Division of Parasitic Diseases, Centers for
Disease Control and Prevention, Atlanta, Georgia
30341-3724,1 and Department of Pathology
and Laboratory Medicine, University of British Columbia and British
Columbia Centre for Disease Control Laboratory Services, Vancouver,
British Columbia V5Z 4R4, Canada2
Received 3 October 2000/Returned for modification 11 December
2000/Accepted 9 January 2001
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INTRODUCTION |
Cryptosporidium parvum, a
protozoan parasite that invades the intestinal epithelium of a wide
range of mammalian hosts, causes a self-limiting but sometimes severe
diarrheal illness in immunocompetent humans (2). However,
in those with compromised immune systems, the disease can be
debilitating, chronic, and life threatening (4, 23).
Because of its ubiquitous distribution in the environment, its small
size, and its resistance to standard chlorination techniques, C. parvum contamination of drinking water may pose a significant public health risk (1, 11, 12). Numerous outbreaks have been traced to contaminated water, and waterborne outbreaks have even
occurred in communities served by state-of-the-art water treatment
facilities (3, 8, 9, 15). To conduct epidemiologic studies
to assess the risks of C. parvum infection that may be associated with drinking water and other potential sources of exposure,
new serologic assays that are rapid, sensitive, and specific are needed.
Characteristic serum immunoglobulin G (IgG) antibody responses have
been shown to develop in humans after C. parvum infection. As shown by Western blot analysis of serum samples collected from outbreak patients and human volunteers, the antibody response is
consistently directed toward two low-molecular-weight antigen families:
one in the 27-kDa size range and a second in the 17-kDa size range
(16-19, 25, 26). We recently reported the development of
two enzyme-linked immunosorbent assays (ELISAs) for the detection of
antibodies to the 17- and 27-kDa antigens (25). The ELISA for antibodies to the 27-kDa antigen uses a recombinant form of the
antigen based on the gene sequence reported by Perryman et al.
(22), while the assay for antibodies to the 17-kDa antigen uses a Triton X-114 detergent extract of oocysts that is enriched for
the native 17-kDa antigen. These assays were shown to be both sensitive
and specific for the detection of antigen-specific antibodies compared
with the "gold standard" Western blot assay (25).
Unfortunately, in our preliminary analysis of the new ELISAs,
relatively few serial samples from confirmed cryptosporidiosis patients
were available for study.
In the late spring and summer of 1996, four major outbreaks of
cryptosporidiosis occurred in the province of British Columbia in
western Canada: 29 laboratory-confirmed cases were identified in the
city of Cranbrook (population 18,131) in the East Kootenay region of
southeastern British Columbia; 157 laboratory-confirmed cases were
identified in the city of Kelowna (population 89,442) in the Central
Okanagan region of central British Columbia; 86 laboratory-confirmed
cases were identified in Kamloops (population 100,850); and 138 laboratory-confirmed cases were identified in Penticton (population
39,754), also in the Central Okanagan region (21). In
three of these communities (Cranbrook, Kelowna, and Penticton), the
outbreaks were associated with consumption of water from the municipal
supply. From these outbreaks, 67 volunteers with laboratory-confirmed
cryptosporidiosis were recruited and were asked to provide multiple
serum specimens over the course of a 1.5-year study. In this work, we
examine the IgG antibody responses to the 17- and 27-kDa C. parvum antigens that are found in these serial specimens, and we
compare the results obtained with the new ELISAs to those obtained with
two different Western blot assay formats.
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MATERIALS AND METHODS |
Serum specimens.
Serum specimens were collected from 67 adults with laboratory-confirmed cryptosporidiosis who were infected
during waterborne outbreaks in British Columbia in the summer of 1996. All patients also met a case definition for cryptosporidiosis based on
the occurrence of three or more loose or watery bowel movements within a 24-h period. Informed consent was obtained from volunteers by procedures reviewed and approved by the Clinical Screening Committee for Research Involving Human Subjects at the University of British Columbia. Single specimens were obtained from 26 patients, and sequential specimens (207 sera) were collected from 41 patients at
approximately 3-month intervals. The elapsed time between the date of
symptom onset and the date of serum collection was recorded for each of
the samples collected from the 41 repeat donors and for two of the
samples collected from those who donated only once. Five of the
individuals who provided multiple serum samples donated their first
sample within 15 days of symptom onset. All sera were aliquoted and
stored at 
20°C until tested. This study was carried out
retrospectively on serum specimens that were coded without personal identifiers.
Crude antigen preparation and Western blots.
