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Infect Immun, August 1998, p. 4008-4009, Vol. 66, No. 8
0019-9567/98/$00.00+0
LETTERS TO THE EDITOR
Serologic Response to Human Cryptosporidium
Infections
 |
LETTER |
Okhuysen et al. (9) reported the susceptibilities and serologic
responses of 19 healthy human volunteers rechallenged with Cryptosporidium oocysts. Their finding that less-severe
symptoms were observed when volunteers were rechallenged supports our
interpretation of the Cryptosporidium outbreak literature
and may explain the difficulty in linking reported cases of
cryptosporidiosis with water supplies that are chronically contaminated
with oocysts (3); however, the low correlation observed between serum
antibody response and infection requires clarification. The serological findings are based on an enzyme-linked immunosorbent assay (ELISA) in
which the antibody responses to all antigens obtained from Cryptosporidium oocysts were measured (2). Miller (6),
Voller (11), and others (10) suggested that because of the complex array of parasite antigens, ELISAs for parasite immunodiagnosis should
use immunodominant antigens rather than extracts of the whole parasite.
It is possible that seroconversion, based on either an ELISA which uses
purified antigens or Western blotting (WB), may have more accurately
estimated the rate of Cryptosporidium infection in these
volunteers (7).
We compared serum antibody responses to Cryptosporidium
antigens determined by an ELISA for combined immunoglobulin G (IgG), IgA, and IgM responses (4) and those by a WB assay for IgG responses to
15/17-kDa and 27-kDa antigen groups (8) using 117 serum samples
collected in the spring of 1992 during a cryptosporidiosis outbreak in
Jackson County, Oreg. (5). The intensity of the WB and the optical
density of the ELISA responses were measured and analyzed as a ratio of
the unknown sample and the positive control response (4). Low
correlations were found between the ELISA and WB results: Pearson
correlations for the IgG response to the 15/17- and 27-kDA antigens and
for the IgA response to the 27-kDa antigen were 0.17, 0.42, and 0.35, respectively.
More than half of 34 ELISA-negative individuals had WB responses
greater than 20% of the positive control in one or more bands. If the
ELISA is less sensitive and specific than the WB, then by using the
ELISA, Okhuysen et al. may have misclassified seroconversions as well
as those volunteers who were considered seronegative at the outset of
the study. Some ELISA-seronegative volunteers may have been WB
seropositive. These WB-seropositive individuals may have been less
susceptible to illness and, based on the definition of infection used,
less likely to have become infected upon rechallenge.
Finally, the definition of infection used in the rechallenge study
(being oocyst positive or having illness) differed from that (being
oocyst positive) used in an earlier study by these authors (2), but
neither definition considers the possibility of volunteers being
asymptomatically infected but stool negative for oocysts. The same
authors previously reported that oocyst stool-positive but asymptomatic
volunteers excreted fewer oocysts than did stool-positive and
symptomatic volunteers (1). This possibility suggests that other
asymptomatically infected individuals with low numbers of oocysts in
their stools may have been misclassified as uninfected. By using the
occurrence of illness to identify 10 of 13 infected persons in this
study, the authors may have inadvertently mingled risk factors for
infection with risk factors for illness from an infection.
| |
REFERENCES |
| 1.
|
Chappell, C. L.,
P. C. Okhuysen,
C. R. Sterling, and H. L. DuPont.
1996.
Cryptosporidium parvum: intensity of infection and oocyst excretion patterns in health volunteers.
J. Infect. Dis.
173:232-236[Medline].
|
| 2.
|
DuPont, H. L.,
C. L. Chappell,
C. R. Sterling,
P. C. Okhuysen,
J. B. Rose, and W. Jakubowski.
1995.
The infectivity of Cryptosporidium parvum in healthy volunteers.
N. Engl. J. Med.
332:855-859[Abstract/Free Full Text].
|
| 3.
|
Frost, F. J.,
G. F. Craun,
R. L. Calderon, and S. A. Hubbs.
1997.
So many oocysts, so few outbreaks.
J. Am. Water Works Assoc.
89(12):8-9.
|
| 4.
| Frost, F. J., A. A. de la Cruz, D. M. Moss, M. Curry,
and R. L. Calderon. Comparisons of ELISA and Western blot assays
for detection of Cryptosporidium antibody. Epidemiol.
Infect., in press.
|
| 5.
|
Leland, D.,
J. McAnulty,
W. Keene, and G. Sterens.
1983.
A cryptosporidiosis outbreak in a filtered-water supply.
J. Am. Water Works Assoc.
85:34-42.
|
| 6.
|
Miller, H. R.
1990.
Immunity to internal parasites.
Rev. Sci. Tech. Off. Int. Epizoot.
9:301-344.
|
| 7.
|
Moss, D. M.,
S. N. Bennett,
M. J. Arrowood,
S. P. Wahlquist, and P. J. Lammie.
1998.
Enzyme-linked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter.
Am. J. Trop. Med. Hyg.
51:110-118.
|
| 8.
|
Moss, D. M.,
S. N. Bennett,
M. J. Arrowood,
M. R. Hurd,
P. J. Lammie, and S. P. Wahlquist.
1994.
Kinetic and isotypic analysis of specific immunoglobulins for crew members with cryptosporidiosis on a U.S. Coast Guard cutter.
