The Immunology and Serology of Leishmaniasis. I. The Fluorescent Antibody Staining Technique.
01 Sep 1965-Transactions of The Royal Society of Tropical Medicine and Hygiene (Oxford University Press)-Vol. 59, Iss: 5, pp 535-544
TL;DR: It is concluded that the fluorescent antibody test is not useful for the detection of past or present infection with L. mexicana.
Abstract: The indirect fluorescent antibody technique was used to stain smears of the leptomonads of 10 strains of Leishmania spp. including L. donovani, L. infantum, L. tropica, L. braziliensis braziliensis, L. braziliensis pifanoi and L. mexicana, and the Leishman-Donovan bodies of some strains. The sera used successfully and giving a strong reaction were from rabbits artificially immunized by leptomonads of all the above strains, and human sera from patients infected with Mediterranean infantile kala-azar, South American kala-azar, South and Central American muco-cutaneous and cutaneous leishmaniasis. Other sera used successfully but giving a weak reaction were from rabbits artificially immunized by Leishman-Donovan bodies of L. mexicana, human sera from patients infected with leishmaniasis tegumentaria diffusa, uta and British Honduran chiclero's ulcer, and serum from monkeys infected in the laboratory with L. mexicana and L. braziliensis. Reactions were less strong with Leishman-Donovan bodies as slide antigen than with leptomonads. No differentiation of strains was possible by serial dilution of sera used in the test, as all sera stained all antigen preparations at more or less the same intensity, indicating a group antigen-antibody reaction in reference to this method. Specific absorption of sera completely eliminated the reaction regardless of the antigen used for absorption. Sera sent from British Honduras from patients with chiclero's ulcer, non-immune subjects and others, were tested for antibodies by the fluorescent antibody technique. No correlation was obtained between disease or possible exposure and positive fluorescent antibody results. It is concluded that the fluorescent antibody test is not useful for the detection of past or present infection with L. mexicana.
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TL;DR: A newly developed direct agglutination test (DAT) for visceral leishmaniasis, IFAT and ELISA were applied to sera of patients with visceral leisiasis, African and American trypanosomiasis, other parasitic infections and healthy controls and it is recommended for sero-epidemiological field work on visceral leishingmaniasis.
Abstract: A newly developed direct agglutination test (DAT) for visceral leishmaniasis, IFAT and ELISA were applied to sera of patients with visceral leishmaniasis, African and American trypanosomiasis, other parasitic infections and healthy controls. The sensitivities of the 3 tests were comparable (96.3% to 100%); excluding patients with African and American trypanosomiasis, the specificities of DAT and IFAT were 100% and ELISA 87.3%. When trypanosomiasis sera were included, the specificities were 72.6%, 94.3% and 79.4% in DAT, IFAT and ELISA respectively. In 273 sera from a leishmaniasis endemic area (Baringo District, Kenya), the sensitivity was 80% in DAT and IFAT and 60% in ELISA, specificities being 99.6% (DAT), 98.5% (IFAT) and 62.5% (ELISA). As the new DAT is economical and easy to perform, it is recommended for sero-epidemiological field work on visceral leishmaniasis.
115 citations
Journal Article•
TL;DR: It is concluded that this model provides an experimental counterpart of human cutaneous leishmaniasis and that it is suitable for the analysis of the role of cell-mediated specific immunity in resistance to intracellular infection.
Abstract: This paper describes the course of infection and development of immunity in guinea-pigs after intradermal inoculation of Leishmania enriettii, and the use of in vivo and in vitro techniques to characterize the immunological response to infection and artificial immunization.
Inoculation of 106 amastigotes into the ear produced a nodule which ulcerated in 2–3 weeks and healed in 8–16 weeks. 8% of animals developed cutaneous metastases which healed with the original lesions. Histology of the primary lesions showed epidermal necrosis overlying a mass of parasitized macrophages which, after 4–6 weeks, became surrounded and infiltrated by lymphocytes. Histological changes in the draining lymph node began after 3 days and proceeded for 6 weeks; both germinal centres and paracortical areas were hyperplastic and the medulla contained many plasma cells. Superinfection produced an `isophasic' lesion, but reinfection after healing elicited only a delayed hypersensitivity response. Artificial immunization with soluble and insoluble antigenic extracts of L. enriettii in Freund's complete adjuvant partially protected against infection; extracts of other leishmanial species failed to protect. Immunological paralysis, attempted with intravenous injections of soluble antigen, increased the severity of subsequent infection.
