CMV

Question 1

CMV structure & classification

Answer 1

Herpesviridae
Betaherpesvirus subfamily

Enveloped, ds-DNA with an icosahedral capsid

Baltimore: I

Question 2

CMV immunological response

Answer 2

High frequencies of specific cytomegalovirus CD4+ and CD8+ T cell responses have been demonstrated in immunocompetent hosts

Question 3

CMV transmission routes

Answer 3

Sexual
Close contact
Blood or tissue
Occupational - day care, nursing
Perinatal

Question 4

CMV clinical syndrome

Answer 4

1. IMN - an absolute lymphocytosis with greater than 50 percent mononuclear cells and the presence of more than 10 percent atypical lymphocytes on peripheral blood smear
2. GI disease - While CMV colitis is almost always secondary to the reactivation of latent infection in immunosuppressed patients, CMV colitis in the immunocompetent host can occur in the setting of primary infection. Bloody diarrhoea and fever.

well-demarcated ulcerations (50 percent), ulceroinfiltrative changes (25 percent), and pseudomembrane formation (25 percent) [52]. The presence of abnormal mucosal surfaces prior to CMV infection may increase the risk of this infection. As an example, evidence of gastrointestinal CMV in patients with pre-existing inflammatory bowel disease has been described

3. Hepatitis
4. Encephalitis, GBS
5. Pneumonitis
6. Retinitis

Question 5

CMV reactivation & prophylaxis

Answer 5

In critically ill & immunocompromised patients.

in a prospective blinded study of 120 critically ill immunocompetent patients who were CMV seropositive, CMV reactivation was detected by real-time polymerase chain reaction (PCR) in 39 patients (33 percent) and was independently associated with continued hospitalization or death by 30 days after admission to the ICU

To date, there is no evidence that prophylaxis or suppression of CMV viremia in critically ill patients leads to improved outcomes. As an example, in a phase II trial, the ganciclovir/valganciclovir for prevention of cytomegalovirus reactivation in acute injury of the lung (GRAIL) study, 160 CMV-seropositive immunocompetent patients with critical illness due to sepsis or trauma, patients were randomly assigned to receive prophylaxis with intravenous (IV) ganciclovir for five days followed by IV ganciclovir or oral valganciclovir or to receive placebo [145]. Antiviral prophylaxis was associated with a reduced incidence of CMV reactivation (12 versus 39 percent). However, there was no difference between the groups in interleukin (IL)-6 levels (the primary outcome) nor were there differences between the groups in incidence of secondary bacteremia or fungemia, intensive care unit (ICU) length of stay, or mortality.

Question 6

CMV diagnostics

Answer 6

1. Enzyme immunoassays and indirect and anticomplement immunofluorescence assays are the most commonly used serologic tests in clinical laboratories; complement-fixation techniques, immune adherence hemagglutination, indirect hemagglutination, and neutralization tests are performed in a limited number of diagnostic laboratories

Recent or acute infection — A diagnosis of recent or acute CMV is considered probable (though not definite) in the following circumstances:

●The detection of CMV-specific IgM antibodies (suggesting recent seroconversion)
●A fourfold or greater increase in CMV-specific IgG titers in paired specimens obtained at least two to four weeks apart

2. Quantitative RT-PCR:

●COBAS AmpliPrep/COBAS TaqMan CMV test – The COBAS AmpliPrep/COBAS TaqMan CMV test is a real-time PCR test that targets the polymerase gene and is calibrated to the World Health Organization (WHO) international standard to quantify the CMV load in plasma with a reported range from 137 to 9,100,000 international units/mL

●Alinity m CMV assay – The Alinity m assay is a real-time PCR test for use with the automated Alinity m System, which targets the UL34 and UL80.5 genes and is calibrated to the WHO international standard to quantify the CMV load in plasma with a reported range from 30 to 100,000,000 international units/mL.

3. CMV antigenemia assays permit the rapid and direct detection of CMV proteins (pp65) in peripheral blood leukocytes. This technique employs fluorescently labeled monoclonal antibodies specific to the pp65 lower matrix protein of CMV in peripheral blood polymorphonuclear leukocytes. Positive results are reported as the number of cells with staining per total number of cells counted. Results are generally available within 24 hours, and the test performs well in HIV-infected patients and recipients of solid organ transplants [20-22]. In these patients, antigenemia appears to correlate with viremia [4].

