Production of Monoclonal Antibodies Directed against the Microsporidium Enterocytozoon bieneusi

JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010

Dec. 1999, p. 4107–4112 Vol. 37, No. 12

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Production of Monoclonal Antibodies Directed againstthe Microsporidium Enterocytozoon bieneusi

ISABELLE ACCOCEBERRY,* MARC THELLIER, ISABELLE DESPORTES-LIVAGE,ABDERRAHIM ACHBAROU, SYLVESTRE BILIGUI, MARTIN DANIS, AND ANNICK DATRY

Unite INSERM 511, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier-Universitairede la Pitie-Salpetriere, 75013 Paris, France

Received 24 March 1999/Returned for modification 24 June 1999/Accepted 7 September 1999

Several hybridomas producing antibodies detected by indirect immunofluorescence antibody test (IFAT)were established by fusion of mouse myeloma SP2/O with spleen cells from BALB/c mice immunized againstwhole spores (protocol 1) or chitinase-treated spores (protocol 2) of Enterocytozoon bieneusi and were clonedtwice by limiting dilutions. Two monoclonal antibodies (MAbs), 3B82H2 from protocol 1, isotyped as immu-noglobulin M (IgM), and 6E52D9 from protocol 2, isotyped as IgG, were expanded in both ascites and culture.IFAT with the MAbs showed that both MAbs reacted exclusively with the walls of the spores of E. bieneusi,strongly staining the surface of mature spores, and produced titers of greater than 4,096. Immunogold electronmicroscopy confirmed the specific reactivities of both antibodies. No cross-reaction, either with the spores ofthe other intestinal microsporidium species Encephalitozoon intestinalis or with yeast cells, bacteria, or anyother intestinal parasites, was observed. The MAbs were used to identify E. bieneusi spores in fecal specimensfrom patients suspected of having intestinal microsporidiosis. The IFAT was validated against standardstaining methods (Chromotrope 2R and Uvitex 2B) and PCR. We report here the first description andcharacterization of two MAbs specific for the spore wall of E. bieneusi. These MAbs have great potential for thedemonstration and species determination of E. bieneusi, and their application in immunofluorescence identi-fication of E. bieneusi in stool samples could offer a new diagnostic tool for clinical laboratories.

Microsporidia are obligate intracellular protistan parasitesthat infect a variety of cells from a wide range of invertebrateand vertebrate hosts. Also identified in humans, more espe-cially in immunocompromised patients, microsporidia appearto be major opportunistic pathogens. These parasites wereshown to be the prevalent cause of intestinal infections re-ported in patients with AIDS and diarrhea (17, 21) in indus-trialized countries. Significantly, the introduction of antiretro-viral combination regimens including human immunodeficiencyvirus (HIV) protease inhibitors has resulted in a decrease inthe number of cases of AIDS-related microsporidiosis (10, 12).

The first documented case of intestinal infection was causedby Enterocytozoon bieneusi (7), the microsporidian speciesmost commonly found in humans. This parasite is usually ob-served in HIV-infected patients with CD4 lymphocyte countsof less than 50 cells/mm3 who complain of chronic diarrhea,nausea, malabsorption, and severe weight loss (4, 24). Enceph-alitozoon intestinalis (14) also causes intestinal infections fre-quently associated with nephritis, sinusitis, or bronchitis (17,21). These parasites are also pathogenic in subjects with im-munodeficiency due to causes other than AIDS. Cases of in-testinal microsporidiosis have been detected in organ trans-plant recipients (25, 28). The two species E. bieneusi and E.intestinalis also appear to be responsible for cases of diarrheain immunocompetent subjects (11). Most of them are pre-sented by travellers returning from tropical areas (26, 27, 29,34). Less expected is the increasing number of HIV-seroneg-ative and asymptomatic individuals found to be infected withmicrosporidia (8, 15, 32).

Over the past 10 years, different diagnosis methods, based

on the detection of the parasites’ spores in stools and otherbiological samples, have been proposed (3, 16, 31, 39). Al-though PCR appears to be the most sensitive method (6, 9, 11,23), immunological tools remain helpful for diagnosis and forepidemiological survey or experimental investigation. Specificmonoclonal antibodies (MAbs) against E. intestinalis isolateseasily obtained through in vitro systems have been produced(5). To date, such systems are still lacking for E. bieneusi.However, spores of this species extracted from fecal samplesenabled the production of the MAbs described in the presentstudy.

