A Monoclonal Antibody for Distinction of Invasive and ... - Europe PMC

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 2807-2813

Vol. 30, No. 11

0095-1137/921112807-07$02.00/0

Copyright © 1992, American Society for Microbiology

A Monoclonal Antibody for Distinction of Invasive and Noninvasive Clinical Isolates of Entamoeba histolytica ARMANDO GONZALEZ-RUIZ,1* RASHIDUL HAQUE,2 TAYYAB REHMAN,1 AURA AGUIRRE,3 CARLOS JARAMILLO,3 GUADALUPE CASTANON,4 ANDREW HALL,2t FELIPE GUHL,3 GUILLERMO RUIZ-PALACIOS,4 DAVID C. WARHURST,' AND MICHAEL A. MILES1 Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, United Kingdom1; International Centre for Diarrhoeal Diseases Research, Bangladesh, Dhaka 1,000, Bangladesh2; Laboratorio de Microbiologia y Parasitologia, Departamento de Ciencias Biol6gicas, Universidad de los Andes, Apartado Ae'reo 4976, Bogotd, Colombia3; and Departamento de Infectologia, Instituto Nacional de la Nutricion "Salvador Zubirdn," C P. 14000, MeXico City, Mexico4 Received 8 June 1992/Accepted 4 August 1992

Approximately 10% of the world population is infected with Entamoeba histolyfica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolatespecific monoclonal antibody was 100%o. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively. Entamoeba histolytica is the etiological agent of human amebiasis. The infection is distributed worldwide, although the majority of cases are found in developing countries where fecal-oral transmission of E. histolytica cysts is facilitated by poor hygiene and the lack of clean water. The spectrum of clinical manifestations observed in infected individuals ranges from asymptomatic infection to intestinal and extraintestinal invasive disease. In 1984, it was estimated that approximately 10% of the world population was infected with E. histolytica and that only 10% of the infected subjects progressed to invasive amebiasis (47). The discrepancy between the number of infected persons and the occurrence of invasive amebiasis seems to be due to the existence of two genetically different but morphologically indistinguishable parasites within the species. Brumpt (5) made this suggestion after infecting kittens with E. histolytica strains from asymptomatic carriers and observing no development of disease. He proposed a new nomenclature for the noninvasive and the invasive strains: Entamoeba dispar and Entamoeba dysenteriae, respectively. A few years later, Simic (36) infected kittens with E. histolytica strains from asymptomatic carriers and then passed these strains on to human volunteers. Neither the kittens nor the humans developed symptoms attributable to the infection. More recently, Martinez-Palomo et al. (21) showed that only invasive strains of E. histolytica agglutinated in the

* Corresponding author. t Present address: Department of Pure and Applied Biology, Imperial College, London SW7 2BB, United Kingdom.

presence of the lectin concanavalin A, giving evidence for a phenotypic difference residing on the parasite membrane. Later, Sargeaunt and coworkers provided strong support for Brumpt's suggestion by showing, with more than 3,000 E. histolytica isolates, that zymodeme analysis clearly distinguished isolates cultured from asymptomatic patients and those cultured from patients with symptomatic amebic infection (31-33). On the basis of zymodeme characterization, other markers, such as resistance to complement-mediated lysis by human serum (28), release of a neutral thiol proteinase (27), overexpression of and structural differences between homologous cysteine proteinases (42), and recognition by specific monoclonal antibodies (MAbs) (22, 24, 38, 40) and by specific DNA probes (4, 7, 13, 39, 41, 42), have been described for invasive strains. Restriction fragment length polymorphisms and sequencing of the small subunit of the rRNA of invasive and noninvasive E. histolytica strains have shown an estimated sequence divergence of 2.2% (6), evidence which reaffirms Brumpt's suggestion that the two parasites are sufficiently genetically distinct to merit the status of independent species. Zymodeme analysis is time-consuming and expensive, and so simpler diagnostic techniques are needed for the identification of invasive strains of E. histolytica. Here we report the production and applications of an invasive isolate-specific MAb against E. histolytica for the identification of invasive clinical isolates from several regions where the organism is endemic and for the preliminary characterization of the corresponding antigen, which has not previously been described. 2807

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GONZALEZ-RUIZ ET AL.

