Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are large double-stranded DNA viruses of the Herpetoviridae family, alphaherpetovirinae sub-family 1 . HSV-1 and HSV-2 share a similar genome structure, with 40% of sequence homologies reaching 83% homology of their protein-coding regions, explaining numerous biological similarities and antigenic cross-reactivity between the two types. HSV-1 and HSV-2 genomes each encode at least 80 different structural and non-structural polypeptides including at least 10 different viral glycoproteins of which most are embedded in the viral envelope (gB, gC, gD, gE, gG,gH, gI, gL, gM, gN) 1 . The majority of the antibody response to HSV infection is raised against these surface glycoproteins. Glycoprotein gB, gC, gD and gE trigger potent immune responses. Some epitopes present on these glycoproteins are shared by HSV-1 and HSV-2, and are causing a significant degree of cross reactivity. However, no cross reactivity between glycoprotein gG1 in HSV-1 and gG2 in HSV-2 can be detected 2 which is why antibodies to this glycoprotein are used for type-discriminating serology. While the type common gB and gD display high similarity (85%) the homology betweengG-1 and gG-2 is much lower, presenting an overall amino acids (aa) identity of <30%. The reason for this is that gG-1 of HSV-1 contains 238 aa, while gG-2 of HSV-2 comprises 699 aa 2 . Furthermore, the envelope glycoprotein G (gG-2) of HSV-2 is cleaved into a membrane-bound portion (mgG-2) and a secreted portion (sgG-2). However, the epitopes for the type-specific antibodies against mgG-2 are not located in the portion of mgG-2 which is lacking in gG-1 but in a region with aa similarity to gG-1. This sequence, located between aa 560 and 573 for HSV-2 gG and between aa 80 and 93 for HSV-1 gG, carries nine identical residues between gG-1 and mgG-2 and five type-specific residues that induce significant structural differences. This results in different exposure of key residues utilized for recognition and explains the lack of cross-reactivity 3 .

Other similarities and differences between the genomes of HSV-1 and HSV-2 are used also for genera- or type-specific molecular assays, including genes coding for some envelope glycoproteins or the DNA polymerase. The conserved gene coding for DNA polymerase is often used for the detection or quantitation of both types and based on a few mismatches between HSV-1 and HSV-2 sequences which may also be used for typing 4 5 .

HSV-1 and HSV-2 are ubiquitous, affecting both urban and remote populations worldwide 6 . HSV-1 seroprevalence reaches 50 to 70% in developed countries and 100% in developing countries and HSV-2 seroprevalence varies from 10 to 40% and may reach 60–95% in HIV-infected individuals and female sex workers.

The classical pattern of HSV-1 and HSV-2 infections associated with oral or genital diseases, respectively, remains the rule in certain parts of the world such as sub-Saharan Africa where HSV-1 infection remains a mandatory community acquired disease in childhood, and HSV-2 infection a sexually transmitted infection (STI) in adults. In contrast, the differentiation of HSV-1 from HSV-2 based on anatomical site of infection is far from absolute in developed countries, the proportion of genital ulcers associated with HSV-1 infection has become predominant in some developed countries 6 7 . This is the result of both the delay in acquisition of oral HSV-1 infection early in life in developed countries (rendering a significant proportion of young adults always susceptible to genital HSV-1 infection at initiation of sexual activity) and the oro-genital sexual practices. This feature is concerning in regards to neonatal herpes given that the risk of HSV vertical transmission is higher during primary infection than during reactivation 8 and that HSV-1 appears more readily transmissible to the neonate than HSV-2 9 . It should be noted that genital HSV-1 infection does not prevent any risks of genital HSV-2 acquisition 9 .

Worldwide, HSV-2 remains the main cause of genital herpes and is the major etiology of genital ulcer disease. In addition, HSV-2 infection has been proven to be an independent cofactor of HIV sexual transmission. In turn HIV-1 infection increases the frequency of HSV-2 reactivations and mucosal shedding, as well as the quantity of shed viruses 7 . In severely immunocompromised HIV-1-infected patients and transplant patients, HSV infections frequently present as chronic, necrotic, extended, and confluent mucocutaneous ulcerations.

Most primary genital infections with HSV-1 and HSV-2 are asymptomatic and all are followed by latent infection of neuronal cells in the dorsal root ganglia and only 10–25% of people with HSV-2 antibodies are aware of their genital herpes. However, a large proportion of seropositive patients present asymptomatic shedding episodes that contribute to the spread of these infections 10 11 .

