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Infection and Immunity, December 2003, p. 6899-6905, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.6899-6905.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Intestinal Antilectin Immunoglobulin A Antibody Response and Immunity to Entamoeba dispar Infection following Cure of Amebic Liver Abscess
Jonathan I. Ravdin,1* Mohamed D. Abd-Alla,1 Seth L. Welles,2 Selvan Reddy,3 and Terry F. H. G. Jackson3
Department
of Medicine,1
School of Public
Health, University of Minnesota, Minneapolis, Minnesota
55455,2
South Africa Medical Research
Council (NATAL), Durban-Overport 4067, South
Africa3
Received 11 June 2003/
Returned for modification 16 July 2003/
Accepted 10 September 2003
 |
ABSTRACT
|
|---|
We
followed 93 subjects with amebic liver abscess (ALA) and 963 close
associate controls at 3-month intervals for 36 months to characterize
intestinal and humoral antibody responses to the amebic
galactose-inhibitable lectin and to determine whether immunity
developed to Entamoeba histolytica or Entamoeba
dispar infection following cure of ALA. We found that ALA subjects
had a higher prevalence and level of intestinal antilectin
immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant
lectin protein) IgA and IgG antibodies, P < 0.01 and
P < 0.05, respectively, compared to controls. The
intestinal antilectin IgA antibody response was sustained over a longer
time period in ALA subjects (71.8% remained positive at 18
months and 52.6% at 36 months, P < 0.001
compared to 17.6% and 10.3% of controls, respectively).
ALA subjects were highly immune to E. dispar infection
throughout the study (0% infected at 6 and 36 months, compared
to 6.5% and 4.9% of control subjects, respectively,
P < 0.05). Upon entry into the study, 6.3% of
ALA subjects were infected with E. histolytica; the incidence
of new E. histolytica infections in controls (as determined by
culture) was too low (1.4%) to determine whether ALA subjects
exhibited immunity to new infections. We found that stool cultures
every 3 months markedly underestimated the occurrence of new E.
histolytica infections, as 15.3% of controls seroconverted
after 12 months of follow-up. Unfortunately, under the field conditions
present in Durban, South Africa, enzyme-linked immunosorbent assay for
detection of lectin antigen in stool yielded unreliable results. In
summary, subjects cured of ALA exhibited sustained mucosal IgA antibody
responses to the amebic galactose-inhibitable lectin and a high level
of immunity to E. dispar infection. Determination of immunity
to E. histolytica following cure of ALA will require the use
of more sensitive and reliable diagnostic
methods.
|
INTRODUCTION
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|---|
One of the major questions in amebiasis research is whether cure of
invasive disease is followed by development of immunity to new
intestinal infections and, thus, recurrence of disease. The enteric
protozoan Entamoeba histolytica is one of the leading
parasitic causes of death worldwide. Disease results from the
parasite's ability to invade the colon, causing amebic colitis, or
spreading via the portal venous system to the liver, resulting in
formation of an amebic liver abscess (ALA). Amebic liver abscesses are
more common in adult men and were thought to be fatal if untreated
(7). A recent study in
Hue, Vietnam, revealed that ALA is even more common than previously
realized and may occur frequently in a subclinical manner
(10). One large
noncontrolled study reported that the rate of recurrence of amebic
liver abscesses over 5 years in a high-risk population was less than
expected compared to historical controls
(14). In a
cross-sectional study, the point prevalence of Entamoeba
species intestinal infection was lower in subjects who possessed serum
antiamebic antibodies
(13).
The E.
histolytica galactose-inhibitable lectin
(12,
22,
26,
27) appears to have a
crucial role in colonization of the gut and parasite invasion. The
lectin mediates attachment of E. histolytica trophozoites to
colonic mucins (11,
12), host epithelial
cells and immune effector cells
(22,
30).