MATERIALS AND METHODS Parasite strains and culture conditions. The axenic E.

histolytica strains HK9 (14), NIH:200 (43), and HM-1:IMSS, formerly known as ABRM (8) (ATCC 30015, 30458, and 30459, respectively), which were originally isolated from patients with invasive intestinal amebiasis, were cultured in Diamond's medium TPS-1 (9) at 37°C. Entamoeba moshkovskii CST (ATCC 30131), a free-living ameba isolated from sewage, the reptilian ameba Entamoeba invadens TRM (23), and a clinical isolate of Pentatrichomonas hominis, an intestinal commensal flagellate (1), were cultured in biphasic Robinson's medium (30) at room temperature (RT). Blastocystis hominis, an organism of uncertain taxonomic status associated with diarrhea in humans (1), was cultured in biphasic Robinson's medium at 37°C. Giardia intestinalis Portland 1 was cultured in Diamond's medium TYI-S-33 (9) at 37°C. The polyxenic E. histolytica invasive isolates TE and SI and the noninvasive isolates C29, 8672, and SAW 1734 were cultured from patients with symptomatic and asymptomatic amebic infections, respectively (38). All the clinical isolates of E. histolytica cultured from patients in areas where amebiasis is endemic were initially isolated and maintained in biphasic Robinson's medium at 37°C. Some isolates were also mass cultured in liquid Robinson's medium (37) at 37°C. These isolates were from the International Centre for Diarrhoeal Diseases Research, Bangladesh (ICDDRB), in Dhaka, Bangladesh, the Laboratorio de Microbiologia y Parasitologia de la Universidad de los Andes (UNIANDES) in Bogota', Colombia, and the Instituto Nacional de la Nutricion "Salvador Zubiran" (INNSZ) in Mexico City, Mexico. Isolates from ICDDRB came either from patients of all age groups seen at the hospital for diarrheal diseases or from children less than 14 years old living in an urban slum area in Dhaka. Isolates from UNIANDES were obtained from patients in areas where amebiasis is endemic or from patients referred to UNIANDES for parasitological diagnosis. Isolates from INNSZ were recovered from a suburban community on the outskirts of Mexico City and, within that community, mainly from children 5 years of age and under. Stool samples. Recently collected stools were examined macroscopically for the presence of blood and mucus; a smear of feces in 0.9% saline was examined microscopically for the presence of erythrocytes, leukocytes, and E. histolytica trophozoites; the presence of erythrocytes within E.

histolytica trophozoites was also recorded. Feces were concentrated by the formalin-ether concentration technique (29) for parasite cysts and ova, and the concentrate was examined microscopically. Feces were also inoculated into Robinson's medium within 6 h of collection, and E. histolytica-positive cultures were subcultured every 48 h. Zymodeme characterization. The zymodeme characterization of the E. histolytica isolates was performed in the country where the isolates were obtained. At ICDDRB, cellulose acetate plates and a Zip Zone chamber with a Titan Plus electrophoresis power supply (no. 1501; Helena Laboratories, Beaumont, Tex.) were used as previously described (15); at UNIANDES, thin-layer starch-gel electrophoresis was employed according to the methods of Sargeaunt and Williams (32) and Sargeaunt et al. (33) for phosphoglucomutase (EC 2.7.5.1), L-malate:NADP+ oxidoreductase (oxaloacetate decarboxylating) (EC 1.1.1.4.0), and glucose phosphate isomerase (EC 5.3.1.9) and according to the methods of Farri et al. (10) for hexokinase (EC 2.7.1.1); and at INNSZ, the method was that of Kollaritsch et al. (18) with automated isoenzyme isoelectrofocusing on PhastSystem

