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Infect Immun. Jul 2006; 74(7): 3897–3903.
PMCID: PMC1489685

Mucosal Immunity to Asymptomatic Entamoeba histolytica and Entamoeba dispar Infection Is Associated with a Peak Intestinal Anti-Lectin Immunoglobulin A Antibody Response

Abstract

We monitored 93 subjects cured of amebic liver abscess (ALA) and 963 close associate controls in Durban, South Africa, and determined by enzyme-linked immunosorbent assay that the intestinal immunoglobulin A (IgA) antibody response to the Entamoeba histolytica galactose-inhibitable adherence lectin is most accurately represented by a complex pattern of transitory peaks. One or more intestinal anti-lectin IgA antibody peaks occurred in 85.9% of ALA subjects over 36 months compared to 41.6% of controls (P < 0.0001). ALA subjects exhibited a greater number of anti-lectin IgA antibody peaks (P < 0.0001) than controls. In addition, their peak optical density values were higher (peak numbers 1 to 3, P < 0.003), peaks were of longer duration (for peaks 1 and 2, P ≤ 0.0054), and there was a shorter time interval between peaks (between 1 and 2 or 2 and 3, P ≤ 0.0106) than observed for control subjects. A prior E. histolytica infection was associated with the occurrence of an anti-lectin IgA antibody peak (79.1%, P < 0.0001) more so than for Entamoeba dispar infection (57.2%, P < 0.001). The annual number of anti-lectin IgA antibody peaks in ALA subjects was 0.71 per year, compared to just 0.22 in controls (P<0.0001), indicating a higher rate of exposure to the parasite than previously appreciated. Anti-lectin IgA antibody peaks were of higher amplitude following a E. histolytica infection compared to E. dispar (P = 0.01) and, for either, were of greater height in ALA subjects than controls (P < 0.01). ALA subjects demonstrated greater clearance of amebic infection after an anti-lectin IgA antibody peak compared to controls, and only 14.3% remained with a positive culture after the peak, compared to 38.9% in controls (P = 0.035). In summary, this prospective controlled longitudinal study elucidated the dynamic nature of the human intestinal IgA antibody response to E. histolytica and E. dispar infection and revealed that ALA subjects exhibit heightened intestinal anti-lectin IgA antibody peaks that are associated with clearance of E. histolytica and E. dispar infection.

As with other enteric organisms, antigen-specific secretory immunoglobulin A (IgA) antibodies have been found to mediate protection against intestinal infection by Entamoeba species (12, 24). Colonic mucins are rich in galactose-containing carbohydrates and have been demonstrated to be the high-affinity receptor for the Entamoeba histolytica galactose-inhibitable surface lectin (9). In general, intestinal IgA antibodies prevent microbial binding to epithelial surfaces and promote clearance of pathogenic enteric organisms by agglutination and blocking of surface receptors essential for microbial pathogenesis and invasion (1). Accumulating evidence from field studies in Bangladesh (12) and South Africa (24) demonstrate that intestinal IgA antibodies to key epitopes of the galactose-inhibitable lectin heavy subunit (2, 14) provide immunity to intestinal infection by E. histolytica and Entamoeba dispar. E. dispar, which is morphologically identical to E. histolytica, is a nonpathogenic (noninvasive) and distinct Entamoeba species (10), yet it contains functional galactose-binding surface lectin molecules that are genetically 86% homologous (17, 22, 23) and share numerous antigenic epitopes with E. histolytica. Infection by E. dispar can induce a low-level intestinal anti-lectin IgA antibody response (24). Previous research of anti-amebic and anti-lectin intestinal IgA antibody responses have only reported the point prevalence of antibodies on a population basis (3, 4, 13-24), their accumulated incidence over time (12), or the prevalence of antibodies in different populations of subjects during long periods of follow-up (24). In addition, the duration of the human intestinal anti-lectin IgA antibody response has been reported to be as short as a mean of 17 days (12, 13) to as long as 36 months in 53% of amebic liver abscess (ALA) subjects (24). Numerous groups are working on a galactose-inhibitable lectin-based amebiasis subunit vaccine (15, 18, 20, 29, 30) designed to induce a protective mucosal or cellular immune response. Without further characterization of the human intestinal immune response and a better understanding of the dynamics (intensity and duration over time) of this response, it will be impossible to rationally develop and design a lectin-based amebiasis subunit vaccine for study in animal models and humans.

