![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, September 2001, p. 913-921, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.913-921.2001
Comparison of Serologic Assays for Detection of
Antibodies against Human Herpesvirus 8
Jose L.
Corchero,1,
Eng-Chun
Mar,1
Thomas J.
Spira
,2
Philip E.
Pellett,1 and
Naoki
Inoue1,*
Division of Viral and Rickettsial
Diseases1 and Division of AIDS, STD, and
TB Laboratory Research,2 National Center for
Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia 30333
Received 9 February 2001/Returned for modification 11 April
2001/Accepted 9 May 2001
 |
ABSTRACT |
Improvement of serologic assays for detection of antibodies
against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and
lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest
virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These
assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as
with other lytic antigen-based assays, including an IFA based
on induced PEL cells, a commercial ELISA based on purified virions, and
ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel
of 180 serum specimens obtained from three groups expected to have
high, intermediate, and low HHV-8 prevalences. Using three
different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent
antigen-based assays, and (iii) in general, IFAs were more sensitive
than ELISAs based on the same open reading frame. We also found that
serum specimens from healthy individuals contained antibodies
cross-reactive with HHV-8 glycoprotein B that can potentially cause
false-positive reactions in lytic PEL-based IFAs. Although this is not
a substantial problem in most epidemiologic studies, it may confound
the interpretation of data in studies that require high assay
specificity. Because the K8.1-based IFA provides sensitivity similar to
that of lytic PEL-based IFAs and improved specificity, it can be a
useful alternative to the PEL-based IFAs.
| |
INTRODUCTION |
Human herpesvirus 8 (HHV-8) was
identified in AIDS-associated Kaposi's sarcoma (KS) by
representational difference analysis (7). Subsequent
studies demonstrated the association of this virus with all four forms
of KS, primary effusion lymphoma (PEL), and multicentric Castleman's
disease (reviewed in references 3, 37, and
38). Both sexual and nonsexual avenues of transmission of
HHV-8 have been identified. Among men who have sex with men (MSM),
HHV-8 is predominantly sexually transmitted (24, 26). Transmission through transplantation and intravenous drug use has been
reported (6, 15, 22, 30, 35). It is likely that HHV-8
primary infection also occurs during childhood in areas of endemicity
(5, 25, 33). Because HHV-8 is frequently secreted into
saliva in MSM (2, 4,18, 32, 41), it may be transmitted
from saliva as is Epstein-Barr virus (EBV).
Since HHV-8 is not readily isolated in cell culture, infection is
usually determined by either PCR or serology. Although PCR detects
HHV-8 DNA in almost all KS lesions, viral DNA loads in peripheral blood
mononuclear cells are frequently not high enough to be detected
(29, 39, 40, 42). To complement PCR and to better
understand the natural history of HHV-8 infection, several serologic
assays have been established. PEL cell lines harboring the HHV-8 genome
were used as antigens in immunofluorescence assay (IFA) and
immunoblotting (IB) (12, 20, 28, 39). HHV-8 ORF73 is the
major component of latent nuclear antigens detected in these latent
PEL-based assays. Seropositivity determined by these assays correlated
well with KS development (17, 24). After treatment with
12-O-tetradecanoylphorbol-13-acetate (TPA), 10 to 30% of
PEL cells produce cytoplasmic antigens that correspond to viral
proteins associated with lytic infection. Although IFAs using
TPA-treated PEL cells (lytic PEL-based IFAs) are the most sensitive
assays (20) and have been used widely, they are possibly confounded by cross-reaction with antibodies against other
herpesviruses. The lack of a matched HHV-8-negative PEL cell line
hinders discrimination of nonspecific reactions against cellular
components. We recently developed new IFAs based on HHV-8 antigens
expressed with the recombinant Semliki Forest virus (rSFV) system
(14). Expression of HHV-8 antigens at a high level and the
availability of true negative controls in this system increased the
specificity and sensitivity of the IFAs. IFA using rSFV
K8.1-infected BHK21 cells (K8.1SFV IFA) was as
sensitive as lytic PEL-based IFA, and the correlation coefficient of
end-point titers and concordance between these two assays were >0.92
and 97% (
= 0.93), respectively. Because K8.1 has no homolog
in other herpesviruses, there is no potential cross-reaction with
antibodies against other herpesviruses in this assay.
To obtain a high-throughput assay, an enzyme-linked immunosorbent assay
(ELISA) based on purified HHV-8 virions was established (9), and it is commercially available. To increase
sensitivity and specificity, several studies have examined the utility
of defined HHV-8 antigens, including synthetic oligopeptides and recombinant proteins. The most useful antigens for serologic assays are
derived from ORF65, ORF73, and K8.1 (13, 16, 19, 31, 39, 40,
43).
Assay performance greatly influences measured seroprevalence in
low-risk populations. For example, seroprevalence in healthy populations in the United States has ranged from 0% in latent antigen-based assays to 20% in lytic PEL-based IFAs (reviewed in
reference 8). A blinded comparison of three ELISAs and
four PEL-based IFAs demonstrated their utility for epidemiologic
investigations and limitations for diagnosing individuals
(34). This made clear the need for both new assay
development and standardization. Because the lack of a "gold
standard" assay for HHV-8 has hampered comparison of assays newly
developed in different laboratories, it is important to compare assays
by using a single set of defined serum samples (23).
In this study, we developed rSFV-based ELISAs and compared them with
previously reported assays. We also studied the specificity of the
lytic PEL-based IFA.
| |
MATERIALS AND METHODS |
Plasmids.
pSCAx, pSCA
, pSCA-K8.1A, pSCA-orf73
, and
pSCA-Helper were described previously (14). pSCA-orf65 was
constructed by PCR amplification of the orf65 gene with primers
5'-GATGAGGATCCTTCCAACTTTAAGGTGAGAGAC-3' and
5'-CGCGGATCCAATCGTTGCCTATTT-3'
(orf65-derived sequences are underlined, and BamHI
sequences are in boldface), followed by cloning into pSCAx. pSCA-gB,
which carries the entire HHV-8 glycoprotein B (gB) open reading frame
in pSCAx, was constructed from pcDNA3.1-gB1b-4 (1). For
the expression of amino and carboxyl domains of ORF73, pBAD-orf73N and
-orf73C were constructed by cloning fragments from the orf73 gene
encoding amino acids 1 to 330 and 935 to 1162, respectively, into
pBAD-Topo (Invitrogen, Carlsbad, Calif.). To obtain these fragments,
the following primer sets were used: for orf73N,
5'-GACGACGACAAGATGGCGCCCCCGGGAATGCGCCTGAGG-3' and 5'-CTGACTTTCCTTGCTAATCTC-3'; for
orf73C,
5'-GACGACGACAAGGAGCCCATAATCTTGACAGGGTCGTCA-3' and
5'-TTAATGATGATGATGATGATGTGTCATTTCCTGTGGAGAGTCC-3'
(orf73-derived sequences are underlined, and the
His6 tag sequence is in italics).
rSFV production, IFAs, and IB.