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equipment (no. 18-1600-01; Pharmacia, Uppsala, Sweden). Briefly, E. histolytica trophozoites were harvested from 2-day, well-grown Robinson's cultures when the number of organisms was approximately 5 x 104/ml (31), washed twice with sterile 0.9% saline, resuspended in 500 pl of distilled water containing enzyme stabilizers (1 mM EDTA-1 mM dithiothreitol-1 mM I-aminocaproic acid), lysed by freezing and thawing (three times), and centrifuged at 14,000 x g for 1 h at 4°C. The supematants were collected, aliquoted, and stored in liquid N2 until used. After electrophoresis of the lysates, the enzymes were developed in the dark by using an agar overlay incorporating substrate solutions according to standard methods (10, 34). Production of E. histolytica NP-40 protein extract. Axenic and polyxenic E. histolytica strains were harvested from liquid Robinson's medium by chilling the culture tubes in ice-water for 15 min, washed (three times) in cold phosphatebuffered saline (PBS), pH 7.2, and centrifuged at 250 x g for 5 min at 4°C. The pelleted cells were lysed by the addition of 3 volumes of lysis buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.75% [wt/vol] Nonidet P-40 [NP-40] [no. 56009; BDH Chemicals, Poole, Dorset, United Kingdom]) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [no. P7626; Sigma Chemical Co., Poole, Dorset, United Kingdom], 1 mM iodacetamide [no. 441812H; BDH], and 1 ,g of tosyl-L-lysine chloromethyl ketone [no. T7254; Sigma]) per ml with continuous stirring on ice for 15 min. The mixture was centrifuged at 16,000 x g for 15 min at 4°C, and the supernatant was recovered and stored at -20°C after the protein content was measured by the BCA protein assay reagent method (no. 23225; Pierce, Rockford, Ill.). Production of MAbs. MAbs were produced according to the method of Galfre and Milstein (12) with some modifications. Two BALB/c mice were immunized intraperitoneally with 50 ,ug of NP-40 protein extract of E. histolytica NIH:200 emulsified with an equal volume of complete Freund's adjuvant. Mice were given intraperitoneal booster injections of the same extract and incomplete Freund's adjuvant (three times) at 2-week intervals. A final booster of 50 ,ug of protein diluted in 200 ,ul of PBS was injected into the tail vein. The antibody response was assessed by indirect enzyme-linked immunosorbent assay (ELISA) (46) by coating the microplate wells with the E. histolytica antigen used for immunization. Four days after the final booster, the spleen of one mouse was removed and the spleen cells were fused with the SP2/O-Agl4 myeloma cell line (35) by using 45% polyethylene glycol 4000 (no. 9727; Merck, Darmstadt, Germany) (11), pH 8.1 (48). Hybridomas secreting MAbs against whole E. histolytica antigen were screened by ELISA and cloned twice by limiting dilution by using BALB/c peritoneal macrophages as feeder cells. A third screening of nine selected hybridoma cell lines (see Results) was performed by an indirect immunofluorescence assay (IFA) (see below) with reference invasive and noninvasive E. histolytica polyxenic strains as antigens. Ascites were produced by the inoculation of hybridomas intraperitoneally into pristane-primed mice. MAbs were isotyped by ELISA with a commercial kit (Sigma ISO-2). The specificity of MAbs against other intestinal parasites (see Results) was determined by ELISA and IFA. Immunofluorescence analysis of fixed parasites. Axenic and polyxenic parasite strains cultured in Diamond's or liquid Robinson's medium were harvested by chilling culture tubes in ice-water for 15 min and washed twice with cold PBS, and 30 ,ul of a suspension of 105 cells per ml was spotted onto each well of multispot polytetrafluorethene-coated slides

