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 herpesMany patients with unrecognized disease “become symptomatic” once they receive adequate counseling1,2

1. Lowhagen GB, et al. Acta Derm Venereol 2005;85(3):248-252. 2. Wald A, et al. N Engl J Med 2000;342(12):844-850.

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

HSV-2 infection (WB)

+ -

Clinical + 60 14

Diagnosis - 95 2224

Sensitivity = 39% PV+ = 81%

Specificity = 99% PV- = 96%

Langenberg, NEJM, 1999

Diagnostic method must be tailored to clinical presentation

Recognized symptomatic

20%

Asymptomatic 20%

Undiagnosed 60%

SerologySerology

Culture, PCR, Culture, PCR, antigen detectionantigen detection

Lesion Evaluation

Sensitivity of Virus Detection By Culture

Culture

Vesiculopustular 70-80

Ulcers 30-40

Crusted lesions 20-30

Lesion EvaluationViral 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 1.5-4 times as sensitive

as viral culture False negatives

possible Not available in some

settings

Differences in HSV-1 and HSV-2 Genital Infection

HSV-1Infrequent recurrences1

Infrequent asymptomatic shedding2

Continued risk for HSV-2 acquisition1

HSV-2Frequent recurrences1

Frequent asymptomatic shedding2

Low risk of HSV-1 acquisition1

1. Corey and Wald. In: Sexually Transmitted Diseases. 1999. 2. 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 blotgG ELISA*gG-membrane tests*gG immunoblot*

*Commercial tests.

Glycoprotein gG tests

Western blotgG 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-2HerpeSelect Immunoblot Focus HSV-1 and HSV-2BiokitHSV-2 Fisher HSV-2Captia Elisa Trinity HSV-1 and HSV-2

Recognized symptomatic

20%

Asymptomatic 20%

Undiagnosed 60%

SerologySerology

Serologic Testing:Type-Specific Glycoprotein G

Antibody Assays Based on type-specific antibody response to glycoprotein G

(gG) Recommended gG commercial tests

for HSV-21

Test Company Sensitivity (%) Specificity (%)

HerpeSelect-2 ELISA Focus 96-100 97-100

HerpeSelect Immunoblot Focus 97-100 98

Captia Select-HSV-2 Trinity 90-92 91-99

Bioelisa HSV-2 IgG Biokit 100 > 98

Wald A. In: Current Clinical Topics in Infectious Diseases. 2002.

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 anHSV-2 Primary Episode

40 days

21 days

Days from HSV-2 primary episodePro

bab

ility

of

rem

ain

ing

se

ron

eg

ati

ve

Full Western blotFocus

150100500

0.0

0.2

0.4

0.6

0.8

1.0

Morrow et al. J Clin Microbiol. 2003

HSV Inhibition Assay of 497 ELISA-Positive Samples (>60% Positive Cutoff)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

ELISA index

Inh

ibit

ion

(%

)

atypical

WB negative

WB positive

Mean = 91.0%SD = 10.6%

Mean - 3SD = 60%

Mean - 2SD = 70%

Atypical

WB negative

WB positive

100

90

80

70

60

50

40

30

20

10

0

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 ELISA270 (86%) confirmed by WB for HSV-2 43 (14%) not confirmed by WB for HSV-2

Median index of confirmed: 8.1 (1.36-25.5)

Median index of unconfirmed: 2.5 (1.2-14.2)

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. 2005

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 Serolog

yInterpretation

- + 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 testingReassure patients that if they are diagnosed,

they have many available management options and resources

Offer HSV type-specific testingProvide 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