Tissue Amastigotes Research Articles

In Situ Detection of Dormant Trypanosoma cruzi Amastigotes Using Bioluminescent-Fluorescent Reporters.

Chagas disease agent, Trypanosoma cruzi, is capable to persist after prolonged drug treatment using effective drugs. The reason of treatment failure is not known, but recent development of highly sensible bioluminescence imaging coupled to tissue clarification techniques has made possible the detection of individual amastigotes within chronically infected murine tissues and the study of their replicative status. In this chapter, we provide a step-by-step explanation for these protocols that allowed the visualization of nonproliferating amastigotes in tissues of chronically infected mice for the first time.

Dipyridamole potentiated the trypanocidal effect of nifurtimox and improved the cardiac function in NMRI mice with acute chagasic myocarditis.

BACKGROUND As chronic Chagas disease does not have a definitive treatment, the development of alternative therapeutic protocols is a priority. Dipyridamole (DPY) is an alternative to counteract the pathophysiological phenomena involved in Chagas cardiomyopathy.OBJECTIVE To evaluate the therapeutic efficacy of DPY associated with nifurtimox (Nfx) in epimastigote axenic cultures and in mice with acute Chagas disease.METHODS NMRI adult male mice were divided into nine groups: three healthy and six Trypanosoma cruzi-infected groups. Mice received vehicle, Nfx or DPY, alone or combined. The doses assayed were Nfx 10 and 40 mg/kg and DPY 30 mg/kg. The treatment efficacy was evaluated by clinical, electrocardiographic, parasitological, biochemical and histopathological methods.FINDINGS In vitro, DPY and Nfx had a trypanocidal effect with IC50 values of 372 ± 52 and 21.53 ± 2.13 µM, respectively; DPY potentiated the Nfx effect. In vivo, Nfx (40 mg/kg) with or without DPY had a therapeutic effect, which was reflected in the 84-92% survival rate and elimination of parasitaemia and heart tissue amastigotes. Nfx (10 mg/kg) had a subtherapeutic effect with no survival and persistence of amastigotes, inflammation and fibrosis in heart tissue; adding DPY increased the survival rate to 85%, and all tested parameters were significantly improved.MAIN CONCLUSION DPY has a trypanocidal effect in vitro and enhances the Nfx therapeutic effect in an in vivo murine model.

The Transcriptome of Leishmania major Developmental Stages in Their Natural Sand Fly Vector.

ABSTRACTThe life cycle of the Leishmania parasite in the sand fly vector involves differentiation into several distinctive forms, each thought to represent an adaptation to specific microenvironments in the midgut of the fly. Based on transcriptome sequencing (RNA-Seq) results, we describe the first high-resolution analysis of the transcriptome dynamics of four distinct stages of Leishmania major as they develop in a natural vector, Phlebotomus duboscqi. The early transformation from tissue amastigotes to procyclic promastigotes in the blood-fed midgut was accompanied by the greatest number of differentially expressed genes, including the downregulation of amastins, and upregulation of multiple cell surface proteins, sugar and amino acid transporters, and genes related to glucose metabolism and cell cycle progression. The global changes accompanying post-blood meal differentiation of procyclic promastigotes to the nectomonad and metacyclic stages were less extensive, though each displayed a unique signature. The transcriptome of nectomonads, which has not been studied previously, revealed changes consistent with cell cycle arrest and the upregulation of genes associated with starvation and stress, including autophagic pathways of protein recycling. Maturation to the infective, metacyclic stage was accompanied by changes suggesting preadaptation to the intracellular environment of the mammalian host, demonstrated by the amastigote-like profiles of surface proteins and metabolism-related genes. Finally, a direct comparison between sand fly-derived and culture-derived metacyclics revealed a reassuring similarity between the two forms, with the in vivo forms distinguished mainly by a stronger upregulation of transcripts associated with nutrient stress.