Transfection, production, and
infection of rSFV, IFAs using rSFV-infected cells, and IB were
performed as described previously (14), except that rSFV
infection of BHK21 cells for preparation of ELISA antigens was done at
a multiplicity of infection of 0.5 to 1.0. Monoclonal antibody-enhanced
IFAs (mIFAs) using untreated and TPA-induced BCBL-1 cells (latent and
lytic mIFAs, respectively) were performed as described previously
(20). Human serum specimens were coded, and two readers
read the IFA slides independently. Antibodies against HHV-8 ORF65 were
obtained from rabbits immunized with a bacterially expressed ORF65
protein (40). Antibodies against EBV gp110 (gB) and HHV-8
gB, ORF73, and K8.1 were described previously (1, 14, 36).
rSFV-based ELISAs.
BHK-21 cells infected with rSFV in one
10-cm-diameter dish were lysed in 2 ml of phosphate-buffered saline
(PBS) containing 0.5% sodium deoxycholate (Dox) until they detached
from the plate (
10 min). The lysed cells were collected, sonicated
twice for 1 min each (50% duty cycle, power 10; model W-375; Heat
Systems-Ultrasonic, Inc., Farmingdale, N.Y.), and then centrifuged (10 min, 16,000 × g) to remove cell debris. Supernatants
containing HHV-8 antigens and -galactosidase were used as antigens
for ELISA.
rSFV-based ELISAs were performed as follows. Antigens were diluted in
carbonate buffer (0.1 M
Na2HCO3-NaH2CO3,
pH 9.4) to a final concentration of 3 µg/ml; 100 µl of the solution
was incubated in each well of ELISA plates (Immulon 2HB; Dynex) at
4°C overnight. After coating, the plates were washed once with PBS
and incubated with PBS containing 5% skim milk at 4°C for 2 h.
Sera were diluted in PBS-5% skim milk with a 30-µg/ml concentration
of a BHK-21 cell extract that was prepared in the same manner as the
extracts from rSFV-infected cells, incubated with agitation at room
temperature (RT) for 2 h to reduce cross-reaction with cellular
components, and then reacted in the ELISA plate for 2 h at 37°C.
After washing with PBS containing 0.05% Tween 20 (PBST), bound
antibodies were incubated with peroxidase-conjugate goat anti-human
immunoglobulin G (Kirkegaard & Perry Laboratories, Gaithersburg,
Md.) for 1 h. The color reaction was developed for 1 h at RT
with tetramethylbenzidine after PBST washing. Reactions were stopped
with 50 µl of 1 M H2SO4. Plates were read at 450 nm against 630 nm with an ELISA plate reader.
Optical density (OD) values specific to HHV-8 antigens were obtained by
subtracting the OD values for the -galactosidase extracts from those
for the HHV-8 antigens.
Bacterial expression of ORF73.
Bacteria containing
pBAD-orf73N or pBAD-orf73C were grown to mid-log phase. ORF73
expression was induced by addition of L-(+)-arabinose (final concentration of 0.05%) for 3 h. Cells from 1 liter of culture were harvested and sonicated. After centrifugation (27,000 × g for 1 h), the supernatant was passed through a
0.4-µm-pore-size filter and mixed with a 1.5-ml bed volume of Talon
resin (Clontech, Palo Alto, Calif.). ORF73 antigens were eluted with a
buffer containing 0.5 M imidazole and dialyzed against carbonate
buffer. About 50 µg of partially purified antigens with 90%
purity was obtained per liter of culture.
ELISA using bacterially expressed ORF73 as antigen
(ORF73bac ELISA).
Microtiter plate wells were coated
with 60 ng of partially purified antigen (4°C, overnight) and then
blocked with 5% skim milk in PBST for 1 h at RT. Serum specimens
were diluted 1:100 and applied to wells. Plates were incubated at RT
for 2 h. Secondary antibody and color reactions were done as
described for rSFV-based ELISAs.
Virion ELISA.
An ELISA kit using purified HHV-8 virions was
purchased from a commercial source (Advanced Biotechnologies, Inc.
Columbia, Md.) and used according to the manufacturer's instructions.
Human sera.
Serum specimens analyzed in two previous studies
(14, 40) were used. The collection included sera from 56 human immunodeficiency virus (HIV)-positive, KS-positive
(HIV+ KS+) patients; 61 HIV+ KS
patients; 3 HIV KS+ patients, and 10 HIV individuals with some risk factors, such as
MSM (n = 2) and patients attending sexually transmitted
disease clinics (n = 8). Fifty serum specimens obtained
as out-of-date material from the Atlanta American Red Cross blood bank
were used as healthy controls. Among the 61 HIV+
KS patients, 13 developed KS within 5 years
(Later KS), 25 did not develop KS in the 5 years after specimen
collection, and 23 had no follow-up study. We refer to the
HIV+ KS+,
HIV KS+, and
HIV+ Later KS patients as the KS group
(n = 72); the HIV+
KS patients and individuals with risk factors
as the intermediate group (n = 58); and blood
donors as the control group (n = 50).
Statistical analyses.
Correlations between titers obtained
by two different assays were evaluated by Pearson's correlation
coefficient. The degree of concordance between two assays was assessed
by the kappa statistic, which measures agreement beyond chance. Kappa
() equals 1.0 for perfect agreement; 0.4 to 0.6 and 0.6 to 0.8 were
considered to represent fair and good agreements, respectively.
Maximum-likelihood estimates of the parameters for receiver-operating
characteristic (ROC) and drawing of ROC curves were obtained
with ROCKIT (0.9B, IBM-compatible version) (27) and
PlotROC software, respectively. The software was downloaded from a
website of the Department of Radiology, University of Chicago
(http://www-radiology.uchicago.edu/krl/top page11.htm). The area under
an ROC curve equals the probability of correctly answering the
two-alternative forced-choice problem. In other words, the area is
proportional to the quality of the performance (accuracy) of a
diagnostic test; an area of 0.5 represents a worthless test, and an
area of 1.0 represents a perfect test.
| |
RESULTS |
Expression of HHV-8 antigens.
