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Cytomegalovirus
Reproductive Infectious Disease Seminars
February 15, 2005
Natali Aziz, MD, MS
Reproductive Infectious Disease and Maternal-Fetal Medicine Fellow
Department of Obstetrics, Gynecology and Reproductive Sciences
University of California, San Francisco
Overview
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Microbiology
Pathogenesis
Virulence
Epidemiology
Congenital CMV
Clinical Manifestations
– Immunocompetent Hosts
– Immunocompromised Hosts
– Congenital/Perinatal Disease
• Diagnosis
• Treatment
– Immunocompromised
• Prophylaxis
– Immunocompromised
• Vaccines
Microbiology
• Double-stranded linear DNA
enveloped virus
• Member of Herpesviridae family
– Alpha-herpesvirus subfamily
• HSV-1 and 2, VZV
– Beta-herpesvirus subfamily
• CMV (HHV5), HHV6, HHV7
– Gamma-herpesvirus subfamily
• EBV, HHV8 (KS)
www.biosciences.bham.ac.uk
Microbiology
• CMV
– Icosahedral nucleocapsid
containing dS DNA viral
structure
– 162 hexagonal protein
capsomeres
– Additional layer of
surrounding protein
(tegument)
– Outer membrane envelope
with glycoprotein complexes
www.biografix.de
Microbiology
• Largest member of
herpesviridae family
– 230-240 kilobase pairs
• Large cytomegalic cells
with enlarged nuclei
• Class E genome
• Unique and inverted repeats
that include the existence of 4
genome isomers caused by
inversion of L-S genome
components (class E).
– Violaceous intranuclear
inclusions surrounded by a
clear halo
– Basophilic stippling may be
present in the cytoplasm
• Replication cycle
– Immediate early: 4 h
– Early: 4-24 h
– Late: 24 h
www.som.tulane.edu
Pathogenesis
• Lytic virus with cytopathic effect
• Initial infection
– Epithelial cells of the salivary gland persistent infection and viral
shedding
– Genitourinary system
• Proximal tubules near cortical areas
– Ultimately can be found in several tissues (salivary gland, lung, liver,
kidney, intestine, adrenal gland and CNS)
• Other pathogenic mechanisms
– Granulomatous reaction, particularly in liver
– Immune complex formation
– Vasculitis
• Establishes a latent host infection
– May reactivate during a period of immunosuppression secondary to
drugs or concurrent infection (eg. HIV)
Pathogenesis
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Incubation period: 28-60 days
Primary infection symptoms: 9-60 days
Viremia: 2-3 weeks
IgM response: 30-60 days
Peak viral titers: 4-7 weeks post infection
Virulence
• US2, US11, US3, US6
– Gene products interfere with MHC class I function and antigen processing
• UL33, US27, US28
– Subvert normal inflammatory process
– Promote tissue dissemination of virus
• MHC class I homologue
– Evades host defense
• UL144
– Encodes TNF homolog and may thereby escape immune clearance
• Antivirals interfere with early gene products
– Ganciclovir: targets UL54 and is phosporylated by UL97 protein
• Genetic differences among viruses
– Multiple strains of CMV
– Differences in genotypes may be associated with differences in virulence
– Dual infection with different strains possible
Schleiss and McVoy, 20004
Immunology
Humeral Immunity
• Primary infection
• CMV IgM antibodies may
be found as early as 4-7
weeks
Cell-Mediated Immunity
• Most important factor
controlling infection
– CD4 and CD8
– Persist as long as 16-20 weeks
after initial infection
• Majority of neutralizing Ab
directed against envelope
glycoprotein gB and gH
– >50% of neutralizing activity
in convalescent serum
attributable to glycoprotein
gB
• Virion tegument proteins
pp150, pp28, and pp65
evoke strong and durable
antibody responses
Glycoprotein B (gpUL55) mediated
morphogenesis of infectious CMV particles
www.biografix.de
Epidemiology
• 50-85% prevalence in US by 40 years
of age
• As high as 100% prevalence in some
populations
• CMV prevalence increases with age
• Risk factors
– Work at day care/contact with
children
– Blood transfusion
– Multiple sexual partners
– Unprotected intercourse
– Parity
– Abnormal cervical cytology
– Lower SES/underdeveloped nations
– Born outside US
– First pregnancy before 15 years of
age
– Infection with STI
• Transmission: transplanted organ,
breast milk, urine, saliva, tears,
stool, sexual contact, blood,
transplacental
• Seldom associated with mortality
in immunocompetent hosts (<1%)
• Significant morbidity and
mortality in immunocompromised
(solid and BM transplant, AIDS,
etc.)