J. Eukaryot. Microbiol.
41:52S-455S[Medline].
|
| 9.
| Okhuysen, P. C., C. L. Chappell, C. R. Sterling, W. Jakubowski, and H. L. DuPont. Susceptibility and Serological
Response of Healthy Adults to Reinfection with Cryptosporidium
parvum. Infect. Immun. 66:441-443.
|
| 10.
|
Venkatesan, P., and D. Wakelin.
1993.
ELISAs for parasitologists: or lies, damn lies and ELISAs.
Parasitol. Today
9:225-239.
[Medline] |
| 11.
|
Voller, A.
1993.
Immunosassays for tropical parasitic infections.
Trans. R. Soc. Trop. Med. Hyg.
87:497-498[Medline].
|
| | | | |
Floyd J. Frost
Southwest Center for Managed Care Research
2425 Ridgecrest SW
Albuquerque, New Mexico 87108
|
| | | | |
Gunther F. Craun
Gunther F. Craun and Associates
Professional Building
101 West Ferderick Street
Staunton, Virginia 24401
|
| |
AUTHORS' REPLY |
Frost and Craun question the sensitivity and relevance of the ELISA in
estimating the rate of infection in our volunteer studies and suggest
that immunobloting could offer a more sensitive indicator of infection.
The objectives of our study were to describe the susceptibility to
reinfection as defined by clinical outcomes and oocyst shedding in
volunteers after reexposure to Cryptosporidium parvum and,
secondly, to evaluate in a prospective fashion the serologic responses
to a crude antigen preparation. We believe that to use seroconversion
to define an outcome (infection) would have been inappropriate in this
particular study.
Regarding the relevance of ELISA seropositivity, the proportion of
immunoglobulin G (IgG) seroconversion was low after primary exposure
but increased upon reexposure. This finding has significant epidemiologic implications. In a recent study (1), the 50% infective
dose for volunteers with preexisting antibodies (by ELISA) was found to
found be 20-fold higher than that for antibody-negative volunteers (1).
Thus, the presence of antibodies to C. parvum as determined
by ELISA provides a useful marker for resistance to subsequent low-dose
exposures.
ELISA and immunoblot discrepancies may be due to differences in the
array or conformation of antigens available for binding in each system.
We believe that both methods will continue to be valuable, although the
information that each provides addresses different issues. In
collaboration with colleagues at the Centers for Disease Control and
Prevention, immunoblot studies were conducted with sera collected from
ELISA-negative volunteers prior to challenge (2), and
low-molecular-weight antigens were recognized in 93%. This high
positivity rate in a defined population in the absence of any recent
diarrheal illness or known recent outbreak in the Houston, Tex., area
suggests that Cryptosporidium antigens may contain
cross-reactive epitopes revealed by Western blotting. Furthermore, 17 of 18 volunteers who became infected after oocyst challenge had
prechallenge Western blot reactivity.
In their letter, Frost and Craun report Western blot results conducted
with samples from convalescent patients following a Cryptosporidum outbreak. Their definition of seroconversion
was determined by comparing the intensity of the reaction versus that of a reference as opposed to the same individual over time as was done
in our study. The utilization of Western blotting in a retrospective
fashion in the absence of microbiological data to determine infection
may be fraught with bias, given the high proportion of individuals with
background positivity and the fact that the number of exposures and the
time of last exposure are not known.
Frost and Craun further suggest that some individuals who experienced
asymptomatic infection and had no detectable oocysts may have been
misclassified as uninfected. The definition of C. parvum
infection in our study included clinical and microbiological criteria
to encompass subjects experiencing illness, some of whom may have been
excreting oocysts below the level of detection by direct
immunoflorescence assay. It is possible that a number of individuals in
whom oocytes are not detectable may experience asymptomatic infections.
Clearly, improved detection methods are needed for the diagnosis of
Cryptosporidium infection.
| |
REFERENCES |
| 1.
| Chappell, C. L., P. C. Okhuysen, C. R. Sterling, C. Wang, W. Jakubowski, and H. L. DuPont. Infectivity of
Cryptosporidium parvum in healthy adults with pre-existing
anti-C. parvum IgG. Am. J. Trop. Med. Hyg., in press.
|
| 2.
| Lammie, P. J., C. L. Chappell, D. M. Moss, P. C. Okhuysen, A. W. Hightower, M. J. Arrowood, and H. L. DuPont.
Immunoblot analysis of antibody reactivity from volunteers
experimentally exposed to C. parvum. J. Infect. Dis., in
press.
|
| | | | |
Pablo C. Okhuysen
Cynthia L. Chappell
Division of
Infectious Diseases
The University of Texas Medical School
Houston, Texas
|
| | | | |
Herbert L. DuPont
St. Luke's Episcopal Hospital
Houston, Texas
|
| | | | |
Charles R. Sterling
School of Veterinary Medicine
University of Arizona
Tucson, Arizona
|
| | | | |
Walter Jakubowski
Environmental Protection Agency
Cincinnati, Ohio
|
Infect Immun, August 1998, p. 4008-4009, Vol. 66, No. 8
0019-9567/98/$00.00+0
This article has been cited by other articles:
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Muller, T. B., Frost, F. J., Craun, G. F., Calderon, R. L., Chappell, C. L., Okhuysen, P. C., Dann, S. M.
(2001). Serological Responses to Cryptosporidium Infection. Infect. Immun.
69: 1974-1975
[Full Text]
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Frost, F. J, Muller, T., Craun, G. F, Fraser, D., Thompson, D., Notenboom, R., Calderon, R. L
(2000). Serological analysis of a cryptosporidiosis epidemic. Int J Epidemiol
29: 376-379
[Abstract]
[Full Text]
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