Infecciones por protozoos hemoflagelados: leishmaniasis, enfermedad de Chagas y tripanosomiasis africana

Leishmaniasis is an infection caused by protozoa of the genus Leishmania and transmitted by sandflies. Clinical manifestations are usually divided into three syndromes: visceral leishmaniasis, cutaneous leishmaniasis and mucosal leishmaniasis. In order to confirm the parasitological diagnosis it is necessary to visualize the amastigotes in tissue or promastigotes in culture or detection of Leishmania DNA by CRP. In most cases the immunological diagnosis is complementary. The optimal drug therapy in each geographical region and each principal syndrome should be defined in terms of effectiveness in the region and the available resources.Chagas disease is caused by the parasite Trypanosoma cruzi. It is a zoonosis whose main route of transmission is carried out by a vector insect subfamily triatominae. The cell cycle is complex. During the cell cycle the parasite goes through four morphological stages. The clinical manifestations will depend on the phase or stage in which the patient is. There is an intense phase, the initial phase, which may present with varied symptoms, but in most cases is asymptomatic. The chronic phase is characterized by cardiac and/or gastrointestinal affectation. Essentially, the diagnosis is based on the use of techniques that detect the parasite existence directly or through techniques that detect antibodies that are produced in response to infection. The treatment of choice is benznidazole.

Natural infection of Didelphis aurita (Mammalia: Marsupialia) with Leishmania infantum in Brazil

BackgroundThe opossum Didelphis have been considered as natural hosts of Leishmania parasites in the New World, suggesting an important role in the epidemiology of Visceral Leishmaniasis (VL). Among six extant species that belong to the genus Didelphis, only two (D. marsupialis and D. albiventris), have been mentioned as natural hosts of Leishmania infantum in Brazil and Colombia. In the present paper, it is reported for the first time, the observation of intracellular parasites (amastigotes) in tissues of Didelphis aurita naturally infected with Leishmania infantum in Brazil. We also discuss some aspects associated to the relationship between L. infantum and the geographical distribution of some species of the genus Didelphis.MethodsThe opossums studied were caught by wire traps (Tomahawk) in Barra de Guaratiba, a peri-urban area in Rio de Janeiro. The opossums were killed with an overdose of Thiopental sodium.At necropsy, macroscopic alterations were examined and samples from liver, spleen, lymph nodes, ear, abdominal skin, scent glands and bone marrow were collected for parasitological and molecular diagnoses.ResultsForty-eight opossums were captured in an AVL endemic region, 30 being caught in a mangrove area and eighteen animals in a forest area near to some residential-yards. Among the thirty opossums trapped in the mangrove area, all of them were negative by both imprint and sera samples assayed on Dipstick Tests, that is a test based on a combination of protein-A colloidal gold conjugate and rk39 Leishmania antigen to detect anti-Leishmania antibody in serum or plasma. At the macroscopic examination one out of eighteen opossums, caught close to the forest, presented alterations compatible with spleen hypertrophy and three were positive by Dipstick Tests (16.6%) and presented amastigotes in the spleen and in one of them, the parasites were also observed in a submandibular lymph node. Leishmania infantum infections were confirmed through dot blot hybridization using a L. infantum-specific biotinylated probe.ConclusionsIn the present paper we present the first report of amastigotes in the tissues of Didelphis aurita (Mammalia: Marsupialia) naturally infected with Leishmania infantum. We also attempt to claim the particular role of some opossum species as hosts of Leishmania infantum, contributing at least in part on the description of potential sylvatic reservoirs.

