A new look at genital herpes: the critical role of the laboratory in diagnosis and management

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LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:

1. Discuss the cause, prevalence, and characteristics of genital herpes infections.
2. Compare and contrast characteristics and clinical features related to HSV1 and HSV2 infections.
3. Identify and discuss tests for the detection of HSV1 and HSV2.
4. Compare and contrast advantages and disadvantages of various tests for the detection of HSV.
5. Discuss various considerations for making decisions on the selection of HSV tests.

The diagnosis of genital herpes infection is a cause of dismay for many people. However, recent advances in herpes virus diagnosis and management provide tools for both more accurate diagnosis and for management to help patients with this all-too-common and often misdiagnosed sexually transmitted disease, which also has particular implications for women. Caused by herpes simplex virus type 1 (HSV1) or type 2 (HSV2), genital herpes is a chronic, lifelong infection with nearly continuous, low-level viral shedding and intermittent clinical recurrences characterized by transmission through sexual contact.¹

A major contributor to the continuation of an ongoing epidemic of genital herpes is that most people with the infection are unaware that they have it. According to the Centers for Disease Control and Prevention (CDC), at least 50 million people in the United States are infected.² The American Social Health Association (ASHA) estimates that there are 1.5 million new cases a year.³ Women are more susceptible to HSV2 infection than men. About one in five women 14 to 49 years of age is infected, compared to about one in nine men in that age range.⁴ Despite these staggering figures, only one in ten people with the infection is aware that he or she has it.³ Classically, genital herpes causes recurrent genital sores; however, most genital herpes cases are not “classic.” The infection often goes unrecognized by infected persons, and even experienced clinicians are incorrect in the clinical diagnosis of genital herpes about 20% of the time, mistaking other common genital problems for herpes.⁵

Whether recognized or not, however, genital herpes carries significant health risks. While the severity of symptoms may vary, genital herpes generally causes psychological distress in people when they become aware of the infection. An infected pregnant woman can transmit the disease to her newborn during birth, resulting in disseminated infection, central nervous system complications, or even death.¹ Genital herpes has been associated with a three- to five-fold increased risk of sexually acquired HIV.⁶ HSV is also known to cause proctitis in men who have sex with other men.⁷

As mentioned above, genital herpes often remains undiagnosed due to lack of symptoms or inaccurate clinical diagnosis. It is estimated that up to 90% of people with herpes are unaware that they have the disease. Consequently, genital herpes is most often transmitted by infected people who are unaware that they have the infection or are asymptomatic at the time of transmission.

Although genital herpes has previously been more often associated with HSV2, it can also be caused by HSV1, the virus more traditionally associated with orolabial herpes (cold sores). Over the past decade the proportion of genital herpes caused by HSV1 has increased, and today in some settings HSV1 accounts for at least half of first episodes of genital herpes.¹ Clinical features of initial genital HSV1 and HSV2 outbreaks are similar, but once infection has occurred the prognoses are quite different (Table 1). Genital HSV1 infection recurs less frequently (on average less than once a year) compared to HSV2, which typically recurs three to four times annually. Thus, about 70% to 90% of people with symptomatic HSV2 will have a recurrence within the first year, compared to fewer than 25% of people with symptomatic HSV1.¹ People with HSV2 are also more likely to shed the virus asymptomatically than those with HSV1, increasing the likelihood of transmission.^1,2

Table 1. Clinical features of genital herpes ⁸

A case for laboratory testing

Cumulative evidence of HSV’s health impact supports the importance of laboratory diagnosis of genital herpes so that infected people can be identified and managed to mitigate health risks as well as to reduce transmission.⁵ Experts agree that a diagnosis based on history and physical examination alone is often inadequate and that even “classical” genital herpes should be confirmed with laboratory testing. Furthermore, identifying the type of infection (HSV1 vs. HSV2) is helpful in assessing prognosis and counseling patients.^1,2 The CDC now recommends molecular methods for HSV testing. Similarly, the International Union Against Sexually Transmitted Infections (IUSTI) recommends using molecular methods for typing all patients with first-episode genital herpes.⁹

With the availability of new in vitro diagnostic tests for HSV, laboratories have an opportunity to contribute to the management and containment of genital herpes infections by offering HSV testing and typing. Following is an overview of the alternatives available, including culture, molecular testing, serology, antigen detection, and cytology.