Both infection and immunization were accompanied by delayed hypersensitivity which could be transferred passively by lymphoid cells. Cell-mediated immunity was studied in vitro by the ability of soluble leishmanial antigens to transform lymphocytes, to inhibit macrophage migration, and to induce the production of lymphokine factors from lymphocytes of sensitized animals. A target cell system was devised in which sensitized lymphocytes destroyed monolayers of parasitized macrophages. Cross reactivity of leishmanial with mycobacterial antigens was shown in skin tests and in target cell destruction, but not in cell transfer or in the other cell culture systems. The phagocytic activity of peritoneal macrophages from recovered animals was increased for homologous but not for heterologous species of Leishmania; the growth of ingested organisms was not however reduced.
Circulating antibodies were not demonstrated by passive cutaneous anaphylaxis, or by agglutination of antigen coated sheep erythrocytes, in the sera of infected or convalescent animals, although some convalescent animals showed active cutaneous anaphylaxis. However, antibodies were demonstrated by both these techniques in immunized animals, which also showed anaphylactic and Arthus hypersensitivity when skin tested with the soluble antigens.
The results are taken to indicate that cellular mechanisms are prominent in the development of immunity of the guinea-pig against L. enriettii, and ways in which the host may eliminate the parasite are discussed. It is concluded that this model provides an experimental counterpart of human cutaneous leishmaniasis and that it is suitable for the analysis of the role of cell-mediated specific immunity in resistance to intracellular infection.
106 citations
TL;DR: An enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis is developed that could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity.
Abstract: Leishmaniasis causes significant morbidity and mortality in areas where it is endemic. In areas where it is nonendemic, global travel and increased incidence of the disease in human immunodeficiency virus and intravenous-drug user populations are also causes for concern. The unavailability of rapid and reliable tests for diagnosis of the various leishmaniases makes patient management difficult. We have developed an enzyme-linked immunosorbent assay (ELISA) that can detect immunoglobulin M (IgM) and IgG antibodies in patients with visceral and cutaneous leishmaniasis. These practical assays are based on soluble antigens from promastigotes cultivated in a protein-free medium. In preliminary studies, 129 visceral (Brazil, Italy, North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis patients with controls were tested. Overall, the tests showed a sensitivity of 95.1%. In addition, the ELISA correctly identified 42 sera from Brazilian dogs with canine leishmaniasis and 10 healthy controls. Serological tests for the various clinical manifestations of leishmaniasis could be useful epidemiological and patient management tools in populations of areas of endemicity and nonendemicity.
94 citations
Cites background from "The Immunology and Serology of Leis..."
...Excreted factor, a component of these antigens, is a negatively charged carbohydrate-like substance which was shown to precipitate antibody from homologous sera of promastigote-infected rabbits (8, 13)....
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TL;DR: Successful agglutination tests could be performed consistently with any of these particles provided formalin treatment was omitted, and prior treatment with formalin rendered erythrocytes difficult to sensitize.
Abstract: Naked sheep erythrocytes, tanned sheep erythrocytes, tanned human erythrocytes of Group O, tanned and formolized sheep erythrocytes and latex particles were sensitized with antigens derived from 8 strains of Leishmania spp. and used in agglutination tests with various antisera and immune sera. Sensitized erythrocytes were formolized and also used in agglutination tests. Successful agglutination tests could be performed consistently with any of these particles provided formalin treatment was omitted. Prior treatment with formalin rendered erythrocytes difficult to sensitize. Formalin treatment of sensitized erythrocytes caused auto-agglutination. The tests with various sera and various absorbed sera revealed serological differences between the strains used. A strain of Leishmania from Panama was shown to be quite distinct from L. mexicana of British Honduras and L. braziliensis of Brazil, and also to be distinct from 2 strains of Leishmania from Costa Rica. The two Costa Rican strains also represented distinct serotypes. Serological differences were detected between L. donovani from India and L. donovani from Kenya. With these tests no serological difference was detected between L. donovani (post-kala-azar dermal leishmanoid) from India and L. tropica from Israel, or between L. braziliensis of Brazil and one strain of so-called L. pifanoi from Brazil.