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A MONOCLONAL ANTIBODY FOR INVASIVE E. HISTOLYTICA

(no. PH-001; Loughton, Hendley, Essex, United Kingdom). Strains grown in biphasic Robinson's medium were harvested after 2 days by pipetting out the bottom starch layer from the 5-ml container and spotting out 30 ,ul of the mixture containing parasites, bacteria, and starch as described above. Slides were air dried, fixed in methanol for 5 min, and stored at -20°C. Slides were warmed at RT for 30 min, and twofold serial dilutions of MAb-containing culture supernatant or mouse ascites were prepared in PBS-2% casein (PBS/C). A 40-,ul sample of each dilution was spotted per well across the slides in duplicate, and the slides were incubated at RT for 30 min in a humid chamber. After the slides were washed twice in a PBS bath for 5 min at RT with continuous rocking, 40 ,lI of a 1/50 dilution in PBS/C of goat (at the London School of Hygiene and Tropical Medicine [LSHTM]) or rabbit (at ICDDRB) anti-mouse immunoglobulins conjugated to fluorescein isothiocyanate (no. F1010; Sigma, and Dakopatt A/S, Copenhagen, Denmark, respectively), with 1/10,000 (wt/vol) Evans blue for counterstaining, was spotted per well and the slides were incubated for another 30 min at RT. The slides were washed as described above, mounted in 50% glycerol in PBS, and examined at ICDDRB in an Olympus fluorescent microscope (model BH-2; Olympus Optical Co. Ltd., Tokyo, Japan) and at LSHTM in a Leitz Diaplan microscope with epifluorescent light (Leitz, Wetzlar, Germany). The titers of positive slides were recorded. Initially, preimmune and immune mouse sera were used as negative and positive controls, respectively, at a 1/200 dilution. Later, slides with known invasive and noninvasive strains were also included as controls when unknown clinical isolates were tested. Immunofluorescence analysis of living parasites. Immunofluorescence analysis of live E. histolytica trophozoites was carried out as previously described by Blakely et al. (2) with some modifications. Briefly, axenic HM-1:IMSS trophozoites were harvested and washed in cold 0.9% saline with 0.2% glucose and 2 mM CaCl2-1 mM ascorbic acid (no. 10303; BDH) and 6 mM L-cysteine (no. C-7880; Sigma). Aliquots of 105 cells were tested with mouse ascites containing invasive isolate-specific MAb diluted 1/20 in the same saline solution for 1.5 h at 37°C, and duplicate aliquots were kept at 4°C. After being washed (twice), the parasites were exposed to a 1/20 dilution of fluorescein isothiocyanateconjugated goat anti-mouse immunoglobulin (no. F1010; Sigma) for 30 min at 37°C, with duplicate samples exposed at 4°C. After being washed again, trophozoites were examined as wet preparations on a Leitz Diaplan fluorescence microscope. Controls included cell aliquots fixed with 1% glutaraldehyde and tested as described above and trophozoites tested with buffer alone, without conjugate, or with nonimmune ascites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (immunoblotting). NP-40 protein extracts from axenic and polyxenic strains cultured in liquid Robinson's medium were mixed with sample buffer (62.5 mM Tris-HCl [pH 6], 2% SDS, 5% P-mercaptoethanol, 10% glycerol, 0.002% bromophenol blue). Polyxenic E. histolytica strains cultured in biphasic Robinson's medium were harvested and mixed with sample buffer, and parasite proteins were solubilized by boiling for 4 min. After boiling, samples were centrifuged at 8,500 x g for 1 min at RT and supernatants were loaded for electrophoresis by using 25 pg of protein from the parasites cultured in liquid medium and 30 ,ul of the mixture from parasites cultured in biphasic medium per gel track. A 5-,ul sample of a solution of molecular mass standards (as follows [molecular masses in

