Herpes Simplex Virus and Varicella Zoster Virus PCR

Real time PCR for herpes simplex virus 1 and 2 (HSV-1, HSV-2) and varicella zoster virus (VZV) detects viral DNA in swabs from skin vesicles, ulcers and genital lesions, ocular samples (corneal scrapings, aqueous or vitreous humour), CSF, bronchoalveolar lavage, blood or plasma, amniotic fluid and tissue.

It has replaced viral culture and direct fluorescent antibody testing because of far higher sensitivity (95 to 100% for lesion swabs, against 50 to 70% for culture) and rapid turnaround (1 to 4 hours). Typing separates HSV-1 from HSV-2, which matters for prognosis, recurrence and counselling. Quantitative viral load is used in neonates, immunocompromised patients and ocular disease.

HSV-1 infects about 60% of the world's population under 50 years and HSV-2 about 13%, with the highest HSV-2 rates in sub-Saharan Africa and among people with HIV. Genital HSV-1 is increasingly common in young adults. VZV causes chickenpox in childhood and zoster in about 25% of people during life (30% by age 85 years), with postherpetic neuralgia in 10 to 20% of those with zoster.

HSV causes encephalitis (the commonest sporadic viral encephalitis, with mortality over 70% untreated), neonatal infection (1 in 3000 to 20 000 births) and keratitis, and VZV causes meningitis, vasculopathy, retinal necrosis and congenital varicella syndrome.

Indications. Genital or oral ulcers and vesicles of unclear cause. Atypical or healed lesions. Suspected HSV encephalitis, meningitis or myelitis, and VZV central nervous system disease. Neonates with sepsis like illness, skin vesicles or seizures. Keratitis, acute retinal necrosis and uveitis.

Zoster sine herpete and atypical zoster, including multidermatomal or disseminated disease. Immunocompromised patients with visceral or mucocutaneous disease. Pregnancy with genital lesions near delivery. Hepatitis or pneumonitis of unknown cause in an immunocompromised host.

Specimen. Lesion: unroof the vesicle, rub the base firmly with a swab (a dry lesion is moistened first) and place it in viral transport medium. CSF 0.5 to 1 mL in a sterile tube. Ocular fluids 0.1 to 0.2 mL. Plasma or whole blood in EDTA for disseminated disease. Keep at 2 to 8 °C and send within 48 hours; freeze at minus 70 °C for longer storage. Swabs with calcium alginate or wooden shafts inhibit PCR and are avoided.

Reference values. HSV-1, HSV-2 and VZV DNA not detected in CSF, blood and sterile fluids. Lesion swabs are negative in normal skin. Asymptomatic shedding of HSV from genital and oral mucosa occurs in 10 to 30% of seropositive people.

Interpretation.

• Positive HSV PCR on a lesion swab: active herpes infection, typed as HSV-1 or HSV-2. First episode genital disease is mostly HSV-1 in young adults in high income regions and HSV-2 in others.
• Positive HSV PCR in CSF with compatible illness: HSV encephalitis (usually HSV-1) or meningitis (usually HSV-2, including Mollaret recurrent meningitis). Sensitivity is 96 to 98% and specificity 99% after the first 72 hours of illness.
• Negative CSF HSV PCR within 72 hours of onset, with high clinical suspicion: repeat the lumbar puncture after 24 to 72 hours and continue aciclovir. Low viral loads, blood in CSF and early sampling cause false negatives.
• Positive VZV PCR in CSF: VZV meningitis, encephalitis, myelitis or vasculopathy, with or without rash. Anti-VZV IgG in CSF adds sensitivity for vasculopathy when PCR is negative.
• Positive VZV PCR on a vesicle swab: confirms chickenpox or zoster, and separates VZV from HSV in atypical rashes, with distinction between wild type and vaccine strain by genotyping.
• Quantitative plasma HSV or VZV DNA: disseminated disease in neonates and the immunocompromised, with falling load showing response.
• Positive aqueous or vitreous PCR in uveitis or retinal necrosis: HSV, VZV or cytomegalovirus aetiology, enabling targeted antivirals.
• Positive PCR in amniotic fluid or neonatal blood: congenital or intrapartum infection.
• Low level positives in blood in healthy people reflect reactivation and need clinical correlation.

Critical values and findings needing immediate action.

• Fever, headache, confusion, seizures or focal signs with a positive HSV PCR in CSF: intravenous aciclovir at high dose for 14 to 21 days, MRI brain, and repeat CSF PCR at the end of treatment to show clearance.
• Neonate with vesicles, seizures, sepsis or hepatitis: immediate intravenous aciclovir, with PCR from surface sites (skin, mouth, nasopharynx, conjunctivae, rectum), blood and CSF.
• Acute retinal necrosis or herpetic keratitis with deep involvement: urgent ophthalmology review, intravitreal and systemic antivirals.
• Disseminated zoster or visceral VZV in the immunocompromised, or varicella in pregnancy: intravenous aciclovir, with immunoglobulin in selected exposures.
• Maternal genital lesions at delivery: PCR confirmation and a plan for caesarean section and neonatal surveillance.
• Resistance suspected in a patient not responding to aciclovir: send for genotypic resistance testing (thymidine kinase and DNA polymerase mutations) and change to foscarnet.

Interferences and pitfalls.

• A negative lesion swab does not exclude infection, because of poor sampling, old or crusted lesions, topical antivirals and low viral load.
• Blood, haemoglobin breakdown products, heparin, calcium alginate swabs and sodium polyanethol sulphonate inhibit amplification, and an internal control shows inhibition.
• Contamination by amplicon carry over causes false positives, and strict laboratory separation of steps is required.
• A positive HSV PCR in CSF with a normal cell count can occur early in encephalitis, so the test is not limited to pleocytosis. Persistence of PCR positivity during aciclovir for the first few days is expected.
• Detection of virus in genital lesions does not date the infection, and serology (type specific IgG) helps to separate primary from recurrent disease.
• Asymptomatic detection in the respiratory tract of intubated patients is usually shedding and does not mean pneumonitis.
• Typing is clinically relevant, but misclassification can occur if primers do not cover variants.
• Aciclovir resistant HSV occurs in 5 to 10% of severely immunosuppressed patients and in under 1% of immunocompetent patients.
• Postherpetic neuralgia and ocular zoster are managed on clinical grounds, and PCR does not predict them.

Special groups.

• Neonates: HSV acquired at delivery presents as skin, eye and mouth disease, central nervous system disease or disseminated disease. Prognosis depends on early treatment.
• Pregnancy: primary genital HSV in the third trimester carries a 30 to 50% risk of neonatal transmission, and VZV in the first half of pregnancy can cause congenital varicella syndrome.
• Immunocompromised patients, including transplant recipients and people with HIV: severe, prolonged, atypical and resistant disease, and disseminated infection.
• Older adults: zoster, zoster ophthalmicus, vasculopathy and encephalitis.
• Regions: HSV-2 is the commonest cause of genital ulcer disease in sub-Saharan Africa and boosts HIV acquisition about three fold.

Follow up tests. CSF cell count, protein and glucose, MRI brain and electroencephalogram, repeat CSF PCR, type specific HSV serology, anti-VZV IgG and IgM in serum and CSF, HIV test, syphilis serology and other genital ulcer pathogens (Treponema pallidum, Haemophilus ducreyi, Chlamydia trachomatis L serovars), cytomegalovirus and Epstein-Barr virus PCR in immunocompromised hosts,

liver enzymes in neonates and visceral disease, antiviral resistance genotyping, and ophthalmology review.