Overview of methodologies

Viral culture is the traditional test for evaluation of patients with genital ulcers or other mucocutaneous lesions and, until recently, was the gold standard for HSV testing. For viral culture, vesicular fluid or cellular material from the base of a genital lesion is collected, placed in culture media, and transported expeditiously to the laboratory for cell culture. Following culture, HSV is most often detected using antibody staining and fluorescence microscopy. For distinguishing between HSV1 and HSV2, positive cultures may be stained and viewed under a fluorescence microscope using type-specific antibody reagents. Viral culture is most sensitive during the early stages of the disease, when lesions are typically vesicular. Sensitivity declines rapidly as lesions ulcerate and crust during the healing phase. Furthermore, viral culture is substantially less sensitive for diagnosis of recurrent as opposed to initial infections. Traditional culture methods take about five days, although a newer methodology can provide results within 24 hours.

Molecular testing is the emerging gold standard for virologic diagnosis of genital herpes, gaining popularity for lesion testing because it is more sensitive and faster compared to culture and can provide dependable results even when a healing lesion is sampled.^2,5 Currently, there are two commercially available HSV tests using molecular methodologies. One uses Strand Displacement Amplification (SDA) technology for the direct, qualitative detection and differentiation of HSV1 and HSV2 DNA in clinician-collected external anogenital lesion specimens from symptomatic female and male patients. Another method is polymerase chain reaction (PCR)-based qualitative test for HSV1 and HSV2 detection and typing of vaginal lesion swab specimens from symptomatic female patients. Molecular testing of genital herpes is also available as laboratory-developed tests (LDTs) at some reference laboratories.

Serologic testing detects HSV antibodies, markers of prior or current infection in serum samples, and has been valuable in epidemiological surveys. Accurate type-specific HSV serologic assays, based on the IgG response to the HSV-specific glycoproteins G1 (HSV1) and G2 (HSV2), are available. In contrast, IgM tests tend to be nonspecific, do not reliably differentiate recent from established infection, and are plagued by frequent false-positive results. Most experts recommend against use of IgM serologic tests for HSV. The CDC cautions providers to specify serologic type-specific assays when ordering such tests for their patients, since not all serologic tests can accurately distinguish between HSV1 and HSV2 antibodies. One disadvantage of serologic tests is that they cannot identify the site of infection. This is less problematic when HSV2-specific antibody is detected, since HSV2 positivity implies anogenital infection. The presence of HSV1 antibodies is more difficult to interpret since a positive test may represent the presence of either orolabial or genital infection. Also, false-negative results are more frequent during early stages of infection, as development of a positive IgG antibody response following initial HSV infection may take up to six months. False-positive results can also occur and are common when serologic testing for HSV antibodies is performed in lower-risk populations.

Direct detection of HSV antigen employing enzyme-specific immunoassays (EIA) or direct fluorescent antibody staining (DFA) is sometimes used. Direct antigen detection is less sensitive than culture for early lesions, but it may be superior to culture for detecting HSV in healing lesions.

Cytology (Tzanck smear) is sometimes performed on lesion scrapings from patients with active genital lesions. However, its clinical utility is limited by poor sensitivity and poor specificity. It also cannot distinguish between HSV1 and HSV2. Similarly, a Pap smear has limited utility for HSV testing. These tests are no longer recommended for diagnosis of suspected HSV infection.

Culture or molecular: the laboratory’s decision

For the reasons discussed above, serologic testing, direct antigen testing, and cytology are less commonly used in HSV testing for the purpose of supporting clinical diagnosis. Thus, the following discussion will focus on culture and molecular methods, the primary choices for genital herpes testing.

Today’s clinical laboratory considers multiple factors in selecting testing methodologies (Table 2). First and foremost is whether the test provides the necessary information for clinical management. Sensitivity and specificity are critical. In the case of HSV testing, the timely availability of virus type in infected people is also important, since knowledge of whether a patient has HSV1 or HSV2 influences patient management and counseling. The timeliness of test results is also an important consideration. The sooner the clinician receives the test result, the sooner patient management can begin. Third is the relative cost vs. benefit of the methodology being considered, including considerations of instrumentation and reagent costs, labor costs, and reimbursement. Other considerations include the cost of false positives or false negatives, which may be difficult to quantify, the value of superior service to clinician clients, and the laboratory’s reputation for quality and service.

Table 2. Comparing culture and molecular testing

Impact on clinical decisions. Most experts agree that when possible, amplified molecular testing is preferable to culture for diagnosis and to guide management decisions for people with possible genital herpes. Molecular testing is significantly more sensitive than culture methods. Recent CDC guidelines include both culture and molecular methods for HSV testing, but note that “the sensitivity of viral culture is low, especially for recurrent lesions, and declines rapidly as lesions begin to heal.”² The IUSTI 2010 “European Guideline for the Management of Genital Herpes” recommends molecular methods for typing all patients with first-episode genital herpes to guide counseling and management.⁷

Molecular testing also provides results more quickly, within hours rather than days. In some molecular testing both HSV1 and HSV2 are tested simultaneously, so the typing results are included in the initial reporting, saving time.¹⁰ In contrast, viral culture requires a second step, typically using DFA, to identify the HSV type once the specimen tests positive. This further extends the time to results.

