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The HerpeSelect
ELISA has a high degree of concordance with the Western blot assay
for the detection of type-specific antibodies to HSV type 2.
Detection of herpes
simplex virus type 2-specific immunoglobulin G antibodies in African
sera by using recombinant gG2, Western blotting, and gG2 inhibition.
Hogrefe W, Xin S, Song J, Ashley R,
Kong L.
Journal of Clinical
Microbiology 2002;40:3635-3640.
Summary:
Question
How do an ELISA assay using recombinant gG2 antigen, the Western blot
assay, and a gG2 inhibition assay compare for the detection of HSV 2
antibodies in sera collected from various geographic locations in Africa?
Design
Sera from four African countries were used to compare the performances of
the HerpeSelect HSV-2 ELISA and Western blotting (WB) for the detection of
HSV-2 antibodies. An HSV inhibition assay was developed to evaluate the
discordant sample results between HerpeSelect and WB.
Participants
Serum panels consisting of: 1) 231 samples from 150 HIV-negative and 81
HIV-positive women with a median age of 26 years attending an outpatient
clinic in Mombasa, Kenya; 2) 51 samples from a blood bank in Kampala,
Uganda, collected in 1989 for HIV serology studies; 3) 176 samples from
HIV-negative women between the ages of 18 to 35 attending a family
planning clinic in Uganda; 4) 150 samples collected from healthy
individuals for HIV screening in South Africa; and 5) 173 samples from
healthy, HIV-negative women aged 18 to 35 attending an STD clinic in
Harare, Zimbabwe, for an HIV seroprevalence study were analyzed.
Description of Tests and Diagnostic
Standard
HerpeSelect HSV-2 ELISA (Focus Technologies, Cypress, CA) was used to
screen all sera for the presence of HSV-2 antibody according to the
package insert. The assay antigen is a column-purified, baculovirus-derived,
truncated glycoprotein G from HSV-2. An index value was obtained for each
sample based on the absorbance of the patient sample divided by the
absorbance of a cutoff calibrator supplied by the kit. The Western blot
assay was performed on all sera, as previously described by researchers at
the University of Washington (Seattle, WA). An inhibition assay was
developed to evaluate the discordant sample results between HerpeSelect
and WB. For the gG2 inhibition assay, HSV-1 and HSV-2 were harvested from
tissue culture, washed, lysed in detergent, and either HSV-1 or HSV-2
lysate was added to an equal volume of a 1:50 dilution of serum sample.
After incubation for 1 h, the samples were then added to the ELISA wells.
The percent inhibition due to the preincubation with the lysates was
determined by using the following formula: [1 - (index of HSV-2 lysate
well/index of HSV-1 well)] X 100. Any ELISA-positive sample with an
inhibition value of <60% was considered a false-positive ELISA result.
Main Outcome Measures
The sensitivity and specificity of the HerpSelect ELISA as determined by
the Western blot and the gG2 inhibition assay were calculated.
Main Results
The results of the ELISA and WB assays performed on the 781 serum samples
from four African countries are shown in Table 1. All of the 37
WB-negative, ELISA-positive samples were from Kenyan and Ugandan panels.
| Table 1.
Comparison of HSV-2 results determined by HerpeSelect ELISA and WB |
| Western
blot |
HerpeSelect
ELISA |
| Positive |
Negative |
Equivocal |
Total |
| Positive |
451 |
0 |
0 |
451 |
| Negative |
37 |
0 |
5 |
42 |
| Atypical |
9 |
2 |
0 |
11 |
| Total |
497 |
279 |
5 |
781 |
The percent inhibition was determined for
417 of 497 ELISA positive samples. Eighty HSV-2 concordant samples from
the HIV-positive panel from Kenya were not analyzed in the inhibition
assay. At the 60% inhibition level, 402 of 417 (96.4%) ELISA-positive
samples were confirmed to be positive. Three samples that were WB and
ELISA positive were considered HSV-2 negative by the inhibition assay.
Twenty-five (68%) of the 37 WB-negative, ELISA-positive samples and all 9
WB-atypical, ELISA positive samples were confirmed to be HSV-2. These
discrepant samples tended to have lower antibody levels and mean percent
inhibition compared to the concordant samples. The sensitivity and
specificity of the ELISA results, based on 765 samples with definitive
results, versus both WB and the inhibition assay are shown in Table 2.
| Table 2. Sensitivity
and specificity of the HerpeSelect ELISA versus WB and the
inhibition assay |
| Parameter |
%
Sensitivity or specificity versus: |
| WB
at indexa: |
Inhibition
assay at index: |
| 1.1 |
1.5 |
2.1 |
2.5 |
3.1 |
1.1 |
2.1 |
| Sensitivity |
99.6 |
98.0 |
95.9 |
93.7 |
90.5 |
100 |
92.8 |
| Specificity |
88.0 |
93.0 |
94.9 |
96.5 |
97.8 |
94.9 |
96.9 |
Authors' Conclusions
The inhibition assay shows a very high
concordance with WB and provides a sound approach for evaluating unclear
HSV results. The HerpeSelect has a very high concordance with both the WB
(95.2%) and the inhibition assay.
Source of funding: None given.
For correspondence: Wayne Hogrefe,
Focus Technologies, 5785 Corporate Ave., Cypress, CA 90630. E-mail
address: whogrefe@focusanswers.com.
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