Galactose-inhibitable lectin binding is an absolute requirement for
trophozoites to exhibit a lytic effect on host cells
(25). The purified lectin
in native and recombinant forms is a highly conserved antigen. In over
95% of samples obtained from hundreds of patients cured of
amebic colitis or liver abscess studied worldwide, native lectin
protein purified from a single cloned E. histolytica isolate
is recognized by serum immunoglobulin G (IgG), IgM, and IgA antibodies
(1,
3,
5,
6,
21,
32). The same has been
found from subjects with noninvasive asymptomatic E.
histolytica intestinal infection
(28,
31).
Monoclonal
antibodies raised to the lectin's carbohydrate-binding domain
completely inhibit parasite binding to colonic mucins in vitro
(11,
12), suggesting that
intestinal antilectin IgA antibodies could prevent parasite
colonization of the gut. In a prospective follow-up study of children
in Bangladesh, there was a delay in the onset of E.
histolytica intestinal infections when intestinal antilectin IgA
antibodies were present
(17). The lectin in
native and recombinant form has been demonstrated to be efficacious as
a subunit vaccine in the gerbil model of amebic liver abscess
(24,
32).
In Durban,
South Africa, E. histolytica and Entamoeba dispar
infections are highly endemic
(16,
20). E. dispar
is a distinct species that is morphologically identical to E.
histolytica but is not known to cause disease
(15). E. dispar
trophozoites possess functional galactose-binding lectin molecules that
are 85% homologous with the E. histolytica
lectin (25) and have many
common epitopes as determined by studies with murine monoclonal
antibodies raised to the E. histolytica lectin
(23). The purpose of our
study was to characterize over time the human mucosal and humoral
antilectin antibody responses and to determine whether intestinal
immunity to infection exists following cure of invasive amebiasis.
These findings provide information that is crucial for the development
of an effective lectin-based amebiasis subunit vaccine.
We
conducted a prospective cohort study of 93 subjects treated for ALA and
963 controls who were family members or closely associated neighbors.
All subjects were enrolled prospectively and followed for at least 36
months. The demographics, risk factors for infection by
Entamoeba species, and prevalence of infection with other
intestinal parasites will be reported
elsewhere.
|
MATERIALS AND
METHODS
|
|---|
Subject recruitment and study
enrollment.
Subjects with
ALA were recruited at King Edward VIII Hospital and other regional
hospitals and clinics in the area around Durban, South Africa. Nurses
fluent in Zulu obtained informed consent in English or Zulu. Control
subjects included nuclear family members, individuals residing in the
same household, and close neighbors living in the same environment.
Control subjects were recruited by study nurses through contact with
the index case; we sought at least 10 close associate controls for each
index case. No criteria for age or gender were applied except age
16 years. All subjects provided blood by venipuncture, feces,
and throat washings at entry into the study (1 week after commencing
treatment of the ALA index case) and at 3-month intervals for a total
of at least 36 months of follow-up. Over the duration of the study,
only seven of the 100 family groups were lost to follow-up. At the
first visit, study nurses filled out a detailed epidemiologic
questionnaire based on an oral history obtained from each of the
subjects enrolled in the study. The University of Minnesota and
University of Natal's institutional review boards for human
subjects approved the consent form, questionnaire, and all aspects of
the study.
Assays performed included fecal microscopy, stool
culture, and zymodeme determination for E. histolytica or
E. dispar, enzyme-linked immunosorbent assay (ELISA) for serum
anti-LC3 (recombinant cysteine-rich section of the lectin heavy
subunit) (32) IgA and IgG
antibodies, ELISA for fecal antilectin and anti-LC3 IgA antibodies, and
a monoclonal antibody-based antigen capture ELISA to detect E.
histolytica- and E. dispar-specific lectin antigen in
feces (1,
2,
3,
5,
6).
Stool
culture.
