Prevalence of antibody to Trypanosoma cruzi in three populations in Belize.

Cross-sectional studies were conducted to determine the prevalence of antibody to Trypanosoma cruzi, the agent of Chagas' disease among three populations in Belize. Specimens were tested using a second-generation enzyme-linked immunoassay (EIA). Confirmatory testing with three single-antigen EIAs and a radioimmunoprecipitation assay (RIPA) were performed. Five (0.5%) of 962 blood donors at the Belize City Hospital were reactive including four (6%) of 65 donors from countries known to be endemic for Chagas' disease and one (0.1%) of 861 from Belize (P < 0.001). Among 467 healthy members of the Belize Defense Force, none were reactive. The third group included workers and families primarily from other Central American countries living on a banana plantation in a rural area of the country. Twenty-seven (6.1%) of 442 sera were reactive. The prevalence was 5.3% of 75 < 15 years of age, 4.2% of 236 15-34 years of age, and 9.7% of 124 > or = 35 years of age (P = 0.11, by chi-square for trend). The prevalence was similar in males (6.7% of 280) and females (5.8% of 154). The prevalence of those born in Belize (4 of 56, 7.1%) was similar with that of those born in El Salvador (9 of 110, 8.2%), Guatemala (6 of 117, 5.1%), and Honduras (8 of 129, 6.2%). Of the four persons with reactive sera who were born in Belize, the immigrant mother of one was also reactive, suggesting possible congenital transmission. Among 31 sera repeatedly reactive by EIA to T. cruzi that were further studied, 22 (71%) were reactive by at least two of three single-antigen confirmatory EIAs and 29 (94%) by RIPA. Additional studies should focus on the epidemiology of T. cruzi and ways to reduce risk of transfusion-related infections in Belize.

Detection of antibodies to Trypanosoma cruzi among blood donors in the southwestern and western United States. I. Evaluation of the sensitivity and specificity of an enzyme immunoassay for detecting antibodies to T. cruzi.

BACKGROUND: Chagas' disease or American trypanosomiasis, caused by infection with Trypanosoma cruzi, is a significant health problem in Latin America. In the United States, transfusions of T. cruzi-contaminated blood from Latin American immigrants may represent the major source of Chagas' disease. STUDY DESIGN AND METHODS: A new enzyme immunoassay (EIA) for the detection of antibody to T. cruzi was evaluated in the sera of blood donors from the southwestern and western regions of the United States. Serum samples had been screened and were negative for all tests required. Specimens that were repeatedly reactive in the Chagas antibody EIA were analyzed for seroreactivity by a confirmatory EIA and by radioimmunoprecipitation assay. RESULTS: Fourteen of the 13,309 donor samples (0.105%) were confirmed as being positive for antibody to T. cruzi. The Chagas antibody EIA showed improved sensitivity over the Chagas IgG enzyme-linked immunosorbent assay and two indirect hemagglutination assays. The Chagas antibody EIA had a specificity of 99.98 percent with negative samples. The sensitivity of the Chagas antibody EIA was 100 percent (80/80) in xenodiagnosed specimens and 100 percent (50/50) in specimens positive by consensus (i.e., reactive in EIA, indirect hemagglutination assay, and immunofluorescence assays). CONCLUSION: This Chagas antibody EIA meets the need for accurate and rapid identification of seroreactive samples in low-prevalence or endemic populations.

Detection of antibodies to Trypanosoma cruzi among blood donors in the southwestern and western United States. II. Evaluation of a supplemental enzyme immunoassay and radioimmunoprecipitation assay for confirmation of seroreactivity.

BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is endemic to Latin America and may be transmitted in the United States via blood donated by infected immigrants. Blood-borne pathogens such as T. cruzi require supplemental testing for confirmation of seroreactivity. STUDY DESIGN AND METHODS: A study was undertaken to determine an optimal scheme for confirmation of seroreactivity in repeatedly reactive samples identified by the Chagas antibody enzyme immunoassay (EIA). The procedure for initial confirmation involves three purified antigens coated onto three separate polystyrene beads and uses an EIA format. If the sample is reactive with two of three or three of three antigens, it is confirmed as seroreactive. If none or one of three beads is reactive, the sample is indeterminate and subjected to a radioimmunoprecipitation assay (RIPA). The RIPA must demonstrate characteristic bands at 32, 34, and 90 kDa. RESULTS: When tested with sera from persons with potentially cross-reactive diseases (n = 39) or against a presumed negative population from southeast Wisconsin (n = 289), the confirmatory EIA had a specificity of 100 percent. Sensitivity was 100 percent (28/28) with xenodiagnosis-positive sera and 97.6 percent (80/82) with chagasic sera from Latin America. The RIPA showed a specificity of 100 percent in EIA-nonreactive samples (n = 100) and a sensitivity of 100 percent with both xenodiagnosis-positive (28/28) and chagasic (82/82) sera. CONCLUSION: The confirmatory EIA and the RIPA together provide a highly specific and sensitive means of confirming seroreactivity for antibodies to T. cruzi.

