Laboratory Diagnosis of HSV Infection Peter Leone, MD Associate Professor of Medicine University of North Carolina

Why Diagnose Genital Herpes?  Epidemic  Most HSV-2 seropositive persons are symptomatic  Transmission occurs from undiagnosed persons  HSV-2 increases risk of HIV acquisition and transmission  Pregnancy management

Underrecognition by Clinicians and Patients: What Should We Do?  Recognize that prevalence within our practices is higher than anticipated  Appreciate that genital HSV-2 does not discriminate  Elevate our “index of suspicion” in all sexually active patients  Provide patient education about signs and symptoms of genital herpes  Many patients with unrecognized disease “become symptomatic” once they receive adequate counseling 1,2 1. Lowhagen GB, et al. Acta Derm Venereol 2005;85(3): Wald A, et al. N Engl J Med 2000;342(12):

Diagnosing Herpes … The clinical diagnosis of HSV is no longer considered an adequate method for diagnosis of genital herpes. Both virologic tests and type-specific serologic tests for HSV should be available in clinical settings that provide care for patients with STDs or those at risk for STDs. –2002 CDC STD Treatment Guidelines

Accuracy of clinical diagnosis of genital herpes Langenberg, NEJM, 1999

Diagnostic method must be tailored to clinical presentation Recognized symptomatic 20% Asymptomatic 20% Undiagnosed 60% Serology Culture, PCR, antigen detection

Lesion Evaluation

Sensitivity of Virus Detection By Culture

Lesion Evaluation Viral Culture vs. PCR  Inexpensive  Type-specific identification has prognostic significance  2 – 5 days for results  High rate of false- negatives; false positives rare  Not available in some settings  Cost varies  Type-specific identification  Rapid turnaround possible  times as sensitive as viral culture  False negatives possible  Not available in some settings

Differences in HSV-1 and HSV-2 Genital Infection  HSV-1  Infrequent recurrences 1  Infrequent asymptomatic shedding 2  Continued risk for HSV-2 acquisition 1  HSV-2  Frequent recurrences 1  Frequent asymptomatic shedding 2  Low risk of HSV-1 acquisition 1 1. Corey and Wald. In: Sexually Transmitted Diseases Ashley RL and Wald A. Clin Microbiol Rev 1999;12(1):1-8.

Serologic Evaluation

Lesion Evaluation and Serologic Evaluation Lesion Evaluation  With viral culture  Typing can be performed  False negative results are common  With PCR  Highly sensitive  Typing can be performed  Cost may be higher than with other tests Serologic Evaluation  Use only glycoprotein G (gG)-based, type-specific tests  Highly sensitive and specific  Seroconversion period with incident infection  If lesion present, can have true/true and unrelated results  Useful during intra-lesional period Centers for Disease Control and Prevention. MMWR Recomm Rep 2002;51(RR-6):1-78.

Glycoprotein gG tests Western blot gG ELISA* gG-membrane tests* gG immunoblot* *Commercial tests. Envelope: gB, gC, gD, gE, gG, gH, gI, gK, gL, gM Tegument: VP16 Nucleocapsid: VP5, ICP35 DNA core HSV virus Accurate Type-Specific HSV Serology Ashley R. Herpes. 1998;5:33-38.

Performance and interpretation of serologic tests  What is the Gold Standard?  Interpretation of Western Blot is still part art  Discrepant analysis  Time to seroconversion

Western Blot  “Gold standard”  Complicated  Expensive  Limited availability  Not FDA approved

Discordant Results Between the ELISA and Western blot.  In pre-selected serum panels, 31 of 96 WB negative sera were HSV-2 positive when tested by an inhibition assay; therefore, using the WB to confirm positive results may overestimate false positive rates in the original ELISA. Hogrefe et al., IHMF 2005

Type-specific gG-based Serology Commercial Kits FDA Approved Tests HerpeSelect  ELISA Focus HSV-1 and HSV-2 HerpeSelect  Immunoblot Focus HSV-1 and HSV-2 Biokit  HSV-2 Fisher HSV-2 Captia Elisa Trinity HSV-1 and HSV-2 Recognized symptomatic 20% Asymptomatic 20% Undiagnosed 60% Serology

Serologic Testing: Type-Specific Glycoprotein G Antibody Assays  Based on type-specific antibody response to glycoprotein G (gG)  Recommended gG commercial tests for HSV-2 1 TestCompanySensitivity (%)Specificity (%) HerpeSelect-2 ELISA Focus HerpeSelect ImmunoblotFocus Captia Select-HSV-2Trinity Bioelisa HSV-2 IgGBiokit100> 98 Wald A. In: Current Clinical Topics in Infectious Diseases