False-positive results of a rapid K39-based strip test and Chagas disease

The definitive diagnosis of visceral leishmaniasis (VL) requires invasive procedures with demonstration of amastigotes in tissue or promastigotes in culture. Unfortunately, these approaches require laboratory materials not available in poor countries where the disease is endemic. The correct diagnosis of VL is important, and made more difficult by the fact that several common tropical diseases such as malaria, disseminated tuberculosis, and enteric fever share the same clinical presentation. Serological tests have been developed to replace parasitological diagnosis in the field. A commercially available K39-based strip test for VL has been developed for this purpose. The endemic area of leishmaniasis in Brazil overlaps the endemic area of Chagas disease, a disease that can cause false-positive serological test results. The aim of this study was to evaluate the incidence of false-positive exams using a rapid test for VL in patients with Chagas disease. A rapid test based on the recombinant K39 antigen of Leishmania was used in: (1) 30 patients with confirmed Chagas disease, (2) 30 patients with a serological diagnosis of Chagas disease by ELISA, indirect immunofluorescence, indirect hemagglutination, and chemiluminescence, (3) 30 healthy patients from a non-endemic area as the control group, (4) 30 patients with confirmed VL, and (5) 20 patients with proved cutaneous leishmaniasis. The sensitivity and specificity of the rapid strip test were 100% when compared with healthy volunteers and those with confirmed Chagas disease. One false-positive result occurred in the group with Chagas disease diagnosed by serological tests (specificity of 96%). The rapid test based on recombinant K39 is a useful diagnostic assay, and a false-positive result rarely occurs in patients with a serological diagnosis of Chagas disease.

Functional characterization of nucleoside transporter gene replacements in Leishmania donovani

Leishmania donovani express two nucleoside transporters of non-overlapping ligand selectivity. To evaluate the physiological role of nucleoside transporters in L. donovani, homozygous null mutants of the genes encoding the LdNT1 adenosine–pyrimidine nucleoside transporter and the LdNT2 inosine–guanosine transporter were created singly and in combination by single targeted gene replacement followed by selection for loss-of-heterozygosity. The mutant alleles were verified by Southern blotting, and the effects of gene replacement on transport phenotype were evaluated by rapid sampling transport measurements and by drug resistance profiles. The Δldnt1, Δldnt2, and Δldnt1/Δldnt2 mutants were all capable of proliferation in defined culture medium supplemented with any of a spectrum of purine nucleobases or nucleosides, except that a Δldnt2 lesion conferred an inability to efficiently salvage exogenous xanthosine, a newly discovered ligand of LdNT2. Each of the three knockout strains was viable as promastigotes and axenic amastigotes and capable of maintaining an infection in J774 and bone marrow-derived murine macrophages. These genetic studies demonstrate: (1) that L. donovani promastigotes, axenic amastigotes, and tissue amastigotes are viable in the absence of nucleoside transport; (2) that nucleoside transporters are not essential for sustaining an infection in mammalian host cells; (3) that the phagolysosome of macrophages is likely to contain purines that are not LdNT1 or LdNT2 ligands, i.e., nucleobases. Furthermore, the Δldnt1, Δldnt2, and Δldnt1/Δldnt2 knockouts offer a unique genetically defined null background for the biochemical and genetic characterization of nucleoside transporter genes and cDNAs from phylogenetically diverse species and of genetically manipulated LdNT1 and LdNT2 constructs.

AXENIC PROMASTIGOTE FORMS OF LEISHMANIA (VIANNIA) LAINSONI AS AN ALTERNATIVE SOURCE FOR LEISHMANIA ANTIGEN PRODUCTION

The present study demonstrates that axenic cultures of Leishmania (Viannia) lainsoni produce larger cell masses in NNN-LIT medium, as well as higher amounts of total proteins in cell extracts, than Leishmania (Leishmania) amazonensis. Antigenicity of L. (V.) lainsoni whole promastigotes is similar to that of L. (L.) amazonensis, as demonstrated by an indirect immunofluorescence diagnostic test using sera from human patients and dogs infected with visceral leishmaniasis. Infectivity of the L. (V.) lainsoni strain used in the present work was demonstrated by the detection by transmission-electron microscopy of tissue amastigotes in skin lesion samples from an experimentally infected hamster. Incubation of lesion fragments in NNN-LIT medium allowed us to obtain promastigote forms, which could be cultivated successfully in vitro. lsoenzyme analysis of such promastigotes confirmed the parasite strain as L. (V.) lainsoni, as compared to other Leishmania reference strains. Our data indicate that L. (V.) lainsoni is a useful alternative source for antigen production as well for use in assays that depend on large cell volumes of Leishmania spp. parasites.