The biosynthesis, processing, and immunolocalization of Leishmania pifanoi amastigote cysteine proteinases.

Biosynthesis, enzymatic processing, and immunocytochemical localization of an abundant developmentally regulated cysteine proteinase of Leishmania pifanoi, Lpcys2, were investigated employing axenic cultured amastigotes and monoclonal antibodies specifically recognizing either the mature proteinase or the carboxy-terminal extension domain. Pulse labeling and protein sequence data indicated that a 45-kDa precursor is processed to a 40-kDa intermediate, which is further cleaved to generate the 27-kDa mature enzyme and a 15-kDa COOH-terminal domain. Evidence indicates that proteolytic activity is associated with the intermediate form as well as the mature proteinase. Treatment with selected cysteine but not aspartic acid proteinase inhibitors arrested proteolytic processing of Lpcys2 in vivo and inhibited parasite cell division. Electron microscopic immunolocalization of both catalytic and COOH-terminal domains in L. pifanoi and Leishmania amazonensis amastigotes showed intense labeling of megasomes, indicating that cleavage of the COOH-terminal domain probably occurs in the megasome. A low level of the mature proteinase was also associated with the flagellar pocket and plasma membrane; consistent with this observation, low level secretion of Lpcys2 into the culture medium was detected. Lpcys1, a related, less abundant amastigote-specific cysteine proteinase lacking a comparable COOH-terminal domain, was localized to the flagellar pocket and megasomes. Consequently, enzyme sorting to megasomes does not appear to depend upon the COOH-terminal domain; hence this region of Lpcys2 may not be essential for its intracellular targeting.

Developmental life cycle of Leishmania--cultivation and characterization of cultured extracellular amastigotes.

The biochemistry and immunology of Leishmania promastigotes has been extensively studied; this is due primarily to the facility with which this stage, in contrast to the amastigotes stage, can be maintained in axenic culture. Several attempts to axenically culture lines of Leishmania amastigotes have been reported in the literature. This paper summarizes methods of adaptation (low pH, elevated temperature and culture medium) and characterization of several axenic lines of Leishmania amastigotes. Based on morphological, biological, immunological and biochemical evidence, these organisms appear to resemble amastigotes from infected macrophages or tissue. The axenically cultured amastigotes appear to be distinct from shocked (heat, serum deprivation, stressed) Leishmania promastigotes in the plethora of proteins synthesized, growth (multiplication) in culture, and developmental regulation observed. These data suggest that Leishmania organisms have a significant developmental response to certain signals (pH, temperature) mimicking their in vivo macrophage milieu. The response to other environmental parameters characteristic of the host-macrophage remain to be determined. These axenically cultured amastigotes should be of interest for further immunological, biochemical and developmental investigations of the disease-maintaining stage of this parasite.

Clinical evaluation of an EIA for the sensitive and specific detection of serum antibody to Trypanosoma cruzi (Chagas' disease).

A commercial EIA for the detection of antibody to Trypanosoma cruzi was clinically evaluated. The primary use of this test is in the diagnosis and screening of donated blood in Latin America. When compared with sera positive by xenodiagnosis, the assay had a clinical sensitivity of 100%. When tested against matched hemagglutination (HA) and immunofluorescence (IFA) results (i.e., when both tests gave negative results) the EIA had a specificity of 99.03% (305/308). The cross-reactivity of this test was determined using sera from malaria and leishmaniasis patients (obtained from Africa, ensuring that the sera did not contain Chagasic antibodies) and from schistosomiasis, toxoplasmosis, tuberculosis, syphilis, and systemic lupus erythematosus samples. The EIA was 100% specific whereas IFA or commercially available HA kits from Latin America cross-reacted with several of the samples. In this investigation, the EIA appeared to be at least as sensitive and more specific than IFA or HA in the serodiagnosis of Chagas' disease.