A preparation
of crude antigen was made from oocysts of the Iowa strain of C. parvum by sonication and freeze-thawing as previously described
(20). By using the discontinuous buffer system of Laemmli
(13), proteins from this crude preparation were resolved under nonreducing conditions on either a large-format 10 to 22.5% gradient polyacrylamide gel (dimensions, 160 by 200 by 0.75 mm; 78 µg
of antigen in a 130-mm-long preparative well) (25) or on a
sodium dodecyl sulfate (SDS)-polyacrylamide (15%) minigel (dimensions, 73 by 102 by 0.75 mm; 4 µg of antigen in a 70-mm-long preparative well) (5, 7). The proteins were then
electrotransferred onto polyvinylidene difluoride (PVDF) membranes
(Immobilon P; Millipore Corp., Bedford, Mass.).
The large-format Western blot assay was conducted in the Atlanta
laboratory by the method of Priest et al. (25), while the minigel-format blot assay was conducted in the British Columbia laboratory by the method of Frost et al. (5, 7). Briefly, the membrane from the large-format gel was cut into approximately 60 2-mm-wide strips that were then incubated overnight at 4°C with 2 ml
of a 1:100 dilution of serum in phosphate-buffered saline (PBS) (0.85%
NaCl and 10 mM Na2PO4 at pH
7.2) containing 0.3% Tween 20. One strip on each tray was incubated
with a known positive control serum, and one strip was incubated in
buffer only. Bound IgG antibodies were visualized with a biotin-labeled
mouse monoclonal anti-human IgG antibody (1:1,000 dilution in 0.3%
Tween 20-PBS for 1 h at room temperature) (clone HP6017; Zymed
Laboratories, South San Francisco, Calif.) and streptavidin-labeled
alkaline phosphatase (1:1,000 dilution in 0.3% Tween 20-PBS for
1 h at room temperature) (Life Technologies, Rockville, Md.) as
previously described (25). Nitro blue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate were used as chromogens.
The membrane from the minigel was placed into a 20-channel Bio-Rad Mini
Protean II Multiscreen apparatus (Bio-Rad, Hercules, Calif.), and the
membrane contained in the channels was incubated overnight at 4°C
with 400 µl of a 1:50 dilution of serum in Tween 20-PBS or with a
reagent blank. Two of the channels on each minigel blot were incubated
with a known positive control serum, and one channel was incubated with
a negative control serum. Bound IgG antibodies were visualized with a
biotin-labeled mouse monoclonal anti-human IgG antibody (clone HP6017)
(1:500 dilution in 0.3% Tween 20-PBS for 30 min at room temperature)
and streptavidin-labeled alkaline phosphatase (1:1,000 dilution in
0.3% Tween 20-PBS for 30 min at room temperature). Membranes were
developed with the chromogen described above.
Both the large gel and minigel blots were read independently by members
from the British Columbia and Atlanta laboratory groups, and a
laboratory consensus was reached for each format for the presence or
absence of antibodies to the 17- and 27-kDa antigens. Blots were only
considered positive if the positions, patterns, and relative
intensities of the bands in the 27- and 17-kDa regions corresponded
with those found in the positive control. In cases in which a consensus
could not be reached among the readers in a laboratory group, the assay
was repeated. If a consensus still could not be reached, the blot
result for that sample was considered negative for purposes of
analysis. An overall consensus for the presence or absence of
antibodies in each sample was determined from the British Columbia and
Atlanta laboratory interpretations of the large-gel-format and
minigel-format blots. For a serum sample to be considered positive for
antibody to one of the antigens, at least two of the four laboratory
consensus blot results for that antigen had to be positive.
ELISAs.
Serologic assays for IgG antibodies against the 27- and 17-kDa C. parvum antigens used either a recombinant form
of the 27-kDa antigen (Cp23) or a partially purified native antigen
fraction isolated from oocysts by Triton X-114 detergent extraction
(Triton antigen), respectively (25). The Triton antigen
fraction has been shown to be greatly enriched for the 17-kDa antigen.