MATERIALS AND METHODS

Sources of parasites. (i) E. bieneusi. In the absence of an in vitro cultivationmodel and due to the invasive procedures needed for collecting epithelium orfluid samples from the gastrointestinal tract, human stools were the source of E.bieneusi spores. Fecal specimens were obtained from HIV-infected patients.Microsporidian spores were detected by fluorochrome Uvitex 2B stain (31) andWeber’s chromotrope-based modified trichrome stain (16). Fecal samples con-taining numerous small oval spores were homogenized and suspended in asolution of phosphate-buffered saline (PBS; Sigma Laboratories, Saint-Quentin-Fallavier, France). The samples were processed for transmission electron mi-croscopy (TEM) and tested by PCR to confirm the identification of the speciesand to exclude a concomitant E. intestinalis infection.

(ii) TEM. The fecal samples were fixed at room temperature in 2.5% glutar-aldehyde in 0.1 M Na cacodylate buffer (pH 7.2) for 60 min, rinsed in buffer, andthen postfixed in ferriosmium [1% (wt/vol) OsO4 and K3Fe(Cn)6 in cacodylatebuffer] for 60 min. After ethanolic dehydration, the samples were embedded inSpurr resin. Thin sections, stained with uranyl acetate and lead citrate, wereexamined with a JEOL JEM 100 CX transmission electron microscope.

(iii) PCR amplification. The PCR assay was performed as described previouslyby Ombrouck et al. (23). The primers V1 (59-CACCAGGTTGATTCTGCCTGAC-39) and EB450 (59-ACTCAGGTGTTATACTCACGTC-39) described byZhu et al. (38) were used to amplify E. bieneusi DNA. The primers V1 and SI500(59-CTCGCTCCTTTACACTCGAA-39) described by Weiss et al. (37) wereused to amplify E. intestinalis DNA.

(iv) E. intestinalis. Spores were obtained from cultures in rabbit kidney cells(RK13), as described by van Gool et al. (30). Parasite spores were harvestedweekly, counted in a hemocytometer, resuspended in PBS, and stored at 4°Cuntil used.

* Corresponding author. Mailing address: Laboratoire de Parasi-tologie-Mycologie, CHU de Bordeaux, 1, rue Jean Burguet, 33000Bordeaux, France. Phone: 33 5-56 79 58 37. Fax: 33 5-56 79 58 79.E-mail: [email protected].

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Antigen preparation procedures. (i) Spore concentration. The stool suspen-sion was filtered through a graded series of six nylon sieves (pore diameters were100, 50, 30, 20, 10, and 5 mm, respectively). The filtration was facilitated by theaddition of 1,000 to 2,000 ml of PBS. The final filtrate was centrifuged at 500 3g for 6 min to eliminate large particles, and the sieved spores in the supernatantwere pelleted by centrifugation at 2,500 3 g for 20 min. The pellet was resus-pended in PBS (1/3 [vol/vol]).

(ii) Spore purification. Density gradient centrifugation was performed withvarious concentrations of Percoll (19). The discontinuous gradient consisted of10 ml of stock isotonic Percoll solution (prepared by mixing 10 ml of 10-fold-concentrated PBS and 90 ml of Percoll [Sigma Laboratories] to yield a pH of 7.4and an osmolarity of 335 mosM), 10 ml of 67.5% stock Percoll diluted with PBS,10 ml of 45% stock Percoll diluted with PBS, and 10 ml of 22.5% stock Percolldiluted with PBS. Five milliliters of the spore suspension was layered over thegradient into a 50-ml Falcon centrifuge tube. After centrifugation at 2,500 3 gfor 30 min at 15°C, four distinct bands were formed. The clearly defined ring atthe 90 to 67.5% Percoll interface, previously determined to contain whole sporesby light microscopy and TEM (results not shown), was collected, washed threetimes in PBS, pelleted at 2,500 3 g for 20 min, and resuspended in PBS (1/3[vol/vol]).