Economic considerations. Viral culture is the predominant method for HSV testing today. Most laboratories cite low cost as a primary reason for its use; however, sometimes such estimates fail to consider all costs of cell culture diagnosis of HSV. A quick calculation of the cost of viral culture supplies, including shell vials, stains, shipping, and miscellaneous supplies, shows that it is comparable to reagent and disposable costs associated with molecular methods. However, tube cultures cost significantly less compared to cultures using shell vials because culture tubes cost significantly less.

Sometimes labor is overlooked as a key cost component. Viral culture requires manual intervention, while most molecular methods use automated instruments. Viral culture results are read manually, using light and fluorescence microscopes, whereas molecular testing results are reported electronically. Furthermore, human intervention may contribute to errors in result interpretation. Also often overlooked in cost considerations is the laboratory’s workflow. Since the sensitivity of viral culture is highly dependent on the quality of the specimen, special handling and immediate attention to incoming culture specimens are required, whereas arriving specimens for molecular testing can be held and integrated into the laboratory’s workflow without compromising results. Another economic consideration is the opportunity to leverage an existing molecular platform and add HSV testing without acquiring new instrumentation.

Last but not least, both culture and molecular testing of HSV1 and HSV2 are reimbursed. Although the exact amount of reimbursement varies by payer, molecular testing generally receives higher reimbursement.

Looking forward

Diagnosis and treatment of HSV1 and HSV2 infections are a first step toward managing genital herpes. Emerging issues focus on controlling transmission to minimize spread of the disease and ultimately on disease prevention. Some advocate the use of HSV serologic screening of select populations, such as people who have a partner with genital herpes. Prevention of neonatal herpes, often a result of infant exposure to HSV in the birth canal at delivery, is built on identifying and counseling women at risk of HSV acquisition and, in the case of infected mothers, protecting the infant from exposure to HSV during labor and delivery. Elective caesarean section in the presence of lesions is the standard of care in many countries today. A third area of interest is HSV/HIV interactions. Studies have demonstrated that HSV2 infection is an independent risk factor for HIV infection. The synergy between HSV and HIV, as demonstrated by co-infection of the same cell and up-regulation of HIV-1 replication by HSV proteins, is of interest not only in therapeutics development but also in prevention strategies in couples in which one partner is infected.¹ In all of these scenarios, the intervention strategy begins with accurate and timely diagnosis of genital herpes, a critical role for the clinical laboratory.

Editor’s note: While Dr. Hook presents a thorough and fair analysis of the two primary choices for HSV testing-culture and molecular-he clearly suggests that the latter is the preferable choice for most clinical labs. We would be pleased to publish concurring or dissenting opinions on this important issue. Please feel free to write me at [email protected] to express what your lab’s experience has been with either of these two alternatives.

References

1. Gupta R, Warren T, Wald A. Genital herpes. Lancet. 2007;370:2127-2137.
2. Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2010. MMWR Recomm Rep. 2010;59:1-110.
3. American Social Health Association. STD/STI statistics. http://www.ashastd.org/std-sti/std-statistics.html. Accessed May 16, 2012.
4. Centers for Disease Control and Prevention. Genital herpes fact sheet. http://www.cdc.gov/std/herpes/stdfact-herpes.htm. Accessed May 16, 2012.
5. Van Wagoner NJ, Hook EW 3rd. Herpes diagnostic tests and their use. Curr Infect Dis Rep. 2012;14(2):175-184.
6. Freeman EE, Weiss HA, Glynn JR, et al. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS. 2006;20:73-83.
7. Klausner JD, Kohn R, Kent C. Etiology of clinical proctitis among men who have sex with men. Clin Infect Dis. 2004;38:300-302.
8. Lafferty WE, Coombs RW, Benedetti J, Critchlow C, Corey L. Recurrences after oral and genital herpes simplex infection. N Engl J Med. 1987;316:1444-1449.
9. International Union Against Sexually Transmitted Infections. 2010 European guideline for the management of genital herpes. http://www.iusti.org/regions/europe/pdf/2010/Euro_Guideline_2010_herpes.pdf. Accessed May 16, 2012.
10. BD ProbeTec™ Herpes Simplex Virus (HSV 1 & 2) Qx Amplified DNA Assays [package insert]. Sparks, MD. BD Diagnostics, 2010.

Edward W. Hook III, MD, is a Professor of Medicine, Microbiology and Epidemiology at the University of Alabama at Birmingham.