Transcript HSV-1 infection

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


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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
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
H S V -2 in fe c tio n (W B )
+
-
C lin ic a l
+
60
14
D ia g n o s is
-
95
2224
+
S e n s itiv ity =
39%
PV = 81%
S p e c ific ity =
99%
PV = 96%
Langenberg, NEJM, 1999
-
Diagnostic method must be
tailored to clinical presentation
Asymptomatic
20%
Recognized
symptomatic
20%
Culture, PCR,
antigen detection
Serology
Undiagnosed
60%
Lesion Evaluation
Sensitivity of Virus Detection By
Culture
Culture
Vesiculopustular
70-80
Ulcers
30-40
Crusted lesions
20-30
Lesion Evaluation
Viral Culture vs. PCR





Inexpensive
Type-specific
identification has
prognostic significance
2 – 5 days for results
High rate of falsenegatives; false
positives rare
Not available in some
settings


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
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-1
Infrequent recurrences1
Infrequent asymptomatic shedding2
Continued risk for HSV-2 acquisition1
HSV-2
Frequent 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
Serologic Evaluation
Lesion Evaluation
 Use only glycoprotein G
 With viral culture
(gG)-based, type-specific
 Typing can be
tests
performed
 False negative results  Highly sensitive and specific
 Seroconversion period with
are common
incident infection
 With PCR
 If lesion present, can have
 Highly sensitive
true/true and unrelated
 Typing can be
results
performed
 Useful during intra-lesional
 Cost may be higher
period
than with other tests
Centers for Disease Control and Prevention. MMWR Recomm Rep 2002;51(RR-6):1-78.
Accurate Type-Specific HSV
Serology
HSV virus
Envelope:
gB, gC, gD, gE,
gG, gH, gI, gK,
gL, gM
Tegument:
VP16
Nucleocapsid:
VP5, ICP35
Ashley R. Herpes. 1998;5:33-38.
DNA core
Glycoprotein gG tests
Western blot
gG ELISA*
gG-membrane tests*
gG immunoblot*
*Commercial tests.
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
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“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
HerpeSelect Immunoblot
BiokitHSV-2
Captia Elisa
Focus HSV-1 and HSV-2
Fisher HSV-2
Trinity HSV-1 and HSV-2
Asymptomatic
20%
Recognized
symptomatic
20%
Serology
Undiagnosed
60%
HSV-1 and HSV-2
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 (%)
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
HerpeSelect-2 ELISA
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.
Probability of remaining seronegative
Time to Seroconversion Following an
HSV-2 Primary Episode
1.0
0.8
0.6
Focus
Full Western blot
40 days
0.4 21 days
0.2
0.0
0
Morrow et al. J Clin
Microbiol. 2003
50
100
Days from HSV-2 primary episode
150
HSV Inhibition Assay of 497 ELISAPositive Samples (>60% Positive Cutoff)
100%
100
90
90%
Mean = 91.0%
SD = 10.6%
80
80%
Inhibition (%)
70
70%
Mean - 2SD = 70%
60
60%
Mean - 3SD = 60%
50
50%
40
40%
30
30%
20
20%
atypical
Atypical
WB negative
WB
negative
WB positive
WB
positive
10
10%
0
0%
0.0
2.0
4.0
6.0
8.0
ELISA index
10.0
12.0
14.0
16.0
Performance of the 2 Generation Focus
HerpeSelect HSV-2 IgG ELISA on
Selected Serum Panels

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.
Hogrefe et al., IHMF
2005
Confirmation of HerpeSelect® HSV-2
ELISA Positive Results (N=313)
Worldwide study: women (33% prevalence)
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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 (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
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Median index of confirmed:
Median index of unconfirmed:
Golden et al Sex Transm Dis Dec. 2005
8.0
2.0
Interpretation of ELISA in
Low Prevalence Population
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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
Serolo Interpretation
gy
+
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
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Work-up genital lesions
Confirm all clinical diagnosis with Typespecific 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