Porcine antibody responses to Taenia solium antigens RGP50 and STS18VAR1

Abstract

Cysticercosis, a disease caused by the larval form of Taenia solium, is diagnosed by detection of specific antibodies or by imaging techniques. Our preferred immunologic assay for cysticercosis is the enzyme-linked immunoelectrodifusion transfer blot, or immunoblot, using the lentil lectin bound antigens from larval cysts. Antibody reactivity with any one of seven glycoproteins is diagnostic for cysticercosis. To develop a simple antibody detection assay for field use, we have synthesized an 8-kD diagnostic antigen, sTs18var1 (a secreted protein with a mature size of 67 amino acids), and expressed a 50-kD membrane protein antigen, rGp50. We used these two diagnostic proteins in a quantitative Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) to measure the antibody responses in Peruvian pigs with cysticercosis. Three study designs were used. First, we followed the kinetics of antibody responses against these two diagnostic proteins in pigs with cysticercosis that were treated with oxfendazole. Second, we measured antibody response in naive experimentally infected pigs. Third, we followed the maternal antibodies against rGp50 and sTs18var1 in piglets born from sows with cysticercosis. These studies showed that antibody responses against the two diagnostic proteins in the FAST-ELISA are quantitatively correlated with infection by viable cysts, with anti-sTs18var1 activity being most responsive to the status of infection.

Full text

PORCINE ANTIBODY RESPONSES TO TAENIA SOLIUM ANTIGENS RGP50
AND STS18VAR1
SUKWAN HANDALI, ARMANDO E. GONZALEZ, KATHY HANCOCK, HECTOR H. GARCIA,
JACQUELIN M. ROBERTS, ROBERT H. GILMAN,
AND VICTOR C. W. TSANG
Immunology Branch, Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and
Prevention, Atlanta, Georgia; School of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, Lima, Peru; Department
of Microbiology, Universidad Peruana Cayetano Heredia, Lima, Peru; Cysticercosis Unit, Instituto de Ciencias Neurológicas, Lima,
Peru; Department of International Health, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland
Abstract. Cysticercosis, a disease caused by the larval form of Taenia solium, is diagnosed by detection of specific
antibodies or by imaging techniques. Our preferred immunologic assay for cysticercosis is the enzyme-linked immuno-
electrodifusion transfer blot, or immunoblot, using the lentil lectin bound antigens from larval cysts. Antibody reactivity
with any one of seven glycoproteins is diagnostic for cysticercosis. To develop a simple antibody detection assay for field
use, we have synthesized an 8-kD diagnostic antigen, sTs18var1 (a secreted protein with a mature size of 67 amino acids),
and expressed a 50-kD membrane protein antigen, rGp50. We used these two diagnostic proteins in a quantitative
Falcon assay screening test–enzyme-linked immunosorbent assay (FAST-ELISA) to measure the antibody responses in
Peruvian pigs with cysticercosis. Three study designs were used. First, we followed the kinetics of antibody responses
against these two diagnostic proteins in pigs with cysticercosis that were treated with oxfendazole. Second, we measured
antibody response in naive experimentally infected pigs. Third, we followed the maternal antibodies against rGp50 and
sTs18var1 in piglets born from sows with cysticercosis. These studies showed that antibody responses against the two
diagnostic proteins in the FAST-ELISA are quantitatively correlated with infection by viable cysts, with anti-sTs18var1
activity being most responsive to the status of infection.
INTRODUCTION
Taenia solium (pork tapeworm) has a two-host life cycle,
with humans as the only definitive host carrying the adult
tapeworm in the intestine and pigs as the intermediate host
harboring the larval cyst in muscles or brain. Humans also
occasionally become the intermediate host by harboring the
metacestode/cysticerci of T. solium after ingesting the eggs of
the tapeworm, a condition known as cysticercosis. Taenia so-
lium cysticercosis is endemic in many developing countries.
1,2
It increases the prevalence of epilepsy in humans and causes
significant economic loss because of infested pork.
