Varicella-zoster virus (VZV) genotyping and phenotyping

Purpose of the test

Clinical use

Detect antiviral resistance in patients with Varicella-zoster virus (VZV) infections.

Clinical background

Varicella-zoster virus (VZV) is a ubiquitous human herpesvirus, belonging to the alpha-herpesvirinae subfamily, affecting populations worldwide. VZV is commonly acquired during childhood and causes a primary infection, varicella or chickenpox. After the initial infection, the virus establishes lifelong latency in sensory ganglia, with a risk of subsequent reactivation resulting in the development of herpes zoster (HZ) or shingles, a painful skin rash also known as shingles. Incidence and severity of HZ are higher with impaired specific cell-mediated immunity, mainly as a result of advanced age, malignancy, hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT), immunodeficiency, or immunosuppressive drug therapy. In particular, HZ remains a significant cause of morbidity among patients undergoing heart transplantation compared to other SOT recipients with liver, kidney, or lung transplantation. These patients have an increased risk of primary infection, reactivation, viral dissemination with visceral involvement, associated bacterial superinfection and frequent recurrences. VZV infections can also cause debilitating neuralgia among severely immunocompromised patients.

Drugs currently licensed to prevent or treat VZV-associated diseases target the viral DNA polymerase. Acyclovir and its prodrug valacyclovir are considered as the first-line therapy, whereas the use of foscarnet or cidofovir is limited for therapy of acyclovir and/or foscarnet resistant infections. The mechanism of action and mode of resistance for drugs used to treat VZV infections is similar to those used to treat HSV infections. Nucleoside analogs are first phosphorylated by the VZV thymidine kinase [open reading frame 36 (ORF36)], followed by activation by cellular kinases to produce the triphosphate active forms that block the viral DNA polymerase (ORF28). The helicase-primase inhibitor amenamevir is approved for the treatment of shingles only in Japan. Because of its efficacy against acyclovir-resistant isolates of VZV and HSV, amenamevir can be used as a feasible alternative for patients failing the first-line treatment. The drug is only available for compassionate use as an alternative treatment in case of refractory/resistant HSV or VZV infections.

Criteria for performing this test in the context of reference activities

Acyclovir resistance VZV infections have been reported in immunocompromised, but not in immunocompetent individuals. For patients showing therapeutic failure within 7 to 10 days of antiviral therapy, VZV genotyping should be evaluated, and alternative drugs such as foscarnet and cidofovir, both inhibitors of DNA polymerase acting independently of the viral thymidine kinase are recommended.

Clinically refractory and/or resistant VZV infections affecting the eye or the central nervous system should be genotyped in both immunocompetent and immunocompromised patients. Eye samples and cerebrospinal fluid (CSF) should be tested rapidly for detection of mutations, deletions, and/or substitutions in specific codons of the VZV genome to evaluate emergence of resistance to acyclovir to allow adjustment of antiviral therapy in function of the VZV genotyping.

VZV resistance testing is mainly based on sequence analysis of the viral thymidine kinase (ORF36) and DNA polymerase (ORF28) genes, since virus cultivation is rarely successful since the cell free virus is quite unstable. Because compartmentalization of viral VZV mutants often occurs, specimens from different body sites (such as various cutaneous lesions, CSF, bronchoalveolar lavage, eye) should be investigated for emergence of drug-resistance longitudinally to also allow evaluation of the viral dynamics.

Test details – VZV genotyping

Includes:

1. (Val)acyclovir resistance: mutations in the VZV ORF36 thymidine kinase and ORF28 DNA polymerase genes.
2. Cidofovir resistance: mutations in the VZV ORF28 DNA polymerase genes.
3. Foscarnet resistance: mutations in the VZV ORF28 DNA polymerase genes.

Test description – VZV genotyping

1. Isolation of DNA from the sample.

2. Amplification of the VZV genes involved in drug-resistance by PCR according to the therapy administered to the patient.

VZV gene	VZV encoded protein	Function / drug target	Codons sequenced (partial or complete gene sequence)	Drug(s) for which resistance is predicted
ORF36	Thymidine kinase	Involved in activation of acyclovir, penciclovir, ganciclovir, and brivudin	341 (complete sequence)	(Val)acyclovir, penciclovir, famciclovir, (val)ganciclovir, brivudin
ORF28	DNA polymerase	Target of acyclovir, penciclovir, ganciclovir,  brivudin, foscarnet, cidofovir	425-1000 (partial sequence)	(val)acyclovir, penciclovir, famciclovir, (val)ganciclovir, brivudin, foscarnet, cidofovir

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 VZV strain OKA 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 VZV infection is due to viral drug-resistance.

Interpretation of the VZV genotypic 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 VZV 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 genotypic tests for VZV drug-resistance

• Test results fail to detect mutations that are present in 20-30% of the viral population.
• Because genotypic artifacts may occur, in particular in mixed mutant populations from specimens with low viral load, retesting in a new sample is advised.
• Some variants in the thymidine kinase and DNA polymerase genes have not yet been phenotypically characterized and the impact of these novel changes can difficult to predicted. The combination of genotypic and phenotypic testing is necessary to diagnose  whether the novel changes in the viral genes are associated with drug-resistance or with a natural polymorphism.

Test details – VZV phenotyping

1. Growth of viral sample in human embryonic lung (HEL) fibroblasts until 100% cytopathic effect (CPE) is reached.
2. Preparation of the viral stock using infected cells.
3. Antiviral assay performed in HEL cells using at least two different viral inoculums and the VZV reference strain OKA.
4. Determination of the EC₅₀ (50% effective concentration) or drug concentration required to inhibit viral CPE by 50% for each antiviral drug.
5. 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 VZV phenotypic results

Results include EC₅₀ values and fold-resistance for different classes of anti-HSV drugs, DNA polymerase inhibitors, including i.e. nucleoside analogues (acyclovir, penciclovir, ganciclovir, brivudin, trifluridine), nucleotide analogues (cidofovir and adefovir), and pyrophosphate analogues (foscarnet).

Limitations of phenotypic tests for VZV drug-resistance

• Phenotypic tests cannot be performed for CSF, ocular fluid, or e-swab specimens because of failure to grow the virus 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.
• Viral isolation is very difficult for VZV because the virus remains cell-associated and therefore, the specimen needs to contain viable VZV-infected cells.

Instructions for samples and transport

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

 Unacceptable requests 

• Insufficient sample
• Incorrect transport or storage of the sample
• Test request form not 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: 3-5 working days for genotypic testing

                             10-20 working days for phenotypic testing

Reporting of test results

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