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kilodaltons]: myosin, 200; phosphorylase b, 97.4; bovine serum albumin, 69; ovalbumin, 46; carbonic anhydrase, 30; trypsin inhibitor, 21.5; lysozyme, 14.3; no. RPN.756; Amersham International plc, Amersham, Buckinghamshire, England) was mixed with an equal volume of sample buffer, boiled as described above, and loaded in one track of each gel. SDS-PAGE was performed according to Laemmli's method (19) with 12% separating and 4% stacking gels in a Mini-Protean II Dual Slab Cell (no. 165-2940; Bio-Rad Laboratories Ltd., Hemel Hempstead, Hertfordshire, United Kingdom) at 200 V for 45 min. Electrophoresed proteins were transferred to nitrocellulose (no. 401-196; Schleicher & Schuell, Dassel, Germany) by the procedure of Towbin et al. (45) using a semidry electroblotting apparatus (Sartoblot II-S, no. SM 17556; Sartorius Ltd., Epsom, Surrey, United Kingdom) (4 mA/cm2, 30 min). Replica gels were stained with Coomassie brilliant blue. Nitrocellulose was blocked with PBS-0.05% Tween 20-2% casein (PBST/C) (60 min at RT), washed (three times for 10 min each at RT in PBS0.05% Tween 20), and probed with supernatant (1/2 dilution in PBST/C) or mouse ascites (1/100 dilution 1 h, RT) containing invasive isolate-specific MAb. After repeated washing, as described above, the membrane was exposed to a 1/2,000 dilution of peroxidase-conjugated rabbit anti-mouse immunoglobulin (no. 315-036-003; Jackson Immunoresearch Inc., Philadelphia, Pa.) in PBST/C (1 h, RT) and washed again (three times), and the protein bands were developed in 50 mM Tris-HCl (pH 7.4)-0.2 M NaCl-0.8% diaminobenzidine (no. 13033; BDH)-5% 4-chloro-1-naphthol (no. C-8890; Sigma)-0.006% H202 for 2 to 3 min. RESULTS Selection of an invasive isolate-specific MAb against E. histolytica. Nine of seventeen hybridoma cell lines initially selected by ELISA detected one or more bands on Western blots (Fig. 1A). These nine cell lines were screened by IFA on fixed E. histolytica trophozoites, two of which (NIH:200 and SI) were from an invasive zymodeme and two of which (C29 and 8672) were from noninvasive zymodemes. MAb 20/7D, an immunoglobulin Gl mouse immunoglobulin, which detected a single band on the Western blots, in IFA recognized only the invasive E. histolytica trophozoites. This prompted us to test a larger number of reference (HM1:IMSS and SAW 1734) and clinical isolates from different areas of endemicity (Table 1). MAb 20/7D reacted with all the invasive E. histolytica isolates and with none of the noninvasive isolates, including those isolates which were characterized by zymodeme and the four isolates which were characterized on clinical grounds only. The fluorescent patterns observed varied from patchy to homogeneous in the cytoplasm and were more intense in the nucleus and its membrane (Fig. 2). The culture conditions (axenic or polyxenic isolates and liquid or biphasic medium) and the different methods of IFA slide preparation (parasites harvested from liquid medium or spotted directly from biphasic Robinson's medium with its accompanying mixture of starch and bacteria) did not affect the recognition of invasive E. histolytica by MAb 20/7D (Table 1). Interestingly, with some polyxenic isolates, such as BOG-3, not all trophozoites on the slide reacted with the MAb, suggesting the presence of a heterogeneous cell population infecting the patient (Fig. 2). MAb 20/7D did not react in IFA with trophozoites of E. moshkovskii, E. invadens, G. intestinalis, P. hominis, and B. hominis (data not shown). Preliminary characterization of the antigen recognized by

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A

ites (data not shown). This suggests that the epitope recognized by MAb 20/7D is not exposed on the surface of the parasite. In SDS-PAGE and Western blotting, MAb 20/7D recognized a single band with estimated molecular masses of 84 kDa in axenically grown and 81 kDa in polyxenically grown invasive E. histolytica (Fig. 1B). This band appeared clearly in samples from all the invasive isolates tested and as a defined or very faint band in samples from the noninvasive isolates SAW 1734, BOG-1, and C29 (data not shown).