Since the pig is the intermediate host and the only reservoir
source for this parasite, porcine cysticercosis is critical for the
development of strategies to control cysticercosis and will
benefit the rural poor by preventing pork condemnation.
3–5
Prevalence of porcine cysticercosis is a reliable indicator of
active transmission. In Peru, we use sentinel pigs to monitor
transmission and measure efficacy of control programs. We
prefer to use native sentinel pigs rather than importing ani-
mals from the city, even though seronegative animals are
rarer, but native pigs are less expensive and more resistant to
local pathogens than city pigs.
3
Unfortunately, the use of na-
tive sentinel pigs is hampered by the longevity of transferred
maternal antibodies from colostrum that can last up to eight
months.
4,6
Development of a quantitative, rapid, serologic as-
say such as the Falcon assay screening test–enzyme-linked
immunosorbent assay (FAST-ELISA),
7,8
to differentiate ac-
tive from inactive infections will be better suited for field use.
To develop a highly sensitive and specific FAST-ELISA, we
have cloned, sequenced, and produced two of the seven lentil
lectin-bound glycoprotein diagnostic antigens used in the en-
zyme-linked immunoelectrodifusion transfer blot (EITB).
9,10
The first one is a recombinant glycoprotein known as Gp50
(rGp50), a membrane-bound protein that we have expressed
in insect cells,
11
and the second one is an 8-kD protein (with
a mature size of 67 amino acids) known as T. solium 18 var 1
(sTs18var1) that we have chemically synthesized.
12
These two
antigens represents two of three dominant antigens of the
original diagnostic glycoproteins used the EITB.
In this study, we report the use of the FAST-ELISA with
rGp50 and sTs18var1 to quantify the progress of infection in
porcine cysticercosis and to differentiate between active and
inactive infections. Understanding the progress of infection in
porcine cysticercosis will allow us to learn more about the
progress of infection of human cysticercosis, where the only
available indicator for therapeutic successes is neuroimaging
techniques that are not available in many parts of the world
where the disease is endemic.
13
METHODS AND MATERIALS
Sera samples. We used samples from three studies. The
first was an oxfendazole (OFZ) treatment study designed to
prove that infected pigs that were treated and cured with
OFZ become immune to future infections.
5
Briefly, 19 in-
fected pigs (based on positive EITB and tongue inspection
and purchased from an endemic village) were treated with
single oral dose of 30 mg/kg of OFZ, a cysticercidal drug. The
pigs were kept 20 weeks after treatment to ensure that all
muscle cysts were killed. Each treated pig was then matched
by age and sex with two uninfected pigs and send back to the
endemic village. Three months later, all animals were repur-
chased from villagers, transported back to our specific patho-
gen-free facility in Lima, and kept for an additional three
months, a sufficient time for cysts to achieve full maturity.
Pigs were then killed in an abattoir and a detailed dissection
of the carcasses was performed to determine parasite burden.
Serologic status of all pigs was determined by the FAST-ELISA.
In the second study, 16 naive pigs were fed oncospheres or
eggs (in proglottids). Sera were collected weekly for 12 weeks
and the pigs were then killed as described in the first study.
Four naive pigs were used as uninfected controls. Serologic
status of all pigs was determined by the FAST-ELISA.
Am. J. Trop. Med. Hyg., 71(3), 2004, pp. 322–326
Copyright © 2004 by The American Society of Tropical Medicine and Hygiene
322

In the third study, 34 piglets from 4 sows with cysticercosis
were followed for up to 145 days. Serologic status of all piglets
and sows were determined by the FAST-ELISA.
Serology. Sera collected were analyzed by the FAST-ELISA
as previously described.
7,8
To ensure comparability of day-to-
day measurement, we prepared a standard serum pool from
the sera of five Peruvian pigs with confirmed cysticercosis and
with high antibody activities against rGP50 and sTs18var1.
We defined 1 ␮L of the standard serum pool as having 100
units of activity. A reference curve constructed from the stan-
dard serum pool was included with each assay. The diluent
used was a negative United States pig serum pool obtained
from Equitech (Kerville, TX).