We studied the relationship of intestinal anti-lectin IgA antibodies, as determined by enzyme-linked immunosorbent assay (ELISA), to E. histolytica and E. dispar infection in a longitudinal prospective controlled follow-up study of adults in an area of high endemicity. Study subjects were chosen within 1 week of treatment for amebic liver abscess (ALA cases) or were asymptomatic close associate controls who resided in the same or neighboring home of the index ALA patients within areas of Durban, South Africa. We previously reported the mean prevalence of intestinal anti-lectin IgA antibodies within the two populations aggregated over 6- or 9-month periods (0 to 6 months, 9 to 18 months, 21 to 27 months, and 30 to 36 months) (24). In a separate report, we defined the epitope specificity of the human intestinal anti-lectin IgA antibodies (2). This report reveals a specific and dynamic pattern of individual peak intestinal IgA antibody responses that is heightened among subjects cured of ALA and associated with clearance of asymptomatic intestinal infection by E. histolytica and E. dispar.

MATERIALS AND METHODS

Subject recruitment and study enrollment.

Adult 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 were asymptomatic, had no history of ALA or amebic colitis, and 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 patient; we sought at least 10 asymptomatic controls for each index case. No criteria for age or gender were applied except a minimum age of 16 years. All subjects provided blood by venipuncture and feces upon entry into the study (1 week after commencing treatment of ALA in the index cases) and at 3-month intervals for a total of at least 36 months of follow-up. Over the duration of the study, only 7 of the 100 family groups were lost to follow-up; all within-family group samples for ALA and control subjects were collected on the same day at the same time in the same location and handled identically. The University of Minnesota and University of Natal's institutional review boards for human subjects approved the consent forms and all aspects of the study. Assays performed included fecal microscopy, stool culture with zymodeme determination for E. histolytica or E. dispar, and ELISA for fecal anti-lectin IgA antibodies (3, 4).

Stool culture.

Fecal samples were cultured in Robinson's medium (26) 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 KH2PO4, 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% 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 two-thirds 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 (28). 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 (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 (30 μg/ml) (Sigma), and ATP (1.3 mM) (Sigma) in 0.1 M Tris-HCl, pH 7.4.

Detection of fecal anti-lectin and anti-LC3 IgA antibodies by ELISA.

Native E. histolytica galactose-inhibitable lectin protein (19) was purified as described previously and used in ELISA for detection of fecal anti-lectin IgA antibodies (4). ELISA was developed by use of checkerboard titration and standardization of dilutions of feces and buffer as described previously (5). Each plate includes negative and positive reference samples as controls. Briefly, flat-bottom 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 anti-human 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 and each plate included known positive- and negative-control samples (25).

All analyses were performed as optical density (OD) ratios, calculated as the ratio of a sample's measured OD value and a noise threshold. The noise threshold is the mean plus 2 standard deviations of OD values from control samples measured in the same batch as collected samples. A peak was defined as one or more consecutive samples with an OD ratio of 2 or greater. The number of peaks for each study participant was tabulated. Additionally, three quantities were calculated to characterize the observed peaks: mean peak height, peak duration, and the peak-to-peak interval of time. The OD ratios of consecutive samples in a peak were averaged to give a mean peak height. Each consecutive sample contributed 3 months to the peak duration, and peak-to-peak intervals were calculated as the number of months between the last sample of a leading peak and the first sample of a trailing peak.

