Clinica di Malattie Infettive e Tropicali

Università degli Studi dell’Insubria –

Ospedale di Circolo e Fondazione Macchi, Varese

Laboratory Tests to Monitor CMV

Infection in Transplant Patients

Paolo Grossi

IHMF Annual Meeting 2007

Dubrovnik, 10th -11th October 2007
 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
 CMV infection in transplant recipients
• Primary infection
– occurs when an allograft from a seropositive
individual is transplanted into a seronegative
recipient; >90% 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 1–3 months posttransplant
• In contrast, antiviral prophylaxis extends the
incubation period
 Pre-tx HCMV serology in a cohort of Heart
and Lung Transplant recipients

Heart n=649 Lung n=187

HCMV neg
HCMV neg 13,4%
10,6%

HCMV pos
89,4% HCMV pos
86,6%

Median age=52 Median age=47

(range 8-71) (range 13-68)

P.Grossi, unpublished data

HCMV associated clinical syndromes

Mononucleosis like syndrome

Gastrointestinal disease
Hepatitis - cholangitis
Neurological disease
Retinitis
Pneumonia
Graft rejection ?
 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.
 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
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).
 CMV Prophylaxis in Solid Organ Transplant Recipients
Efficacy Comments

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

i.v. immunoglobulin Reduction of CMV No benefit with the Very high cost
disease exception of Kidney
transplant
4-6 weeks Reduction of CMV No benefit Delayed onset
i.v. ganciclovir disease

≥ 12 weeks Reduction of High cost;

i.v. ganciclovir CMV disease bacterial
superinfections;
≥ 12 weeks Reduction of Selection of
oral ganciclovir CMV disease resistant strains.
≥ 12 weeks Reduction of Delayed onset;
oral valganciclovir CMV disease Selection of
resistant strains ?
P.Grossi, 2007
 PV16000: Time to CMV Disease Up
To 6 Mo (EC, ITT)
100

90
% Patients with no CMV Disease

80

70

60
364 D+/R- SOT patients
Investigator treated
50
CMV
40

30 valganciclovir
20
ganciclovir
10
Prophylaxis period
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 2004;4:611-620
 Late CMV Disease: Definition
• CMV disease occurring >3 months
post-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
Occurrence of late CMV disease at day 101–365 in
R+ and D+/R- patients by type of transplant

Limaye AP, et al. Lancet 2000; 356: 645–49

 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 2003;52:324–
 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
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.
 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
 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
 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
 Viremia
Quantification of PBL carrying infectious virus

18 h cocolture

PBL shell vial p72

isolation preparation
peripheral
blood
HCMV p72 staining
(immunofluorescence
or immunoperoxidase)

Antigenemia
Quantification of pp65-positive PBL

peripheral PBL cytospin

blood separation preparation HCMV pp65 staining
(immunofluorescence
or immunoperoxidase)

RNAemia
Detection (or quantification) of HCMV mRNAs (IE or Late)
DNAemia
Quantification of HCMV DNA in PBL, plasma or whole blood

RNA PBL isolation

extraction mRNA
cDNA RT-PCR
plasma separation
Nucleic Acid
Sequence Based whole blood
Polymerase Chain Reaction
Amplification (NASBA)
peripheral blood
Peripheral ds DNA DNA extraction
blood 1 2 3 4 5 6 7 8

external standard sample

ssRNA
(amplification product)
chemiluminescent hybridization
internal standard
revelation (gel electrophoresis ds DNA (amplification product)
gel electrophoresis and densitometric analysis)

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

 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
 Preemptive Antiviral Therapy
Starting Criteria

CMV seronegative first positive antigenemia

CMV seropositive antigenemia >=100/200,000

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 1995;59:847‑851.

 HCMV Infection in 303 Thoracic Organ
Transplant Recipients
Heart (n=211) Lung (n=92)
D+/R- = 22, 10.4% D+/R- = 13, 14.1%
(n=108)
51.2% (n=23)
25.0%
(n=44)
47.8%
(n=52)
24.6%

(n=7) (n=22)
(n=44) 23.9% (n=3)
3.3%
20.9% 3.3%

Preemptive therapy Infection not eligible

No infection Protocol violation

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

 Incidence of CMV infection and disease in
heart (n=211) and lung (n=92) 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 1998;17:50

 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. 2005;143:870-880

 Recurrence of HCMV infection in HCMV D+/R-
Thoracic Organ Transplant Recipients
Early Preemptive p-value
symptomatic therapy (n=21)
therapy (n=21)
# of patients with 8 (38) 17 (80.9) 0.011
recurrence%
# of episodes 12 25 0.0196

# of treated 8 ( 66.6) 18 (72) n.s.

episodes (%)
# of symptomatic 0 2 (8.6)
recurrence (%)
P.Grossi, unpublished data
Incidence of CMV Infection and Treatments
According to the Treatment Arm

Antigenemia Nasba p-value

(n=42) (n=40)
HCMV infection 39 (92.9%) 30 (75.0%) 0.03

Treated 15 (38.5%) 25 (83.3%) 0.02

preemptively

N. of pts with 0 0
diseases

G.Gerna, P.Grossi, et al. TRANSPLANTATION 2003;75:1012–1019.

Peak DNAemia levels as
determined by
quantitative PCR (QPCR)
in solid organ transplant
recipients 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 2004;73:412–418

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. , 2004;73:412–419.

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: 2356–2364

Virologic and immunologic follow-up of two heart
transplant recipients. G.Gerna, et al. AJT 2006;6:2356–2364
 Studies in organ transplant recipients using
valganciclovir as prophylaxis or preemptive therapy

Singh N. Rev. Med. Virol. 2006; 16: 281–287.

Strippoli GF, et al. 2006 The Cochrane Collaboration.
 Prophylaxis vs Pre-Emptive Therapy
Prophylaxis Pre-emptive
Patients’s selection All (100%) Targeted to high risk
patients ~30%
Duration of administration 90-100 days 14-21 days

Cost of surveillance testing None 500-1000 US$/pt

Drug acquisition cost 6294 US$/pt 1762 US$/pt
Logistic Easy Difficult
Potential for toxicity High Low
Late onset disease ++ -
Resistance High Low

Results from a large randomized trial are needed!

 Conclusions
o CMV infection continue to be a significant cause
of morbidity in transplant recipients
o Studies have demonstrated the limitation of
antiviral prophylaxis
o Pre-emptive therapy based on virological
monitoring has been proven to reduce the risk
of viral disease in immunocompromised hosts
o 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.
 Conclusions
o
single cutoff may be safely used for initiation of pre-
emptive antiviral therapy both in primary and
reactivated CMV infection
o
CMV 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.
•
imultaneous monitoring of HCMV infection and HCMV-
specific T-cell immunity predicts T-cell mediated
control of HCMV infection.