●The antigenemia test is more sensitive than culture for the detection of CMV in blood [20-23], and studies have shown that a higher number of positive cells correlates with risk of developing active disease (10 to 20 positive cells/200,000 leukocytes) [24,25].
●The limitations of the antigenemia test include lack of stability of the whole-blood specimen. insensitivity when the patient has a low neutrophil count (<1000 cells/microL), and more difficulty standardizing test results

4. Histologic examination of tissue biopsies is useful for the diagnosis of CMV tissue-invasive disease. Diagnosis is based on the presence of inclusion bodies, typically basophilic intranuclear inclusions, although eosinophilic cytoplasmic inclusions may also be seen; the diagnosis of CMV in tissue sections can be confirmed with immunohistochemical staining [26]. Some experts recommend immunohistochemical staining of biopsy specimens in which inclusions bodies are not seen as a method to improve diagnostic sensitivity [26]. Detection of CMV inclusions in a lung biopsy or in a bronchoalveolar lavage specimen supports the diagnosis of tissue invasive disease and increases the predictive value of a positive culture.
5. Using human fibroblast cultures, CMV can be isolated from multiple specimen types, including the blood, cerebrospinal fluid (CSF), throat washings, bronchoalveolar lavage fluid, urine, and biopsy specimens. Shell vial culture has largely replaced conventional culture in clinical virology laboratories, since the former method is much faster than the latter method.

Shell vial culture — Shell vial culture is a rapid culture method based on low-speed centrifugation and detection of CMV early antigen prior to the development of characteristic cytopathic effects in tissue culture, thus accelerating the time to diagnosis. Following centrifugation of clinical samples (eg, urine, bronchial washings, tissue) to increase the absorption of virus, cell monolayers are exposed to monoclonal antibodies against early antigen. Binding of antibodies is indicative of “early” CMV replication within the cells. Results are typically available within two to three days after specimens are obtained.

Question 7

CMV resistance testing

Answer 7

Markers suggestive of CMV resistance include a rising viral load, rebounding viral load, and a persistently elevated viral load in the setting of antiviral therapy.

In the past, resistance testing was performed using phenotypic methods, which required isolation of the virus in cell culture, determining the viral titer, and then culturing the virus in the presence of drug

Development of resistance usually occurs after prolonged drug exposure (>6 weeks)

Resistance testing - sequencing of UL97 & UL54 & UL 56

Five drug classes are approved for the treatment and/or prophylaxis of CMV disease, four of which target the DNA polymerase: ganciclovir (valganciclovir), foscarnet, cidofovir, and maribavir. Letermovir inhibits the CMV viral terminase complex.

The initial phosphorylation of ganciclovir requires the viral UL97 kinase. Resistance to ganciclovir can occur due to mutations in the UL97 phosphotransferase gene or the UL54 polymerase gene, whereas resistance to foscarnet and cidofovir is due to mutations in the UL54 polymerase gene. Resistance to maribavir is due to mutations in the UL97 gene.

Resistance to letermovir is rare, but if it occurs, it is due to mutations in the UL56 gene

CMV isolates possessing both UL97 and UL54 mutations often have high-level ganciclovir resistance. The ganciclovir resistance conferred by UL54 mutations cannot be overcome by increasing the dose of ganciclovir. UL54 DNA polymerase mutations confer various combinations of resistance to ganciclovir, foscarnet, and/or cidofovir. Mutations in the UL97 gene conferring resistance to ganciclovir and maribavir are mostly distinct from each other. Therefore, maribavir is effective treatment for ganciclovir resistant CMV due to UL97 gene mutations.