The mean number of peaks per person was compared between the ALA and control groups using a two-sample t test. We compared the distribution of the number of peaks between the two groups with the Jonckheere-Terpstra test. Mean peak heights of the ALA and control groups were compared with a two-sample t test. Peak durations were analyzed using Kaplan-Meier curves that estimate the proportion of peaks that exceed any given duration. The curves for ALA and control study participants were compared using the log-rank test. Peak durations were censored if the last observed sample was a peak. Peak-to-peak interval durations were analyzed similarly to the peak durations using Kaplan-Meier curves and the log-rank test. These analyses account for right censoring in which some intervals and some peaks were of unknown duration due to the study ending before the end of a peak or a peak-to-peak interval was observed.

RESULTS

Study of intestinal anti-lectin IgA antibody responses in individual subjects over time (Fig. (Fig.1)1) revealed many patterns of OD readings and what appeared to be different patterns in subjects cured of ALA compared to infected or uninfected asymptomatic controls. ALA subjects appeared to have more frequent intestinal anti-lectin IgA antibody peaks (Fig. (Fig.1,1, top), while asymptomatic control subjects had fewer peaks despite the occurrence of E. dispar or E. histolytica infections (Fig. (Fig.1,1, middle and bottom). By standardizing the definition of an anti-lectin IgA antibody peak (twofold above the baseline) and recalculating all of the readings as a ratio above the baseline, we were able to quantitatively evaluate the pattern of the intestinal anti-lectin IgA antibodies in each of the 1,053 subjects studied prospectively every 3 months over the course of 3 years (13 potential observations per subject).

FIG. 1.
Patterns of individual intestinal anti-lectin IgA responses over time and the occurrence of asymptomatic E. histolytica (or E. dispar) infection in ALA (top) or control subjects (middle and bottom). Open triangles indicate negative cultures, shaded triangles ...

A greater percentage of the ALA subjects (85.9%) than of the asymptomatic controls (41.6%; P < 0.0001) exhibited at least one intestinal anti-lectin IgA antibody peak over the 36 months (Fig. (Fig.2).2). ALA subjects also exhibited a greater average number of intestinal anti-lectin IgA antibody peak responses than did controls, as determined by distributional shift analysis (Jonckheere-Terpstra) test, P < 0.0001) (Fig. (Fig.2).2). As ALA subjects are predominantly male (83%) and controls are predominately female (76%), we repeated this analysis comparing male-to-male and female-to-female subjects. Gender was not a confounding variable, as male ALA subjects exhibited a greater number of intestinal IgA antibody peaks than male controls (77% compared to 24%, respectively; P < 0.0001), and comparable results were demonstrated for female subjects (P < 0.0001). Peak intestinal anti-lectin IgA antibody levels were of higher amplitude, on average, in ALA subjects than in controls for all subjects exhibiting one, two, or three antibody peaks over the 36 months (P < 0.0005) (Table (Table1).1). Alternatively, comparison of the amplitude of all first or second anti-lectin IgA antibody peaks across both populations revealed that ALA subjects had higher OD values than controls (P < 0.002) (Table (Table11).

FIG. 2.
Percentages of control and ALA subjects with zero to five intestinal anti-lectin IgA antibody peaks. The difference between control and ALA subjects for the distribution of number of peaks is significant (P < 0.0001), as determined by the Jonckheere-Terpstra ...
TABLE 1.
ALA subjects have intestinal anti-lectin IgA antibody peaks of greater amplitude than asymptomatic controls during 36 months of follow-up

Peak intestinal IgA antibody responses were of longer duration in ALA subjects than in controls, as determined by mean peak duration (Fig. (Fig.3,3, top) or by hazard ratio for the time from the beginning to the end of a peak (Fig. (Fig.3,3, bottom) (peaks one and two, P < 0.002); there were no differences in the duration of peaks three through five. Lastly, the time interval between peak IgA antibody levels (troughs) was, as expected, less in ALA subjects than in control subjects (P < 0.0001).

FIG. 3.
Mean duration of peak intestinal anti-lectin IgA antibody responses for ALA and control subjects. Antibody peaks 1 through 3 last longer in ALA subjects than in control subjects, as determined by differences of the means (top) (P < 0.0001 for ...

