Cytomegalovirus

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

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) www.biosciences.bham.ac.uk

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 www.biografix.de

5. Largest member of herpesviridae family 230-240 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: 4-24 h Late: 24 h 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). Microbiology www.som.tulane.edu

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: 9-60 days • Viremia: 2-3 weeks • IgM response: 30-60 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. Humeral Immunity Primary infection CMV IgM antibodies may be found as early as 4-7 weeks 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 Cell-Mediated Immunity Most important factor controlling infection CD4 and CD8 Immunology Glycoprotein B (gpUL55) mediated morphogenesis of infectious CMV particles www.biografix.de

10. 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 Epidemiology www.cdc.gov

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: 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

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: 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

13. Congenital CMV and Pregnancy www.aafp.org

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 www.crprc.ucdavis.edu

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 www.5mcc.com

19. 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

20. Transplant patients: Interstitial pneumonitis, GI disease, retinitis, hepatitis, encephalitis, myeloradiculopathy, and CMV syndrome HIV patients Retinitis and CNS involvement more common Clinical Manifestations Immunocompromised CMV Esophagitis CMV Cecal Ulcer www.vh.org/

21. 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 Clinical ManifestationsCongenital/Perinatal www.med.nagoya-u.ac

22. Clinical ManifestationsCongenital/Perinatal

23. Clinical ManifestationsCongenital/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 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 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 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. 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 Maternal DiagnosisCongenital CMV www.dpcweb.com

30. 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 CMV detected in amniotic fluid by culture or PCR Culture sensitivity: 50-69% PCR sensitivity: 77-100% Combined sensitivity: 80-100% Comparable PPV and NPV Sensitivity of amniotic fluid testing markedly lower if performed before 21 weeks GA Severity not predicted by amniotic fluid assessment Fetal DiagnosisCongenital CMV

31. 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 ConsiderationsCongenital CMV

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 • 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. Behavioral Modification Avoidance infectious saliva, urine, bodily fluids Careful hygiene practices Condom use Prevention www.med.nagoya-cu.ac

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. 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 Vaccines