1. 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

2. Overview Microbiology Pathogenesis Virulence Epidemiology
Congenital CMV
Clinical Manifestations
Immunocompetent Hosts
Immunocompromised Hosts
Congenital/Perinatal Disease
Diagnosis
Treatment
Immunocompromised
Prophylaxis
Vaccines

3. 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)

4. 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

5. Microbiology Class E genome Largest member of herpesviridae family
Unique and inverted repeats that include the existence of 4 genome isomers caused by inversion of L-S genome components (class E).
Largest member of herpesviridae family
kilobase pairs
Large cytomegalic cells with enlarged nuclei
Violaceous intranuclear inclusions surrounded by a clear halo
Basophilic stippling may be present in the cytoplasm
Replication cycle
Immediate early: 4 h
Early: h
Late: 24 h

6. 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)

7. Pathogenesis Incubation period: 28-60 days
Primary infection symptoms: days
Viremia: 2-3 weeks
IgM response: days
Peak viral titers: 4-7 weeks post infection

8. 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

9. Immunology Cell-Mediated Immunity Humeral Immunity Primary infection
Most important factor controlling infection
CD4 and CD8
Humeral Immunity
Primary infection
CMV IgM antibodies may be found as early as 4-7 weeks
Persist as long as 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

10. 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

11. 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: %
Primary infection: %
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

12. 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: %
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

13. Congenital CMV and Pregnancy

14. Congenital CMV and Pregnancy

15. Congenital CMV and HIV
Congenital CMV is NOT more common in infants of HIV-infected 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)

16. Clinical Manifestations
Immunocompetent host disease
Immunocompromised host disease
Congenital/Perinatal disease
lbmi.org/pathologyimages

17. Clinical Manifestations Immunocompetent
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

18. Clinical Manifestations Immunocompromised
Significant disease in the immunocompromised host
Pneumonia
Hepatitis
Encephalitis
Colitis/GI ulcerations
Uveitis
Retinitis
Neuropathy
CMV syndrome
Normal Retina
CMV Retinitis

19. Clinical Manifestations Immunocompromised
Excretion of CMV in saliva and urine common
Highest incidence for CMV infection in BMT recipients is 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

20. 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

21. Clinical Manifestations Congenital/Perinatal
Clinical Findings
Petechiae/Purpura(71-76%)
Jaundice (67%)
Hepatosplenomegaly (60%)
Microcephaly (53%)
IUGR (50%)
Retinitis
Cerebral calcifications
Hepatitis
Non-immune hydrops
Laboratory Findings
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

22. Clinical Manifestations Congenital/Perinatal

23. Clinical Manifestations Congenital/Perinatal
Ventriculomegaly and Periventricular Calcifications

24. 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].

25. 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 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 fluorescein-labeled 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 anti-Ig.
Not as sensitive as traditional tissue culture

26. Diagnostic Studies CMV antigenemia (pp65)
forums.gardenweb.com
CMV antigenemia (pp65)
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

27. 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

28. 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 low-level viremia versus a higher level and CMV disease

29. 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

30. Fetal Diagnosis Congenital CMV
CMV detected in amniotic fluid by culture or PCR
Culture sensitivity: 50-69%
PCR sensitivity: %
Combined sensitivity: %
Comparable PPV and NPV
Sensitivity of amniotic fluid testing markedly lower if performed before 21 weeks GA
Severity not predicted by amniotic fluid assessment
Ultrasound findings
Abdominal and liver calcifications
Lateral ventricle calcifications in lateral border
Hydrops
Echogenic bowel
Ascites
Hepatosplenomegaly
Ventriculomegaly
?Thickened nuchal translucency not associated with maternal infection (Sebire et al 1997)
Ultrasound may initially be normal
CNS involvement poorer prognosis

31. Considerations Congenital CMV
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
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

32. 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

33. Treatment Ganciclovir Valganciclovir Foscarnet Cidofovir
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

34. Treatment: CMV Retinitis

35. Prevention Behavioral Modification
Avoidance infectious saliva, urine, bodily fluids
Careful hygiene practices
Condom use

36. 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

37. 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