Sticks (transferable solid phase screening, catalog # 445497;
Nunc, Roskilde, Denmark) were sensitized with 1 ␮g/mL of
rGp50 and 2 ␮g/mL of sTs18var1, respectively, in 96-well
plates (Catalog # 269620; Nunc) for two hours at room tem-
perature on a shaker (Titer Plate Shaker; Laboratory-Line
Instruments, Melrose Park, IL) at speed 5. The diluent used
was 0.01 M phosphate-buffered saline (PBS), pH 7.2. After
sensitization, the sticks were washed with 0.01 M PBS, pH 7.2,
containing 0.3% polyoxyethylene sorbitan monolaurate 20
(Tween 20; Sigma Chemical Company, St. Louis, MO) using
a standard plastic garden sprayer. Sera to be assayed for an-
tibodies against rGp50 and sTs18var1 were dispensed into the
wells, and 4.5 ␮L of serum were diluted to a total volume of
150 ␮L in 0.01 M PBS, pH 7.2/0.3% Tween 20/5% nonfat dry
milk. Sera with activities above the maximum standard curve
were diluted further using the United States normal pig serum
until the activities were within the boundaries of the standard
curve. Sticks were exposed to the sera for fives minutes at
room temperature with mixing, washed again as in the previ-
ous step, and then exposed to a home-made antibody/enzyme
conjugate (150 ␮L of affinity-purified goat anti-pig IgG
[heavy and light chain activity] labeled with horseradish per-
oxidase) and diluted 1:1,000 in PBS/Tween 20. After exposure
to the conjugate for five minutes at room temperature with
mixing, and washed as before, the sticks were immersed in
150 ␮L of SureBlue 3,3⬘,5,5⬘-tetramethylbenzidine substrate
(Kirkegaard and Perry Laboratories, Gaithersburg, MD) for
five minutes at room temperature with mixing. The sticks
were then removed and discarded. The absorbance of each
well was measured at 650 nm using a ThermoMax microtitra-
tion plate (Molecular Devices, Sunnyvale, CA). The activity
unit of each serum sample was derived from the standard
curve and was expressed in units/microliter. All samples were
run in triplicate.
Data analysis. The FAST-ELISA for rGp50 and sTs18var1
performance index (Youden J index) was determined.
14
In
principle, the J index is sensitivity (number of positive
samples detected in the infected group divided by number of
known positive samples) plus specificity (number of negative
samples detected in the uninfected group divided by number
of known negative samples) minus 1. The highest J index
obtainable is 1.0 and occurs only when there is 100% sensi-
tivity and 100% specificity.
7,14
Optimizing the J index will,
therefore, allow us to determine what assay cut-off point to
use to differentiate between positive and negative samples.
The mean number of weeks at which time antibody responses
decreased below and increased above the detection level was
determined by calculating Kaplan-Meier product limit esti-
mates of the survival function. Pearson correlation coeffi-
cients were used to assess the association between infection
and antibody response. All statistical analysis were performed
using SAS version 8.02 (SAS Institute, Cary, NC).
15
RESULTS
Determination of the J indices and the test cut-off
points. The results from the assay of the pretreatment sera
from the 19 OFZ experiment pigs (pigs positive for cysticer-
cosis) and 27 control pigs gave a J index of 0.86 with a cut-off
point of 0.40 unit/␮L with rGp50 and a J index of 1 with a
cut-off point at 0.70 unit/␮L with sTs18var1, respectively.
Therefore, sera with activity units ⱖ 0.40 for rGp50 and
ⱖ 0.70 for sTs18var1 were considered positive.