Question 8

CMV infection vs disease

Answer 8

●CMV infection refers to virus isolation or detection of viral proteins (antigens) or nucleic acid in any body fluid or tissue specimen regardless of symptoms or signs [1,2].
●CMV disease refers to evidence of CMV infection with attributable symptoms or signs; CMV disease may manifest as either a viral syndrome (eg, fever, malaise, leukopenia, neutropenia, atypical lymphocytosis, thrombocytopenia) or as tissue-invasive disease

Question 9

CMV PCR to remember

Answer 9

1 log difference between whole blood & plasma

Use same assay in same lab - cannot compare across labs

BAL PCR suggestive of pneumonitis or
benign secretion

Question 10

CMV in pregnancy & cCMV

Answer 10

ECCI guidance

Global prevalence of cCMV - 0.64%
20% risk of permanent sequelae in infected children

2 major changes:

1. Demonstration of efficacy of oral valacyclovir in preventing vertical transmission
2. Evidence indicating risk of major sequelae is limited to infection in 1st trimester

Primary prevention & maternal infection diagnosis:

1. CMV serology universal screening recommended if there is capacity.
2. IgM & IgG preferred for diagnosis of maternal infection.
3. Avidity to exclude recent primary infection. Second avidity if first avidity value indeterminate.
4. PCR only recommended in case if isolated IgM in previously seronegative women.

Secondary prevention & foetal infection diagnosis:

1. Oral valaciclovir 8g/day (2g QDS) in 1st trimester maternal infection ASAP until PCR on amniotic fluid. Stop if PCR negative, complete until delivery if PCR positive.
2. IVIG May be considered in primary infection in 1st trimester.
3. PCR on amniotic fluid from 17 weeks AFTER 8 weeks from maternal infection.
4. Foetal USG & MR in 3rd trimester of maternal infection.

Question 11

Neonatal CMV

Answer 11

1. Test Saliva or Urine < 3 weeks from birth. DBS for retrospective diagnosis.
2. IgM not recommended.
3. Testing indicated in maternal primary CMV in pregnancy, IUGR, Foetal abnormalities, very preterm (<32 weeks) or <1500g, lab abnormalities.
4. 6 months valganciclovir treatment in confirmed neonatal CMV with significant symptoms at birth started ASAP.
5. 6 weeks treatment in isolated hepatitis or isolated thrombocytopenia.
6. Regular follow up including eye, ear, and neuro assessments at regular intervals.

Age cut off to start treatment: 1 month

Question 12

CMV newer drugs & MOA

Answer 12

Letermovir inhibits the terminal phase of the virus lifecycle by targeting the subunit pUL56 of the terminase enzyme complex. Antiviral activity is highly specific to CMV, so additional prophylactic antivirals are required if prevention of HSV is required.

Resistance mutations occur most commonly in UL56 or UL89 rather than at the viral polymerase, and there is no cross-resistance between letermovir and other CMV agents

Cyclosporine in particular increases letermovir exposure; if coadministered, 240 mg/day of letermovir is suggested rather than the typical 480-mg daily dose

Maribavir also possesses a mechanism of action unique from other antivirals, acting as a competitive inhibitor of adenosine triphosphate (ATP) binding to pUL97, a protein kinase responsible for phosphorylating itself and other proteins essential to the viral replication cycle. Because pUL97 is required for the phosphorylation and antiviral activity of ganciclovir, coadministration of maribavir and ganciclovir is antagonistic [12]. Synergy, however, was observed when maribavir was administered with foscarnet, cidofovir, or letermovir [12, 15, 23]. Maribavir has in vitro antiviral activity against CMV and EBV but not against HSV 1 and 2, VZV, HHV-6, and HHV-8: requires a concurrent agent for prophylaxis against other herpes viruses.

Mutations in UL97 in the vicinity of the ATP-binding domain and UL27 have been shown to confer resistance to maribavir. The most frequent UL97 mutations did not confer changes in the ganciclovir-binding domain, and thus maribavir-resistant variants maintained susceptibility to ganciclovir and vice versa

400 mg twice daily for 8 weeks

Question 13

CMV in SOT

Answer 13

Offer Valganciclovir prophylaxis to people receiving ALL SOTs for at least 3 months following transplantation if either:

1. The recipient is seronegative for CMV and receives an allograft from a CMV seropositive donor (D+/R-)
   OR
   The recipient has received T-lymphocyte depletion therapy with Antithymocyte gobulin (ATG) or Alemtuzumab (Campath®), where donor: recipient serostatus is D+/R+ or D-/R+

Do not routinely offer Valganciclovir prophylaxis if donor and recipient are seronegative for CMV (D-/R-)

