Purpose of the test
Clinical use
Detect antiviral resistance in patients with adenovirus (AdV) infection.
Clinical background
Adenoviruses (AdV) are ubiquitous human pathogens that generally cause self-limiting disease, although they are responsible for severe disease among children and immunocompromised individuals. Symptoms and disease manifestations largely depend on the adenovirus type, host immune status, and site of infection. More than 60 types of genetically distinct human AdVs (divided into species A-G) exist, each type preferentially infecting certain tissues and causing a spectrum of distinct illnesses.
Hematopoietic stem cell and solid organ transplant (HSCT and SOT) recipients, individuals with congenital immunodeficiencies, or patients with uncontrolled HIV infections, may suffer from colitis, hepatitis, encephalitis, or disseminated disease, and have an increased risk for mortality due to AdV infection. Among immunocompromised persons, AdV disease can be related not only to primary infection, but also to reactivation from a latent pool of virus already present in the individual or from the graft. After primary infection, AdV remains latent in the lymphoreticular system and in immunocompromised patients, reactivation augments morbidity and mortality rates.
Currently, no approved antiviral therapies specific to AdV exist and current therapeutic options are limited and includes preemptive suppression of AdV replication using antivirals such as cidofovir and/or immunotherapy.
Criteria for performing this test in the context of reference activities
In vitro studies have shown the emergence of AdV cidofovir-resistance. Therefore, AdV drug-resistance could emerge in the clinic following prolonged treatment with this broad-spectrum anti-DNA virus drug.
Test details – AdV typing/genotyping
Includes:
- Adenovirus typing, since specific primer sets need to be used to amplify DNA polymerase, which is partially conserved among distinct AdV types.
- Cidofovir resistance: mutations in the AdV E2B DNA polymerase gene.
Test description — AdV typing / genotyping
Adenovirus typing
- Isolation of viral DNA from the sample.
- Amplification of the adenovirus hexon gene by PCR.
- Direct sequencing of the hexon gene.
- Obtained sequences are compared to the NCBI database using the BLAST software. Homologous highly similar sequences of previously published HAdVs with the highest scores are considered as identification results of human adenovirus type.
Adenovirus genotyping
1. Amplification of the DNA polymerase (E2B) gene by PCR
AdV gene |
AdV encoded protein |
Function / drug target |
Codons sequenced (partial or complete gene sequence) |
Drug(s) for which resistance is predicted |
E2B |
DNA polymerase |
Target of cidofovir |
Complete sequence |
Cidofovir |
2. Direct sequencing of the DNA polymerase gene by Sanger dideoxy sequencing, which has a limit of detection of viral mutant subpopulations of 20%-30% of the amplicons.
3. Sequence alignment (derived sequences of patient isolate are aligned against reference adenovirus strain).
4. 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 AdV infection is due to viral drug-resistance.
Interpretation of the results - AdV typing/genotyping
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 viral 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 genotyping tests for AdV 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.
Test details – AdV phenotyping
- Growth of viral sample in human embryonic lung (HEL) fibroblasts until 100% cytopathic effect (CPE) is reached.
- Preparation of the viral stock.
- Antiviral assay performed in HEL cells using at least two different viral inoculums and reference AdV strains from different types.
- Determination of the EC50 (50% effective concentration) or drug concentration required to inhibit viral CPE by 50% for each antiviral drug.
- Comparison of EC50 values between the reference strain and patient sample(s) and determination of the fold-resistance (Ratio EC50 patient sample/EC50 reference strain).
Interpretation of the AdV phenotypic results
Results include EC50 values and fold-resistance for different classes of anti-HSV drugs, DNA polymerase inhibitors, including i.e. nucleotide analogues (cidofovir), pyrophosphate analogues (foscarnet), as well as ribavirin.
Limitations of phenotypic tests for AdV drug-resistance
- Phenotypic tests cannot be carried out for cerebrospinal fluid, ocular fluid, blood, or e-swab specimens because of failure to grow the virus in cell culture from these sample types.
- For successful viral isolation, the sample should be collected immediately in a virus transport medium, stored refrigerated at 2-8°C, and transported refrigerated within 48 hours of sample collection.
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 (typing / genotyping).
10-20 working days (phenotyping).
Reporting of test results
Results will be sent via e-mail according to the requesting laboratory or physician’s wishes.