Oxfendazole study. For analyzing the antibody responses
after treatment with OFZ, only data from infected pigs will be
reported. A total of 19 infected and treated pigs were ob-
served in the study; 4 pigs (21%, 4 of 19) were found to have
viable cysts at the end of the study and the rest of the pigs
(79%, 15 of 19) had only degenerated or no remnants of cysts
found. In those pigs with viable cysts, all viable cysts were
found only in the brain. In all infected and treated pigs, the
antibody responses against rGp50 and sTs18var1 showed
similar patterns: increased antibody responses against rGp50
and sTs18var1 immediately after treatment and then mostly
a decrease to below detection levels at a mean ± SE of 40.9 ±
1.6 weeks for rGp50 and 20.7 ± 2.5 weeks for sTs18var1
(Figures 1 and 2). At end of the study, in pigs with only
degenerated cysts, 40% (6 of 15) and 13% (2 of 15) of the
antibody responses against rGp50 and sTs18var1, respec-
tively, were above the cut-off point. For pigs with viable cysts,
50% (2 of 4) of the antibody responses against rGp50 and
sTs18var1 were above the cut-off point by end of the study.
The two pigs that tested negative were animals with only
minimal numbers of cysts in the brain. In all instances, the
ratio of rGp50/sTs18var1 was > 1.
Experimental infection. In this study, 20 pigs were inocu-
lated by several methods to produce porcine cysticercosis. In
four pigs that received no infection (control pigs), all were
free from cysticercosis and antibody responses against rGp50
and sTs18var1 were below detection limit. In 16 pigs that
received infective eggs, two pigs had only degenerated cysts
and they showed some increase of antibody responses to
rGp50 and sTs18var1, but by end of the study, the antibody
responses decreased below the cut-off point, except in one pig
against sTs18var1.
There were 14 pigs with living cysts by end of the study
(range ⳱ 1–590 alive cysts and 6–601 total cysts). The anti-
body responses against rGp50 and sTs18var1 followed the
same patterns, and could be detected as early as three weeks,
with mean ± SE times for detection of 7.6 ± 0.8 weeks for
rGp50 and 6.1 ± 0.6 weeks for sTs18var1 (Figures 3 and 4). By
the end of the study, 70% of the pigs with viable cysts had a
positive antibody response against rGp50 and 93% had a
positive antibody response against sTs18var1. In this experi-
ment, the ratio of rGp50/sTs18var1 was < 1. The correlations
between antibody responses at the time of necropsy and num-
ber of viable cysts were 0.62 and 0.86 for rGp50 and
sTs18var1, respectively.
Sow-piglet study. In this study, 34 piglets from 4 sows with
cysticercosis were followed for 145 days. The relationship be-
PORCINE ANTIBODY RESPONSES TO RGP50 AND STS18VAR1 323

tween antibody responses against rGp50 and sTs18var1 in
sows and their piglets at time of delivery is shown in Table 1.
Sows with higher antibody responses against rGp50 and
sTs18var1 had piglets with higher levels of maternal antibod-
ies. Maternal antibodies against rGp50 and sTs18var1 in pig-
lets decreased below detection levels at a mean ± SE of
74.4 ± 4.8 days and 31.1 ± 4.4 days, respectively (Figure 5).
DISCUSSION
The results of this study suggested that we could use anti-
body data from the FAST-ELISA for rGp50 and sTs18var1 to
quantitatively follow the progress of infection, treatment of
cysticercosis in pigs, and the fate of maternal antibodies in
piglets. Antibody responses against rGp50 and STs18var1 did
not show significant differences. The only clear difference was
that in pigs with established infections, the ratio of rGp50/
sTs18var1 is >1, but it was reversed in the early infections,
such as the experimentally infected pigs. Antibody responses
against rGp50 could be detected at seven weeks post-
infection instead of at six weeks with sTs18var1 and waned
later at 41 weeks instead of 21 weeks. Also, in piglets, mater-
nal antibodies against rGp50 waned at nine weeks compared
with four weeks against sTs18var1.
Data from OFZ-treated pigs with viable cysts showed that
positive antibody responses against rGp50 and sTs18var1
could be detected in only 50% of the pigs. The viable cysts
were found in only in the brain. We speculated that viable
cysts in the brain did not induce high antibody responses due
to the fact that the brain is considered a privileged site for
immune functions.