(iii) Sterilization. To monitor for bacterial and fungal contaminants, the iso-late of spores was mixed with an antibiotic solution of ceftriaxone (20 mg/ml),vancomycin (10 mg/ml), amikacin (10 mg/ml), and amphotericin B (0.25 mg/ml)and placed at 4°C. Antibiotics were added daily at the same concentration untilsterilization of the preparation as determined by aerobic and anaerobic cultiva-tion was achieved. After 3 to 5 days of this regimen, the sterile concentrate wascentrifuged at 2,500 3 g for 20 min and washed twice in sterile PBS.

The pellet was then divided into two aliquots of 1 ml each in sterile PBS, in oneof which spores were incubated for 60 min at room temperature with 50 ml of aconcentrated (5 IU/ml) chitinase from Serratia marcescens (Sigma Laboratories),treated by two freeze-thaw cycles, centrifuged at 2,500 3 g for 20 min, andexamined after fluorochrome Uvitex 2B stain.

Aliquots were resuspended and diluted in sterile 0.15 M NaCl (1/3 [vol/vol]).Spore counts were performed by using 2-ml droplets applied to 5-mm-wide wellson multiwell slides, stained according to the Uvitex 2B method.

Production of MAbs. Adult (6-week-old) female BALB/c mice, for hybridomadevelopment and ascites production, were purchased from Charles River Lab-oratories (Saint-Aubin-les-Elbeuf, France). Two protocols of immunization, us-ing two groups of six mice each, were carried out. In protocol 1, animals receivedwhole spores of E. bieneusi; in protocol 2, they received chitinase-treated spores.All the animals were immunized intraperitoneally (i.p.) four times at 3-weekintervals with 5.6 3 107 spores per 100 ml emulsified at a 1:1 ratio in Freund’scomplete adjuvant (Sigma Laboratories) for the first inoculation and in Freund’sincomplete adjuvant (Sigma Laboratories) for the other three inoculations. Twocontrol mice were not injected.

Seven days after each immunization, sera were screened by indirect immuno-fluorescence as described below to determine which mice had the highest para-site-specific antibody responses. Mouse serum adsorption experiments were per-formed with an antigen preparation of enteropathogenic bacteria and yeastsisolated from human stool samples and grown on aerobic culture. The prepara-tion was added to serum samples prediluted in PBS, which subsequently wereshaken at room temperature for 120 min and spun down (10,000 3 g, 5 min).Measurements were done by using the supernatants. Sera were stored at 280°Cand used as positive controls during all immunoassays.

Two of the immunized mice, one in each group, were selected to receive afurther intravenous dose of 2 3 107 spores in 100 ml of sterile 0.15 M NaCl, andtheir spleens were used for the fusion protocol 3 days later (one fusion protocolper immunization protocol). Cells of the murine myeloma line SP2/O were fusedwith spleen cells from the donor mouse at a 1:5 ratio in 50% polyethylene glycol(Sigma Laboratories) (13). Stable hybrids were selected by growth in Dulbecco’sminimum essential medium containing 20% fetal bovine serum, hypoxanthine,aminopterin, and thymidine as previously described (33). Supernatant culturemedium was screened by an indirect immunofluorescence antibody test (IFAT).Hybridoma cultures whose supernatants showed antibody activity against E.bieneusi were expanded onto 24-well plates and cloned twice by limiting dilutions(13). Pristane-primed female BALB/c mice were injected i.p. with 2 3 106 cellsfrom each hybridoma line, and ascitic fluid was collected 10 to 15 days later,centrifuged (400 3 g for 15 min) to remove cells, aliquoted, and stored at 280°Cuntil used (13). Culture supernatants of the different hybridoma lines were alsocollected. MAbs were purified from ascites or supernatants with DynabeadsM-450 rat anti-mouse immunoglobulin M (IgM) and M-450 rat anti-mouseIgG2a (Dynal, Compiegne, France), according to the manufacturer’s instruc-tions.

IFAT. The IFAT was performed with (i) washed whole spores of E. bieneusi,which were used for the immunization in protocol 1, suspended in PBS to obtain108 spores per ml and (ii) E. intestinalis spores from tissue culture supernatantsresuspended in PBS at 109 spores per ml, as antigens. Antigen slides wereprepared by depositing 2-ml volumes of the spore suspension onto each well of18-well slides, which were then air dried and fixed in ice-cold acetone for 10 min.