Is IgM Useful in Distinguishing New vs. Recurrent GH Infection? No! Do not order IgM antibodies to diagnose new vs. recurrent GH infection. Often laboratories automatically do IgM test Why aren’t IgM tests helpful in determining the recency of GH infection? - IgM tests are not type-specific – IgM could be from HSV-1 or HSV-2! - Each of the many episodes of viral reactivation can produce new IgM and IgG, making it difficult to interpret results as to acuity of infection. IgM has role in Dx of neonatal HSV Ashley RL. Herpes 1998;5:33–38.

Time to Seroconversion Following an HSV-2 Primary Episode 40 days 21 days Days from HSV-2 primary episode Probability of remaining seronegative Full Western blot Focus Morrow et al. J Clin Microbiol. 2003

HSV Inhibition Assay of 497 ELISA- Positive Samples (>60% Positive Cutoff) Atypical WB negative WB positive

Performance of the 2 Generation Focus HerpeSelect HSV-2 IgG ELISA on Selected Serum Panels Hogrefe et al., IHMF 2005  The 2 generation HerpeSelect HSV-2 ELISA reduced the number of false positive results by ~40% when the WB used as the gold standard respectively.

Confirmation of HerpeSelect ® HSV-2 ELISA Positive Results (N=313) Worldwide study: women (33% prevalence)  Positive samples by HerpeSelect HSV-2 ELISA 270 (86%) confirmed by WB for HSV-2 43 (14%) not confirmed by WB for HSV-2  Median index of confirmed: 8.1 ( )  Median index of unconfirmed: 2.5 ( )  Majority of unconfirmed are between 1.1 and 2.0

Confirmation of HerpeSelect ® HSV-2 ELISA Positive Results (N=103) Seattle STD clinic: men (13% seroprevalence)  Positive samples (106) by HerpeSelect HSV-2 ELISA 80%(80) confirmed by WB for HSV-2 16%(17) not confirmed by WB for HSV-2  Median index of confirmed: 8.0  Median index of unconfirmed: 2.0 Golden et al Sex Transm Dis Dec. 2005

Interpretation of ELISA in Low Prevalence Population  In low-prevalence populations (<10%), should consider selectively using a higher index (2.2 or 3.5) value to define positivity based either on the presence or absence of clinical findings suggestive of genital herpes or clinical risk history.  Confirmation either by WB or by Biokit (increased PPV 80% to ~96%) Golden et al Sex Transm Dis Dec Laeyendecker et al., J Clin Microbiol 2004 Morrow BMC Infectious Diseases 2005

Interpretation of Test Results  In patients with culture-positive or PCR-positive genital lesions  You have a confirmed type-specific, site-specific diagnosis  If seronegative for the type identified on culture, assume new infection  In pregnant patients, it is important to distinguish new infection from established infection  IgM-based tests are not reliable for distinguishing new infection from established infection and should never be used for this purpose

Interpretation of Serologic Test Results  In patients with culture-negative or PCR-negative genital lesions  You must rely on the type-specific serology results HSV-1 Serolo gy HSV-2 Serolo gy Interpretation -+ Genital HSV-2 infection +- HSV-1 infection; site unknown. Repeat HSV-2 serology in 8 to 12 weeks. Reswab subsequent lesions. ++ Genital HSV-2 infection; probable orolabial HSV-1 infection -- Repeat HSV-1 and HSV-2 serology in 8 to 12 weeks. Reswab subsequent lesions.

Undiagnosed Patients: What Should We Do?  Inform patients about the importance of testing  Reassure patients that if they are diagnosed, they have many available management options and resources  Offer HSV type-specific testing  Provide patient-sensitive and timely follow-up care after testing is performed

Candidates for Serologic Testing  Patients  With recurrent genitourinary symptoms  With a culture-negative lesion or clinical diagnosis only  Presenting for STI screening or requesting herpes testing  Diagnosed with an STI  With a current or past partner with genital herpes  With HIV-infection  Who are pregnant? (not in ACOG guidelines) Centers for Disease Control and Prevention. MMWR Recomm Rep 2002;51(RR-6):1-78.

Summary  Work-up genital lesions  Confirm all clinical diagnosis with Type- specific test  Don’t be afraid to use Type –specific serology  When screening for GH, keep in mind clinical history and local prevalence with low (1.1 to 2.0 or 3.0) serologic ELISA index assay