B 1 2

12 3 4 5

200 . 97.4 F 69 . 46 30 -21.5

_

3

200 °i

97.4-F 69 46 30 21.5

_

Lr

DISCUSSION FIG. 1. Western blot of axenic and polyxenic clinical invasive isolates of E. histolytica. NP-40 protein extract of the axenic strain HK9 and the mixture of starch, bacteria, and parasites from the bottom layer of well-grown polyxenic Robinson's cultures were

subjected to SDS-PAGE and transferred to nitrocellulose. (A) Nitrocellulose was cut into individual strips and probed with either culture supernatant diluted 1/2 or serum from the mouse that was used for the fusion diluted 1/200. Lanes: 1 and 2, two different MAbs which were not specific for invasive E. histolytica and which represent the nine hybridoma lines selected by Western blotting for screening by immunofluorescence with invasive and noninvasive E. histolytica trophozoites; 3, invasive isolate-specific MAb 20/7D; 4, polyclonal mouse serum; 5, secondary anti-mouse peroxidase-conjugate only. (B) Nitrocellulose was probed with ascites containing the invasive isolatespecific MAb 20/7D diluted 1/100. Lanes: 1, axenic invasive E. histolytica HK9; 2 and 3, polyxenic invasive clinical isolates BOG-2 and BOG-3 cultured from patients in Colombia with amebic dysentery. Numbers on the left, molecular weight markers (in thousands).

MAb 20/7D. MAb 20/7D reacted with axenic and polyxenic invasive E. histolytica trophozoites fixed with methanol (Fig. 2) and with HM-1:IMSS trophozoites fixed with glutaraldehyde but failed to react with live HM-1:IMSS trophozo-

Tiype and

We have produced a MAb capable of distinguishing invasive strains of E. histolytica from noninvasive strains by IFA. So far, we have tested 88 E. histolytica isolates, comprising 6 reference and 82 clinical isolates, 78 characterized by zymodeme and 4 by clinical information only (see below). The clinical isolates were recovered from patients with symptomatic or asymptomatic infections from Bangladesh, Colombia, and Mexico, countries in which amebic infection is highly endemic (47) and which are widely separated geographically. Overall, the sensitivity and specificity of the IFA based on the invasive isolate-specific MAb 20/7D were both 100%. The 78 clinical isolates characterized by zymodeme represent three different zymodemes, two invasive and one noninvasive, from a total of the six parent zymodemes reclassified by Blanc and Sargeaunt (3). These authors suggested such a reclassification after showing that the amount of starch in the culture medium produced variability in the isoenzyme electrophoresis patterns; this variability produced second-order zymodemes derived from the parent zymodemes. This variability together with the cost and time needed for the zymodeme analysis supports the use of

TABLE 1. Reactivity of MAb 20/7D with reference and clinical isolates of E. histolytica No. with diagnosisZeodemed Culture D ACeme Origin' conditions'

no. of isolatesADDC

IFA

titer

Reference 3

U.K.

U.K.

Ax (2), Xe (1) Xe; L, B (1)

3

3

Clinical 2 8 3 3 16 4 1

Ban Ban Ban Ban Ban Ban

Xe Xe Xe Xe Xe Xe

Col

Xe; L, B

Col U.K.