Both assays were performed in Atlanta and British Columbia as
previously described (25). Briefly, antigens were diluted
in 0.1 M NaHCO3 buffer at pH 9.6 to
concentrations of 0.2 µg/ml (Cp23) and 0.28 µg/ml (Triton antigen)
and were used to coat 96-well plates overnight at 4°C (50 µl/well;
Immunlon 2; Dynatech Industries, McLean, Va.). Plates were washed with
0.3% Tween 20-PBS for 1 h at 4°C and then washed four times
with 0.05% Tween 20-PBS. Sera from the British Columbia outbreak
patients were diluted 1:50 in 0.05% Tween 20-PBS, loaded in duplicate
(50 µl/well), and incubated for 2 h at room temperature. Serial
samples collected from individual patients were assayed on the same
plate. Three positive control sera, two negative control sera, and two
buffer blanks were run in duplicate on each plate. A twofold serial
dilution (1:50 to 1:12,800) of a strong positive control was also
included on each plate to be able to generate a standard curve. The
bound antibodies were quantitated with a biotinylated mouse monoclonal
antibody against human IgG (1:1,000 in 0.05% Tween 20-PBS) (clone
HP6017) and alkaline phosphatase-labeled streptavidin (1:500 in 0.05%
Tween 20-PBS) with p-nitrophenylphosphate substrate (Sigma
Chemical Co., St. Louis, Mo.) as previously described (25). A405s were measured
with a Molecular Devices (Sunnyvale, Calif.) UVmax kinetic microplate reader.
Antibody levels of the unknown samples (from the mean of duplicate
wells) were assigned a unit value based upon the nine-point positive
control standard curve with a four-parameter curve fit and were
expressed per microliter of serum. The 1:50 dilution of the positive
control serum was arbitrarily assigned a value of 6,400 U. Samples that
were found to have values above 6,400 arbitrary units (AU) were further
diluted and reassayed until they fell within the range of the curve
values. If the standard deviation was greater than or equal to 15% of
the mean value for the duplicate wells, the assay for that sample was
repeated (unless both values were considered negative). Cutoff values
were determined by using a relative operating characteristic curve to
maximize the sensitivity and specificity of the ELISAs compared with
those of the Western blot consensus. A positive ELISA response for
antibodies to the 27-kDa antigen was defined as one above 86 AU with
the recombinant Cp23 protein, and a positive ELISA response for
antibodies to the 17-kDa antigen was defined as one above 35 AU with
the Triton-extracted antigen. When applied to an unrelated set of 220 serum samples, these cutoff values compared favorably with the Western
blot results when the same standard curve was used (sensitivity and
specificity of >87% for the Triton antigen assay and >89% for the
Cp23 antigen assay) (J. W. Priest, B. W. Furness, and P. J. Lammie, unpublished observations).
All samples were assayed on three separate occasions with freshly made
serum dilutions: twice in the Atlanta laboratory (2 months apart) and
once in the British Columbia laboratory. During one of the assay runs
in the Atlanta laboratory, the duplicate serum dilutions were placed in
random locations on the ELISA plate to eliminate the possibility of
bias as a result of the use of adjacent wells. Samples were considered
positive for antibodies to the 17- and 27-kDa antigens if at least two
out of three of the respective ELISA values were above the threshold.
Statistical analysis.
The sensitivities and specificities of
the ELISAs were calculated from a relative operating characteristic
curve by using the consensus Western blot result as the gold standard.
Spearman's rank order correlation coefficients for the comparison of
assay results were calculated with SigmaStat for Windows, version 2.03 (SPSS, Inc.).
Antibody half-lives were calculated by probit analysis of the
cumulative percent decrease in antibody levels when the number of
nonzero responses was sufficient to produce 95% fiduciary limits. When
samples were limited to two nonzero decreasing antibody responses, the
half-life was calculated by interpolating the number of days after
onset at which response decreased by 50%. Antibody responses among the
different ELISAs during specified intervals after exposure were
compared by the Kruskal-Wallis test. Unless otherwise stated, all
statistical procedures were performed with SAS version 8 statistical software (SAS Institute, Inc., Cary, N.C.).
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RESULTS |
Western blot analysis.
Sera from 67 laboratory-confirmed
cryptosporidiosis patients were assayed for antibodies to the 27- and
17-kDa sporozoite surface antigens with the large-format Western blot,
and a consensus interpretation of the blot results was reached by the
Atlanta laboratory group. Of the 26 patients who donated only a single serum specimen, 24 (92%) were positive for antibodies against both
antigens by large-format Western blot (data not shown). One of the two
patients who lacked antibodies had a serum collection date less than 15 days after symptom onset and therefore may not have had sufficient time
to develop an antibody response. The symptom onset date for the other
negative patient was not known. Of the 41 patients who donated more
than one serum specimen, all were positive for antibodies to one or
both antigens at some point during the first 183 days of the study. As
represented by the large-format Western blots shown in Fig.
1, the 41 patients who donated more than
one serum specimen could be grouped into five general categories.