Undiluted supernatants of hybridoma cultures or the ascitic fluid, seriallydiluted twofold in 0.1% bovine serum albumin (BSA) in PBS starting from a 1:2dilution, were transferred to the 18-well slides (20 ml of each dilution per well),

and the slides were incubated at room temperature for 30 min in a moistchamber. The slides were then washed three times in PBS at 10-min intervals,and each well was then covered with 20 ml of fluorescein isothiocyanate (FITC)-labelled goat anti-mouse IgG-IgM-IgA (Sigma Laboratories) at a dilution of1:200 containing Evans blue as the counterstain. The slides were incubated atroom temperature for 30 min and washed three times as described above,coverslips were added with buffered glycerol mounting fluid, and the slides wereexamined with a Leitz Laborlux fluorescence microscope equipped with epiflu-orescence illumination. PBS and conjugate controls were included with eachslide.

Characterization of anti-E. bieneusi MAbs. (i) Isotyping. The MAb isotypeswere determined with a dipstick isotyping kit (Sigma Laboratories) according tothe instructions enclosed.

(ii) Ultrastructural immunolocalization. Stool samples from patients with anE. bieneusi infection were purified by gentle filtration and Percoll discontinuousgradient as described earlier. Then they were fixed at room temperature in 4%paraformaldehyde–0.5% glutaraldehyde in 0.15 M Na cacodylate buffer (pH 7.2)for 45 min and rinsed three times at 10-min intervals in 0.1 M ammoniumchloride in cacodylate buffer and one time for 10 min in cacodylate buffer alone.After ethanolic dehydration, the material was embedded in LR WHITE resin.The sections were collected on gold or nickel grids. They were incubated at roomtemperature in 1% BSA in PBS for 45 min to block unbound sites and then for120 min with ascitic fluid containing MAbs (1:512). After a series of six washings(5 min each), in 0.25% BSA in PBS, sections were incubated for 60 to 120 minwith goat anti-mouse affinity-purified IgM or goat anti-mouse affinity-purifiedIgG labelled with 10-mm gold particles (Sigma Laboratories) used as secondantibody. Controls consisted of sections incubated with the second antibodyalone. After being washed in sterilized water, samples were examined with aJEOL JEM 100 CX transmission electron microscope.

(iii) SDS-PAGE and Western blot analysis. Western blot analysis was per-formed with E. intestinalis spores used as antigens. Parasite proteins were sepa-rated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (18), with a 5% stacking gel and a12% resolving gel. Intact spores in 1 ml of sample buffer containing 5% b-mer-captoethanol were boiled for 5 min and centrifuged at 10,000 3 g to removeparticulate materials. Each preparative slab gel (16 by 20 cm) was loaded with2 3 109 parasites. After electrophoresis, the separated polypeptides were elec-trophoretically transferred onto a nitrocellulose membrane (pore diameter, 0.22mm; Bio-Rad, Ivry-sur-Seine, France) which was then incubated with 5% (wt/vol)nonfat dry milk (Regilait) in PBS for 60 min to block unbound sites, washed inPBS containing 0.05% Tween 20 (PBS-Tween) for 20 min and cut into 3-mm-wide longitudinal strips. The strips were incubated for 60 min with either undi-luted supernatants or ascitic fluid containing MAbs (1:512), murine immunesera, preimmune murine sera, or a specific E. intestinalis IgG3 6C12C11 MAbpreviously developed in our laboratory (unpublished data) as a positive control.After being washed in PBS-Tween, strips were incubated for 60 min with affinity-purified peroxidase-labelled goat anti-mouse IgG-IgM antiserum (Sigma Labo-ratories) diluted 1:2,000 and developed with 4-chloro-1-naphthol (Sigma Labo-ratories) after being washed with three changes of PBS-Tween. After colordevelopment for 30 min, the strips were rinsed in distilled water, dried, andstored in the dark.

(iv) Cross-reactivity studies. The reactivity of the MAbs with bacteria, para-sites, and fungi was assessed by IFAT.

