapy both in primary
  and reactivated CMV infection
• HCMV DNA quantification on whole blood is the
  elective assay for monitoring viral load, since
  directly correlating with viral replication and
  clinical symptoms However, standardization of
  the different methods is mandatory.
• Simultaneous monitoring of HCMV infection and
  HCMV-specific T-cell immunity predicts T-cell
  mediated control of HCMV infection.