49 citations
TL;DR: Two established immunodiagnostic techniques, immunofluorescence and indirect haemagglutination, were compared with ELISA using intact promastigotes as antigen for the detection of specific antibodies against Leishmania in the serum of patients with visceral or mucosal leishmaniasis from the Sudan.
Abstract: Two established immunodiagnostic techniques, immunofluorescence and indirect haemagglutination, were compared with ELISA (enzyme-linked immunosorbent assay) using intact promastigotes as antigen for the detection of specific antibodies against Leishmania in the serum of patients with visceral or mucosal leishmaniasis from the Sudan. The ELISA was found to be more sensitive and more specific than either of the other two tests.
37 citations
References
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TL;DR: Dermal leishmaniasis in British Honduras is clearly indicated to be a zoonosis, with the infected animals acting as reservoirs for the human disease, it follows that a large proportion of the population will remain constantly exposed to infection.
Abstract: 1. 1) During a 3-year study on the epidemiology of dermal leishmaniasis in British Honduras, about 150 human cases were seen among which 46 were examined in detail. The parasite, Leishmania mexicana , was isolated in NNN cultures in 27 instances. 2. 2) Race, sex, age and physical condition of the individual appear to have little bearing on the susceptibility of human beings to the disease which is strictly sylvatic in origin and principally restricted to chicleros, mahogany hunters, and other forest workers. 3. 3) The disease is almost always limited to a single dermal lesion. No evidence could be found of any cutaneous spread of infection or visceral involvement. The ear is the most commonly affected part of the body (40 per cent. of the 50 lesions studied), but the hands and arms are also frequently involved (28 per cent.). Lesions on the torso and legs are uncommon. This differential distribution of lesions is thought due to a combination of at least three main factors: firstly the head and arms are the most frequently exposed parts of the body, secondly there is a marked tendency for body lesions to heal spontaneously, and finally L. mexicana appears to proliferate more readily in ear tissue than in that of other parts of the body. 4. 4) Development of the human lesion is described from a study of experimental insect transmission by Phlebotomus , natural infections of known duration and syringe-induced infections in volunteers. The incubation period following the introduction of leptomonads into the skin varied from 2 to 4 weeks. An account of the histology of the developing lesion is given. 5. 5) Three men with infections of short duration resisted attempts to super-infect them with massive intradermal inoculations of leptomonads of L. mexicana . This early premunition contrasts with the much more slowly developed immunity in the case of L. tropica infection. 6. 6) The culture of material from lesions in NNN medium proved the most reliable method of diagnosis. Hamsters and white mice are readily infected both with leptomonads from cultures and L.D. bodies taken directly from human lesions. In one instance the Montenegro skin-test was shown to give equally good reactions with antigens prepared from leptomonads of L. mexicana and L. tropica . 7. 7) Three human volunteers were readily infected with strains of L. mexicana isolated from the wild forest rodents, Ototylomys, Nyctomys and Heteromys species. 8. 8) Sixteen human cases of “Bay-Sore” were successfully treated with Stibophen (pentasodium antimony-biscatechol-3: 5-disulphonate). 9. 9) The medical and economic importance of dermal leishmaniasis in British Honduras is discussed. Two-thirds of the Colony is still under forest from which the main natural resources of chicle, timber and some agricultural products are derived, and one-third of the working population is employed in forest work of some kind. As we have clearly indicated dermal leishmaniasis in British Honduras to be a zoonosis, with the infected animals acting as reservoirs for the human disease, it follows that a large proportion of the population will remain constantly exposed to infection.