16
To answer the question of any difference in antibody re-
sponses against rGp50 and sTs18var1 between pigs with vi-
able and non-viable cysts, data from the experimentally
infected pig study showed that infection or cysts viability
correlated well with antibody responses against sTs18var1
(r ⳱ 0.86), but not against rGp50 (r ⳱ 0.62). In the sow-piglet
study, we found that although the piglets were not infected,
they do have maternal antibodies against rGp50 and
sTs18var1. The antibody against sTs18var1 however, waned
after 31 days; this observation is important because it enables
one to differentiate pigs with real infections from those ma-
ternal antibodies, an effort that is quite difficult with the
EITB.
4
FIGURE 1. Antibody responses against rGp50 in oxfendazole-
treated pigs detected by the Falcon assay screening test–enzyme-
linked immunosorbent assay (FAST-ELISA). A, Pigs with degener-
ated cysts. B, Pigs with viable cysts. Samples were tested in triplicate
and the average absorbance at 650 nm was converted into units of
activity based on a standard curve. The horizontal dashed line rep-
resents the cut-off value of the FAST-ELISA.
FIGURE 2. Antibody responses against sTs18var1 in oxfendazole-
treated pigs detected by the Falcon assay screening test–enzyme-
linked immunosorbent assay (FAST-ELISA). A, Pigs with degener-
ated cysts. B, Pigs with viable cysts (all in the brain). Samples were
tested in triplicate, and the average absorbance at 650 nm was con-
verted into units of activity based on a standard curve. The horizontal
dashed line represents the cut-off value of the FAST-ELISA.
HANDALI AND OTHERS324

There are several implications of this study. First, it is pos-
sible to use a simple, fast, quantitative FAST-ELISA to fol-
low the progress of infection in porcine cysticercosis, corre-
late antibody responses with viability of cysts, and monitor
the progress of maternal antibodies in piglets. We showed
that the FAST-ELISA for sTs18var1 could give us more in-
formation than that for rGp50. The reasons are that antibody
against sTs18var1 disappears early in treated pigs and piglets,
is found in 93% of the infected pigs, and has a higher corre-
lation with cyst viability (r ⳱ 0.86) and a J index of 1. Second,
for the purpose of cysticercosis control programs, the EITB is
important for estimating the burden of exposure (total expo-
sure to cysticercosis),
17
but the FAST-ELISA for sTs18var1
will give a better estimate of the true prevalence of cysticer-
cosis (ability to detect infected pigs with live cysts and thus
potential sources of infection) and also confirm treatment
efficacy in the frame of intervention control programs. Com-
bination of the EITB and FAST-ELISA for sTs18var1 will
give a better picture of the cysticercosis problem.
FIGURE 4. Antibody responses against sTs18var1 in experimen-
tally infected pigs detected by the Falcon assay screening test–
enzyme-linked immunosorbent assay (FAST-ELISA). Samples were
tested in triplicate, and the average absorbance at 650 nm was con-
verted into units of activity based on a standard curve. The horizontal
dashed line represents the cut-off value of the FAST-ELISA.
FIGURE 5. Antibody responses in piglets against A, rGp50 and B,
sTs18var1. Pig sera of piglets from the sow-piglets study were tested
by the Falcon assay screening test–enzyme-linked immunosorbent
assay (FAST-ELISA). Samples were tested in triplicate, and the av-
erage absorbance at 650 nm was converted into units of activity based
on a standard curve. The horizontal dashed line represents the cut-off
value of the FAST-ELISA.
FIGURE 3. Antibody responses against rGp50 in experimentally
infected pigs detected by the Falcon assay screening test–enzyme-
linked immunosorbent assay (FAST-ELISA). Samples were tested in
triplicate, and the average absorbance at 650 nm was converted into
units of activity based on a standard curve. The horizontal dashed line
represents the cut-off value of the FAST-ELISA.
