Cytomegalovirus (CMV) genotyping

Purpose of the test

Clinical use

Detect antiviral resistance in patients with cytomegalovirus (CMV) infection.

Clinical background

CMV is a member of the Herpesviridae family (beta-herpesvirinae subfamily) infecting about 60% of the adult population in Western countries. After primary infection, CMV remains latent indefinitely and can reactivate when the immune system is weakened. CMV infections are usually asymptomatic or mild in healthy individuals, though are more serious in patients with an immunocompromised patients, such as transplant recipients, HIV seropositive people, individuals receiving immune suppressive therapy for autoimmune diseases, or patients undergoing radiotherapy and/or chemotherapy. In these patients, CMV primary infection, re-infection, or  reactivation  can cause severe morbidity and mortality.

CMV infection can usually be prevented or treated with antiviral drugs. Drugs used to manage CMV diseases include six licensed drugs: Ganciclovir (and its oral prodrug valganciclovir), maribavir, foscarnet, cidofovir, and letermovir. These drugs inhibit the viral UL97 protein kinase (maribavir), DNA polymerase [(val)ganciclovir, cidofovir, and foscarnet] or terminase (letermovir) through various mechanisms. Over time, as CMV replicates under drug pressure, viral genes can acquire mutations that may confer resistance to one or more drugs.  Emergence of specific mutations in viral genes can make the drug(s) ineffective and is associated with increased morbidity and mortality. Therefore, it is important to detect resistance as rapidly and accurately as possible to adapt antiviral therapy.

Criteria for performing this test in the context of reference activities

Approximately 5% to 12% of transplant recipients receiving ganciclovir develop drug resistance, being the prevalence higher in certain patient populations (i.e., lung and intestinal transplant recipients, patients heavily immunosuppressed, transplant recipients with CMV serostatus donor-positive, recipient-negative CMV, patients undergoing prolonged exposure to subtherapeutic doses of anti-CMV drugs).

According to the guidelines from The Transplantation Society International CMV Consensus Group, antiviral drug-resistance should be considered for refractory HCMV infection in patients receiving appropriate doses of antiviral therapy. CMV infection is considered refractory when CMV viral load fails to decrease by >1 log10 or CMV disease fails to improve , after ≥2 weeks of appropriately dosed antiviral therapy. In patients with suspected refractory or resistant CMV infection, antiviral resistance should be investigated  by genotypic resistance testing.

The same blood/plasma specimen used for CMV viral load quantification is usually analyzed for CMV drug-resistance testing. To unravel viral compartmentalization and dynamics, it is highly recommended to investigate longitudinally in addition to blood specimens, the sites of CMV disease (e.g., grafted organ, end-organ disease, cerebrospinal fluid, bronchoalveolar lavage) for detection of resistance mutations.

Test details

Includes:

1. (Val)ganciclovir resistance: mutations in the CMV UL97 protein kinase and UL54 DNA polymerase genes.
2. Cidofovir resistance: mutations in the CMV UL54 DNA polymerase gene.
3. Foscarnet resistance: mutations in the CMV UL54 DNA polymerase gene.
4. Maribavir resistance: mutations in the CMV UL97 protein kinase and UL27 genes.
5. Letermovir resistance: mutations in the CMV UL51, UL56, and UL89 terminase subunit genes.

Test description

• 1. Isolation of DNA from the sample.
• 2. Amplification of the HCMV genes involved in drug-resistance by PCR according to the therapy administered to the patient.

• 3. Direct sequencing of the amplicons by Sanger dideoxy sequencing, which has a limit of detection of viral mutant subpopulations of 20%-30%).
• 4. Sequence alignment (derived sequences of the patient sample are aligned with the reference strain AD-169 sequences).
• 5. Detected mutations are compared to a database of known mutations associated with drug-resistance or natural occurring polymorphisms (inter-strain variability) to determine whether clinical resistance CMV infection is due to viral drug-resistance.

Interpretation of the results

Results include a list of the detected mutations and their association with  resistance to each specified drug, inter-strain variability, or unpredicted significance (novel changes). In function of the CMV genotype, alternative therapeutic options are suggested.

A result of unavailable or incomplete genotyping indicates that not all viral amplicons could be amplified and sequenced. This can be due to insufficient viral load, quality of the sample (storage and transportation conditions, age of the sample), and/or presence of polymerase chain reaction inhibitors.

Limitations of the genotypic tests for CMV drug-resistance

• Test results fail to detect mutations that are present in 20-30% of the viral population.
• Tests are most reliable if the CMV viral load in the specimen is at least 3 log₁₀ IU/ml (1,000 IU/ml). Results for specimens with lower viral loads should be interpreted with caution and should be confirmed within a new sample.
• Because genotypic artifacts may occur, in particular in mixed mutant populations from specimens with low viral load, retesting in a new sample is advised.

Instructions for samples and transport

https://rega.kuleuven.be/regavir/shipping

 Unacceptable requests 

• Inappropriate or insufficient sample.
• Improper transport or storage of the sample.
• Test request form not fully completed without specifying the virus for which drug-resistance needs to be investigated.

Turnaround time (and frequency of analysis)

Frequency of analysis: every working day, during working hours.

Response time: 3-7 working days.

Reporting of test results

Results will be sent via e-mail according to the requesting laboratory or physician’s wishes.