Expression of ORF65 and gB in
the rSFV system was examined by IB and IFA (Fig.
1 and 2).
Although ORF65 protein was barely detectable in TPA-induced BCBL-1
cells, it was easily detected as a band with an apparent molecular mass
of 20 kDa both in 293T cells transfected with pSCA-orf65 and in BHK-21
cells infected with rSFV-orf65 (Fig. 1A). The molecular mass was
consistent with that in a prior report (21). Although the
rabbit antibody detected nonspecifically a band of 30 kDa (Fig. 1A,
lanes 3 to 6), KS+ human sera did not detect the
band (data not shown). Cytoplasmic localization of ORF65 was observed
in the rSFV-infected cells (Fig. 2A and C). Two forms of gB,
corresponding to the full-length and cleaved forms (1),
were detected in 293T cells transfected with pSCA-gB and in BHK-21
cells infected with rSFV-gB as well as in TPA-induced BCBL-1 cells
(Fig. 1B). HHV-8 gB was detected in the cytoplasm and on cell surfaces
of BHK-21 cells infected with rSFV-gB (Fig. 2D and F) (data with
paraformaldehyde fixation not shown), which was consistent with its
expression in BCBL-1 cells (1). As shown here and
previously (14), the expression levels of every HHV-8
antigen in rSFV-infected BHK-21 cells were significantly higher than in
those in plasmid-transfected 293T cells or in TPA-induced BCBL-1 cells.
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 1.
rSFV expression of HHV-8 ORF65 and gB. Cell lysates
obtained from equal numbers (5 × 104 and 4 × 103 cells/lane in panels A and B, respectively) of the
following cells were separated by sodium dodecyl sulfate-15% (A) or
-8% (B) polyacrylamide gel electrophoresis: BCBL-1 cells untreated
(lanes 1) or treated with TPA (lanes 2); BHK-21 cells infected with
rSFV-gal (lanes 3), rSFV-orf65 (lane 4 in panel A), or rSFV-gB (lane
4 in panel B); and 293T cells transfected with pSCA-gal (lanes 5),
pSCA-orf65 (lane 6 in panel A), or pSCA-gB (lane 6 in panel B). ORF65
and gB proteins were detected with rabbit antibodies against a
bacterially expressed ORF65 protein ( -ORF65) and against an
oligopeptide containing gB amino acids 828 to 845 (-gB),
respectively.
|
|
View larger version (76K):
[in this window]
[in a new window]
|
FIG. 2.
Localization of HHV-8 ORF65, and gB in rSFV-infected
cells. BHK-21 cells infected with rSFV-ORF65 (A and C) and with rSFV-gB
(D and F) were reacted with the same rabbit antibodies described for
Fig. 1 (A, B, D, and E) and with KS+ human sera (C
and F). BHK-21 cells infected with rSFV-gal were used as a negative
control (B and E).
|
|
The amino (ORF73N) and carboxyl (ORF73C) domains of ORF73 were
expressed in Escherichia coli and affinity purified (data
not shown). Because all KS+ human sera that
reacted with ORF73C reacted with ORF73N in an ELISA, ORF73C was used
for further assay comparisons.
Optimization of rSFV-based ELISAs.
To optimize antigen
preparation from rSFV-infected cells, we tested various solubilization
detergents. Of 17 detergents tested, Dox reproducibly resulted in the
highest signal-to-noise ratio in an ELISA format (data not shown).
Lysis of cells directly in the culture dish with Dox-containing PBS
gave more consistent and efficient antigen preparation than harvesting
the cells by trypsinization and subsequent Dox
treatment (data not shown). The HHV-8 antigens were found mainly in the
soluble fractions of the lysates (K8.1, >95%; ORF73, 85%).
Therefore, the soluble fractions prepared from the lysates were used as
the ELISA antigens.
Because an amount of coating antigen of greater than 300 ng per well
led to signal saturation, this amount (equivalent to 2 × 103 rSFV-infected cells) was used for subsequent
experiments. Twenty 96-well ELISA plates can be prepared from a single
10-cm-diameter culture dish of cells.
Comparison of the assays.
The following 11 assays were
compared: IFAs using untreated and TPA-treated BCBL-1 cells
(latent and lytic mIFAs), rSFV-based IFAs
(K8.1SFV, ORF73SFV, and
ORF65SFV IFAs), a commercial ELISA using HHV-8 virions (virion ELISA), ELISAs using
oligopeptides with major immunogenic epitopes of ORF65 and
K8.1
(ORF65pep and K8.1pep ELISAs), rSFV-based ELISAs (ORF73SFV and K8.1SFV ELISAs), and ORF73bac ELISA. Among
these 11 assays, latent mIFA, ORF73SFV IFA
and ELISA, and ORF73bac ELISA are latent
antigen-based assays, and the others are lytic antigen-based assays.
Human serum specimens from three groups, i.e., the KS, intermediate,
and control groups, were used for the assay comparison. The prevalence
of HHV-8 infection in these groups was expected to be very high,
intermediate, and very low, respectively.
The results of assay comparisons were analyzed first in the context of
interassay agreement and then in comparison to defined standards as
described below.
Pairwise interassay agreements were evaluated in terms of two
statistical values: the correlation coefficient (r) of OD
measurements (or IFA end-point titers) between two assays and the
concordance kappa statistic () of serologic status (seropositive or
seronegative) determined by two assays (Table
1). An arbitrary cutoff for each ELISA
was defined as the mean plus five standard deviations (SD) of OD values
obtained from a panel of healthy blood donors whose HHV-8 serologic
status was negative (end-point titer of <40) in K8.1SFV IFA (n = 49). As shown in
Table 1, the ORF73-based ELISAs had good correlation and
concordance. The K8.1- and virion-based ELISAs also showed very good
correlations and concordances, which were better than those between
these assays and ORF65pep ELISA. Lower
concordances between K8.1SFV IFA and the lytic
antigen-based ELISAs can be explained by the different serum dilutions
in ELISAs (1:100) and IFAs (starting from 1:20), because
their correlations were better for the sera with IFA end-point titers
of >80 (Fig. 3). Some of the ELISAs were
also evaluated with 1:40 serum dilutions; increased background obscured
any possible sensitivity increase (data not shown).
View larger version (35K):
[in this window]
[in a new window]
|
FIG. 3.
