Herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) genotyping and phenotyping

Purpose of the test

Clinical use

• Detect antiviral resistance in patients with herpes simplex virus 1 (HSV-1) or herpes simplex virus 2 (HSV-2) infections.

Clinical background

HSV is responsible for recurrent orolabial and genital infections. The most common clinical manifestations of HSV-1 affect the orofacial and ocular areas and the anogenital region for HSV-2. However, an increased number  of genital infections are being caused by HSV-1. In immunocompetent individuals, the symptoms associated with both viruses are usually self-limiting, and antiviral therapy is aimed at accelerating lesion healing and preventing transmission. Both HSV-1 and HSV-2 can also cause, though infrequently, serious diseases, such as encephalitis and disseminated neonatal infections. In immunocompromised patients, particularly those with defects in cell-mediated immunity, the herpetic lesions are more extensive, persist for longer periods, have the potential to disseminate and recurrences are more frequent atypical in appearance. Also, HSV can lead to disseminated visceral and meningoencephalitis in the immunocompromised hosts.

Antiviral drugs available for the treatment of HSV-1 and HSV-2 include acyclovir (and its oral prodrug valacyclovir), penciclovir (and its oral prodrug famciclovir), cidofovir, foscarnet, and brivudin. Ganciclovir and trifluridine are only used topically for the treatment of HSV eye infections. Acyclovir and its prodrug are also commonly used prophylactically. All these drugs ultimately inhibit viral DNA synthesis by targeting the HSV DNA polymerase (encoded by the UL30 gene). The nucleoside analogs acyclovir, penciclovir, ganciclovir, and brivudin must first be phosphorylated by the viral thymidine kinase, before undergoing additional phosphorylation by cellular kinases and finally inhibiting the viral DNA polymerase resulting in inhibition of viral genome replication. Trifluridine is also a nucleoside analogue that can be initially phosphorylated  by the viral thymidine kinase but also by cellular kinases. Cidofovir is a nucleoside phosphonate analog that does not require activation by the viral thymidine kinase to become bioactive. Although cidofovir is not FDA-approved for the treatment of HSV infections, it is often used off-label in either intravenous (IV) or topical forms to manage acyclovir and/or foscarnet-resistant infections. Foscarnet is a pyrophosphate analog that directly inhibits the HSV DNA polymerase and is FDA-approved for the treatment of acyclovir-resistant HSV infections.

Reduced susceptibility to nucleoside analogues is usually mediated by mutations in the HSV UL23 gene encoding for the thymidine kinase. The most common types of mutations in this viral gene conferring resistance include additions or deletions in G/C homopolymer strings, leading to premature stop codons or frameshifts. Single amino acid substitutions in conserved regions of the ATP-binding site or nucleoside-binding site of the viral thymidine kinase are less frequently responsible for drug-resistance. Reduced sensitivity to acyclovir can also be mediated by mutations in UL30 gene (encoding for the DNA polymerase), which can also confer cross-resistance to foscarnet and, more rarely, to cidofovir.

The new drug pritelivir acts on the viral helicase primase complex composed of three subunits UL5, UL8, and UL52 genes. It is still undergoing evaluation in Phase 3 clinical trials and can be obtained through an expanded access program for compassionate use to treat HSV infections resistant to acyclovir and/or foscarnet.

Criteria for performing this test in the context of reference activities

The management of HSV infections in immunocompromised individuals can be challenging due to antiviral resistance. Rates of resistance to nucleoside analogues in immunocompetent persons are very low (<1%), except for infections occurring in immune-privileged sites such as the eye (herpetic keratitis) and the central nervous system (HSV encephalitis). Resistance to acyclovir is increasingly being recognized in immunocompetent individuals with herpetic keratitis. In immunocompromised patients, resistance rates vary from 0% to 14% in hematopoietic stem cell transplant (HSCT) recipients and 0% to 3% in solid-organ transplant (SOT) recipients. Refractory and/or resistant (R/R) HSV infections often occur among patients with advanced human immunodeficiency virus (HIV) disease (prevalence rates of 3%–7%), although R/R HSV infections are much less frequently seen in patients receiving effective antiretroviral therapy.

Clinically refractory and/or resistant HSV infections in the immunocompromised host  and infections of the eye and the central nervous system in all patients should be tested for mutations, deletions, and/or substitutions in specific codons of the HSV genome. These tests  evaluate emergence of resistance to acyclovir and/or foscarnet to implement alternative antiviral therapies in function of the HSV genotypic and phenotypic tests. Because compartmentalization of viral HSV mutants often occurs, specimens from different body sites (such as various mucocutaneous lesions, cerebrospinal fluid, end-organ disease, bronchoalveolar lavage, eye) should be investigated for drug-resistance longitudinally to also allow evaluation of the viral dynamics.

Test details – HSV-½ genotyping

Includes:

1. (Val)acyclovir resistance: mutations in the HSV-½ UL23 thymidine kinase and UL30 DNA polymerase genes.
2. Cidofovir resistance: mutations in the HSV-½ UL30 DNA polymerase genes.
3. Foscarnet resistance: mutations in the HSV-½ UL30 DNA polymerase genes.
4. Pritelivir and amenamevir resistance: mutations in the HSV-½ UL5, UL8, UL52 helicase-primase genes.

Test description – HSV-½ genotyping

1. Isolation of DNA from the sample.

2. Amplification of the HSV-½ genes involved in drug-resistance by PCR according to the therapy administered to the patient.

HSV-½ gene	HSV-½ encoded protein	Function / drug target	Codons sequenced (partial or complete gene sequence)	Drug(s) for which resistance is predicted
UL23	Thymidine kinase	Involved in activation of acyclovir, penciclovir, ganciclovir, and brivudin	276 (complete sequence)	(Val)acyclovir, penciclovir, famciclovir, (val)ganciclovir, brivudin, trifluridine
UL54	DNA polymerase	Target of acyclovir, penciclovir, ganciclovir,  brivudin, foscarnet, trifluridine cidofovir	310-1100 (partial sequence)	(val)acyclovir, penciclovir, famciclovir, (val)ganciclovir, brivudin, trifluridine, foscarnet, cidofovir
UL5	Helicase activity	Target of pritelivir and amenamevir	881 (HSV-1), 882 (HSV-2) (complete sequence)	Pritelivir and amenamevir
UL8	Regulatory subunit		755 (HSV-1), 752 (HSV-2) (complete sequence)
UL52	Primase activity		1058 (HSV-1), 1066 (HSV-2) (complete sequence)

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 HSV-1 strain Kos or HSV-2 strain G sequences).

5. Detected mutations are compared to a database of known mutations associated with drug resistance or naturally occurring polymorphisms (inter-strain variability) to determine whether clinical resistance HSV-½ infection is due to viral drug-resistance.

Interpretation of the HSV-½ 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 HSV-½ 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 HSV-½ 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 cannot be predicted. Therefore, 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 – HSV-½ 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.
3. Antiviral assay performed in HEL cells using at least two different viral inoculums and the corresponding reference strain (HSV-1 Kos strain or HSV-2 G strain).
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 HSV-½ 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), as well as the helicase-primase inhibitor pritelivir.

Limitations of phenotypic tests for HSV-½ 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.
• Antiviral drug-resistance mutations can confer a significant reduction in viral fitness. Therefore, in cases of mixed mutant populations, viruses with reduced fitness will be overgrown in cell culture by the virus with a superior replication capacity. Isolation of viral clones from the original sample is then necessary to evaluate the phenotype of each mutant virus.

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.