Fecal samples were
cultured in Robinson's medium
(29) for detection of
E. histolytica and E. dispar parasites. Primary
cultures were performed by adding a small piece of fecal material to a
Bijoux bottle containing an agar slope, to which was added 10 mg of
starch, 4 drops of 20% erythromycin, and 10 ml of BR medium
(Escherichia coli strain B incubated in R medium for
48 h at 37°C). Stock R medium contains 125
g of NaCl, 50 g of citric acid, 12 g of
KH2 PO4, 12.4 g of ammonium sulfate,
1.25 g of magnesium sulfate (7H2O), and 100 ml of
lactic acid, diluted to 2.5 liters with distilled water. For use, 100
ml of stock was diluted with 7.5 ml of 40% NaOH and 2.5%
of bromothymol blue, adjusted to 1 liter with distilled water at pH
7.0, and autoclaved.
After 24 h, the supernatant was
removed, leaving the starch-fecal layer. The supernatant was replaced
about 2/3 of the way up the slope with BRS medium (equal volume of BR
and sheep serum incubated for 24 h at 37°C) diluted
1:4 with phthalate solution (10.2% potassium phthalate,
2% NaOH, pH 6.3).
After 48 h of incubation at
37°C, a drop from the starch layer was mixed with double
strength Lugol's iodine and examined microscopically. A second
reading was performed after an additional 48 h of incubation.
Positive cultures were subcultured every 2 or 3 days with a fresh
slope.
Hexokinase isoenzyme
electrophoresis.
Entamoeba species were
differentiated by electrophoretic migration of hexokinase isoenzymes
(16). Briefly,
trophozoite lysates were separated in 1% agarose (SeaKem LE,
Rockland. Maine) by electrophoresis at 80 V, 22 mA for 1 h at
room temperature. The enzyme was stained with phenzin methosulfate
(PMS) (10 µg/ml) (Sigma) solution containing NADP (300
µg/ml) (Sigma), glucose (1 mg/ml), glucose-6-phosphate
dehydrogenase (1 unit/ml) (Sigma), MgCl3 (7.18 mM) (Fisher
Scientific, Itasca, Ill.),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT; 30 µg/ml) (Sigma), and ATP
(1.3 mM) (Sigma) in 0.1 M Tris-HCl, pH
7.4.
Detection of serum anti-LC3 IgG and
IgA antibodies by ELISA.
Detection of serum anti-LC3 IgG and
IgA antibodies by ELISA was performed as described previously
(7,
28,
32). Recombinant 52-kDa
LC3 protein was purified as described by Song et al.
(32); 96-well microtiter
flat-bottomed polystyrene ELISA plates were coated with LC3 protein,
and nonreactive sites were blocked with 1% bovine serum albumin.
Serum samples were analyzed by ELISA at a 1:1,000 dilution for IgG and
at 1:500 for IgA antibodies in phosphate-buffered saline-Tween
(1% bovine serum albumin). Following incubation for 2
h at room temperature, alkaline phosphatase-conjugated goat anti-human
IgG (Sigma) or IgA antibodies (ICN Biomedicals; Costa Mesa, Calif.)
were utilized at 1:5,000 for IgG and 1:2,000 for IgA in
phosphate-buffered saline-Tween (1% bovine serum albumin) for
2 h at room temperature. Reading the plates and correcting
the results for nonspecific background binding were performed as
described
(28).
Detection
of fecal lectin antigen by ELISA.
The ELISA for detection of 170-kDa
lectin antigen was performed as described
(3). Briefly, 96-well
flat-bottomed microtiter polystyrene ELISA plates (Costar, Corning,
N.Y.) were coated with monoclonal antibody 3F4, which recognizes
epitopes present in both E. histolytica and E. dispar
lectin, or the 8C12 antibody, which is specific for epitopes present
only in E. histolytica lectin
(23). Feces were mixed in
an equal volume of phosphate-buffered saline containing 2 mM
phenylmethylsulfonyl fluoride (USB, Cleveland, Ohio). Fecal samples
were added at 100 µl per well and incubated for 2 h
at room temperature or overnight at 4°C. Alkaline
phosphatase-conjugated antilectin monoclonal antibodies 8A3
(recognizing both E. histolytica and E. dispar
lectin) and 1G7 (specific for E. histolytica)
(23) were added at
1:1,000 dilution and incubated in developing buffer for 2 h
at room temperature. Plate reading with correction for nonspecific
background was performed as described
(28).