Xe Xe Xe Xe

2 7 3 2 6 1 1 1

1 2 1 31 2 7 1

Col Ban Col Mex Col

Xe, L, B Xe Xe

3

1 1 9 3

1

2e 1

27

4 2 7 1

II I XIV XIV XIV II II II II II ND ND I I

1:1,600

Negative 1:1,280

1:640 1:320 1:1,280

1:640 1:320

1:3,200 1:1,600 1:1,600 1:1,600 Negative Negative

INegative ND

Negative

a Ban, Bangladesh; Col, Colombia; Mex, Mexico; U.K., United Kingdom. The axenic strains HK9 and HM-1:IMSS were isolated in Korea and Mexico and maintained in culture at LSHTM. respectively, b Ax, axenic isolates cultured in Diamond's medium; Xe, polyxenic or monoxenic isolates with Escherichia coli (all isolates cultured in biphasic Robinson's medium unless otherwise stated); L, liquid medium; B, biphasic medium; numbers in parentheses, numbers of isolates cultured under the conditions stated. c AD, amebic dysentery (erythrocytes observed inside E. histolytica trophozoites by microscopy); D, diarrhea (loose or liquid stools); AC, asymptomatic carrier (formed stools). d XIV and II, invasive zymodemes; I, noninvasive zymodeme; ND, not done. e One isolate recovered from the stools of a patient suffering from an amebic liver abscess.

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simpler laboratory techniques such as MAb-based IFA for the characterization of clinical isolates of E. histolytica (38, 40). Recently developed objective attachments allow fluorescence microscopy to be performed with a standard light microscope in field conditions (20, 26). We have also adapted MAb 20/7D to a capture ELISA for the detection of E. histolytica antigen in feces (14a). Thus, if the IFA assay results are confirmed worldwide with all the zymodemes, zymodeme analysis can be reserved for reference laboratories. The nonuniform reactivity of some clinical polyxenic isolates in IFA agrees with the concept that some patients can be infected with mixtures of E. histolytica strains, which requires cloning of the cultured parasites for reliable zymodeme analysis (31). Direct visualization in the IFA eliminates the need for cloning. Alternatively, this nonuniform reactivity could be the result of differential or developmental regulation of the expression of the epitope by invasive strains in culture. Considering the evolutionary distance between invasive and noninvasive strains (6), it is not surprising to find another antigenic difference that separates them. Nevertheless, MAb 20/7D recognizes an epitope with interesting features. The 81- and 84-kDa protein bearing the epitope is not equivalent to those already described (2, 22, 24, 27, 40, 44). Furthermore, the fact that it is present in both invasive and noninvasive isolates, as shown by Western blotting, but differentially recognized by IFA suggests that this epitope is expressed and processed in such a way that it is accessible to MAb 20/7D only in nondenatured E. histolytica antigens of the invasive zymodemes. Once the protein has been denatured in SDS-PAGE, the epitope in noninvasive strains also becomes accessible to the MAb. Similar examples of cryptic epitopes unmasked by solubilization of proteins have been seen with the Rho(D) antigen of human erythrocytes (25) and membrane antigens of the parasites Leishmania tropica (16) and Trypanosoma cruzi (17). MAb 20/7D has been used to isolate a clone from a cDNA expression library of the E. histolytica invasive strain NIH: 200 (14a). This finding suggests that the reactivity of the epitope with the MAb does not depend on glycosylation of the protein, although glycosylation differences could explain the observed difference in molecular weights between the axenic and the polyxenic isolates studied. The binding of MAb 20/7D might reflect different biological properties such as pathogenicity, although it does not imply major structural differences, as the epitope is present in both strains. Quantitative studies on the expression of the protein in invasive and noninvasive E. histolytica strains and further comparisons of these antigens could give a better insight into the basis of their distinction by the 20/7D MAb. The conservation of the epitope recognized by MAb 20/7D

FIG. 2. Immunofluorescent photomicrographs of methanol-fixed E. histolytica trophozoites probed with mouse ascites containing the invasive isolate-specific MAb 20/7D diluted 1/50, followed by goat anti-mouse fluorescein-conjugated antiserum diluted 1/50 containing Evans blue diluted 1/10,000 for counterstaining. (A) Axenically grown invasive strain HM-1:IMSS; (B) polyxenically grown invasive clinical isolate BOG-3; (C) polyxenically grown noninvasive clinical isolate BOG-1. Axenic and polyxenic trophozoites were cultured in TPS-1 (Diamond's) and Robinson's media, respectively. The polyxenic invasive and noninvasive clinical isolates were isolated in Colombia from a patient with amebic dysentery and an asymptomatic cyst carrier, respectively.