Correlations between lytic antigen-based assays. IFA
end-point titers or OD measurements were compared among the indicated
assays. (A) K8.1SFV IFA versus virion ELISA. (B)
K8.1SFV IFA versus K8.1SFV ELISA. (C)
K8.1SFV ELISA versus virion ELISA. (D) K8.1SFV
ELISA versus K8.1pep ELISA. Closed squares, blood donors;
gray triangles, intermediate group; open circles, KS group. Broken
lines indicate the arbitrary cutoffs for ELISAs (the mean plus 5 SD of
OD values from a panel of 49 healthy blood donors) and the cutoff for
IFAs (serum dilution of 1:40, based on the results shown in Fig. 4B).
Broken lines divide a set of serum specimens into four groups:
specimens in the upper right quadrant of each panel were positive in
both assays, the ones in the lower left quadrant were negative in both
assays, the ones in the lower right quadrant were positive in one assay
(x axis) but negative in the other assay
(y axis), and the ones in upper left quadrant were
negative in one assay (x axis) but positive in the other
assay (y axis).
|
|
Correlation coefficients are independent of cutoffs, because they are
based on comparison of signal strengths. In contrast, values are
dependent on how the cutoffs are determined, because the value
represents interassay agreement of serologic status that is classified
based on the cutoffs. Interestingly, the and r values
correlated well, suggesting that arbitrary cutoff determination does
not change the overall interpretation (Fig. 4A). The lowest correlations were between
latent antigen- and lytic antigen-based assays (Fig. 4A; Table 1).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
Interassay comparisons. (A) Correlation coefficients
(r) and concordance kappa statistics () between two
lytic antigen-based assays ( ), between two latent antigen-based
assays ( ), and between latent and lytic antigen-based assays ( ).
Latent antigen-based assays include ORF73SFV IFA and
ORF73SFV and ORF73bac ELISAs. Lytic
antigen-based assays include K8.1SFV IFA and virion,
K8.1SFV, K8.1pep, and ORF65pep
ELISAs. A dashed line shows a linear regression between the
r and values. (B) Specificity and sensitivity of
K8.1SFV IFA using different serum dilutions as cutoffs to
determine whether each specimen is seronegative or seropositive. To
calculate specificity and sensitivity, the following conditions were
used as a standard: (i) seropositive in one of the four lytic
antigen-based assays (virion, K8.1SFV, K8.1pep,
and ORF65pep ELISAs) or KS positive (including current KS
and KS developed within 5 years) (solid line) or (ii) seropositive in
two of the lytic antigen-based assays or KS positive (dashed line). (C)
ROC analyses of ELISAs. The serologic status of each specimen
determined by K8.1SFV IFA (cutoff serum dilution of 1:40)
was used as a defined standard, based on the results shown in panel
B.
|
|
Next, we evaluated each assay in comparison to a defined standard in
two ways. In the first, on the assumption that all KS group sera
contain antibodies against HHV-8, the sensitivity of each assay was
calculated as a percentage of positives among the KS group serum
specimens. HHV-8 antibodies were detected in 95.8, 94.4, and 83.3% of
KS group specimens by K8.1SFV IFA using cutoff serum dilutions of 1:20, 1:40, and 1:80, respectively. The lytic antigen-based ELISAs detected antibodies in 78 to 88% of KS specimens, and latent antigen-based assays identified HHV-8 infection in less than
two-thirds of KS specimens, using the mean plus 5 SD of control OD
values as the cutoff (Table 2). Even when
using the mean plus 2 SD in place of 5 SD as the cutoff, only 64 to 65% of KS specimens were positive in ORF73-based ELISAs, while 86 to 93% were positive in the lytic antigen-based ELISAs.
ORF65SFV IFA detected antibodies in only 41% of
KS specimens at a serum dilution of 1:20.
Alternatively, serologic status determined by
K8.1SFV IFA was used as a standard to evaluate
each assay, because K8.1SFV IFA is the most
sensitive assay that uses a defined antigen. First, we employed
ROC-type analysis to identify a cutoff that maximizes both sensitivity
and specificity of K8.1SFV IFA. For this purpose, serologic results obtained by K8.1SFV IFA at
different serum dilutions were compared with the combined results
obtained from four different lytic antigen-based assays (virion,
K8.1SFV, K8.1pep, and
ORF65pep ELISAs), plus information on whether the
specimen was obtained from the KS group (KS or Later KS) patients. As
shown in Fig. 4B, the cutoff that gave the best combination of
sensitivity and specificity (shortest distance to the top left corner)
was a serum dilution of 1:40 when applying the standards of either (i)
being positive in at least one of the four assays or in the KS group or
(ii) being positive in at least two of the assays or in the KS group.
Then, using the serologic status determined by
K8.1SFV IFA at a serum dilution of 1:40 as the
defined standard, the sensitivity and specificity of each assay were
calculated. As shown in Table 2, the lytic antigen-based ELISAs, the
latent antigen-based IFAs, the latent antigen-based ELISAs, and
ORF65SFV IFA had sensitivities of 70 to 81%, 51 to 54%, 42 to 43%, and 35%, respectively, with >95% specificity,
using the mean plus 5 SD of control OD values as the cutoff for the
ELISAs. Even when using 2 SD in place of 5 SD for the cutoff, the
ORF73-based ELISAs still had only 53 to 54% sensitivity. The order of
sensitivities of the assays was similar to that determined by the
positivity in the KS group based on each cutoff setting.
Finally, the ELISAs were analyzed by ROC analysis using the
serologic status determined by K8.1SFV IFA as the
standard (Fig. 4C). ROC analysis eliminates bias due to the
arbitrarily determined cutoffs. Areas under the ROC curves of
K8.1-based ELISAs, virion and ORF65pep
ELISAs, and ORF73-based ELISAs were 92%, 89 to 90%, and 79%,
respectively, with the values for ORF73-based ELISAs being
significantly lower than the others (Table 2).
Antibodies recognizing HHV-8 gB in healthy blood donors.
The
prevalence of antibodies against HHV-8 gB was determined by
gBSFV IFA. While only 52% of the
KS+ specimens were positive in this assay, other
groups, including healthy controls, had similar levels of anti-HHV-8 gB
seroprevalence. We determined the gBSFV IFA
end-point titers of 30 blood donor specimens. Approximately 25% had
titers of 
80 (Fig. 5A; an example of
IFA with a blood donor specimen is shown in Fig. 5B), although only
four specimens were positive in at least one of the 11 other HHV-8
serologic assays. Interestingly, three out of four
specimens that had end-point titers of 320 were positive in
lytic mIFA, with only one of them being positive in
K8.1SFV IFA. Anti-EBV VCA antibodies in most
blood donor specimens (27 out of 30) were positive at a serum dilution
of 1:250, with no significant correlation between EBV VCA and HHV-8 gB
end-point titers (data not shown). The four serum specimens with HHV-8
gB end-point titers of 320 had intermediate EBV VCA titers (250 to
2,000).