Detection
of fecal antilectin and anti-LC3 IgA antibodies by ELISA.
Native E. histolytica
galactose-inhibitable lectin protein
(22) and recombinant LC3
protein (32) were
purified as described and used in ELISA for detection of fecal
antilectin IgA antibodies
(7). Briefly,
flat-bottomed microtiter plates were coated with lectin protein (0.125
µg/well) and the nonreactive sites were blocked with
1% bovine serum albumin. Fecal samples were mixed with an equal
volume of phosphate-buffered saline-2 mM phenylmethylsulfonyl
fluoride and added at 100 µl/well for incubation at
room temperature for 2 h or overnight at 4°C.
Alkaline phosphatase-conjugated goat antihuman IgA antibodies (Sigma)
were added at a 1:5,000 dilution in phosphate-buffered saline-Tween
containing 1% bovine serum albumin, for incubation at room
temperature for 2 h. Plate reading with correction of results
for nonspecific background binding was performed as described
(7).
Treatment
of data.
Assays to detect
humoral (anti-LC3 IgG and IgA) or mucosal (fecal anti-LC3 and
antilectin IgA) antibody responses utilized a continuous optical
density (OD) scale, with a cutoffs for positivity of two standard
deviations above a culture-negative control used as a laboratory
standard. A subject was considered antibody positive if he had at least
two consecutive positive readings at baseline or on follow-up. The same
standard was applied to determine whether a subject was E.
histolytica or E. dispar culture positive except, as
shedding of the parasite may be intermittent, one negative culture
between two positive results was accepted as a duplicate positive
criterion. Finally, a person was considered negative for any of these
immunologic or infective markers if he had at least two consecutive
negative assays. Subjects who did not meet the criteria for being
positive or negative were excluded from the analyses for that assay or
comparison. The baseline period included months 0 to 6, with the
follow-up period being months 9 to 18, 21 to 27, and 30 to 36 months;
these time periods were used to track longitudinal changes in antibody
prevalence or culture results.
Analytic
methods.
For continuous
data, differences of distributions between groups were evaluated with
Wilcoxon rank-sum tests. Accordingly, tests results are presented with
median levels of these variables, along with associated
25%-75% interquartile ranges. Contingency table analysis
was used to compare proportions (yes/no) between groups: chi-square or
Fisher's exact tests (for data that were spare or nonnormally
distributed) were used to evaluate differences. It should be noted that
while we enrolled ALA cases and close associate controls, the study
design is essentially a prospective cohort, with exposure groups
defined by disease status as a proxy for prior infection with E.
histolytica at baseline (ALA versus control). Similarly, subjects
could be scored by culture positivity at baseline, and followed over
time to evaluate antibody responses or the occurrence of new
infections. Results for all tests were aggregated at baseline or
follow-up for all subjects for comparison between ALA and their control
subjects.
|
RESULTS
|
|---|
Results from 36 months
of follow-up are available for all subjects who completed the study, 93
ALA and 963 control subjects. Data are aggregated by baseline and
follow-up periods. The average age of the ALA subjects was 41.5 years;
the gender distribution was 83% male and 17% female. The
control subjects were 25% male and 75% female, with an
average age of 36.5 years.
Intestinal and serum
antilectin antibody responses.
The prevalence in ALA subjects and
close associate controls of intestinal antilectin IgA antibodies at
baseline (0 to 6 months) and follow-up (9 to 36 months) is illustrated
in Fig.
1. ALA subjects had a higher prevalence of intestinal antilectin IgA
antibodies at each time period studied compared to controls
(85.7%, 67.2%, 56%, and 52.6% positive
ELISAs versus 16.3%, 17.6%, 11.8%, and
10.3% in controls, respectively) (P < 0.001 for
each time period; Fig. 1).