Figure 1A (patient 1) is representative of the 21 patients (51% of
those who donated more than one specimen) who were positive for
antibodies to both the 27- and 17-kDa antigens at each time point. A
gradual decline over time in the total antibody response was apparent
from the intensities of the bands on the blots. Of the five patients
(12%) who donated their first serum sample 
15 days after symptom
onset, all were initially positive for antibodies to the 27-kDa
antigen, but negative for antibodies to the 17-kDa antigen, and all had
a detectable peak antibody response to the 17-kDa antigen by the second
time point (3 to 4 months later) (represented by patient 2 in Fig. 1B).
Ten patients (24%) represented by patient 3 in Fig. 1C were initially
positive for antibodies to both antigens, but later became negative for one of the antibodies. Three patients (7%) who had an antibody response to at least one of the antigens later became completely negative (represented by patient 4 in Fig. 1D). Finally, Fig. 1E
(patient 5) is representative of two patients (5%) who were positive
for antibodies to the 27-kDa antigen at every time point, but who did
not have a detectable response to the 17-kDa antigen in any of the
specimens tested (average number of tested specimens per patient, 6).
No obvious changes were noted in the intensities of the blot responses
to the 27-kDa antigen for these two patients. In summary, changes in
the blot responses that were consistent with recent exposure to
C. parvum were observed for 39 of the 41 patients (95%) who
donated multiple serum samples.
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FIG. 1.
Large-gel-format Western blots of sera collected at
intervals from five cryptosporidiosis patients. Crude C.
parvum antigens (600 ng of protein/mm of gel width) were
resolved on an SDS-polyacrylamide (10 to 22.5%) gel under nonreducing
conditions and then transferred to a PVDF membrane. Sera that had been
collected from five laboratory-confirmed cryptosporidiosis patients
(patients 1 to 5 represented in panels A to E, respectively) at
intervals of approximately 3 months were diluted 1:100 with 0.3% Tween
20-PBS and incubated (2 ml per well) with 2-mm-wide strips of membrane
overnight at 4°C. Seven serum samples were donated by patients 1 and
2 (Western blots shown in temporal order in lanes 1 to 7 of panels A
and B, respectively), and six serum samples were donated by patients 3, 4, and 5 (Western blots shown in temporal order in lanes 1 to 6 of
panels C, D, and E, respectively). The first sample in each panel
(Western blots shown in lane 1) was collected within the first 3 months
(A), 15 days (B), 3 months (C), and 1 month (D and E) after symptom
onset. The blots were developed with a biotinylated mouse anti-human
IgG monoclonal antibody and alkaline phosphatase-labeled streptavidin
as described in Materials and Methods. The positions of the 27- and
17-kDa antigen families are indicated. Control strips that were
incubated with a positive serum (P) and a buffer blank (B) are shown in
panel F.
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When the sera from the outbreak patients were assayed with the minigel
Western blot format, we observed that some of samples previously
determined to be positive for antibodies to the 27-kDa antigen when the
large-format Western blot was used had responses that appeared to be
negative or were very weak and difficult to interpret. The minigel blot
results for patients 1 through 3, who were shown in Fig. 1 to have
strong antibody responses to the 27-kDa antigen, are presented in Fig.
2. While the 27-kDa antigen responses in
samples from patient 1 were clearly positive by minigel blot (Fig. 2A),
samples 1 and 7 from patient 2 (Fig. 2B) and samples 2 to 6 from
patient 3 (Fig. 2C) were interpreted as negative by both laboratories.
This apparent lack of sensitivity was not limited to these two
patients: 22 patients had at least one sample in which this discrepancy
was observed. Overall, of the 209 samples that were interpreted by both
laboratories as positive for antibodies to the 27-kDa antigen by using
the large gel blot, 61 were considered negative by both laboratories
with the minigel blot. This suggests a sensitivity of the
minigel-format blot of only 71% for the detection of antibodies to the
27-kDa antigen compared with that of the large-gel-blot format. In
contrast, the minigel blot results for antibodies to the 17-kDa antigen for the three patients shown in Fig. 2 and for most of the other patients were in good agreement with large-gel-blot results. Of the 196 samples that were considered positive for antibodies to the 17-kDa
antigen by both laboratories using one blot format, only 11 (6%) were
considered negative by both laboratories with the other blot format.
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FIG. 2.
Minigel Western blot results for sera collected at
intervals from cryptosporidiosis patients 1, 2, and 3. Crude C.
parvum antigens (57 ng of protein/mm of gel width) were
resolved under nonreducing conditions on an SDS-polyacrylamide (15%)
minigel and transferred to a PVDF membrane. Sera from patients 1 (A), 2 (B), and 3 (C) were diluted 1:50 with 0.3% Tween 20-PBS and incubated
(400 µl per channel) with separate regions of the membrane overnight
at 4°C. Blots were developed as described in Materials and Methods.
