Laboratory Tests

The clinical diagnosis of genital herpes is both insensitive and non-specific. Therefore the clinical diagnosis of genital herpes should be confirmed by laboratory testing. Clinical laboratories should offer both virus detection tests and antibody detection tests.

Virus Detection

Virus detection tests begin by vigorously swabbing lesion sites to collect cells containing HSV. Viral detection tests include:

Conventional Culture (viable virus)

Isolation of HSV in cell culture is the preferred HSV test of most healthcare providers. Cell culture requires the collection of live virus samples that require special care in transport to the laboratory to retain viability. When viable samples are used, culture can be highly specific (if typing is performed) and positive results are generally reliable. The sensitivity of culture declines rapidly as lesions begin to heal and for this reason frequently non-positive results are falsely negative. Type-specific serology tests should be used in these cases to confirm a clinical diagnosis of genital herpes.

Modified Culture

• Shell Vial: A centifugation enhanced culture technique used to obtain rapid culture results. Generally less sensitive than conventional culture.
• ELVIS® (Enzyme-Linked Virus Inducible System): A technique which combines cell culture amplification with HSV-activated reporter genes. The test produces results which are equal to conventional culture.

Antigen Detection (virus antigen)

The common methods used in antigen detection are:

• Immunofluorescent (IFA) and immunoperoxidase (IPA) assays which detect HSV antigen in smears or tissues. HSV-specific antibodies are labeled with fluorescent dyes or enzymes (peroxidase). The labeled antibodies are incubated with the specimen and bind to HSV antigens in the specimen, if present. The attached fluorescent dye or enzyme can be visualized in appropriate regions of infected cells under a microscope.
• Enzyme Immunoassays, (EIA or ELISA) are done on fluids or other samples using HSV-specific antibody that is bound to a solid surface. The antibody captures antigen to which anti-HSV antibodies labeled with enzymes are added. These attach to the bound antigen and cause a color change.

These tests are inexpensive and rapid, but have relatively low sensitivity and lack usefulness in asymptomatic patients.

Polymerase Chain Reaction

Polymerase Chain Reaction or PCR (virus nucleic acid, DNA) tests are highly sensitive but expensive to perform and therefore, not suited to routine use. PCR amplifies small amounts of virus type-specific DNA to easily detectable levels using pre-prepared genetic sequences unique to HSV type-1 or HSV type-2. PCR is recommended for use in diagnosing CNS infections of neonates by testing cerebrospinal fluid.

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Antibody Detection

Antibody Detection or serology tests involve the detection of antibodies to HSV in the blood. Both type-specific and nonspecific antibodies to HSV develop during the first several weeks following infection and remain in the blood indefinitely. During primary infection, the first antibodies produced belong to the IgM class. The presence of IgM antibodies in HSV infections is difficult to interpret and may be confusing because unlike IgG, IgM antibodies are not type-specific. The presence of IgM antibodies may indicate a true primary infection, a super-infection with the other serotype, or reactivation of a latent HSV infection, (HSV type-1 or HSV type-2). For this reason, IgM testing is not recommended as a diagnostic tool.

IgG antibody detection, or serology tests, are an important adjunct to virus detection methods in patients with no obvious symptoms. The following tests are in this category:

Type-common (crude antigen) serology tests measure HSV antibodies in the blood but are unable to distinguish between HSV type-1 and HSV type-2 infection though labeled as if they could distinguish. Detection of antibodies can be useful to diagnose HSV infection when culture, virus antigen detection or PCR yield negative results, but are not helpful in making a definitive diagnosis of genital herpes in a patient with prior oral HSV type-1 infection.

Type-specific serology tests are those that accurately distinguish between HSV type-1 and HSV type-2 antibodies.

The advent of recombinant technology has enabled production of highly purified preparations of the HSV type-specific antigens gG1 and gG2, and these antigens can be used to detect HSV type-specific IgG antibodies. Sera from individuals infected with only HSV type-1 will react only with gG1, sera from individuals infected with only HSV type-2 will only react with gG2, and sera from individuals infected with both HSV type-1 and HSV type-2 will react with both gG1 and gG2. Type-specific HSV IgG assessment is a valuable tool for the accurate identification of the type(s) of HSV responsible for infection.

The Western Blot assay for HSV is highly accurate in differentiating HSV type-1 and HSV type-2 antibodies when used with a step to cross-adsorb antibodies to HSV type-1 and HSV type-2 antigens. However, it is expensive to perform, takes several days, and is not commercially available. The provider of Western Blot testing services is the University of Washington.

There are several type-specific ELISA (enzyme immunoassay) tests commercially available that are relatively easy to perform in the laboratory and give results quickly. Type-specific assays for HSV antibodies must be based on the HSV-specific glycoprotein G2 to accurately diagnose infection with HSV type-2 and with glycoprotein G1 to accurately diagnose HSV type-1.

HerpeSelect, from Focus Diagnostics offers two FDA-cleared lab-based assay formats for detecting type-specific HSV IgG antibodies: HerpeSelect Immunoblot uses purified recombinant type-specific gG-1 and gG-2 antigens, and native HSV common antigens immobilized on nitrocellulose membranes. HerpeSelect ELISA consists of two micro-plate assays, one for detecting antibodies to HSV type-1 (gG1), and the other for antibodies to HSV type-2 (gG2). Both assay formats had sensitivity and specificity values ranging from 91-100% in clinical trials with sexually active adults and pregnant women.

Other FDA-cleared gG-based type-specific assays are available. Trinity Biotech manufactures HSV type-1 and type-2 kits, which have recently received FDA clearance. A type-specific HSV-2 assay, formerly available from Diagnology (POCkit^®), is expected to be reintroduced soon by another manufacturer.

Serological tests for HSV only indicate prior infection and do not identify the site of infection. These tests may give false-negative results in the early stages of infection because antibodies may not reach detectable levels for 8 weeks or longer. False-positive results can also occur in patients with low likelihood of HSV infection. Repeat testing or a confirmatory test may be indicated in some cases.

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