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 5.
Antibodies against HHV-8 gB in healthy blood donors. (A)
gBSFV IFA end-point titers against HHV-8 gB of 30 blood
donor serum specimens. Two serum specimens (black bars) were positive
in both K8.1SFV IFA and lytic mIFA (titers of 20 and 40).
Two other serum specimens (gray bar) were positive in lytic mIFA
(titers of 40 and 80) but negative in all other HHV-8 serologic assays.
All of the other 26 specimens (white bars) were negative in all of the
other 11 HHV-8 serologic assays. (B and C) Example of IFA with a
healthy blood donor serum specimen (RC22) that did not contain
detectable antibodies against HHV-8 in any of the 11 other HHV-8
serologic assays. The specimen reacted with rSFV-gB-infected cells (B)
but not with rSFV-gal-infected cells (C).
|
|
| |
DISCUSSION |
Lytic antigen-based assays are more useful than latent
antigen-based assays for detecting HHV-8 infections.
In this
study, to improve HHV-8 serologic assays, we developed several IFAs and
ELISAs, and compared the performance of a total of 11 assays, using a
panel of 180 serum specimens obtained from groups with high,
intermediate, and low HHV-8 prevalence.
Martin et al. (23) described a stepwise calibration using
well-defined serum specimens for evaluation of a new ELISA. Although we
did not take the same approach, we paid careful attention to the
potential problems associated with the arbitrary setting of ELISA
cutoffs. First, we compared the correlation coefficients with
concordance statistics. The generally high correlation between these
two parameters suggests that arbitrary determination of cutoffs may not
be a major contributor to interassay discordance. Instead, we found
that a match of antigen types, latent or lytic, was important to obtain
concordant results. In addition, plots of IFA end-point titers against
ELISA OD measurements suggest that IFAs are more sensitive than ELISAs,
although it is formally possible that at low serum dilutions the IFAs
gave nonspecific results. However, this is unlikely, because comparison
of K8.1SFV IFA with the results from a combined
data set (Fig. 4B) demonstrated that specificities of the IFA were 88 and 91% at serum dilutions of 1:20 and 1:40, respectively, and that
the decrease in sensitivity exceeds the increase in specificity at
higher serum dilution cutoffs.
We evaluated assay performance in three ways: by using (i)
sensitivities in the KS group, (ii) conventional sensitivity and specificity calculations using a defined standard, and (iii) areas under ROC curves. To establish defined standards, we used the K8.1SFV IFA results instead of the PEL-based
assay results because (i) our study indicates that latent mIFA has low
sensitivity, (ii) lytic mIFA is potentially cross-reactive with
antibodies against other herpesviruses, and (iii) the lack of negative
control cell lines in the PEL-based assays introduces a degree of
subjectivity into their interpretation.
Importantly, the three evaluation methods led to the same conclusions:
(i) the performances of the lytic antigen-based ELISAs were almost
equivalent, and (ii) the lytic antigen-based assays were more
sensitive in identifying seropositives than the latent antigen-based
assays. Although K8.1-based ELISAs were slightly better than
virion and ORF65pep ELISAs in the ROC analysis,
an analysis of more specimens will be needed to determine if the difference is statistically significant. In prior work, bacterially expressed K8.1 was found to be a less sensitive antigen than K8.1 expressed in eukaryotic cells (14, 16, 19). This may be attributed to our previous finding that KS+ sera
contain antibodies that react more strongly with the glycosylated form
of K8.1 (14).
In most cases, IFAs performed better than ELISAs when antigens derived
from the same open reading frame were used. The one exception was
ORF65SFV IFA, which performed poorly in
comparison with the corresponding ELISA, ORF65pep
ELISA. Assays using bacterially and recombinant baculovirus-expressed
ORF65 have performed relatively well (13, 39, 43). Since
the ORF65 expression level in rSFV-infected cells was equivalent to
that of K8.1 (data not shown), the amount of protein may not be the
cause of the low sensitivity.
Comparison of several HHV-8 antigens expressed as glutathione
S-transferase fusion proteins in E. coli
suggested that the latent ORF73 antigen performed better than the
tested lytic antigens in ELISAs and IB assays (16). In
other work, latent PEL-based IFAs showed high concordances with virion
ELISA and with an ELISA using bacterially expressed ORF65 antigens
(34, 39). In contrast, IB assays with
baculovirus-expressed ORF65 and K8.1 proteins were more sensitive than
an IB assay with ORF73 (43). Comparison of ELISAs based on
recombinant ORF73 and ORF65 proteins by ROC analysis using
KS+ MSM and HIV female
intravenous drug users as defined true positive and negative standards
showed better performance of the ORF65-based ELISA (13). ROC analysis using KS patients and hemophilia study subjects as defined
positive and negative standards showed that K8.1- and virion-based
ELISAs were significantly better than an ORF73-based ELISA
(11). Because of the high-level ORF73 expression in the rSFV-based system and a strong correlation between
ORF73SFV and ORF73bac
ELISAs, it is unlikely that insufficient antigen or posttranslational modification of ORF73 can explain the poor performance of latent antigen-based assays in this study. Higher sensitivity of IFAs than
ELISAs, methods used to define assay cutoffs, and posttranslation modifications of lytic antigens in mammalian cells may explain the
discrepancies among the prior studies. It is likely that latent antigen-based assays markedly underestimate HHV-8 prevalence.
Our results suggest that there is still room to further optimize the
performance of the ELISAs to bring them up to the sensitivity levels of
IFAs. Sensitivity may be increased by refinement of ELISA conditions
(10), use of combinations of multiple antigens in a single
ELISA or use of combined results from multiple assays (11, 16,
40), or an additional step of signal amplification. Although the
obvious candidates have been evaluated, it is also possible that
additional antigenic proteins will be identified for serologic assays.
PEL-based lytic IFAs have a potential specificity problem.
Sixty percent of blood donor specimens reacted in
gBSFV IFA. However, it is unlikely that these
reactivities were specific to HHV-8 infection for the following
reasons: (i) most blood donor specimens (26 of 30) were negative
in all 11 other HHV-8 serologic assays; (ii) because
gBSFV IFA detected antibodies against HHV-8 gB in
only half of the KS+ specimens, it is unlikely
that gBSFV IFA is more sensitive than the other
assays; and (iii) rabbit antibodies against EBV gB reacted with
TPA-induced BCBL-1 cells at a low serum dilution (data not shown). Because gB is one of the most highly conserved
herpesvirus glycoproteins, the simplest explanation would be that
antibodies against gBs of other herpesviruses cross-react in
gBSFV IFA.