T
ABLE 1
Relationship between antibody responses in sows and maternal an-
tibodies in piglets against rGp50 and sTs18var1 at time of delivery*
Subject†
Antibody responses against
rGp50 (units/␮L)*
Antibody responses against
sTs18var1 (units/␮L)
Sow 1 58.7 17.3
Piglets (4) 58.3 (15–101.9) 13.8 (5.7–24.3)
Sow 2 0.4 3.9
Piglets (8) 0.07 (0.01–0.14) 1.5 (0.8–2.1)
Sow 3 first delivery 40.5 7.2
Piglets (8) 28.2 (10–48) 9.7 (3.8–15.2)
Sow 3 second delivery 47.1 8.6
Piglets (4) 71.3 (55.4–117) 20.4 (12–30.5)
Sow 4 1.6 0
Piglets (8) 14.2 (9.4–18.7) 0.04 (0–0.38)
* Values are the mean (range) activity units of antibody responses of all piglets from the
same sow.
† Values in parentheses are the number of piglets.
PORCINE ANTIBODY RESPONSES TO RGP50 AND STS18VAR1 325

The only disadvantage of this FAST-ELISA system is that
it failed to detect an antibody response in 50% of the pigs
with viable cysts only in the brain. This situation will be cru-
cial when it comes to diagnose human neurocysticercosis, but
this problem exists also with the EITB.
18
In conclusion, the FAST-ELISA for rGp50 and sTs18var1
could follow the progress of infection in porcine cysticercosis
and the antibody responses against rGp50 and sTs18var1 cor-
related well with cysts viability, although the overall perfor-
mance of the FAST-ELISA for sTs18var1 was better than for
rGp50.
Received August 21, 2003. Accepted for publication April 1, 2004.
Financial support: This research was supported by International Col-
laborations in Infectious Disease Research–National Insitutes of
Health (NIH) grant U01 AI-35894, NIH National Institute of Allergy
and Infectious Diseases Tropical Medicine Research Center grant 1
P01 AI-51976-01, Wellcome grant 063109, and Bill and Melinda
Gates foundation grant 23981.
Authors’ addresses: Sukwan Handali, Kathy Hancock, and Victor C.
W. Tsang, Immunology Branch, Division of Parasitic Diseases, Na-
tional Center for Infectious Diseases, Centers for Disease Control
and Prevention, 4770 Buford Highway, Mailstop F-13, Atlanta, GA
30341-3724, E-mails: ahi0@cdc.gov, kyh7@cdc.gov, and vct1@cdc.gov.
Armando E. Gonzalez, School of Veterinary Medicine, Universidad
Nacional Mayor de San Marcos, Cuadra 29 Avenida Circunvalacion
s/n, San Borja, Lima, Peru, E-mail: emico@terra.com.pe. Hector H.
Garcia, Department of Microbiology, Universidad Peruana Cayetano
Heredia, Avenida Honorio Delgado 430, San Martin de Porras, Lima,
Peru and Cysticercosis Unit, Instituto de Ciencias Neurológicas,
Lima, Peru, E-mail: hgarcia@terra.com.pe. Jacquelin M. Roberts,
Data Management Activity, Division of Parasitic Diseases, National
Center for Infectious Diseases, Centers for Disease Control and Pre-
vention, 4770 Buford Highway, Mailstop F-22, Atlanta, GA 30341-
3724, E-mail: jmr1@cdc.gov. Robert H. Gilman, Department of In-
ternational Health, Johns Hopkins University School of Hygiene and
Public Health, 615 North Wolfe Street, Baltimore, MD 21205, E-mail:
gilmanbob@yahoo.com.
REFERENCES
1. Garcia HH, Gilman R, Martinez M, Tsang VC, Pilcher JB, Her-
rera G, Diaz F, Alvarado M, Miranda E, 1993. Cysticercosis as
a major cause of epilepsy in Peru. The Cysticercosis Working
Group in Peru (CWG). Lancet 341: 197–200.
2. Garcia HH, Del Brutto OH, 2000. Taenia solium cysticercosis.
Infect Dis Clin North Am 14: 97–119.
3. Gonzalez AE, Gilman R, Garcia HH, McDonald J, Kacena K,
Tsang VC, Pilcher JB, Suarez F, Gavidia C, Miranda E, 1994.
Use of sentinel pigs to monitor environmental Taenia solium
contamination. The Cysticercosis Working Group in Peru
(CWG). Am J Trop Med Hyg 51: 847–850.