81 citations
TL;DR: The indirect fluorescein-labeled antibody technic was used and evaluated in the serodiagnosis of kala-azar in humans and reliable testing for visceral leishmaniasis fluorescent antibodies was possible even when dried blood smears were used instead of serum.
Abstract: : The indirect fluorescein-labeled antibody technic was used and evaluated in the serodiagnosis of kala-azar in humans. The tests were performed on slides using Leishmania donovani leptomonad forms as antigen. Evans blue, employed as a counter-stain, resulted in improved contrast, thus making the test easier to interpret, yet did not significantly diminish the specific yellow-green fluorescence. Occasional crossreactions were observed with specimens from individuals with viral, bacterial, mycotic, and parasitic infections as well as with degenerative diseases. Although cross-reactions were frequently observed in individuals with mucocutaneous leishmaniasis, negative results were reported from L. tropica patients. Reliable testing for visceral leishmaniasis fluorescent antibodies was possible even when dried blood smears were used instead of serum.
77 citations
TL;DR: Results are summarized of studies in which the FA technique was used to stain fixed blood forms of T. rhodesiense, T. gambiense, and T. cruzi toward the development of a reliable and practical test for the laboratory diagnosis of African and American trypanosomiasis.
Abstract: A fluorescent antibody technique for the serodiagnosis of African and American trypanosomiasis in man is described. The test can be performed simply and rapidly through the use of trypanosomes in blood smears as antigen. There were extensive cross-reactions with sera from patients with other species of trypanosomes. Tests of sera from healthy controls and from patients with nontrypanosomal diseases and disorders revealed relatively few cross-reactions, indicating a high degree of specificity. The finding that dried blood on absorbent paper could be tested successfully with the FA technique may indicate its usefulness for studies in endemic areas. Human infections with African and American trypanosomiasis constitute important clinical and public health problems in large regions of the world. An unequivocal diagnosis of trypanosomiasis is often difficult to obtain since the clinical picture is not always well defined and the organisms frequently cannot be recovered by blood examinations. Consequently, there is a need for reliable, rapid, and inexpensive procedures which could provide the basis for an adequate diagnosis of these infections, especially during the latent and chronic phases of the disease. The fluorescent antibody technique (Coons et al., 1941) is rapidly developing into a practical, sensitive, and specific diagnostic method for several parasitic infections. Fife and Muschel (1959) described a fluorescent antibody technique (FA) for the serodiagnosis of Trypanosoma cruzi infections. T. cruzi cultured on a diphasic blood agar medium was utilized as a source of antigen. This technique, which required all the reactions to be carried out in test tubes to prevent drying of organisms, appeared to be less specific than the complementfixation test using purified T. cruzi antigen. Studies with T. rhodesiense and T. gambiense in experimental animals (Williams et al., 1963) have resulted in a rapid and practical FA technique which can be run on slides using as Received for publication 21 December 1962. antigen thin blood smears from infected rats. A technique for the use of minute amounts of dried blood in the fluorescent antibody test was developed recently (Anderson et al., 1961). This technique, which permitted mailing of specimens under adverse conditions, was found to be particularly useful in epidemiological surveys of schistosomiasis and trichinosis (Sadun et al., 1961, 1962). The current report summarizes results of studies in which the FA technique was used to stain fixed blood forms of T. rhodesiense, T. gambiense, and T. cruzi toward the development of a reliable and practical test for the laboratory diagnosis of African and American trypanosomiasis. The procedures were evaluated with sera obtained from humans in endemic areas. Furthermore, in the present work attempts were made to determine the degree of cross-reactivity of different trypanosome species and to determine whether blood smears dried on absorbent paper could be used in the serological diagnosis of trypanosomiasis. MATERIALS AND METHODS Sera: Human sera were obtained from individuals with well-documented trypanosomiasis infections. The diagnosis of trypanosomiasis in the patients was established by the recovery and identification of organisms from the blood or spinal fluid. To determine the specificity of the FA test, control sera from individuals with viral, bacterial, and parasitic infections other than trypanosomiasis were used. To test whether the presence of auto-
39 citations