Utilization of MAbs in diagnosis and comparison with other methods. Fifteendiarrheal fecal samples containing microsporidial spores and 25 negative stoolsamples were collected and preserved in PBS with 10% Formol (1/3 [vol/vol]).Intestinal microsporidiosis had been previously diagnosed in the 15 patients byclassical staining methods (16, 31). The fecal samples were filtered through a50-mm-pore-diameter filter, and after ether sedimentation by centrifugation at2,500 3 g for 15 min, the pellet was suspended in PBS (1/3 [vol/vol]) and appliedto slides. All the stool samples were coded and blind-tested by IFAT. Thespecificities of the MAbs were evaluated by comparison with PCR and TEM (ifavailable) performed as described previously.

RESULTS

MAb production. Prior to immunization, mouse sera werescreened for antibodies against intestinal microsporidiumspores by IFAT and Western blot analysis, with E. bieneusi andE. intestinalis spores as antigens. Preimmunization sera andhealthy-mouse-control sera did not react with any of the par-asite spores. Because they were immunized with impure anti-gens, BALB/c mice were screened for serum parasite-specificantibody response 7 days after each injection before a finalboost and fusion. All mice began to produce antibodies afterthe second i.p. injection. The highest antibody response wasraised after the fourth injection. Sera from mice 2-1 (protocol1) and 3-2 (protocol 2) produced the better fluorescence (lim-

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iting dilution, 1:100). These two mice were thus selected for thefusion protocol.

For each immunization protocol, a single fusion was per-formed with spleen cells of the donor mouse. A total of 960wells were seeded with fused SP2/O myeloma cells, and theirsupernatants began to be screened by whole spore E. bieneusiIFAT 11 days after fusion. After the third screening, six anti-body-secreting hybridomas still reacted against spores of E.bieneusi. After two cloning procedures by limiting dilutions,two stable clones, clone 3B82H2 from protocol 1 and clone6E52D9 from protocol 2, were obtained. Isotype determina-tion revealed that clone 3B82H2 secreted IgM and clone6E52D9 secreted IgG2a. The two MAbs were expanded inboth ascites and culture and subjected to thorough screening.

IFAT. The two MAbs showed strong indirect immunofluo-rescence after incubation with whole purified E. bieneusispores, almost to the same extent, and yielded titers of greaterthan 4,096. All reacted exclusively with the walls of the spores,which fluoresced brightly and were thus easily recognized witha 31,000 magnification (Fig. 1A and B). 3B82H2 and 6E52D9showed mutual competition in their binding to E. bieneusiwhen the two MAbs were combined in one IFAT. No fluores-cence was observed on E. intestinalis spore walls or filaments orwhen FITC-conjugated second antibody was employed alone.

Characterization of the MAbs. (i) Ultrastructural immuno-localization. The binding of each MAb to E. bieneusi sporeswas studied by TEM which revealed a labelling of the sporewall, more especially when sections of mature spores wereincubated with 3B82H2 or 6E52D9 MAb. Interestingly, differ-ent parts of the spore wall reacted with these MAbs. Goldparticles were exclusively distributed over the exospore in spec-imens treated with the IgG MAb 6E52D9 (Fig. 2A and B),whereas a labelling of the endospore was obtained with theIgM MAb 3B82H2 (Fig. 2C and D). No labelling was observedwhen sections were incubated with the second antibody alone.No reactivity was displayed by E. intestinalis spores collected inculture supernatants or in stool samples. The immunogoldlabelling of the spore wall was consistent with the IFAT results.

(ii) Western blot analysis. Neither of the two MAbs reactedwith E. intestinalis antigens by Western blot analysis, comparedto an IgG3 6C12C11 MAb directed against E. intestinalis wholespores.

(iii) Cross-reactivity studies. By IFAT, MAbs in ascitic fluidwere assessed for cross-reactivity to enteropathogenic bac-teria (Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae,Shigella dysenteriae, Salmonella typhi, Yersinia enterocolitica,Pseudomonas aeruginosa, Enterobacter aerogenes, Enterococcusfaecalis), other intestinal parasites (Giardia intestinalis, Entam-oeba histolytica, Entamoeba coli, Cryptosporidium parvum, Sar-cocystis hominis, Isospora belli, Blastocystis hominis), and yeastsfrom stool samples. There was no cross-reactivity even at the1:128 dilution to any of the organisms evaluated.