4. Gonzalez AE, Verastegui M, Noh JC, Gavidia C, Falcon N, Ber-
nal T, Garcia HH, Tsang VC, Gilman RH, Wilkins PP, 1999.
Persistence of passively transferred antibodies in porcine Tae-
nia solium cysticercosis. Cysticercosis Working Group in Peru.
Vet Parasitol 86: 113–118.
5. Gonzalez AE, Gavidia C, Falcon N, Bernal T, Verastegui M,
Garcia HH, Gilman RH, Tsang VC, Cysticercosis Working
Group in Peru, 2001. Protection of pigs with cysticercosis from
further infections after treatment with oxfendazole. AmJTrop
Med Hyg 65: 15–18.
6. Tsang VC, Brand JA, Boyer AE, 1989. An enzyme-linked im-
munoelectrotransfer blot assay and glycoprotein antigens for
diagnosing human cysticercosis (Taenia solium). J Infect Dis
159: 50–59.
7. Hancock K, Tsang VCW, 1986. Development and optimization of
the FAST-ELISA for detecting antibodies to Schistosoma
mansoni. J Immunol Methods 92: 167–176.
8. Richards FO, Tsang VCW, Brand JA, Hancock K, 1989. Modi-
fication of the FAST-ELISA for field diagnosis of Schistoso-
miasis mansoni with serum of blood samples. Trop Med Para-
sitol 40: 332–334.
9. Greene RM, Wilkins PP, Tsang VC, 1999. Diagnostic glycopro-
teins of Taenia solium cysts share homologous 14- and 18-kDa
subunits. Mol Biochem Parasitol 99: 257–261.
10. Greene RM, Hancock K, Wilkins PP, Tsang VC, 2000. Taenia
solium: molecular cloning and serologic evaluation of 14- and
18-kDa related, diagnostic antigens. J Parasitol 86: 1001–1007.
11. Hancock K, Pattabhi S, Greene RM, Yushak ML, Williams F,
Khan A, Priest JW, Levine MZ, Tsang VC, 2004. Character-
ization and cloning of GP50, a Taenia solium antigen diagnos-
tic for cysticercosis. Mol Biochem Parasitol 133: 115–124.
12. Hancock K, Khan A, Williams FB, Yushak ML, Pattabhi S, Noh
J, Tsang V, 2003. Characterization of the 8-kilodalton antigens
of Taenia solium metacestodes and evaluation of their use in
an enzyme-linked immunosorbent assay for serodiagnosis.
J Clin Microbiol 41: 2577–2586.
13. Garcia HH, Gilman RH, Catacora M, Verastegui M, Gonzalez
AE, Tsang VC, 1997. Serologic evolution of neurocysticercosis
patients after antiparasitic therapy. J Infect Dis 175: 486–489.
14. Youden WJ, 1950. Index for rating diagnostic tests. Cancer 3:
32–35.
15. Elandt-Johnson RC, 1999. Survival Models and Data Analysis.
New York: John Wiley & Sons, Inc.
16. Xiao BG, Link H, 1998. Immune regulation within the central
nervous system. J Neurol Sci 157: 1–12.
17. Garcia HH, Gonzalez AE, Gilman RH, Palacios LG, Jimenez I,
Rodriguez S, Verastegui M, Wilkins P, Tsang VC; Cysticerco-
sis Working Group in Peru, 2001. Short report: transient anti-
body response in Taenia solium infection in field conditions-a
major contributor to high seroprevalence. Am J Trop Med Hyg
65: 31–32.
18. Garcia HH, Herrera G, Gilman RH, Tsang VC, Pilcher JB, Diaz
JF, Candy EJ, Miranda E, Naranjo J, 1994. Discrepancies be-
tween cerebral computed tomography and Western blot in the
diagnosis of neurocysticercosis. The Cysticercosis Working
Group in Peru (Clinical Studies Coordination Board). Am
J Trop Med Hyg 50: 152–157.
HANDALI AND OTHERS326