Seroconversion has been accepted as a gold standard for documenting new infections or exposure to infective agents. We now find that peak intestinal IgA antibody responses are an equivalent finding; E. histolytica infections were immediately followed by a peak IgA antibody response (79.1%; P < 0.0001), a stronger association than for E. dispar infections (57.2%; P < 0.0001). Statistically, any peak anti-lectin IgA antibody response was associated with a positive culture for E. histolytica or E. dispar (P < 0.0001, compared to nonpeak observations). We determined the number of anti-lectin IgA antibody peaks per year per person and found that ALA subjects had, on average, 0.71 anti-lectin antibody peaks per year, a markedly greater annual rate than that of control subjects (0.22; P < 0.0001) The differences between ALA and control subjects were present even if male or female subjects were segregated, with men only at 0.77 to 0.24 and women at 0.60 to 0.23 (P < 0.001, respectively, for each). There was no difference between men and women among ALA subjects or controls.

For all subjects, IgA antibody peaks were of higher amplitude following an E. histolytica infection than an E. dispar infection (P = 0.01) (Table (Table2).2). In addition, ALA subjects responded with a higher anti-lectin IgA antibody peak than controls following an E. histolytica infection (P < 0.01) (Table (Table2).2). We examined all instances in which an anti-lectin IgA peak followed an E. histolytica or an E. dispar infection, and a fecal culture was performed 3 months following the peak. In 21 ALA subjects compared to 72 controls, only 14.3% had an ongoing positive culture for E. histolytica or E. dispar, compared to 38.9% of control subjects (P = 0.035). In this subgroup, the anti-lectin IgA antibody peaks were again found to be greater in the ALA subjects than in controls (difference of 1.107; P = 0.0184).

TABLE 2.
Magnitude of intestinal anti-lectin IgA antibody peaks immediately following a positive fecal culture for E. histolytica or E. dispar in ALA subjects, control subjects, and both groups combined

DISCUSSION

We now report that intestinal anti-amebic IgA antibody responses are most accurately reflected as a dynamic series of peaks lasting 3 to 6 months based on prospective longitudinal following up and analysis of over 1,000 individuals every 3 months for 3 years. Lack of awareness of this more accurate depiction of mucosal immunity may lead to inaccurate and false conclusions regarding the frequency of parasite challenge, identification of immune and nonimmune hosts, and the significance of intestinal anti-lectin IgA antibodies in relationship to immunity to E. histolytica intestinal infection. A significantly increased percentage of ALA subjects exhibited anti-lectin antibody peaks compared to controls, indicating that sensitization from a previously cured invasive infection plays a major role in how humans respond to repeated parasite challenges in areas of endemicity. ALA subjects exhibited a much greater level of immune responsiveness to a high level of endemic exposure to the parasites. It is illogical to interpret this finding as an increased frequency of parasitic exposure for just the ALA individuals, as they live in the same homes or area of endemicity as the control population. It has been demonstrated that asymptomatic infections involve entire family groups in South Africa (11).

Anti-lectin IgA antibody peaks following amebic infection were of higher magnitude following E. histolytica (compared to E. dispar) infection and greater in ALA subjects than in controls for each species. Lastly, anti-lectin IgA antibody peaks in ALA subjects resulted in a greater percentage of clearance of amebic infection than in controls.

We hypothesize that ALA subjects have an enhanced amnestic response, leading to a more rapid clearance of the parasite, thus accounting for the previously reported immunity to reinfection with E. dispar in adults cured of ALA (4). This heightened immune responsiveness following successful treatment of invasive E. histolytica infection was observed compared to control subjects with untreated asymptomatic intestinal infection. The occurrence of asymptomatic amebic liver abscess has recently been reported in the population in Hue, Vietnam, by study of serology and hepatic ultrasound (6). As invasive E. histolytica infection, particularly ALA, can be associated with an antigen-specific suppression of cellular immune responses (27), it is not surprising that subjects with long-term untreated subclinical invasive amebic infection do not exhibit effective mucosal immunity (7). Recurrence of amebic liver abscess has been demonstrated in South Africa in subjects who were not treated with luminal amebicidal agents and were unable to clear an established intestinal infection (16). This was also observed in a subset of ALA subjects in Hue, Vietnam (8); however, intestinal anti-amebic IgA antibodies were not evaluated in these studies.