Genital herpetic infection is mainly diagnosed on clinical grounds, especially when the clinical picture is classical, with the presence of typical papular lesions progressing to vesicle and ulcerative lesions which finally crust, associated with local adenitis and in recurrent cases preceded by prodromal 12 (Figure 1). However, clinical diagnosis of genital herpes may be limited in accuracy. The clinical differentiation of genital HSV infection from other infectious (Treponemapallidum, Haemphilusducreyi) and non-infectious etiologies of genital ulceration is often difficult and laboratory confirmation of the infection should always be sought 13 . Besides classic vesicular lesions, HSV genital infection may be associated with other clinically atypical presentations. These include either unusual sites (extragenital regions: buttocks, thighs) or atypical morphological forms of genital disease (vulvar, penile or perianal fissures, localized recurrent erythema, recurrent radicular or lower back pain, cystitis, urethritis, vaginal discharge without overt genital lesions) 14 15 . Meningitis may be observed during phases of primary infection and reactivation and can also confuse the diagnosis of genital HSV infection 16 . Accordingly, exclusive reliance on clinical diagnosis could lead both to false positive and false negative diagnosis of the condition. Thus, a clinical diagnosis of genital herpes should be confirmed with laboratory tests 6 12 17 18 19 .

The laboratory diagnosis of genital herpes is recommended in various situations:

• Confirmation of clinically suspected genital herpes.

• Variable presentation of genital herpes.

• Extra-genital complications of genital herpes 20 .

• Differential diagnosis with other ulcerative STIs.

• Differential diagnosis with other genital ulcerative dermatoses (Crohn’s disease, Behçet syndrome or fixed drug eruption).

Because HSV-1 has become a frequent etiology of genital herpes, species typing is also a cornerstone of genital herpes diagnosis. Whether genital herpes is caused by HSV-1 or HSV-2 influences prognosis and counseling. Even though up to 50% of first-episode cases of genital herpes are caused by HSV-1, recurrences and subclinical viral shedding are much less frequent for genital HSV-1 infection than genital HSV-2 infection. Thus, information regarding whether one is infected with HSV-1 or HSV-2 can prove useful in discussing risks for recurrence.

Detection of HSV-specific IgG antibodies can be done sensitively by several immunological methods. Serologic diagnosis of HSV infections and HSV type-specific antibody testing are summarized in Table 5, and commercially available assays approved by the Food and Drug Administration (FDA, United States) in Table 6. Accurate type-specific HSV serologic assays are based on the detection of HSV-specific gG1 (HSV-1) and gG2 (HSV-2) antibodies using native, purified or recombinant gG1 or gG2 as antigens. Serological assays based on antigen preparations from whole virus or from crude infected-cell protein mixtures detect predominantly type-common antibodies, may have low sensitivity in detecting HSV-2 antibodies in HSV-1–seropositive patients, or may incorrectly type antibodies in patients with only HSV-1 or HSV-2 infection. Some commercial assays described as “type-specific” are actually based on relative reactivity of serum antibodies to crude preparations of HSV-1 versus HSV-2 antigens. The accuracy of such tests for HSV-2 antibody detection is low compared with glycoprotein G–based tests, and their use is not recommended 48 .

ELISA:Enzyme-linked immunosorbent assay; EIA: Enzyme immunoassay;

UW-WB: Western blot test developed at the University of Washington.

NA: Not Applicable.

Type-specific IgG antibodies are negative in early presentations of herpes disease, and become detectable two weeks to three months after the onset of symptoms and persist indefinitely. Thus, immediately after infection there is a ‘window’ in which testing for antibodies will give a negative result. Consequently, primary HSV infections can be documented by using any serologic methods to show seroconversion with paired sera. HSV IgM testing substantially increased the ability to detect early infection in patients who lack detectable IgG, but may be negative during primary disease. IgM testing can also be positive during reactivation of disease and cannot be used to distinguish primary from recurrent infection. Because of these limitations, HSV IgM testing has limited availability in routine diagnostic settings and cannot be recommended in routine clinical practice.