Biochemical and molecular characterization of Leishmania pifanoi amastigotes in continuous axenic culture.

Inability to culture the disease-producing amastigote form of Leishmania has greatly hampered its study. We have biochemically characterized an axenically cultured amastigote-like form of Leishmania pifanoi. This form closely resembles amastigotes in proteinase, ribonuclease, adenine deaminase and peroxidase activity. It also exhibits comparable rates of growth, transformation, synthesis of DNA, RNA and protein, and metabolism of glucose and linoleic acid. It is distinct from promastigotes in these characteristics. The expression of the genes for beta-tubulin and the P100/11E reductase is developmentally regulated in this axenic form as in amastigotes. These results, combined with previous demonstrations of amastigote morphology and antigenicity in the culture form, confirm that Leishmania amastigotes have been successfully propagated in axenic media. This strain should serve as an excellent model for the study of amastigote biochemistry, pharmacology and immunology, and the molecular genetics of the transformation between amastigote and promastigote forms.

Amastigote and epimastigote stage-specific components of Trypanosoma cruzi characterized by using monoclonal antibodies. Purification and molecular characterization of an 83-kilodalton amastigote protein.

Stage-specific mAb have been produced to amastigotes and epimastigotes of Trypanosoma cruzi (Brazil strain). mAb C-1 through C-6 reacted specifically with T. cruzi strains; no cross-reactions were found with membranes of promastigotes or amastigotes of Leishmania species. One mAb produced against the epimastigote membranes (C-5) was found to be specific against this stage by radioimmune binding assay, immunofluorescence, and radioimmunoprecipitation. mAb C-5 recognized a novel epimastigote protein at Mr (greater than 200,000) on immunoprecipitation with radiolabeled epimastigotes. Three amastigote stage-specific monoclonal antibodies were produced against membrane-enriched preparations of T. cruzi (Brazil strain) amastigotes grown in axenic culture (C-1 through C-3). By indirect immunofluorescence assay, monoclonal antibody C-2 bound only to T. cruzi amastigotes; no reaction with either tissue culture-derived trypomastigotes or epimastigotes was observed. mAb C-1 and C-2 each specifically immunoprecipitated a single protein molecule with Mr 83,000 from [35S]-methionine-labeled amastigotes. mAb C-2 was also used to affinity purify an 83-kDa Ag that was recognized by human Chagasic sera from patients of endemic countries of Latin America in an enzyme immunoassay. Amino acid composition and preliminary sequence data of the 83-kDa protein are presented. These mAb and/or purified Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of Chagas' disease.

Monoclonal antibodies specific for the amastigote stage of Leishmania pifanoi. I. Characterization of antigens associated with stage- and species-specific determinants.

Eight mAb were produced against membrane-enriched preparations of Leishmania pifanoi amastigotes either grown in axenic culture (P-1 through P-6) or isolated from macrophage cell culture (P-7 and P-8). Two mAb produced against promastigote membranes (P-9 and P-10) were found to be specific against this stage. Antibodies P-1 through P-8 on analysis by radioimmune binding only reacted with determinants present on amastigotes. mAb P-2, P-4, and P-8 also reacted with Leishmania amazonensis amastigotes but not promastigotes. No cross-reactions were found on any other species of Leishmania or with membranes of Trypanosoma cruzi epimastigotes or amastigotes. An indirect immunofluorescence assay using mAb P-1 through P-8 confirmed the stage specificity and binding to L. pifanoi axenically grown amastigotes, amastigotes within infected hamster tissue, and amastigotes within J774.1 macrophages. When Western blot analysis of amastigote membranes was conducted, one distinct group of molecules associated with L. pifanoi-specific determinants was identified. mAb P-1, P-3, P-5, P-7, and P-8 bound to molecules Mr 43 and 34 kDa. Promastigote-specific mAb P-9 recognized a diffuse pattern from 88 to greater than 200 kDa, and mAb P-10 localized a second class of proteins with Mr53 kDa. On immunoprecipitation of solubilized [35S]methionine-labeled amastigotes, mAb P-2 recognized a doublet of Mr 35 and 33 kDa and another doublet at Mr 17.5 and 13.5 kDa. mAb P-4 and P-7 each precipitated a band at Mr 34 kDa. These studies indicate that antigenically the axenically cultured amastigote is closely related to macrophage-derived amastigote. These mAb and/or purified protein Ag may be useful in studying stage differentiation, monitoring transformation, and for further taxonomic, epidemiologic, and immunologic studies of New World leishmaniasis.