The positions of the 27- and 17-kDa antigens are indicated. Panel C was
scanned and digitally enhanced to improve the resolution of the 27-kDa
band in lane 1. Control strips that were incubated with a positive
serum (P) and a buffer blank (B) are shown in panel D.
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As described in Materials and Methods, an overall blot consensus was
reached based upon the two laboratories' interpretations of the
responses on the large and minigel format blots. In general, there was
good agreement between the two laboratories in the interpretation of
the blot results. Eighty-seven percent of the large-format Western
blots were assigned the same response for the 17-kDa antigen by both
laboratories, and 95% of the blots were assigned the same response by
both laboratories for the 27-kDa antigen. Similarly, the two
laboratories were in agreement for 96% of the 17-kDa antigen responses
on the minigel blots and for 90% of the 27-kDa antigen responses.
Table 1 summarizes the overall consensus
blot results for the antibody status of the serum samples. Overall, 210 of 233 samples (90%) were positive for antibodies to the 27-kDa
antigen (24 from donors who provided only one specimen and 186 from
donors who provided sequential specimens), and 175 samples (75%) were positive for antibodies to the 17-kDa antigen (24 from single specimens
and 151 from sequential specimens). Of the 41 patients who donated more
than one serum specimen, 38 (93%) were positive by consensus for
antibodies to the 17-kDa antigen, and 40 (98%) were positive by
consensus for antibodies to the 27-kDa antigen (Table 1). A total of 37 patients (90%) were simultaneously positive for antibodies to both
antigens on at least one occasion, and 11 of these became negative for
antibodies to either the 17-kDa antigen (n = 8), the
27-kDa antigen (n = 1), or both antigens (n = 2) during the course of the study. The consensus
blot responses were used as the gold standard for comparisons with the
ELISA results presented below.
ELISA results.
To assess the inter- and intralaboratory
variability in the assays, the levels of antibodies to the 27- and
17-kDa antigens were analyzed independently by ELISA once in the
British Columbia laboratory and twice in the Atlanta laboratory. In one
of the sets of assays conducted in the Atlanta laboratory, the samples were placed in random wells on the ELISA plate so as eliminate the
possibility of positional bias. The mean ELISA responses for the
patients whose blot responses were shown previously in Fig. 1B and C
are shown in Fig. 3A and B, respectively.
As expected from the positive 27-kDa antigen blot responses in Fig. 1B,
the mean ELISA responses for the Cp23 assay (Fig. 3A, dashed line) were
consistently above the 86-AU cutoff value, and except in the first
sample (which was negative for antibodies to the 17-kDa antigen by
blot), the mean responses for the Triton antigen assay (solid line)
were above the 35-AU cutoff value. Similarly, the mean ELISA responses
for patient 3 (blot responses shown in Fig. 1C) were consistently
positive for the Cp23 assay (Fig. 3B, dashed line), and the response
was positive for the initial sample by Triton antigen assay (solid
line), in good agreement with the blot results. The results of the
three sets of assays for all of the patient samples were also in good
agreement with each other: the intralaboratory correlation coefficients
for the Cp23 and Triton antigen ELISAs calculated for all 233 samples
were 0.978 and 0.913, respectively, and the interlaboratory correlation
coefficients were >0.969 and >0.924, respectively. All three assays
gave the same result (positive or negative) for 94% of the Cp23 assays and 88% of the Triton antigen assays.
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FIG. 3.
Mean Triton antigen and Cp23 ELISA responses for sera
collected at intervals from cryptosporidiosis patients 2 and 3. Sera
from cryptosporidiosis patients 2 (A) and 3 (B) were assayed
independently in Atlanta (twice) and in British Columbia (once) with
the Triton antigen and Cp23 ELISAs described in Materials and Methods.
The optical densities were converted into an AU value based upon a
9-point standard curve that was included on each ELISA plate. The means
of the ELISA values for each time point after symptom onset are plotted
for both the Triton antigen ELISAs (solid line) and the Cp23
ELISAs (dashed line). Each bar indicates one standard deviation.
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As summarized in Table 1, the agreement between the consensus Cp23
ELISA results and the consensus Western blot results was excellent for
both the single and sequential specimens. Compared with the blot, the
Cp23 ELISA was 100% sensitive and specific for single specimens and
was 92% sensitive and 100% specific for sequential specimens.
