Polyomavirus typing / genotying

Purpose of the test

Clinical use
Detect antiviral resistance in patients with BK polyomavirus (BKPyV) infection.

Clinical background
Human polyomaviruses (HPyVs) include a group of 14 species that usually cause asymptomatic infection in healthy persons and persist in an asymptomatic state. However, in the immunocompromised host, HPyVs cause a variety of severe disease outcomes, such as cancer (Merkel cell polyomavirus, MCPyV), kidney disease (BK polyomavirus, BKPyV), and fatal infection of the brain (JC polyomavirus, JCPyV).

Infection with the BKPyV is important in kidney transplant or hematopoietic stem cell transplant recipients, in which it can lead to polyomavirus-associated nephropathy (PVAN)  and eventually loss of the transplanted kidney or to hemorrhagic cystitis (HC), respectively. Considering the lack of specific antivirals for BKPyV, reduction of immunosuppression therapy remains the main approach for treating PVAN despite the risk of allograft rejection.

The broad-spectrum anti-DNA virus agent cidofovir, fluoroquinolones, leflunomide, and intravenous immunoglobulins have been considered as adjunctive therapy in PVAN, though their clinical efficacy against BKPyV has not been fully determined. Not all patients respond equally to cidofovir treatment, some may still showing signs of BKPyV infection after cidofovir therapy. Similar pharmacological approaches are used to manage HC though reducing immunosuppression in HSCT patients increases the risk of acute rejection and exacerbates graft versus host disease (GvHD).

Criteria for performing this test in the context of reference activities

Cidofovir resistance in in vitro experiments has been associated with changes in the viral Large T antigen (LTag) for the non-human primate polyomavirus SV40. Therefore, it can be hypothesized that cidofovir failure in patients being treated with this antiviral for BKPyV disease can eventually be associated with acquisition of mutations in the BKPyV genome. Patients not responding to cidofovir therapy for BKPyV disease should be monitored for emergence of drug-resistance.

Test details – PyV detection and typing / BKPyV genotyping

Includes:

1. Detection of JCPyV and BKPyV.
2. BKPyV typing.
3. Cidofovir resistance: mutations in the LTag gene.

Test description – PyV detection and typing / BKPyV genotyping

1. Isolation of DNA from the sample.
2. Amplification of the whole genome for JCPyV and BKPyV.
3. Direct sequencing of VP1 gene by Sanger dideoxy sequencing, which has a limit of detection of viral mutant subpopulations of 20%-30% of the amplicons.
4. Sequence alignment: derived sequences of the patient isolate are aligned to the sequence of the Dunlop reference BKPyV strain.
5. Typing of BKPyV is performed according to the article from Morel et al, 2017 (PMID: 28151406).
6. Alignment of the patient VP1 (major capsid protein) sequence to other BKPyV strains from the same subgroup is carried out to identify the known VP1 sequences that are the most similar to the VP1 sequence of the patient.
7. Direct sequencing of the LTag gene by Sanger dideoxy sequencing, which has a limit of detection of viral mutant subpopulations of 20%-30% of the amplicons.
8. For analysis of the LTag, then the sequences of the LTag of the closest related samples are used as reference to identify the changes potentially associated with cidofovir resistance.

Interpretation of the results — BKPyV typing / genotyping

Results include a list of the detected mutations in the VP1 gene (complete sequence, 362 amino acids) that allow BKPyV typing.

Results also include a list of changes in the LTag (complete sequence, 695 amino acids) potentially linked to cidofovir resistance.

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 genotyping tests for BKPyV drug-resistance

• Test results fail to detect mutations that are present in 20-30% of the viral population.
• Tests are most reliable if the 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 in 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.