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in the axenic strains HK9 (14) and NIH:200 (43), isolated 39 and 43 years ago, respectively, and in the polyxenic cultures recently isolated from infected patients suggests that the protein bearing this epitope may be essential for the organism. The invasive isolate 2788, from Bangladesh, was recovered from an asymptomatic carrier who could be a source for disease transmission if left untreated. Prospective epidemiological studies using MAb 20/7D would define more clearly the geographical distribution and the patterns of transmission of invasive strains and might allow focused intervention studies for particular regions, communities, or individuals. Although the IFA results obtained with the clinical isolates for which zymodemes had not been determined also accorded with the invasive isolate-specific reactivity of MAb 20/7D, it is necessary to perform zymodeme analysis with cultured E. histolytica trophozoites from a more extensive series of isolates. Field studies are in progress to examine such a series of isolates including all known zymodemes. A limited number of direct fecal smears from patients with dysentery have been examined by fluorescence microscopy after being probed with MAb 20/7D, with encouraging results (data not shown). If perfected, this technique would obviate the need to culture the parasite prior to identification of invasive organisms. ACKNOWLEDGMENTS A.G.R. is a recipient of a scholarship from the Consejo Nacional de Ciencia y Tecnologia (CONACYT), Mexico, and A.A. is partially supported by a scholarship from the Sir Patrick Manson Bequest. This work has been supported by the Nestle Nutrition Research Grant Programme (grant no. 87/34), a grant from the Commission of the European Communities (contract no. STD-2 0206-UK), the British Council in Colombia and the United Kingdom, and the WHO Programme on Intestinal Parasitic Infections. We are very grateful to the following people for providing us with parasite strains: L. S. Diamond, E. histolytica HK9, NIH:200, and HM-1:IMSS; J. P. Ackers, E. histolytica isolates TE, SI, C29, and 8672; D. Mirelman, E. histolytica SAW 1734; J. E. Williams, E. moshkovskii, E. invadens, P. hominis, and B. hominis; and A. Goldin, G. intestinalis Portland 1. We also thank I. Frame for his comments on the manuscript. REFERENCES 1. Ash, L. R., and T. C. Orihel (ed.). 1990. Protozoans, p. 82-88. Atlas of human parasitology American Society of Clinical Pathologists, Chicago. 2. Blakely, P., P. G. Sargeaunt, and S. L. Reed. 1990. An immunogenic 30-kDa surface antigen of pathogenic clinical isolates of Entamoeba histolytica. J. Infect. Dis. 162:949-954. 3. Blanc, D., and P. G. Sargeaunt. 1991. Entamoeba histolytica zymodemes: exhibition of a and 8 bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch content in the medium. Exp. Parasitol. 72:87-90. 4. Bracha, R., L. S. Diamond, J. P. Ackers, G. D. Burchard, and D. Mirelman. 1990. Differentiation of clinical isolates of Entamoeba histolytica by using specific DNA probes. J. Clin. Microbiol. 28:680-684. 5. Brumpt, E. 1925. Etude sommaire de l"Entamoeba dispar' n. sp., amibe a kystes quadrinuclees parasite de l'homme. Bull. Acad. Med. 94:942-952. 6. Clark, C. G., and L. S. Diamond. 1991. Ribosomal RNA genes of 'pathogenic' and 'nonpathogenic' Entamoeba histolytica are distinct. Mol. Biochem. Parasitol. 49:297-302. 7. Cruz-Reyes, J. A., W. M. Spice, T. Rehman, E. Gisborne, and J. Ackers. 1992. Ribosomal DNA sequences in the differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica. Parasitology 104:239-246. 8. De la Torre, M., L. Landa, and B. Sepulveda. 1970. Avances en

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