Similarly, the Triton antigen ELISA response for the single specimens
correlated well with the 17-kDa antigen Western blot response
(sensitivity of 96% and specificity of 100%). However, the Triton
antigen ELISA response for the sequential specimens correctly
identified only 74% of the Western blot-positive samples and 87% of
the negative samples
somewhat below the 90% sensitivity and 94%
specificity we described for this assay in an earlier work
(25). The difference may be related to the use of
sequential specimens in the current study. The sensitivity of the
Triton antigen ELISA was higher for samples collected between 16 and 92 days after symptom onset (96%) than for samples collected in any
subsequent 3-month interval (range, 81 to 59%) (data not shown). In
addition, many of the false-negative specimens (14 of 40; 35%) were
donated by the 10 patients who, by overall blot consensus, converted
from positive to negative by Western blot for antibodies to the 17-kDa
antigen, and they were followed in sequence by a true blot-negative
sample. Thus the low sensitivity may be attributed to a response that
rapidly approached the limit of detection for the assay. The low
specificity (87%) may be attributed to the fact that six of the seven
false-positive serum specimens were donated by a single patient.
As can be seen in the graphs in Fig. 3, the antibody responses to the
Cp23 and Triton antigens observed in patients 2 and 3 tended to rise
and decay in parallel, and much of the response was lost within the
first year after symptom onset. The same downward trends that were
observed in the individual patient responses were also apparent when
the responses from the 41 patients who donated multiple samples were
plotted versus the time after symptom onset. Figure
4A shows the number of samples collected
in each 3-month interval after symptom onset. The five samples that
were collected within 15 days of symptom onset were grouped separately because they were collected before these patients had developed an
antibody response to the 17-kDa antigen. As shown in Fig. 4B and C,
when the geometric means for the Triton antigen and Cp23 ELISA
responses were calculated for each time interval after symptom onset, a
steady decline after the 16- to 92-day interval was shown. Figure 4B
and C also give a visual indication of the low levels of intra- and
interlaboratory variation that were observed among the three
independent sets of assays. Although the repeat set of assays performed
in Atlanta yielded a higher geometric mean value for six of nine time
intervals for the Triton antigen assay (open circles, Fig. 4B), a
statistical comparison by time interval of the two sets of responses
from Atlanta and the set from British Columbia did not reveal a
significant difference. The P values for the Triton antigen
ELISA response comparisons were greater than 0.09 for each of the
sample collection intervals (Kruskal-Wallis test with chi-square
approximation), and the P values for the Cp23 ELISA response
comparisons were greater than 0.53 for each of the sample collection
intervals.
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FIG. 4.
ELISA analysis of sera from cryptosporidiosis patients
who donated sera on more than one occasion. Panel A shows the total
number of serum samples collected during each time interval after
symptom onset from 41 confirmed cryptosporidiosis patients who donated
more than one specimen. Triton antigen (B) and Cp23 (C) ELISAs were
performed with these serum samples on two occasions in Atlanta to
assess the intralaboratory variation (Atlanta, random and Atlanta,
repeat) and on one occasion in British Columbia to assess the
interlaboratory variation (British Columbia). For the assays
represented by the "Atlanta, random" results, the duplicate serum
samples were placed in random locations on the ELISA plate to eliminate
the possibility of positional bias. The geometric means (log scale) of
the ELISA responses are plotted for each interval of sample collection,
and the threshold for positivity is indicated in each graph by a dotted
line. Panel D shows the fraction of the samples from each time interval
positive for antibodies in the Triton antigen (black bars) and Cp23
(grey bars) ELISAs. A serum sample was considered ELISA positive if at
least two of the responses from the three independent assays were above
35 AU for the Triton antigen assay and above 86 AU for the Cp23
assay.
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The prevalence of the samples that were positive by Triton antigen
ELISA and Cp23 ELISA versus the time interval of sample collection is
shown in Fig. 4D. The percentage of the patients who were positive by
the Triton antigen ELISA demonstrated a downward trend consistent with
the conversion of some patients from positive to negative for
antibodies to the 17-kDa antigen by Western blot, while the percentage
of patients who were positive by the Cp23 ELISA was relatively
constant, as expected from the Western blot results for the 27-kDa
antigen described earlier.
Antibody half-life determination.
A sufficient number of
nonzero Cp23 ELISA responses were available from 25 patients to allow
the calculation of the antibody half-lives by probit analysis. A mean
half-life of 87 days (median, 72 days; range, 50 to 181 days) was
obtained for Cp23 reactivity. From the responses of 15 patients, a mean
half-life of 84 days (median, 69 days; range, 47 to 215 days) was
estimated for the Triton antigen response. The half-lives for the two
antibody responses were not significantly different (P = 0.514). For those patients' responses that could not be analyzed by
the probit procedure, a half-life estimate was calculated assuming a
linear rate of decay. These estimates (mean of 83 days for the Cp23
assay with 10 patients; mean of 68 days for the Triton antigen assay
with 17 patients) did not differ significantly from those described above.