PEL-based lytic IFAs have been found to give consistently higher
seroprevalence results than other assays. In this study, we found that
three out of four serum specimens with the highest titer in
gBSFV IFA were positive in lytic mIFA, and only
one of them was positive in K8.1SFV IFA. In other
words, 2 out of 28 blood donors were positive by lytic mIFA in the
absence of corroborating evidence from any other assay. Because gB is
expressed at a high level in TPA-induced BCBL-1 cells (1),
it is possible that antibodies cross-reactive with HHV-8 gB
produced false-positive results in lytic PEL-based IFAs. This potential
false positivity is not a substantial problem in most epidemiologic
studies, because lytic PEL-based IFAs generally agree with
lytic antigen-based assays (14, 40, 43). However, it is
essential to further define the specificity of lytic PEL-based IFAs for
studies in which the effect of low specificity can become the main
source of seropositivity and confound data interpretations. This
would include: (i) determination of more precise seroprevalences
in low-prevalence populations; (ii) evaluation of risks associated with
HHV-8 transmission, for example, via transfusions or organ transplantation, because donors are frequently from low-prevalence populations; and (iii) diagnosis of individuals. If lytic PEL-based IFA
is used for such purposes, it would be better to confirm positives with
a second assay. Because K8.1SFV IFA provides
sensitivity similar to that of lytic PEL-based IFAs and is more
specific, it represents a useful alternative to PEL-based assays.
| |
ACKNOWLEDGMENTS |
We thank M. K. Offermann, C. Gunthel, S. Dollard, and the
Atlanta HHV-8 Working Group for serum specimens; S. Dollard and Q. Zheng for use of peptide ELISA data; D. Burns, J. B. Black, C.-P.
Pau, and D. Sanchez-Martinez for rabbit antibodies; Y.-X. Meng for
pcDNA3.1-gB1b-4; and M. Cannon and other members of the Herpesvirus
Section at the Centers for Disease Control and Prevention for useful discussion.
J.L.C. was supported by a fellowship through a Collaborative Research
and Development Agreement between the Centers for Disease Control and
Prevention and Biokit, S.A.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Centers for
Disease Control and Prevention, MS-G18, 1600 Clifton Rd., Atlanta, GA
30333. Phone: (404) 639-4219. Fax: (404) 639-0049. E-mail:
nai0{at}cdc.gov.
Present address: Biokit S.A., Barcelona, Spain.
| |
REFERENCES |
| 1.
|
Baghian, A.,
M. Luftig,
J. B. Black,
Y.-X. Meng,
C.-P. Pau,
T. Voss,
P. E. Pellett, and K. G. Kousoulas.
2000.
Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments.
Virology
269:18-25[CrossRef][Medline].
|
| 2.
|
Blackbourn, D. J.,
E. T. Lennette,
J. Ambroziak,
D. V. Mourich, and J. A. Levy.
1998.
Human herpesvirus 8 detection in nasal secretions and saliva.
J. Infect. Dis.
177:213-216[Medline].
|
| 3.
|
Blauvelt, A.
1999.
The role of human herpesvirus 8 in the pathogenesis of Kaposi's sarcoma.
Adv. Dermatol.
14:167-206[Medline].
|
| 4.
|
Boldogh, I.,
P. Szaniszlo,
W. A. Bresnahan,
C. M. Flaitz,
M. C. Nichols, and T. Albrecht.
1996.
Kaposi's sarcoma herpesvirus-like DNA sequences in the saliva of individuals infected with human immunodeficiency virus.
Clin. Infect. Dis.
23:406-407[Medline].
|
| 5.
|
Bourboulia, D.,
D. Whitby,
C. Boshoff,
R. Newton,
V. Beral,
H. Carrara,
A. Lane, and F. Sitas.
1998.
Serologic evidence for mother-to-child transmission of Kaposi sarcoma-associated herpesvirus infection.
JAMA
280:31-32[Free Full Text].
|
| 6.
|
Cannon, M. J.,
S. M. Dollard,
D. K. Smith,
R. S. Kelin,
P. Schuman,
J. D. Rich,
D. Vlahov, and P. E. Pellett for the HIV Epidemiology Research Study Group..
2001.
Bloodborne and sexual transmission of human herpesvirus 8 in US women with or at risk for human immunodeficiency virus infection.
N. Engl. J. Med.
344:637-643[Abstract/Free Full Text].
|
| 7.
|
Chang, Y.,
E. Cesarman,
M. S. Pessin,
F. Lee,
J. Culpepper,
D. M. Knowles, and P. S. Moore.
1994.
Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.
Science
266:1865-1869[Abstract/Free Full Text].
|
| 8.
|
Chatlynne, L. G., and D. V. Ablashi.
1999.
Seroepidemiology of Kaposi's sarcoma-associated herpesvirus (KSHV).
Semin. Cancer Biol.
9:175-185[CrossRef][Medline].
|
| 9.
|
Chatlynne, L. G.,
W. Lapps,
M. Handy,
Y. Q. Huang,
R. Masood,
A. S. Hamilton,
J. W. Said,
H. P. Koeffler,
M. H. Kaplan,
A. Friedman-Kien,
P. S. Gill,
J. E. Whitman, and D. V. Ablashi.
1998.
Detection and titration of human herpesvirus-8-specific antibodies in sera from blood donors, acquired immunodeficiency syndrome patients, and Kaposi's sarcoma patients using a whole virus enzyme-linked immunosorbent assay.
Blood
92:53-58[Abstract/Free Full Text].
|
| 10.
|
Edelman, D. C.,
F. Ketema,
R. D. Saville,
J. Herman,
A. M. Sill,
P. S. Gill,
W. A. Blattner, and N. T. Constantine.
2000.
Specifics on the refinement and application of two serological assays for the detection of antibodies to HHV-8.
J. Clin. Virol.
16:225-237[CrossRef][Medline].
|
| 11.
|
Engels, E. A.,
D. Whitby,
P. B. Goebel,
A. Stossel,
D. Waters,
A. Pintus,
L. Contu,
R. J. Biggar, and J. J. Goedert.
2000.
Identifying human herpesvirus 8 infection: performance characteristics of serologic assays.
J. Acquir. Immune Defic. Syndr.
23:346-354.
|
| 12.
|
Gao, S.-J.,
L. Kingsley,
M. Li,
W. Zheng,
C. Parravicini,
J. Ziegler,
R. Newton,
C. R. Rinaldo,
A. Saah,
J. Phair,
R. Detels,
Y. Chang, and P. S. Moore.
1996.