– Despite antiviral therapy,
allogenic BMT patients will
have 15-75% interstitial PNA
mortality rate
www.cdc.gov
Congenital CMV and Pregnancy
• Most common congenitally acquired infection
– Occurs 0.2-2% of all neonates
– Approximately 40, 000 infants infected annually in US
• Leading cause of congenital hearing loss in US
• Prevalence in pregnancy
– Seropositive: 50-80%
– Primary infection: 0.7-4%
– Recurrent infection: 13.5%
• Cervical excretion of CMV common during pregnancy
– Not indication for c-section
• CMV in breast milk
– CMV not contraindication to breast feeding
• Vertical transmission
– Transplacental infection: symptomatic
– Exposure to genital tract secretions: usually asymptomatic
– Breast feeding : usually asymptomatic
• Worse disease in premature infants
Congenital CMV and Pregnancy
• Vertical transmission greatest during 3rd trimester
• More serious fetal sequelae when infection in 1st trimester
• Vertical transmission
– Primary maternal infection: 30-40%
– Recurrent maternal infection: 0.15-2%
• Symptomatic congenital CMV disease less likely in women with
pre-existing immune response
– Congenital hearing loss most severe sequelae
• Most infants with congenital CMV are asymptomatic at birth
• 10% of infants infected in-utero will develop CMV signs and
symptoms at birth
– Poor prognosis
– 30% of severely infected die
– 80% of survivors severe neurologic sequelae
• 85-90% of infected infants are asymptomatic at birth
– 10-15% will develop long-term neurologic sequelae, hearing loss
Congenital CMV and Pregnancy
www.aafp.org
Congenital CMV and Pregnancy
Congenital CMV and HIV
• Congenital CMV is NOT more common in infants of HIVinfected mothers (Kovacs 1999, Mussi-Pinhata 1998)
• Perinatal acquisition of CMV higher among HIV-infected
babies (Kovacs 1999)
– Dual infection associated with higher HIV progression (RR 2.59)
and CNS disease
• CMV and HIV potentiate each other in vitro (Skolnik 1988)
Clinical Manifestations
• Immunocompetent host
disease
• Immunocompromised
host disease
lbmi.org/pathologyimages
• Congenital/Perinatal
disease
Clinical Manifestations
Immunocompetent
www.crprc.ucdavis.edu
• Usually asymptomatic or mild flu-like symptoms
• Mononucleosis syndrome
– Fever/chills, malaise, myalgia
– Mild hepatitis, leukocytosis, atypical lymphocytes in blood x 6 weeks
– Less hepatomegaly, splenomegaly, pharyngitis than EBV
– Older patients, longer fever duration, less cervical LAN
– Negative Monospot or heterophile-agglutinin tests
• Meningoencephalitis, pericarditis, myocarditis, thrombocytopenia, hemolytic
anemia, maculopapular rash, GI ulcers, pneumonia less common
• Reactivation possible
– Viremia
– Positive IgM in presence of IgG
– In setting of concurrent infections or stress
Clinical Manifestations
Immunocompromised
• Significant disease in the
immunocompromised host
– Pneumonia
– Hepatitis
– Encephalitis
– Colitis/GI ulcerations
– Uveitis
– Retinitis
– Neuropathy
– CMV syndrome
Normal Retina
CMV Retinitis
www.5mcc.com
Clinical Manifestations
Immunocompromised
• Excretion of CMV in saliva and
urine common
• Highest incidence for CMV
infection in BMT recipients is 30-60
days post transplant
• Viremia in organ transplant patients
– Marker for pneumonia in allogenic
BMT patients
– Viremia c/w 60-70% risk of PNA
– CXR: miliary/interstitial infiltrate,
localized/nodular infiltrates less
common
CMV Pneumonia
Clinical Manifestations
Immunocompromised