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DISCUSSION |
Several different assay formats have been used to assess the
levels of human serum IgG antibodies to C. parvum antigens:
an ELISA that uses a crude antigen preparation (29), a
large-gel-format Western blot that requires the separation of parasite
antigens on a gradient SDS-polyacrylamide gel (6, 17-19,
25), a minigel-format Western blot (5, 7, 10), and
ELISAs that use purified recombinant or native antigens
(25). A number of laboratories have noted that the results
of the large-gel-format Western blot and those of the crude antigen
ELISA were not well correlated (6,18, 19, 25, 28; F. J. Frost and G. F. Craun, Letter, Infect. Immun.
66:4008-4009, 1998). In general, the large-gel-format Western blot appeared to be more sensitive than the crude antigen ELISA
for the detection of IgG antibodies: more than half of the crude
antigen ELISA-negative individuals in the study of Frost et al.
(5; Frost and Craun, Letter) were found to be antibody positive by the large-gel-format Western blot. Unfortunately, because
of its complexity and expense, the large-gel-format Western blot is not
suitable for use in large-scale epidemiologic studies of the prevalence
of C. parvum antibodies in the general population. To
accomplish this type of work in a more reagent- and cost-efficient manner, several groups have turned to the minigel-format Western blot
(5, 7,10). The assay as described by Frost uses
10-fold-less antigen per millimeter of PVDF membrane than the
large-format blot assay originally developed in our laboratory
(17, 18, 25). While the minigel assay compared favorably
in our hands with the large-gel-format blot for the detection of
antibodies to the 17-kDa antigen, it failed to detect 29% of the
samples that were positive for antibodies to the 27-kDa antigen. Recent studies have suggested that roughly one-third to one-half of those individuals who are positive for antibodies by the large-gel Western blot assay only have an antibody response to the 27-kDa antigen (6, 25). Thus the minigel-format blot (at least in the
format currently used) may significantly underestimate the proportion of the population with prior Cryptosporidium parvum
exposure. No data yet exist on whether the minigel assay would perform
better if more antigen was used.
Using a large collection of serial samples from laboratory-confirmed
cryptosporidiosis patients, we have demonstrated that the recombinant
Cp23 and Triton antigen ELISAs can be used to monitor changes in the
antibody responses to two specific C. parvum surface
antigens. The high sensitivities and specificities of the Cp23 and
Triton antigen ELISAs in the analysis of the single specimens (>96%)
and those of the Cp23 ELISA in the analysis of the sequential specimens
(>93%) are similar to what we have previously reported
(25), but the Triton antigen results are somewhat lower for the sequential specimens than previously reported (74 and 87%
versus 90 and 94%, respectively). The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days
of symptom onset (96%) than in subsequent samples, and many of the
false-negative samples were donated by patients who eventually
converted to negative for antibodies to the 17-kDa antigen by Western
blot. We think the lower sensitivity is simply an artifact caused by
the repeat sampling of patients who have borderline-positive Western
blot responses in samples collected many months after the clearance of
the infection. Similarly, the lower specificity of the assay can be
attributed to one patient who donated six of seven false-positive
specimens. Interestingly, this patient had a very high level of
antibodies to the 27-kDa antigen, and a small amount of 27-kDa antigen
is normally found in the Triton extract used in the assay (the amount
of 27-kDa antigen is 10- to 20-fold lower than that of the 17-kDa
antigen) (25). When these sera were checked by Western
blotting with Triton-extracted antigen, only the 27-kDa antigen was
apparent (data not shown). No other bands were visible. Thus, it is
possible that the Triton antigen ELISA is not absolutely specific for
antibodies to the 17-kDa antigen when high concentrations of antibodies
to the 27-kDa antigen are present. In practice, however, very few patients have been found with a high 27-kDa-antigen response and no
17-kDa-antigen response.
We noted that the antibody response to both antigens increased and
decreased in parallel in a time frame consistent with C. parvum infection near the time of symptom onset. The peaks of the
responses most likely occurred between 16 and 92 days of symptom onset,
but, because of the 3-month interval between sample collections, they
could not be further pinpointed. In a recent study in which human
volunteers were fed C. parvum oocysts, the peak of the
response most likely occurred between 11 and 32 days after ingestion of oocysts (19). We conclude that it is highly unlikely that
the antibody responses observed in our study could have resulted from cross-reaction with antibodies from a concurrent or previous infection with another organism. This conclusion is further supported by the
observation that most of the antibody response to these two antigens is
directed against the protein component of the antigen rather than
against a carbohydrate component (24, 25; J. W. Priest and P. J. Lammie, unpublished observations). Robbins et al.