Consider Valganciclovir prophylaxis for at least 3 months after starting treatment for acute allograft rejection if either donor or recipient are CMV positive (D+/R-, D+/R+ or D-/R+)

Monitor Full Blood Count at least every 2 weeks whilst on Valganciclovir

For adults, children and young people who develop CMV infection or disease following solid organ transplantation: Offer treatment with oral Valganciclovir for a duration of at least 2 weeks

In ganciclovir resistance: Offer oral Maribavir for 8 weeks (not licensed in children)
OR
Offer Intravenous Foscarnet for at least 3 weeks

Question 14

CMV in BMT

Answer 14

1. D-/R- preferred
2. D+/R+ preferred in unrelated allogenic HSCT with myeloablation
3. D+/D- suitable for R+ in haploidentical HSCT with cyclophosphamide

Monitoring:

Perform qPCR weekly for first 100 days post transplant. Indication for treatment is load doubling < 2 days.

Monitoring of IFN-gamma producing CMV specific T cells is useful - T spot.

Treatment:

1. Prophylaxis: Letermovir 480mg OD - interferes with cyclosporine, so dose decreased to 240mg for 100 days.

Difficulty in monitoring as PCR will be positive - terminate complex inhibitor. ?CMV RNA testing.

2. Pre-emptive therapy with ganciclovir/valganciclovir/foscarnet.

Ganciclovir - 5mg/kg OD
Valganciclovir: 900mg OD x 100 days
Foscarnet: INDUCTION:
90/mg/kg BD for 2-3 weeks
MAINTANENCE: 90-120mg/Kg OD

Cidofovir is 3rd line. Maribavir or leflunomide are 4th line.

3. Treatment of disease - Ganciclovir. Second line Foscarnet. 3rd like Cidofovir.

Ganciclovir - 5mg/kg BD for 14-21 days followed by maintanence of 5mg/Kg OD if risk of relapse.

Cidofovir: 5mg/kg once weekly for 2 weeks then 5mg/kg every 2 weeks.

4. Adaptive T cell therapy with CMV specific CD8 cells.

Question 15

CMV treatment

Answer 15

1. Ganciclovir. Dosing:

A. Prophylaxis: 6 mg/kg once daily, on 5 days of the week, alternatively 5 mg/kg once daily

B. Preemptive therapy: Initially 5 mg/kg every 12 hours for 7–14 days, then maintenance 6 mg/kg once daily, on 5 days of the week, alternatively maintenance 5 mg/kg once daily.

C. Treatment: Initially 5 mg/kg every 12 hours for 14–21 days, then maintenance 6 mg/kg once daily, on 5 days of the week, alternatively maintenance 5 mg/kg once daily, maintenance only for patients at risk of relapse; if disease progresses initial induction treatment may be repeated.

2. Valganciclovir:

A. Prophylaxis: 900 OD for 100 days post transplant

B. Retinitis: Initially 900 mg twice daily for 21 days, then maintenance 900 mg daily, induction regimen may be repeated if retinitis progresses.

3. Foscarnet:

Initially 60 mg/kg every 8 hours for 2–3 weeks, alternatively initially 90 mg/kg every 12 hours for 2–3 weeks, then maintenance 60 mg/kg daily, then increased if tolerated to 90–120 mg/kg daily, if disease progresses on maintenance dose, repeat induction regimen.

4. Cidofovir: Initially 5 mg/kg once weekly for 2 weeks, then maintenance 5 mg/kg every 2 weeks, maintenance treatment to be started 2 weeks after completion of induction treatment.

Question 16

Neonatal CMV testing SMI

Answer 16

SEE ALBUM

Question 17

Cytotect

Answer 17

CMV specific IgG being trialled in pregnancy to prevent congenital CMV

Given off label in Paeds BMT

Question 18

Posoleucel

Answer 18

Multi-virus specific T cell therapy :

Adeno
BK
CMV
EBV
HHV6
JC

Question 19

CMV pneumonitis

Answer 19

Diagnosis needs biopsy and HPE
Rt-PCR alone not enough

If compatibility clinical & radiology with RT-PCR showing ONLY CMV, then may consider the possibility of

Ganciovir first line treatment

Question 20

CMV canonical mutations

Answer 20

Confer resistance to ganciclovir

M460V/I
H520Q
C592G
A594V
L595S
C603W