• Transplant patients:
– Interstitial pneumonitis, GI disease, retinitis,
hepatitis, encephalitis, myeloradiculopathy, and
CMV syndrome
• HIV patients
– Retinitis and CNS involvement more common
CMV Cecal Ulcer
CMV Esophagitis
www.vh.org/
Clinical Manifestations
Congenital/Perinatal
www.med.nagoya-u.ac
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Clinical Findings
Laboratory Findings
Petechiae/Purpura(71-76%) •
Jaundice (67%)
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Hepatosplenomegaly (60%) •
Microcephaly (53%)
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IUGR (50%)
Retinitis
Cerebral calcifications
Hepatitis
Non-immune hydrops
Elevated LFT’s (83%)
Hyperbilirubinemia (81%)
Thrombocytpenia (77%)
Elevated CSF protein (77%)
Long Term Sequelae
– Hearing loss
– Mental retardation
– Neurologic manifestations
• Associated with higher IgM levels
Clinical Manifestations
Congenital/Perinatal
Clinical Manifestations
Congenital/Perinatal
Ventriculomegaly and Periventricular Calcifications
Diagnostic Studies
• Conventional cell culture
• Serology
• Shell vial culture
• CMV antigenemia (pp65)
• Molecular methods (PCR)
Cytomegalovirus-infected human diploid fibroblast cells in culture.
Modified acridine orange staining [M. Battaglia, unpublished].
Diagnostic Studies
• Shell vial culture
– Early antigen detection with monoclonal antibodies
– Viremia has been shown to be a risk factor for CMV
pneumonia in patients who have received allogenic
marrow transplants
– Shell vial assay reduced identification time to 24-48 hours
– Monitoring of the shell vial assay prior to the onset of
disease
• Practical method for starting early antiviral treatment
– Uses permissive cell line for CMV
• Centrifuged at a low speed and placed in an incubator
• After 24 and 48 hours, the tissue culture medium is
removed and the cells are stained using a fluoresceinlabeled anti-CMV antibody
• The cells are read using a fluorescent microscope
• Alternatively, the cells are stained with an antibody
against CMV, followed by a fluorescein-labeled antiIg.
– Not as sensitive as traditional tissue culture
Diagnostic Studies
• CMV antigenemia (pp65)
forums.gardenweb.com
– Antigenemia: relatively new test developed in the late 1980s
– Recognition of CMV early antigen by a mixture of 2 mouse
monoclonal antibodies, C-10 and C-1
– The detector system is fluorescein-labeled anti-mouse Ig
– Cells are counted using a fluorescent microscope
– Any positive cell confirms the diagnosis of CMV viremia
– The literature has suggested that a higher cell count corresponds
to risk of disease.
– Antigenemia has been used to predict CMV pneumonia in patients
who have received transplants
– A positive antigenemia test result can be used as a trigger to
institute ganciclovir therapy when a preemptive strategy is used
for the prevention of CMV disease in transplant patients
– Available in 24 hours
Diagnostic Studies
• Molecular methods (PCR)
– Qualitative polymerase chain reaction
• PCR has been used to detect CMV in blood and tissue samples
• The PCR test depends on the multiplication of primers specific for a
portion of a CMV gene
– Primers usually bind to the area of virus that codes for early
antigen
• The test is extremely sensitive
• Positive before the antigenemia test in patients with viremia who
have received transplants.
– The PCR test result is not usually positive in patients without
CMV viremia.