Gold standard noncommercial tests for HSV-2 include the immunodot enzyme assay (developed at Emory University, Atlanta, Georgia, United States), the Western blot test (developed at the University of Washington (UW-WB)), and the monoclonal antibody-blocking enzyme immunoassay (developed by the Central Public Health Laboratory, London, United Kingdom) 49 50 . These tests are used in their respective specialized reference laboratories but are not replicable in many settings, thereby limiting their suitability for large-scale epidemiologic studies. The UW-WB test has been used as a gold standard in several studies, including the evaluation of commercial serological assays required for clearance by the FDA, and in the evaluation of the performance of other gold standard tests. Despite its excellent performance, Western blot remains primarily a research tool. At present, Western blot is not FDA approved, and requires a high level of technical ability, time, and expense to perform.

Type-specific HSV glycoprotein G (gG)-based ELISA became commercially available in 1999. The sensitivities of these gG type-specific tests for the detection of HSV-2 antibody vary from 80–98%, and false-negative results might be more frequent at early stages of infection 51 . The specificities of these assays are ≥96%. The tests approved for use in the USA have sensitivity of 97–100% and specificity of 94–98%, when measured in comparison with the Western blot. False-positive results can occur, especially in patients with a low likelihood of HSV infection. Repeat or confirmatory testing might be indicated in some settings, especially if recent acquisition of genital herpes is suspected 51 . Some HSV-2 strains have been identified with mutations or deletions in gG2-gene leading either to the lack of gG-2 expression or the production of truncated forms 52 53 . Infections with such variants caused genital lesions similar to wild HSV-2 infection but immune response to gG-2 were either reduced or absent 52 53 . Negative detection of type-specific HSV-2 antibodies does not eliminate the rare possibility of a HSV-2 infection. HSV-2 DNA detection or HSV-2 isolation in cell culture along with a negative serology beyond the primary infection suggests an infection with a gG-2 deficient HSV-2 strain.

HerpeSelect® ELISA tests (HerpeSelect® 1 ELISA IgG Herpes Simplex Virus-1 (HSV-1) ELISA IgG; HerpeSelect® 2 ELISA IgG Herpes Simplex Virus-2 (HSV-2) ELISA IgG,Focus Technologies, Inc., Cypress, CA [formerly MRL Diagnostics]) are FDA approved, widely available and have been extensively studied 51 54 55 56 57 .

Point-of-care rapid tests can also provide results for HSV-2 antibodies from capillary blood or serum during a clinic visit. These immunoassays are designed to use capillary blood from a finger stick (or serum) and typically employ lateral flow of serum through a membrane containing a dot of gG1 or gG2 antigen. When serum is applied to the kit, a visual color change develops (pink dot) if herpes antibodies are present. Despite a reported inter-operative variability of 5-10% in test interpretation, these point-of-care tests perform relatively well with sensitivities ≥91% and specificities ≥94% 48 . The major benefit of point-of-care assays is that they give results rapidly (potentially while the patient is still in the clinical site,) allowing for more timely patient education and counseling. The major drawback of these tests is their cost relative to herpes ELISA-based systems.

If genital lesions are present, type-specific serology and direct virus testing can help to establish if the episode is a new HSV infection or reactivation (Table 7).

NA: Not applicable.

Type-specific HSV antibodies can take from 2 weeks to 3 months to develop. Thus, in a person with newly acquired herpes the initial absence of IgG antibodies specific for gG and subsequent development of such antibodies after 12 weeks confirms new HSV infection. The distinction between newly acquired HSV and reactivated HSV is helpful for epidemiological studies, and is sometimes helpful clinically for management of psychosocial issues, because it can help clarify the source of infection.

Because nearly all HSV-2 infections are sexually acquired, the presence of type-specific HSV-2 antibody implies anogenital infection; thus, education and counseling appropriate for people with genital herpes should be provided. The presence of HSV-1 antibody alone is more difficult to interpret. The majority of people with HSV-1 antibody have oral HSV infection acquired during childhood, which might be asymptomatic. However, acquisition of genital HSV-1 appears to be increasing, and genital HSV-1 also might be asymptomatic.

Taken together, type-specific HSV serological assays might be useful in the following situations (Table 8):

• Recurrent genital symptoms or atypical symptoms with negative HSV cultures;

• Clinical diagnosis of genital herpes without laboratory confirmation;

• Partner with genital herpes.

In addition, HSV serologic testing should be included in a comprehensive evaluation for STIs among people with multiple sex partners, HIV infection, and men who have sex with men who are at increased risk for HIV acquisition. Screening for HSV-1 or HSV-2 in the general population is not recommended, due to concerns that HSV-2 diagnosis provides no benefit and could lead to psychosocial sequelae (Table 8).