(27) have suggested that serum antibodies to the
carbohydrate epitopes of surface antigens, especially those whose
acquisition is age-related, may result from exposure to cross-reacting
species found on normal enteric and respiratory flora, while serum
antibodies that recognize surface protein epitopes are most often
elicited by infection with the specific pathogen. We believe that the
Cp23 and Triton antigen ELISAs are specific for C. parvum
infection in humans, and despite our efforts and those of other
laboratories, no evidence of cross-reaction has been found to implicate
other human parasites, including Toxoplasma gondii,
Giardia lamblia, and Isospora sp. (14,
29; Priest and Lammie, unpublished).
Our results indicate that antibodies against both the 17- and 27-kDa
antigens are cleared at about the same rate, with half-lives of
approximately 12 weeks each, and that both antibody responses reach a
fairly constant level about 1 year after the infection. This working
estimate will be important for future studies of the prevalence of
cryptosporidiosis in the population. To our knowledge, only one other
group has attempted to establish an estimate of the duration of the
human serum antibody response following Cryptosporidium
infection. In a recent work, Frost et al. (5) collected
paired serum samples from cryptosporidiosis patients from a waterborne
outbreak in Jackson County, Oreg., at 6 months and then again at 2.5 years after the end of the outbreak and analyzed these samples by using
the large-gel- and minigel-format Western blot assays. They observed
that the antibody response to the 27-kDa antigen declined by 46% (as
measured from the blots by densitometry) over this 2-year period, while
the response to the 17-kDa antigens declined by only 9%. Their
conclusions that the response to the 17-kDa antigen may have declined
to baseline levels before the beginning of the serum collection and
that the antibody response to the 27-kDa antigen may remain high for an extended period of time appear to be supported by our results. Indeed,
had sera been collected from the patients in our study 6 months after
symptom onset (184 to 274 days) and again 1 year later (548 to 638 days) (Fig. 4), a pattern similar to that reported by Frost et al.
would likely have been observed, since the largest fluctuations in the
antibody responses appear to occur within the first 6 months after
symptom onset.
Our work supports the suggestion that the postinfection level of the
antibody response is higher (relative to the threshold of antibody
detection) for the 27-kDa antigen than for the 17-kDa antigen: eight
patients with a persistent 27-kDa antigen response became negative by
overall blot consensus for antibodies to the 17-kDa antigen, while only
one patient with a persistent 17-kDa antigen response became negative
for antibodies to the 27-kDa antigen over the course of the study. The
geometric means of the responses to the 27-kDa antigen in the various
time intervals of our study were consistently above the cutoff
threshold, but this was not the case for the responses to the 17-kDa
antigen (Fig. 4). We also observed that the five patients who provided a serum sample <15 days after symptom onset either had a very early
27-kDa antigen response or had a preexisting 27-kDa antigen response
from a previous infection, since no response to the 17-kDa antigen was
apparent until the following time point. In our studies of sera
collected from individuals with no known previous exposure to
Cryptosporidium, we have, in fact, observed that a
significant proportion of the population has an antibody response only
to the 27-kDa antigen (25; Priest and Lammie,
unpublished). We do not yet understand why the response to the 27-kDa
antigen should persist, but it may in some way be related to repeated
exposure to the parasite.
In conclusion, we have demonstrated that the Triton antigen and Cp23
ELISAs are useful for monitoring the changes in antibody responses
associated with infection with C. parvum and that this technology can be transferred to other laboratories. Given that antibodies to the 27-kDa antigen may persist above the limit of detection, we believe that the Cp23 ELISA may be better suited to the
assessment of historic exposure to Cryptosporidium, while the Triton antigen ELISA may be a better choice for the detection of
recent infections. For both assays, a high antibody response is likely
to be indicative of a recent infection.
We thank Delynn Moss for help with the preparation of the antigen
used in this work and James Kwon for assistance in the interpretation of blot strips.
The University of British Columbia gratefully acknowledges that the
American Water Works Association Research Foundation is the joint owner
of some of the technical information upon which this is work is based.
The University of British Columbia thanks the foundation for its
financial, technical, and administrative assistance in funding and
managing the project through which this work was discovered.
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Abstract
Introduction