KSHV antibodies among Americans, Italians and Ugandans with and without Kaposi's sarcoma.
Nat. Med.
2:925-928[CrossRef][Medline].
|
| 13.
|
Goudsmit, J.,
N. Renwick,
N. H. Dukers,
R. A. Coutinho,
S. Heisterkamp,
M. Bakker,
T. F. Schulz,
M. Cornelissen, and G. J. Weverling.
2000.
Human herpesvirus 8 infections in the Amsterdam Cohort Studies (1984-1997): analysis of seroconversions to ORF65 and ORF73.
Proc. Natl. Acad. Sci. USA
97:4838-4843[Abstract/Free Full Text].
|
| 14.
|
Inoue, N.,
E.-C. Mar,
S. C. Dollard,
C.-P. Pau,
Q. Zheng, and P. E. Pellett.
2000.
New immunofluorescence assays for detection of human herpesvirus 8-specific antibodies.
Clin. Diagn. Lab. Immunol.
7:427-435[Abstract/Free Full Text].
|
| 15.
|
Jones, D.,
M. E. Ballestas,
K. M. Kaye,
J. M. Gulizia,
G. L. Winters,
J. Fletcher,
D. T. Scadden, and J. C. Aster.
1998.
Primary-effusion lymphoma and Kaposi's sarcoma in a cardiac-transplant recipient.
N. Engl. J. Med.
339:444-449[Free Full Text].
|
| 16.
|
Katano, H.,
T. Iwasaki,
N. Baba,
M. Terai,
S. Mori,
A. Iwamoto,
T. Kurata, and T. Sata.
2000.
Identification of antigenic proteins encoded by human herpesvirus 8 and seroprevalence in the general population and among patients with and without Kaposi's sarcoma.
J. Virol.
74:3478-3485[Abstract/Free Full Text].
|
| 17.
|
Kedes, D. H.,
E. Operskalski,
M. Busch,
R. Kohn,
J. Flood, and D. Ganem.
1996.
The seroepidemiology of human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus): distribution of infection in KS risk groups and evidence for sexual transmission.
Nat. Med.
2:918-924[CrossRef][Medline].
|
| 18.
|
Koelle, D. M.,
M.-L. Huang,
B. Chandran,
J. Vieira,
M. Piepkorn, and L. Corey.
1997.
Frequent detection of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in saliva of human immunodeficiency virus-infected men: clinical and immunologic correlates.
J. Infect. Dis.
176:94-102[Medline].
|
| 19.
|
Lang, D.,
W. Hinderer,
M. Rothe,
H.-H. Sonneborn,
F. Neipel,
M. Raab,
H. Rabenau,
B. Masquelier, and H. Fleury.
1999.
Comparison of the immunoglobulin-G-specific seroreactivity of different recombinant antigens of the human herpesvirus 8.
Virology
260:47-54[CrossRef][Medline].
|
| 20.
|
Lennette, E. T.,
D. J. Blackbourn, and J. A. Levy.
1996.
Antibodies to human herpesvirus type 8 in the general population and in Kaposi's sarcoma patients.
Lancet
348:858-861[CrossRef][Medline].
|
| 21.
|
Lin, S.-F.,
R. Sun,
L. Heston,
L. Gradoville,
D. Shedd,
K. Haglund,
M. Rigsby, and G. Miller.
1997.
Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.
J. Virol.
71:3069-3076[Abstract].
|
| 22.
|
Luppi, M.,
P. Barozzi,
T. F. Schulz,
G. Setti,
K. Staskus,
R. Trovato,
F. Narni,
A. Donelli,
A. Maiorana,
R. Marasca,
S. Sandrini, and G. Torelli.
2000.
Bone marrow failure associated with human herpesvirus 8 infection after transplantation.
N. Engl. J. Med.
343:1378-1385[Abstract/Free Full Text].
|
| 23.
|
Martin, J. N.,
Z. Amad,
C. Cossen,
P. K. Lam,
D. H. Kedes,
K. A. Page-Shafer,
D. H. Osmond, and B. Forghani.
2000.
Use of epidemiologically well-defined subjects and existing immunofluorescence assays to calibrate a new enzyme immunoassay for human herpesvirus 8 antibodies.
J. Clin. Microbiol.
38:696-701[Abstract/Free Full Text].
|
| 24.
|
Martin, J. N.,
D. E. Ganem,
D. H. Osmond,
K. A. Page-Shafer,
D. Macrae, and D. H. Kedes.
1998.
Sexual transmission and the natural history of human herpesvirus 8 infection.
N. Engl. J. Med.
338:948-954[Abstract/Free Full Text].
|
| 25.
|
Mayama, S.,
L. E. Cuevas,
J. Sheldon,
O. H. Omar,
D. H. Smith,
P. Okong,
B. Silvel,
C. A. Hart, and T. F. Schulz.
1998.
Prevalence and transmission of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in Ugandan children and adolescents.
Int. J. Cancer
77:817-820[CrossRef][Medline].
|
| 26.
|
Melbye, M.,
P. M. Cook,
H. Hjalgrim,
K. Begtrup,
G. R. Simpson,
R. J. Biggar,
P. Ebbesen, and T. F. Schulz.
1998.
Risk factors for Kaposi's-sarcoma-associated herpesvirus (KSHV/HHV-8) seropositivity in a cohort of homosexual men, 1981-1996.
Int. J. Cancer
77:543-548[CrossRef][Medline].
|
| 27.
|
Metz, C. E.,
B. A. Herman, and C. A. Roe.
1998.
Statistical comparison of two ROC-curve estimates obtained from partially-paired datasets.
Med. Decision Making
18:110-121[Abstract/Free Full Text].
|
| 28.
|
Moore, P. S.,
S.-J. Gao,
G. Dominguez,
E. Cesarman,
O. Lungu,
D. M. Knowles,
R. Garber,
P. E. Pellett,
D. J. McGeoch, and Y. Chang.
1996.
Primary characterization of a herpesvirus agent associated with Kaposi's sarcoma.
J. Virol.
70:549-558[Abstract].
|
| 29.
|
Moore, P. S.,
L. A. Kingsley,
S. D. Holmberg,
T. Spira,
P. Gupta,
D. R. Hoover,
J. P. Parry,
L. J. Conley,
H. W. Jaffe, and Y. Chang.
1996.
Kaposi's sarcoma-associated herpesvirus infection prior to onset of Kaposi's sarcoma.