However some data suggest that most people are interested in HSV-2 testing, which may result in safer sex practice. A review examined studies that measured the short and long-term psychosocial effects resulting from serological diagnosis of HSV-2 in persons without recognized symptoms of genital herpes infection 58 . Overall HSV-2 serological testing did not result in long-term psychosocial harm in most people. Recently a study conducted in pregnant women showed that serotesting sexual partners of pregnant women for HSV reduced the frequency of unprotected genital sex acts in pregnant women at known risk of HSV-2 acquisition compare to HSV-2-seronegative women with partners who were negative or not tested 59 .

Long-term prophylaxis and treatment with antiviral drugs targeting the viral DNA polymerase (DNA pol) can result in the development of resistance 60 . The prevalence of acyclovir-resistant HSV is about 1% in immunocompetent individuals and increases in immuncompromised patients, 5% in HIV-seropositive individuals and 30% in hematopoietic stem cell recipients 61 62 . Antiviral drugs such as acyclovir or valacyclovir inhibit the viral DNA pol in triphosphorylated form, the first phosphorylation step ensured by the viral thymidine kinase (TK) and the subsequent steps by host cell kinases. Therefore mutations in both DNA pol and TK may confer resistance to antiviral drugs (Table 9). Because a functional TK may be dispensable but not the DNA pol for HSV replication, there is a higher probability of inducing a viable acyclovir-resistant virus by a mutation in the UL23 gene coding for TK than by a mutation in the UL30 gene coding for DNA pol. Accordingly, 95% of clinical isolates exhibiting acyclovir resistance harbor mutations in UL23 gene 61 62 .

^a The number is the amino acid position in the protein. The two letters correspond respectively to the wild type amino acid and the mutated amino acid.

^b Nucleotide numbering TK: thymidine kinase, DNA pol: DNA polymerase, ACV: aciclovir, FCV: foscarnet, CDV: cidofovir.

Resistance may be suspected when lesions persist for more than 1 week after initiating antiviral treatment or the emergence of new satellite lesions during treatment. A virological confirmation helps health care professionals choose among different treatment options while it avoids the selection of multidrug-resistant strains 63 .

Resistance can be assessed by the detection of specific mutations in UL23 or UL30 genes conferring resistance to antiviral drugs (genotypic assays) or by testing a virus against antiviral agents (phenotypic assays). Because most resistance cases are due to TK deficiency or to defective TK activity, mutations in the UL23 gene should be tested first. Genotypic assays consist of the comparison of UL23 and UL30 genes sequences with the whole panel of mutations described in the literature (Table 9) 61 62 63 64 65 66 67 . To be useful in clinical practice, it is essential to be able to discriminate between random variations (polymorphism) and true drug resistance mutations. Therefore, when possible, it is best to test in parallel strains collected before and on antiviral therapy. Before starting genotypic assays, an estimation of the viral load should be obtained because the amplification may be hampered at low levels, especially for the UL30 gene that has been shown to need more than 4.5 and 5.5 log10 copies/ml for HSV-1 and HSV-2 respectively. Virus isolation in cell culture may be required to increase the input of DNA material 64 . However amplification in cell culture can alter the population balance in the native sample.

Phenotypic assays are based on the measurement of virus growth inhibition in the presence of antiviral drugs. Various concentrations of virus are incubated with various concentrations of antiviral drugs, and the determination of the reduction of virus-induced cytopathic effect or plaque formation compared to a reference strain or the strain isolated before treatment enables the measurement of viral susceptibility to antiviral drugs. The gold standard phenotypic method for the evaluation of HSV susceptibility is the plaque reduction assay 60 62 .

Although TK is not essential for growth in cell culture, it is important for viral pathogenesis, particularly for reactivation from latently infected trigeminal ganglia in animal models 68 69 . This feature has likely minimized the development of TK based resistance in the immunocompetent community. In patients with ACV resistant strain, cessation of antiviral treatment results in reversion of HSV isolates to ACV sensitivity 70 . The most frequent strains reactivated after an episode caused by a resistant HSV strain are thus ACV-sensitive 70 . However reactivation of some TK-negative HSV clinical isolates have been reported 71 72 . Therefore, despite an initial antiviral efficacy, the same resistance will likely be selected as the previous episode and ACV treatment may fail, especially if the immunosuppression condition remained.