Use of the purified native galactose-inhibitable E.
histolytica lectin protein was more sensitive in ELISA for
detection of antigen-specific fecal IgA antibodies than the recombinant
LC3 protein (P < 0.01; 85.7% compared to
55.2% positive at baseline). The prevalence of intestinal
antilectin IgA antibodies decreased over time in ALA and control
subjects, compared to baseline values (P = 0.01 and
P > 0.05, respectively). Seventy-two percent of the
ALA subjects positive for intestinal antilectin IgA antibodies at
baseline remained positive at 18 months, compared to only 37.5%
of comparable controls (P = 0.0007). By 30 to 36
months, 25.9% of ALA subjects who were positive on entering the
study had persistence of intestinal antilectin IgA antibodies, compared
to 10.8% of controls (P < 0.038).
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FIG. 1. Prevalence
of a positive ELISA for intestinal antilectin IgA antibodies in
subjects cured of ALA (light bars) and controls (dark bars). The
prevalence of antilectin IgA antibodies was greater in cases than
controls at baseline (0 to 6 months) and during each follow-up period
(P < 0.0001, for each). There was a significant
decrease in the prevalence of antilectin IgA antibodies between
baseline and follow-up intervals in ALA cases (P <
0.01 for each comparison) and in close associate controls (0 to 6 and 9
to 18 months compared to 21 to 36 months, P <
0.05).
|
|
At
baseline the prevalence of serum anti-LC3 IgA antibodies was greater in
ALA subjects than controls (93.4% compared to 6.9% of
controls, P < 0.001, Fig.
2A). Serum anti-LC3 IgA antibodies remained elevated in ALA subjects
compared to controls at each interval of follow-up (85.5%,
67.2%, and 57.9% compared to 13.6%, 8.4%,
and 6.3%, respectively, P < 0.001 for each,
Fig. 2A). There was an
increase in serum anti-LC3 IgA antibodies in controls at 18 months
(P < 0.05) compared to baseline), indicating new
E. histolytica infections. Interestingly, the prevalence of
serum anti-LC3 IgG antibodies in ALA subjects declined just as rapidly
as that of antilectin IgA antibodies (Fig.
2B, P <
0.001 for each interval compared to baseline). The prevalence of serum
anti-LC3 IgG antibodies was higher in ALA subjects than controls at
each interval studied (P < 0.001, Fig.
2B).
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FIG. 2. Prevalence
of serum anti-LC3 IgA (A) and IgG antibodies (B)
for subjects cured of ALA (light bars) and controls (dark bars). The
prevalence of a positive test for each antibody studied was greater in
ALA subjects than controls at baseline and during each follow-up period
(P < 0.001 for each). The prevalence of serum anti-LC3
IgA antibodies increased in controls only at 9 to 18 months (P
< 0.001); in ALA cases there was a decrease in the prevalence
of anti-LC3 IgG antibodies during each follow-up period (P
< 0.001 compared to the previous
period.
|
|
During the
entire duration of the study an intestinal anti-lectin IgA antibody
response occurred at some point in time in 97.6% of ALA subjects
and 49.7% of controls, P < 0.001, Fig.
3. Of interest, in controls fecal antilectin IgA antibodies had a higher
commulative prevalence than either serum anti-LC3 IgG or IgA antibodies
(49.7% compared to 32.6% and 24.3%, respectively,
P < 0.001, Fig.
3). Therefore, an
intestinal antilectin IgA antibody response must occur on occasion
without seroconversion. In addition, we found that the presence of
fecal anti-IgA antibodies in controls was positively associated
(P < 0.01) with E. dispar infection. Taken
together, this data indicates that E. dispar infection can
induce a transient intestinal but not humoral antilectin IgA antibody
response.