AIDS
10:175-180[Medline].
|
| 30.
|
Parravicini, C.,
S. J. Olsen,
M. Capra,
F. Poli,
G. Sirchia,
S.-J. Gao,
E. Berti,
A. Nocera,
E. Rossi,
G. Bestetti,
M. Pizzuto,
M. Galli,
M. Moroni,
P. S. Moore, and M. Corbellino.
1997.
Risk of Kaposi's sarcoma-associated herpes virus transmission from donor allografts among Italian posttransplant Kaposi's sarcoma patients.
Blood
90:2826-2829[Abstract/Free Full Text].
|
| 31.
|
Pau, C.-P.,
L. L. Lam,
T. J. Spira,
J. B. Black,
J. A. Stewart,
P. E. Pellett, and R. A. Respess.
1998.
Mapping and serodiagnostic application of a dominant epitope within the human herpesvirus 8 ORF 65-encoded protein.
J. Clin. Microbiol.
36:1574-1577[Abstract/Free Full Text].
|
| 32.
|
Pauk, J.,
M.-L. Huang,
S. J. Brodie,
A. Wald,
D. M. Koelle,
T. Schacker,
C. Celum,
S. Selke, and L. Corey.
2000.
Mucosal shedding of human herpesvirus 8 in men.
N. Engl. J. Med.
343:1369-1377[Abstract/Free Full Text].
|
| 33.
|
Plancoulaine, S.,
L. Abel,
M. van Beveren,
D. A. Tregouet,
M. Joubert,
P. Tortevoye,
G. de The, and A. Gessain.
2000.
Human herpesvirus 8 transmission from mother to child and between siblings in an endemic population.
Lancet
356:1062-1065[CrossRef][Medline].
|
| 34.
|
Rabkin, C. S.,
T. F. Schulz,
D. Whitby,
E. T. Lennette,
L. I. Magpantay,
L. Chatlynne, and R. J. Biggar.
1998.
Interassay correlation of human herpesvirus 8 serologic tests. HHV-8 Interlaboratory Collaborative Group.
J. Infect. Dis.
178:304-309[Medline].
|
| 35.
|
Regamey, N.,
M. Tamm,
M. Wernli,
A. Witschi,
G. Thiel,
G. Cathomas, and P. Erb.
1998.
Transmission of human herpesvirus 8 infection from renal-transplant donors to recipients.
N. Engl. J. Med.
339:1358-1363[Abstract/Free Full Text].
|
| 36.
|
Sanchez-Martinez, D.,
J. L. Patton,
J. A. Stewart, and P. E. Pellett.
1995.
Detection of Epstein-Barr virus-specific antibodies by means of baculovirus-expressed EBV gp125.
J. Virol. Methods
52:145-153[CrossRef][Medline].
|
| 37.
|
Sarid, R.,
S. J. Olsen, and P. S. Moore.
1999.
Kaposi's sarcoma-associated herpesvirus: epidemiology, virology, and molecular biology.
Adv. Virus Res.
52:139-232[Medline].
|
| 38.
|
Schulz, T. F.
1999.
Epidemiology of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8.
Adv. Cancer Res.
76:121-160[Medline].
|
| 39.
|
Simpson, G. R.,
T. F. Schulz,
D. Whitby,
P. M. Cook,
C. Boshoff,
L. Rainbow,
M. R. Howard,
S.-J. Gao,
R. A. Bohenzky,
P. Simmonds,
C. Lee,
A. de Ruiter,
A. Hatzakis,
R. S. Tedder,
I. V. D. Weller,
R. A. Weiss, and P. S. Moore.
1996.
Prevalence of Kaposi's sarcoma associated herpesvirus infection measured by antibodies to recombinant capsid protein and latent immunofluorescence antigen.
Lancet
348:1133-1138[CrossRef][Medline].
|
| 40.
|
Spira, T. J.,
L. Lam,
S. C. Dollard,
Y.-X. Meng,
C. P. Pau,
J. B. Black,
D. Burns,
B. Cooper,
M. Hamid,
J. Huong,
K. Kite-Powell, and P. E. Pellett.
2000.
Comparison of serologic assays and PCR for diagnosis of human herpesvirus 8 infection.
J. Clin. Microbiol.
38:2174-2180[Abstract/Free Full Text].
|
| 41.
|
Vieira, J.,
M.-L. Huang,
D. M. Koelle, and L. Corey.
1997.
Transmissible Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in saliva of men with a history of Kaposi's sarcoma.
J. Virol.
71:7083-7087[Abstract].
|
| 42.
|
Whitby, D.,
M. R. Howard,
M. Tenant-Flowers,
N. S. Brink,
A. Copas,
C. Bishoff,
T. Hatzioannou,
F. E. A. Suggett,
D. M. Aldam,
A. S. Denton,
R. F. Miller,
I. V. D. Weller,
R. A. Weiss,
R. S. Tedder, and T. F. Schultz.
1995.
Detection of Kaposi's sarcoma associated herpesvirus in peripheral blood of HIV-infected individuals and progression to Kaposi's sarcoma.
Lancet
346:799-802[CrossRef][Medline].
|
| 43.
|
Zhu, L.,
R. Wang,
A. Sweat,
E. Goldstein,
R. Horvat, and B. Chandran.
1999.
Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells.
Virology
256:381-392[CrossRef][Medline].
|
Clinical and Diagnostic Laboratory Immunology, September 2001, p. 913-921, Vol. 8, No. 5
1071-412X/01/$04.00+0 DOI: 10.1128/CDLI.8.5.913-921.2001
This article has been cited by other articles:
-
Bergallo, M., Costa, C., Margio, S., Sidoti, F., Re, D., Segoloni, G. P., Cavallo, R.
(2007). Human herpes virus 8 infection in kidney transplant patients from an area of northwestern Italy (Piemonte region). Nephrol Dial Transplant
22: 1757-1761
[Abstract]
[Full Text]
-
Trento, E., Castilletti, C., Ferraro, C., Lesnoni La Parola, I., Mussi, A., Muscardin, L., Bordignon, V., D'Agosto, G., Amantea, A., Mastroianni, A., Ameglio, F., Fluhr, J., Cordiali-Fei, P.
(2005). Human Herpesvirus 8 Infection in Patients With Cutaneous Lymphoproliferative Diseases. Arch Dermatol
141: 1235-1242
[Abstract]
[Full Text]
-
Coberley, S. S., Condit, R. C., Herbst, L. H., Klein, P. A.
(2002). Identification and Expression of Immunogenic Proteins of a Disease-Associated Marine Turtle Herpesvirus. J. Virol.
76: 10553-10558
[Abstract]
[Full Text]
Top
Abstract
Introduction