Behavior of Leishmania major metacyclic promastigotes during the course of infection and immune response development in resistant versus susceptible hosts

Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH) in resistant versus susceptible inbred mice. Non-infective (procyclic) promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible) and C57BL/6 (resistant) mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.

American cutaneous leishmaniasis

American cutaneous leishmaniasis is endemic in widespread areas of Latin America. The causative agents include L. (V.) braziliensis, L. (L.) mexicana, L. (V.) panamensis, and related species. The spectrum of disease includes single, localized, cutaneous ulcers, diffuse cutaneous leishmaniasis, and mucosal disease. The main reservoirs for L. (V.) braziliensis and other Leishmania (Vianna) spp. are small forest rodents. The vectors are ground-dwelling or arboreal Lutzomyia sandflies, which are abundant in the forest. Disease is most common in persons working at the edge of the forest and among rural settlers. The incubation period of cutaneous leishmaniasis varies from two weeks to several months. A wide variety of skin manifestations ranging from small, dry, crusted lesions to large, deep, mutilating ulcers may be seen. Ulcerative lesions are usually shallow and circular with well-defined, raised borders and a bed of granulation tissue. In L. (V.) braziliensis infection, regional lymphadenopathy often precedes the development of cutaneous lesions by one to 12 weeks. A definite diagnosis depends on the identification of amastigotes in tissue or promastigotes in culture. Antileishmanial antibodies are present in the serum of some patients with cutaneous leishmaniasis as detected by ELISA, immunofluorescent assays, direct agglutination tests or other assays, but the titers are usually low. The leishmanin skin test result usually becomes positive during the course of the disease. For treatment two pentavalent antimony-containing drugs are used: stibogluconate sodium, and meglumine antimoniate (Glucantime). Amphotericin B deoxycholate is an alternative for persons who fail to respond to pentavalent antimony. Immunoprophylaxis and immunotherapy are promising new approaches to prevention and treatment.

Stage-dependent role of nitric oxide in control of Trypanosoma cruzi infection.

Trypanosoma cruzi, the causative agent of Chagas' disease, is known to be susceptible to nitric oxide (NO)-dependent killing by gamma interferon-activated macrophages. Mice deficient for inducible nitric oxide synthase (iNOS) are highly susceptible to T. cruzi, and inhibition of iNOS from the beginning of infection was reported to lead to an increase in trypomastigotes in the blood and to high mortality. In the present study, we investigated whether NO production is essential for the control of T. cruzi in all phases of the infection. BALB/c mice were treated at different time intervals after T. cruzi infection with an iNOS inhibitor, aminoguanidine or L-N6-(1-iminoethyl)-lysine (L-NIL). Treatment initiated with the beginning of the infection resulted in 100% mortality by day 16 postinfection (p.i.). If treatment was started later during the acute phase at the peak of parasitemia (day 20 p.i.), all the mice survived. Parasitemia was cleared and tissue amastigotes became undetectable in these mice even in the presence of the iNOS inhibitor L-NIL. Inhibition of iNOS in the chronic phase of the infection, i.e., from day 60 to day 120 p.i., with L-NIL did not result in a reappearance of parasitemia. These data suggest that while NO is essential for T. cruzi control in the early phase of acute infection, it is dispensable in the late acute and chronic phase, revealing a fundamental difference in control mechanisms compared to those in infections by other members of the order Kinetoplastida, e.g., Leishmania major.

Animal Models for the Analysis of Immune Responses to Leishmaniasis

This unit focuses on the murine model of cutaneous leishmaniasis and models of visceral leishmaniasis in mice and hamsters. Each basic protocol describes the methods used to inoculate parasites and to evaluate infections with regard to lesion progression and visceralization, and quantification of parasite load. Five support protocols are provided; two for the growth and isolation of metacyclic promastigotes from in vitro culture, one for isolation of tissue amastigotes from infected animals, one for cryopreservation of parasites, and one for the preparation of blood agar plates for quantitation of parasite numbers in infected tissue.

Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells.

Intracellular staining for cytokines and parasites, combined with two-color flow cytometric analyses, were used to examine the frequencies of IL-12-, TNF-alpha- and IL-6-producing macrophages in response to Leishmania major infection and/or activation with IFN-gamma/lipopolysaccharide (LPS). Inflammatory macrophages were obtained from nonimmune granulomas, initiated by the injection of polyacrylamide microbeads (Bio-gel P-100) into subcutaneous pouches of different mouse strains. Infection of inflammatory macrophages in vitro using metacyclic promastigotes produced identical effects on cytokine responses regardless of whether cells from genetically resistant or susceptible mouse strains were used: IL-12 was not produced in response to infection itself, virtually every infected cell lost its ability to produce IL-12 in response to IFN-gamma/LPS, and the IL-6 response was partially inhibited, while the TNF-alpha response of infected cells was unimpaired. Low-multiplicity infection of inflammatory macrophages in vivo using either metacyclic promastigotes or tissue amastigotes also resulted in the complete and selective inhibition of IL-12 responses in infected cells. These data establish the physiologic relevance of prior observations regarding the selective impairment of IL-12 induction pathways in infected macrophages, and suggest a mechanisms for the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease.

Transfected Leishmania expressing biologically active IFN-gamma.

Infection of susceptible BALB/c mice with Leishmania major leads to progressive infection with the failure to expand and activate Th1 CD4+ T cells that elaborate IFN-gamma, a critically implicated cytokine for control of disease. We used the recently described capacity to express foreign genes in trypanosomatids to introduce into Leishmania the murine IFN-gamma gene on a drug-selectable plasmid under the constitutive control of intergenic tubulin sequences. Several clones of L. major were established and demonstrated to contain IFN-gamma DNA and IFN-gamma RNA that was appropriately trans-spliced with the Leishmania-specific leader sequence, and to secrete IFN-gamma into the media. The secreted IFN-gamma was biologically active as assessed by up-regulation of class II MHC Ag and induction of macrophage nitric oxide synthase activity in a macrophage cell line. Infection of nude mice with IFN-gamma-containing organisms resulted in significantly slower progression of disease as compared to infection with organisms containing the empty plasmid, suggesting that biologically important activation of infected macrophages might be occurring in vivo. Infection of genetically susceptible BALB/c mice, however, did not impede the expansion of Th2 cells and the inexorable progression of disease. Despite the demonstration of increased levels of IFN-gamma transcription in vivo, induction of nitric oxide synthase in macrophages and expression of Ly-6, and IFN-gamma-inducible Ag, on CD4+ lymphocytes could not be shown. In all cases, organisms recovered from tissue amastigotes contained the IFN-gamma plasmid and secreted active IFN-gamma. The data confirm earlier studies that IFN-gamma alone is not sufficient to impede activation and maturation of Th2 cells in susceptible mice, even when targeted directly to the infected cell.

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.

The comparative fine structure and surface glycoconjugate expression of three life stages of Leishmania major

The cellular ultrastructure and surface glycoconjugate expression of three life stages of Leishmania major were compared. Noninfective logarithmic phase promastigotes (LP) are immature cells bearing a thin cell coat, short flagellum, small and empty flagellar pocket, and a loose cytoplasm filled with profiles of ER and large Golgi complex. LP also contain subpopulations of maturing cells containing less ER and Golgi and synthesizing cytoplasmic granules of different size, number, and electrondensity. Infective or metacyclic promastigotes (MP) are fully differentiated nondividing forms with a thickened, prominent cell coat, long flagellum, distended flagellar pocket filled with secretory material, and few cytoplasmic organelles other than abundant electron-dense granules. Tissue amastigotes also contain electron-dense cytoplasmic granules, their flagellar pockets are also enlarged and contain secretory material, but they lack a discernable cell coat. Immunogold labeling of GP63 on the cell surface was extensive only on amastigotes. Promastigote GP63 appeared to be masked by the presence of a densely packed lipophosphoglycan (LPG) coat which was extensively labeled on the entire surface of MP and LP. An elongated, developmentally modified form of LPG was abundantly labeled only on MP. LPG was poorly labeled on amastigotes, arguing that the promastigote cell coat is a stage-specific structure which is lost during intracellular transformation.

Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes

This study describes the characterisation of externally oriented surface peptides of both morphological forms of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar). Using 125I surface labelling techniques and peptide extraction in the detergents Triton X-100 and Triton X-114, a major iodinable promastigote peptide at 63 kDa or 65 kDa (depending on detergent used) was identified. This peptide was demonstrated to be the immunodominant membrane peptide of L. donovani and was strongly recognised by human sera from parasitologically confirmed cases of kala-azar. This peptide was not demonstrated on the surface of tissue amastigotes, although in vitro translations of poly(A +) RNA from both promastigotes and amastigotes demonstrated that both forms possessed mRNA that directs the synthesis of a 63 kDa peptide. It is suggested therefore that in amastigotes this peptide may be a processed antigen. We also report the isolation of a recombinant cDNA clone in the bacteriophage vector λ gt10 which encodes a 63 kDa polypeptide that is recognised by human kala-azar sera. It is proposed that this surface peptide could be used in a specific immunodiagnostic test for leishmaniasis.

Cryostat sections with tissue amastigotes of Trypanosoma cruzi as a substrate for Chagas' disease serology

Sera from 30 chronic chagasic patients together with 52 control samples (34 with other pathological conditions and 18 from normal individuals) were titrated by the indirect immunofluorescent technique (IFA) on Trypanosoma cruzi amastigotes. Acetone-fixed cryostat sections of skeletal muscle of Rockland mice 10 days post-infection with the RA isolate of T. cruzi were used as substrate. Results were compared with titres obtained by conventional IFA on epimastigotes. All 52 control sera had amastigote litres ⩽2 double dilutions (dd) as compared with epimastigote values. Out of the 30 chagasic samples, differences were ⩾4 dd (⩾1 log) for 22, 3 dd for 5 and ⩽2 dd for the remaining 3, when comparing amastigote and epimastigote titres. These results show that the use of amastigotes in cryostat sections of infected tissue for performing Chagas' serology is a simple, adequate and sensitive method.

Identification of Leishmania amastigotes and their antigens in formalin fixed tissue by immunoperoxidase staining

An immunoperoxidase indirect sandwich method is described here for the detection of tissue amastigotes in formalin-fixed biopsy material. Tissue sections are incubated with a rabbit anti- Leishmania antiserum, washed, then incubated with a peroxidase conjugated swine anti-rabbit antiserum (Dako). A second wash, followed by incubation with 3,3′diaminobenzidine (DAB) and H 2O 2 substrate results in the deposition of a stable, dark brown reaction product at the site of amastigote antigen. Sections are lightly counter-stained with haematoxylin. Amastigotes are revealed as dark brown stained bodies sharply contrasted against blue stained host tissue with excellent morphological preservation and may be seen easily at the light microscope level without oil immersion. In this preliminary study an anti- L. tropica antiserum (raised against live promastigotes) demonstrated positive staining of L. tropica, L. major, L. mexicana and L. donovani amastigotes in human and mouse tissue. A second antiserum, raised against an extract of L. major promastigotes also gave the same positive results. A possible Leishmania exo-antigen is suggested by positively stained material apparently emanating from amastigotes within a giant cell and by similar positively stained material in the cytoplasm of nonparasitized pinocytotic epithelioid cells. No positive staining was observed when either anti- L. tropica antiserum (absorbed with live homologous promastigotes) or normal rabbit immunoglobulin was incubated with the tissue. The technique is sensitive, simple to perform, free from artefacts, provides a permanent record and may also be adapted for ultrastructural studies. The convenience of using formalin-fixed material recommends this technique for retrospective studies and clinical use where a diagnosis cannot be confirmed by Giemsa stained smears or the culture of tissues and as a research tool in the laboratory for following the distribution and fate of antigen.