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FIG. 3. Percentage
of ALA (light bars) and controls (dark bars) individuals ever having a
positive test over the entire 36 months of the study. ALA subjects had
a higher cumulative positive percentage for each antibody studied
compared to controls, P < 0.01. Of interest, the
cumulative percentage of control subjects with fecal antilectin IgA
antibodies was greater than for either serum anti-LC3 IgA or IgG
antibodies (P <
0.005).
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Intestinal and serum ELISA results were analyzed as a
continuous variable by comparison of OD values at equal dilutions. At
baseline (0 to 6 months), ALA subjects had markedly higher ELISA OD
values at the 25th, 50th, 75th, and 90th percentiles for intestinal
antilectin IgA and serum anti-LC3 IgA antibodies, compared to
ELISA-positive control subjects assayed at identical dilutions of feces
or serum (P = 0.0001 for each percentile, Fig.
4). The same was true when ELISA OD levels for serum anti-LC3 IgG
antibodies were compared between ALA subjects and ELISA-positive
controls (P < 0.001, respectively, for each quartile,
data not shown). During the entire 36-month follow-up period,
antibody-positive ALA subjects had higher median OD values for all of
the antiamebic antibodies studied, compared to ELISA-positive control
subjects (P 
0.04 for each, Table
1).
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TABLE 1. Comparison
of OD values between ELISA-positive ALA subjects and close associate
controls during 36 months of follow-upa
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Occurrence of E. histolytica and
E. dispar intestinal infections.
By stool culture and zymodeme
determination, 6.3% of ALA subjects were infected with E.
histolytica during the baseline period (0 to 6 months), compared
to only 1.2% of controls (P = 0.001, Table
2). The prevalence of E. histolytica infection over time was at
10.0%, 5.9%, and 3.1% in ALA subjects during
follow-up, compared to 2.6%, 0.7%, and 1.0% of
control subjects, respectively (P = 0.005, Table
2). Over the duration of
the study, by culture and zymodeme determination, 15.6% of ALA
subjects and 3.5% of controls were infected with E.
histolytica (P = 0.001, Table
2). In ALA subjects there
were seven E. histolytica infections detected after 6 months;
six of seven were found at 9 to 18 months and only one was found
thereafter (Table 2). The
same pattern was evident in control subjects; 13 to 16 new infections
were detected at 9 to 18 months and only three thereafter (Table
2). In controls, there was
no correlation between being positive for intestinal antilectin IgA
antibodies or serum antilectin IgA or IgG antibodies and the occurrence
or nonoccurrence of new E. histolytica infections (P
> 0.1 for each).
Studies of E. dispar intestinal
infection revealed that at baseline (0 to 6 months), none of the 85 ALA
subjects who submitted adequate fecal samples were infected with E.
dispar, compared to 6.5% of controls (P =
0.007, Table
3). During the follow-up period, only 4 of 81 ALA subjects became
transiently infected with E. dispar compared to 149 of 713
control subjects (4.9% compared to 20.9%, P
< 0.05, Table 3).
At the end of the prospective study, 30 to 36 months, none of the ALA
subjects were infected with E. dispar, compared to 6.5%
of controls (P < 0.05, Table
3). Among the controls,
there was no correlation between having a positive serum or fecal
antibody test at baseline and immunity against the occurrence of a new
E. dispar infection. (P = 0.80).
We
determined that culture of fecal samples at 3-month intervals was not
sensitive enough to detect the majority of new E. histolytica
infections. By sero-conversion criteria, with serum anti-LC3 IgG
antibodies, 15.3% of asymptomatic controls had a new E.
histolytica infection during 12 months of follow-up (baseline
compared to months 9 to 18), yet culture and zymodeme criteria detected
only 1.0% as having a new infection (P >
0.01).
All fecal samples were subjected to a monoclonal
antibody-based ELISA for detection of E. histolytica and
E. dispar-specific lectin antigen, a research assay that we
applied successfully in a number of studies encompassing hundreds of
amebic infections in Cairo, Egypt
(3,
6,
7). However, we found that
the antigen detection ELISA results for all subjects over the duration
of the study did not correlate with stool culture and zymodeme
determination (only 6 of 32 positive cultures for E.
histolytica and 22 of 91 for E. dispar had true positive
ELISAs, Table
4), with clinical group, or with seropositivity (data not shown).