Utilization of MAbs in diagnosis and comparison with othermethods. By Uvitex 2B and Weber’s modified trichrome stain-ing methods, spores with the morphological features charac-teristic of microsporidia (16, 31) were detected in fecal speci-mens from 15 patients. Among these samples, 14 containedsmall oval spores suggestive of E. bieneusi, whereas 1 stoolcontained larger spores suggestive of E. intestinalis.

By IFAT, the two MAbs reacted exclusively with the smalloval spores present in the 14 samples (Table 1). These sporesfluoresced brightly, 41 in a scale of 0 to 4, with a prominentlabelling of the spore walls when smears were treated with3B82H2 or 6E52D9 MAb at 1:512 and 1:1,024 dilutions (Fig.1C). Neither of the two MAbs generated background in for-malin-fixed stool specimens. No fluorescence was observed

either in the stool containing larger spores suggestive of E.intestinalis or in stools from patients without intestinal micro-sporidiosis.

The IFAT was compared for reliability with the PCR andTEM (if available). The results were consistent with those ofIFAT (Table 1). The microsporidian species of each positivesample was confirmed and E. bieneusi but not E. intestinalisspores were recognized by MAbs 6E52D9 and 3B82H2. ByPCR, no signals were observed for the 25 fecal specimensnegative for microsporidia.

FIG. 1. Purified whole spores of E. bieneusi stained by indirect immunoflu-orescence with MAbs 6E52D9 (A) and 3B82H2 (B) in ascitic fluid. MAbsrecognize antigens localized in the spores walls. (C) Formalin-fixed smear of afecal sample, from one of the 14 patients with microsporidia, reacted with a 1:512dilution of MAb 6E52D9. Note the bright fluorescence of spore walls. Bar 5 5mm.

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DISCUSSION

We report here the production, characterization, and reac-tivity of the first MAbs directed against E. bieneusi, the mostcommon microsporidium infecting AIDS patients. Since there

FIG. 2. Immunogold electron micrographs of E. bieneusi mature spores afterincubation with MAb 6E52D9 and MAb 3B82H2. (A) Labelling of the outerlayer of the spore wall (exospore [arrowhead]) with MAb 6E52D9. The arrowindicates the double row arrangement of E. bieneusi polar tube sections. Mag-nification, 3120,000. (B) The MAb selectively labels the exospore (arrowhead).No gold particles are visible at the surface or inside the bacteria (left). Magni-fication, 3100,000. (C) The tangential section of the spore treated with MAb3B82H2 shows the distribution of gold particles in the internal layer of the wall(endospore [arrowhead]). Magnification, 3100,000. (D) Labelling of the endo-spore (arrowhead) with the same MAb. Magnification, 3100,000.

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is no in vitro culture system presently available, it has beenimpossible to produce enough antigen to screen for specificantibodies. We circumvented this problem by developing aprocedure for the isolation, purification, and sterilization ofparasite spores from human stools. Apparently, the best pres-ervation of the spore antigens is obtained when using gentlefiltration, centrifugation in isotonic conditions, and gradualaddition of low concentrations of antibiotics to the final fecalsuspension.

The most difficult part of the study was identification of anMAb with the desired specificity because immunization wasdone with impure antigenic material and assays to detect thedesired MAb were not available. However, the IFAT per-formed with E. bieneusi-purified whole spores as antigens ap-peared to be specific enough to select the MAbs that bound tospore walls, and the results of the immunofluorescence assayscould be confirmed at the ultrastructural level by TEM.

Of the two MAbs, one belongs to the IgM class (protocol 1)and the other belongs to the IgG class (protocol 2). The spe-cific reactivity of the selected antibodies depends on the natureof the immunogen. Significantly, the MAb 3B82H2 reactivewith the endospore known to contain chitin was raised againstthe antigenic fraction which was not treated with chitinase.