• Qualitative PCR has also been used to detect CMV in histological
sections
Diagnostic Studies
• Molecular methods (PCR)
– Quantitative polymerase chain reaction
• Quantitative PCR has been used to detect plasma CMV
• Quantitative PCR is as sensitive as qualitative PCR and provides an
estimate of the number of CMV genomes present in plasma
• Research
– Determine if the number of CMV genomes present in the
plasma correlates with risk of disease in different at-risk
populations
– Number of CMV genomes (ie, viral load) present would
indicate whether therapy is necessary because patients below a
certain cut-off would not develop CMV disease
– However, the level of viremia necessary for CMV disease to
occur may vary depending on host factors and the type of
organ transplant, and this may need to be determined
empirically
– Literature from different organ transplant systems suggests that
this method may be the test that discriminates between lowlevel viremia versus a higher level and CMV disease
Maternal Diagnosis
Congenital CMV
• Serum tested 2-4 weeks apart
• IgG seroconversion or fourfold
increase (ie. 1:4 to 1:16) of IgG
• IgM useful but not always
reliable sign of primary infection
– May persist for months
– Appears in reinfection
– False positive if RA
– >30% of IgG value may
suggest active infection
www.dpcweb.com
Fetal Diagnosis
Congenital CMV
• CMV detected in amniotic fluid by
• Ultrasound findings
culture or PCR
– Abdominal and liver
calcifications
– Culture sensitivity: 50-69%
– Lateral ventricle calcifications in
– PCR sensitivity: 77-100%
lateral border
– Combined sensitivity: 80-100%
– Hydrops
– Comparable PPV and NPV
– Echogenic bowel
• Sensitivity of amniotic fluid testing
– Ascites
markedly lower if performed before 21
– Hepatosplenomegaly
weeks GA
– Ventriculomegaly
• Severity not predicted by amniotic
– ?Thickened nuchal translucency
fluid assessment
not associated with maternal
infection (Sebire et al 1997)
• Ultrasound may initially be normal
• CNS involvement poorer
prognosis
Considerations
Congenital CMV
Therapies
• No current therapies for maternal
or fetal CMV infection
• Ganciclovir crosses placenta in
vitro
• Reported use of ganciclovir and
CMV IgG postnatally for
congenital disease
– Prevention of long-term
neurologic sequelae not
proven
Screening?
• Routine screening not recommended
• IgM not reliable for differentiating
primary infection
• Maternal immunity does not
eliminate fetal infection
• Screening indications
– Symptoms suggestive of CMV
infection (mononucleosis-like
syndrome or elevated LFT’s)
– Exposure to CMV
– Immunocompromised patients
Antiviral Therapy for Congenital and
Perinatal Infection
• A phase II study has investigated intravenous ganciclovir
at 8 and 12 mg/kg per day, divided every 12 hours and
given for six weeks in each case (Whitley 1997)
– Decreased viral excretion during drug administration
– Viruria returned after drug cessation
– 16% stable/improved in hearing at 6 months F/U
– At age two, eight of 33 infants were judged to be
developing normally
• A phase III placebo-controlled trial of ganciclovir is
currently underway by the National Institute of Allergy
and Infectious Diseases Collaborative Antiviral Study
Group (Kimberlin 2000)
– Preliminary results suggest improvement/prevention
of hearing loss during first 6-12 months of life and
premature infants benefit most
Treatment
• Ganciclovir
– Targets UL54 protein
– Mutations in the UL97-encoded CMV phosphotransferase and alterations in
viral DNA polymerase have been associated with resistance
– DNA polymerase alteration also a/w cross-resistance to the nucleotide analog
cidofovir
• Valganciclovir
• Foscarnet
• Cidofovir
– Nucleoside analogue
• Treatment not usually indicated in immunocompetent patients
• Indications
– Treatment of disease in immunocompromised: retinitis, GI disease,
pneumonitis, neurologic disease, viremia
Treatment: CMV Retinitis
Prevention
• Behavioral Modification
– Avoidance infectious saliva,
urine, bodily fluids
– Careful hygiene practices
– Condom use
www.med.nagoya-cu.ac
Prophylaxis
• Ganciclovir used for CMV prophylaxis in solid organ
and allogenic bone marrow transplant patients post
surgery
• AIDS CMV prophylaxis
– CD4 <50
• Ganciclovir not recommended by US PHS due to cost, lack of
survival advantage, neutropenia, high pill burden
• Ophtho exams Q 1-3 months
– Immune reconstitution in response to ARV’s
• Prophylaxis with antivirals x 6 months with CD4 >100
Vaccines
• Live attenuated vaccine
developed (Plotkin 1991)
– Largest trial: Towne 125
strain
– 500 subjects
– Partial efficacy
– Economically beneficial
• Concerns
– Reactivation and infection
of host
– Viral shedding from cervix
or breast milk
– Possible oncogenic
potential of vaccine virus
• Glycoprotein vaccine in guinea
pig model (Bourne 2001)
– Reduced in-utero CMV
transmission
– Improved pregnancy
outcome