Leishmania mexicana: comparative fine structure of amastigotes and promastigotes in vitro and in vivo.

Amastigotes of Leishmania mexicana pifanoi were cultivated by serial transfers in cell-free medium UM-54 at 33 and 35 C. Electron microscopy was used to analyze the structural relationships among promastigotes, axenically cultured amastigotes, and amastigotes in footpads of infected hamsters. These studies revealed very close structural similarities between culture and hamster derived amastigotes. However, both of these amastigotes differed from the promastigotes in the following aspects. The flagellum of promastigotes contained a paraxial rod originating at the axosome level within the flagellar pocket, whereas the flagellum of amastigotes lacks this structure. The flagellar pocket of promastigotes was usually small whereas amastigotes had a distended reservoir. Subpellicular microtubules of promastigotes terminated at the posterior end, whereas those of amastigotes ended subterminally. Membrane bounded vesicles were present only in amastigotes. These results along with the biologic and antigenic comparisons indicate that amastigotes obtained from axenic cultures are related very closely to amastigotes from infected hamster footpads and that their relationship to promastigotes is far more distant.

Leishmania mexicana pifanoi: analysis of the antigenic relationships between promastigotes and amastigotes by gel diffusion, immunoelectrophoresis, and immunoprecipitation.

The antigenic relationships between Leishmania mexicana pifanoi promastigotes, axenically grown amastigotes, and amastigotes isolated from the footpads of infected hamsters or from a J774 macrophage cell line were studied by three serologic methods. Amastigote and promastigote antigens were disrupted by freeze-thawing of intact cells in a lysis buffer. Antisera were prepared in rabbits by repeated subcutaneous inoculations of the parasite antigens in complete Freund's adjuvant and were tested against the homologous and heterologous antigens in a series of gel diffusion experiments. Negative results were obtained in all control experiments. In each instance, the homologous antigen-antiserum reactions yielded the largest numbers of precipitin lines. A pattern of cross-reactivity was also observed in the heterologous systems. Results indicated that the amastigote and promastigote forms had unique and common antigens. The two parasite antigen-antiserum systems were also examined by immunoelectrophoresis. Qualitative and quantitative differences between the promastigote and amastigote antigens were readily demonstrable by this technique. Results indicated that each parasite form had specific and many common antigens. In the homologous system, major proteins, with molecular weights (MW) of 23, 52, and 68 kd, were demonstrated in the promastigotes by immunoprecipitation of lactoperoxidase-catalyzed radioiodinated cells. In a similar (homologous) system, axenically grown amastigotes were found to contain three proteins with MW of 38, 70, and 74 kd and, therefore, different from those demonstrated for the promastigotes. All the results suggested that the three amastigote stages of different origins are antigenically similar to one another, but differ from the promastigote forms.

Further studies on the surface saccharides in Trichomonas vaginalis strains by fluorescein-conjugated lectins.

Fluorescence emitted by individual cells of several Trichomonas vaginalis strains, nearly all of which were cloned, incubated with fluorescein-conjugated lectins in the absence (experimental) or presence (control) of inhibitory sugars, or else in phosphate-buffered saline alone (autofluorescence) was measured with a Leitz MPV Compact microfluorometer. Irrespective of whether the organisms were postfixed in formalin or glutaraldehyde, the relative fluorescence emitted by the cells was closely comparable, provided that appropriate neutral density filters were employed. However, autofluorescence was much higher for glutaraldehyde-fixed trichomonads. Therefore, although better preserved and more amenable to subsequent manipulations, such organisms were found unsuitable for use in "qualitative" titration of the fluorescence emitted by various strains. Provided that the necessary precautions were taken, comparable fluorescence readings were obtained with trichomonads affixed to glass slides by heat (41 degrees C, on a section spreader) or by a cytologic centrifuge (Cytospin 2). Large numbers of concanavalin A (Con A)-binding sites were present on organisms of all strains, irrespective of their virulence for human patients and as estimated by the subcutaneous mouse assay; these sites were shown with the aid of D-mannose to be mannose or mannose-related residues. More binding sites for soybean agglutinin (SBA) were found on the virulent than on avirulent strains. On the basis of inhibition experiments, the sugar residues mainly responsible for these differences appeared to be D-lactose residues. Similar differences were observed with Ricinus communis agglutinin Type I (RCA I), for which D-galactose was employed as the competing sugar. However, with two cloned strains the situation with regard to RCA I binding was reversed - more of the lectin bound to a mild than to a virulent strain. The results obtained with Ricinus communis Type II agglutinin (RCA II) were often similar to those noted for RCA I; however, in most instances the inhibition with N-acetyl-D-galactosamine (GalNAc) was lower. Furthermore, the results noted with the GalNAc-specific agglutinins from Dolichus biflorus and Helix pomatia suggested that only very few GalNAc residues were available for binding on the surfaces of all T. vaginalis strains examined in the course of this study. Statistical analyses of fluorescence emitted by four clones of each, Balt 42 (virulent) and JH31A (avirulent) T. vaginalis strain upon incubation with Con A and SBA revealed homogeneity of these strains with regard to the number of the specific surface saccharide residues, D-mannose and D-lactose.(ABSTRACT TRUNCATED AT 400 WORDS)