Extensive cross-reactivities among different microsporidianspecies have been observed with polyclonal sera from rabbitsimmunized with a single microsporidian species (22). Morerecently, polyclonal antisera raised against Encephalitozoon cu-niculi in rabbits or in mice were used in the IFAT to detect E.bieneusi organisms in deparaffinized tissue sections (36) and instool (3, 39). Using the MAbs described in this study in eitherIFAT, TEM or Western blot analysis, we did not observe anycross-reactions with the other human intestinal microspo-ridium, E. intestinalis, or with other intestinal parasites, yeastcells, or bacteria.

The reference techniques used for the comparative detec-tion of microsporidia directly from fecal specimens were PCR,TEM, and Uvitex 2B and Weber’s modified trichrome stain-ings. Immunofluorescence assay with MAbs appeared to be

highly specific. E. bieneusi spores were identified in the 14 stoolspecimens with complete concordance with the results of PCRand TEM (if available). The sensitivities of the MAbs in de-tecting subclinical infections seemed attractive. Indeed, thediagnosis could be performed even when few spores were ex-creted in stool samples. It is noteworthy that no backgroundwas observed in the fecal specimens which were examined bythe immunofluorescence protocol described herein. Moreover,the application of these MAbs as tools for detecting E. bieneusiin feces does not require either pretreatment of the samples orabsorbing the FITC-conjugated antibodies with formalin-fixedstool sediment, as described by others (3, 5).

Diagnosis of microsporidiosis to the species level is essentialfor the treatment of patients. To date, although different ther-apeutic agents are effective against most microsporidian spe-cies, none can eradicate E. bieneusi except fumagillin, which isstill under investigation (20). Presently, the identification ofhuman microsporidia to the species level requires time-con-suming methods such as electron microscopy and moleculartechniques (6, 7, 9, 11, 23, 35). The two MAbs newly describedin this study are the first to be raised against E. bieneusi. Theirapplication in the immunofluorescence identification of thisorganism could offer a new diagnostic tool for clinical labora-tories. The bright fluorescence of the spore wall facilitates thediagnosis even for the untrained eye. In addition these MAbscould offer new approaches for the study of E. bieneusi. Usedas ligands, they could enable the isolation and purification of E.bieneusi spores with methods such as affinity chromatographyor immunomagnetic separation. Ongoing studies will deter-mine the usefulness of these techniques in our follow-up assaysto develop in vivo (1, 2) or in vitro models of E. bieneusi as wellas in Western blot analysis or enzyme-linked immunosorbentassay.

ACKNOWLEDGMENTS

We thank Jean Jacques Hauw for providing TEM facilities andJacques Breton for revising the manuscript.

This study was supported by grants from SIDACTION.

TABLE 1. Comparison of four diagnostic methods for the 15 patients with microsporidia detected by stained smears of stool samplesa

Caseno. Sexc Age

(yr)HIVstatus

No. ofCD4/mm3

Stool sample results by:

Light microscopyb PCRd

TEMIFAT

Small Large E. bieneusi E. intestinalis 3B82H2 6E52D9

1 M 30 1 44 N 1 2 ND 1 12 M 16 2 737 VN 1 2 E. bieneusi 1 13 M 52 1 0 R 1 2 E. bieneusi 1 14 M 42 1 119 F 1 2 E. bieneusi 1 15 F 26 1 13 N 1 2 E. bieneusi 1 16 M 38 1 35 N 1 2 ND 1 17 M 39 1 23 R 1 2 ND 1 18 M 29 1 10 N 1 2 E. bieneusi 1 19 F 35 1 3 VN 1 2 E. bieneusi 1 110 M 39 1 41 R 2 1 E. intestinalis 2 211 M 49 1 20 N 1 2 ND 1 112 M 50 2 ND N 1 2 ND 1 113 M 52 1 6 N 1 2 E. bieneusi 1 114 M 45 1 40 F 1 2 ND 1 115 M 34 1 15 VN 1 2 ND 1 1

a 1, positive; 2, negative; ND, not done.b Uvitex 2B stain and Weber’s modified trichrome stain were performed for all patients. Spores were classified as either small (diameter, 1 to 1.5 mm) or large

(diameter, 1.2 to 2.2 mm). Classification of spore quantity per microscopic field (magnification, 31,000; oil immersion): VN, very numerous; N, numerous; F, few; R,rare.

c M, male; F, female.d Specific PCR assay for direct detection of intestinal microsporidia.

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