Previous studies have reported that the annual prevalence of E. histolytica or E. dispar infection in most areas of endemicity was 10 to 15%, with asymptomatic infection lasting an average of 12 months (7, 11, 14, 17, 19). Our data indicate that parasite challenge occurs much more frequently and that surveillance by stool culture is very insensitive to indicate the level of exposure to infection in an area of endemicity. We performed an antigen detection ELISA on all fecal samples to detect E. histolytica and E. dispar infection, as successfully applied to field studies in Cairo, Egypt (3-5). Unfortunately, the field conditions in South Africa resulted in longer travel times each day before samples could be processed, resulting in inaccurate assays, presumably due to degradation of the lectin antigen that was to be detected in the ELISA (22). We previously reported that seronegative controls in Durban, South Africa had an annual seroconversion rate of 15%, as determined by ELISA for serum anti-LC3 IgG antibodies (24). In ALA subjects, seroconversion cannot be assessed, as their serologies remain positive for years after treatment. However, anti-lectin intestinal IgA antibody peak responses indicated that oral challenge with an Entamoeba species (E. histolytica or E. dispar) occurred in 70% of the population each year. These repeated parasite challenges at short intervals might help to explain the persistence of positive antiamebic serologies for years after an episode of invasive amebiasis. Therefore, intestinal anti-lectin IgA antibody peak responses appear to be a much more sensitive and accurate epidemiologic tool for assessment of environmental exposure than serology, fecal culture, or even intermittent fecal PCR studies. This report extends our previous findings (22), demonstrating that E. dispar infection is capable of inducing a mucosal immune response, albeit one of lower intensity than E. histolytica. It is not unusual for luminal noninvasive infections to induce mucosal immune responses; for example, this is found in Ascaris and Giardia lamblia (50% asymptomatic) infections (1). Our findings raise issues regarding the occurrence of tolerance to E. histolytica infection, as a significant percentage of controls did not exhibit any peak IgA antibody responses over the entire 36 months. While this could reflect an inability to mount a mucosal immune response, it may also be consistent with some form of innate immunity, as reported in Bangladesh (12), in that approximately 20% of children were found never to be infected over a 24-month period while residing in an area of high endemicity.

Our findings add to the evidence that intestinal anti-lectin IgA antibodies can mediate immunity to asymptomatic infection by E. dispar and E. histolytica. We demonstrated that the higher peaks of anti-lectin IgA antibodies in ALA subjects than in controls were associated with a greater clearance of Entamoeba infection. These findings, combined with earlier studies of epitope analysis (2), indicate that it is the intensity and rapidity of the antilectin mucosal immune response that leads to eradication of infection rather than differential epitope recognition.

In summary, we discovered distinct patterns of intestinal anti-lectin IgA antibody peak responses that provided compelling evidence that subjects cured of ALA exhibit a higher degree of mucosal immunity than asymptomatic controls. This finding correlates with prior reports of immunity to reinfection (12, 14, 24), although the limitations of stool culture methodology did not allow for sufficient sensitivity to determine the duration of immunity to a new E. histolytica infection. Development and experimentation with a lectin-based amebiasis subunit vaccine designed to prevent intestinal infection and subsequent invasion by E. histolytica trophozoites must consider this dynamic mucosal immune process. By enhancing mucosal immune responsiveness, as observed in ALA subjects, a vaccine may limit the occurrence or duration of asymptomatic intestinal parasite infections upon environmental exposure, which serve as “boosters to vaccine-induced mucosal immunity” and thus provide sufficient protection against the occurrence of invasive amebiasis.

Acknowledgments

This research was supported by NIH grants UO1-AI35840 and PO1-AI 36359 to J.I.R. and by support of T.F.G.H.J. by the MRC (South Africa).

We thank Yvette Massey for expert secretarial assistance and Rose Hilk for data technology services.

Notes

Editor: W. A. Petri, Jr.

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