Leishmania mexicana pifanoi: in vivo and in vitro interactions between amastigotes and macrophages.

Macrophages of the cell line J774 were used in a comparative study of virulence involving amastigote stages of Leishmania mexicana pifanoi isolated from macrophages (AMA-M) of the aforementioned cell line, amastigote forms grown in the UM-54-cell-free medium (AMA-C), and promastigote stages. The macrophage cultures were inoculated with AMA-M and AMA-C at the culture cell to parasite ratios of 1:3, 1:5, and 1:10. The macrophages were exposed to either kind of amastigotes for 24, 48, and 72 h. At the end of each of these periods, and for each dilution, the percentages of macrophages harboring the parasites within their cytoplasm and the mean numbers of intracellular parasite/macrophage were estimated on the basis of examination of 200 phagocytes. When either AMA-M or AMA-C were employed, after 24 h, the percentages of infected macrophages were, respectively, 84.5%, 89.0%, and 94.5% for the three aforementioned dilutions, the majority of the phagocytes containing 1-5 parasites. After 48- and 72-h exposures, the macrophages harbored 6-11 and 11-20 amastigotes/cell, respectively. Evidently intracellular multiplication of the amastigotes has taken place. In contrast to the results obtained with amastigote forms, after inoculations of the macrophages cultures with promastigotes at the dilutions previously used for amastigotes, only 48-78 phagocytes were found to contain intracellular stages within their cytoplasm. Many macrophages were parasite-free, especially when exposed to fewer promastigotes. Experiments in which 5 X10(6) promastigotes, AMA-M, or AMA-C were inoculated into the footpads of hamsters yielded the following results with regard to terminal footpad volumes: 1.57, 3.31, and 3.32 cm3, respectively. Evidently both kinds of amastigotes had equal virulence for hamsters; however, the promastigote stages were much les virulent for these experimental hosts.

Leishmania mexicana: serial cultivation of intracellular stages in a cell-free medium.

A cell-free liquid medium has been devised for serial cultivation of Leishmania mexicana pifanoi amastigotes at 33 and 35 C. It consists of tissue culture Medium 199 fortified primarily with a high concentration of water-soluble vitamins, nucleotides, and inactivated fetal bovine serum. The initial pH of the medium is 7.2. Starting with a population of promastigotes as inoculum and serially cultured at 33 or 35 C at 4- to 10-day intervals, the proportion of amastigotes steadily increased and that of promastigotes gradually decreased during the first subculture. By the end of the incubation period in the second subculture, practically all (99%) of the organisms are amastigotes. The amastigotes thus obtained can be cultured indefinitely by serial transfers. In this medium, amastigotes may reach a density of 8 X 10(7)/ml after 10 days of incubation at 33 C, and 5 X 10(7)/ml at 35 C. The medium was modified to have an initial pH of 8.0 by Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer and higher concentrations of sodium bicarbonate. When amastigotes cultured in the original medium at 33 or 35 C are transferred into the modified medium and incubated at 26 C, the amastigotes entirely transformed into promastigotes after three serial passages. These promastigotes could be serially subcultured indefinitely in the modified medium at 4- to 12-day intervals. The promastigotes cultured at 26 C may reach a population density of 7 X 10(7)/ml after 12 days of incubation.