Therefore, under the field conditions and analysis as performed in this
study, antigen detection ELISA technology was found to be
unreliable.
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TABLE 4. Lack
of correlation of ELISA results for fecal lectin antigen detection and
culture with zymodeme determination for E.
histolytica and E. dispara
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DISCUSSION
|
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We now report the
first large prospective, controlled cohort study of E.
histolytica and E. dispar intestinal infection following
cure of ALA. We evaluated 93 subjects starting at 1 week following cure
of ALA and 963 controls (immediate family members and/or neighbors).
Subjects were evaluated at 3-month intervals thereafter with collection
of feces and blood, for a total of 36 months of follow-up.
There
was a high prevalence (>85%) of intestinal antilectin
IgA antibody responses in ALA subjects, which unexpectedly persisted in
over 50% of subjects for 18 months after treatment. The
intensity of the intestinal antibody response as measured by ELISA OD
readings was greater in ALA subjects than in antibody-positive controls
during any time period. To rule out sample coding errors, we utilized a
criterion of at least two positive culture results to identify an
infected subject. A recently published study genotyped a subset of
Entamoeba species isolated from our study subjects and
revealed that, except for two rare transient exceptions, the same
isolate was found over time in each individual studied, even for up to
six positive cultures over 3 years
(34). Therefore, in our
study use of a duplicate positive criterion should not significantly
mask the occurrence of new infections.
Following cure of ALA,
subjects were highly immune to intestinal infection by E.
dispar. There are no luminal amebicidal agents available for use
in South Africa; therefore, we expected a high incidence of E.
histolytica and E. dispar intestinal infection among ALA
subjects upon entry into the study. Stool cultures revealed a lower
than anticipated (16)
prevalence of new E. histolytica infections in controls,
making it impossible to determine whether ALA subjects, once cleared of
their original infection, were immune to new E. histolytica
infections by comparison to controls. Use of seroconversion criteria
revealed that the rate of new asymptomatic E. histolytica
infections in controls was actually sevenfold higher than suggested by
the stool culture data. However, seroconversion criteria cannot be
applied to ALA subjects, as almost all (>93%) were
seropositive upon entry into the study.
Control subjects with
intestinal antilectin IgA antibodies had no history of ALA or colitis;
mucosal antibody responses were most likely due to a relatively recent
asymptomatic E. histolytica or E. dispar intestinal
infection. E. histolytica infections are well documented to
produce humoral antilectin antibody responses
(28,
32). In addition,
intestinal antilectin IgA antibodies were more prevalent than humoral
antilectin IgA or IgG antibodies and positively associated with new
E. dispar infections, suggesting that E. dispar can
induce a mucosal but not a humoral immune response. We were able to
conclude that the low levels of intestinal antilectin IgA antibodies
present in ELISA-positive control subjects are not sufficient to
provide immunity to new E. dispar infections.
We suggest
that the immunity to E. dispar infection found in our
prospective longitudinal study of ALA patients is due to the high
levels of intestinal antilectin IgA antibodies present. Haque et al.
(17) reported that
children previously treated for amebic colitis had a delay in
acquisition of new E. histolytica intestinal infections and
that this relative immunity correlated with the presence of intestinal
antilectin IgA antibodies. Of interest, this immunity was observed only
during the first five months of follow-up
(17). In a subsequent
study, Haque et al. (18)
found that intestinal antilectin IgA antibodies were detectable for an
average of only 31 days; this is in marked contrast to our findings
following either ALA or during follow-up of intestinal IgA in
antibody-positive asymptomatic controls.
Given the 85%
homology in lectin amino acid sequence and shared epitopes between the
E. dispar and E. histolytica lectins
(23), we propose that
immunity to E. dispar may be a surrogate marker for immunity
to E. histolytica. Unfortunately, the insensitivity of stool
cultures every 3 months for E. histolytica infection and the
lack of specificity of the fecal antigen detection ELISA under the
field conditions in Durban, South Africa, prevented our determining
directly whether ALA subjects also exhibited immunity to E.
histolytica infection. We could not independently evaluate the
unique contribution to immunity of high titers of antilectin antibodies
to immunity to E. dispar infection. However, given the
demonstrated role of IgA antibodies in immunity to intestinal bacterial
and parasitic infection
(4) and the in vitro
adherence-inhibitory activity of murine fecal antilectin IgA antibodies
(8), it is likely that
intestinal antilectin IgA antibodies are mediating the immunity
observed.
A commercially available ELISA (DiaTech Labs) for
detection of lectin antigen has been utilized successfully
(19). The conditions for
performance of the ELISAs in this report are not identical to the
commercial assay; in addition, the field conditions in South Africa
resulted in a substantial delay (hours) between collection and
processing of samples. We found an unacceptably low rate of correlation
of lectin antigen positive ELISA results with positive stool cultures
for E. histolytica and E. dispar, and therefore we
could not apply antigen detection technology in this study. Previously,
we successfully performed hundreds of fecal antigen detection ELISAs on
samples collected in Cairo, Egypt, with excellent correlation to
culture results (3,
4,
6). However, other
investigators have found a similar lack of sensitivity and specificity
with earlier generations of the DiaTech assay when applied to field
conditions in the tropics (E. Tannich and T. F.
H. G. Jackson, personal communications). It is important to
emphasize that use of the DiaTech assay per the manufacturer's
instructions was not performed in this study. Studies utilizing PCR for
detection of parasite ribosomal DNA confirm that stool culture with
zymodeme determination underestimated the incidence of E.
histolytica but not E. dispar infection
(9).
In summary, we
found that subjects cured of ALA have a high prevalence and level of
both intestinal and serum IgA antibodies directed against the amebic
galactose-inhibitable adherence lectin. Both E. histolytica
and E. dispar contain functional galactose binding lectin
molecules with multiple shared epitopes
(23,
25). Cure of ALA is
followed by a high level of immunity to E. dispar intestinal
infection for the entire 36 months of our study. Therefore, our data
and those of others (17,
18) indicate that mucosal
antilectin IgA antibodies may mediate immunity in adults to new
intestinal infections by E. dispar or E. histolytica.
Use of a more sensitive and specific diagnostic test (PCR) rather than
use of stool culture and zymodeme determination will allow us to more
directly address this hypothesis. Numerous strategies have been
developed for use of the E. histolytica galactose-inhibitable
lectin as a subunit amebiasis vaccine, especially to elicit protective
intestinal antilectin IgA antibodies
(33). Clearly, if
antilectin IgA antibodies have a role in human immunity to E.
histolytica intestinal infection, as indicated by this study, such
vaccine strategies should continue to be actively
pursued.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by
NIH grants PO1-AI36359-01 and UO1-AI35840 from NIAID and from the MRC
(South Africa).
We thank Shana Brooks for expert secretarial
assistance and Rose Hill for very important data technology
services.
|
FOOTNOTES
|
|---|
* Corresponding author. Mailing address: Department of Medicine, University of
Minnesota, 516 Delaware Street, 14-110 PWB, MMC194, Minneapolis, MN 55455. Phone: (612) 625-3654. Fax: (612) 625-3654. E-mail: ravdi0001{at}umn.edu. 
Editor:
B. B. Finlay
|
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Infection and Immunity, December 2003, p. 6899-6905, Vol. 71, No. 12
0019-9567/03/$08.00+0 DOI: 10.1128/IAI.71.12.6899-6905.2003
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Top
Abstract
Introduction
) compared to antibody-positive
controls (
) (P = 0.001 for each). The same
was true for serum anti-LC3 IgA antibodies (solid line), P
< 0.001 comparing ALA cases (