World Leishmaniasis Research Articles

CD4+T Cells Play a Dominant Role in Protection against New World Leishmaniasis Induced by Vaccination with the P-4 Amastigote Antigen

Immunodepletion studies of P-4-vaccinated mice indicate that CD4+ and not CD8+ T cells are critical for protection against Leishmania pifanoi (Leishmania mexicana complex). Although a moderate CD8+ T-cell response is elicited by vaccination, CD4+ T cells are the dominant responding population in vitro and at the cutaneous site of infection. These protective T cells produce gamma interferon (IFN-gamma), macrophage migration inhibitory factor (MIF), and tumor necrosis factor/lymphotoxin (TNF/LT), each of which significantly contributed to intracellular parasite destruction in vitro. These results indicate that a singular CD4+ T-cell response (IFN-gamma, MIF, and/or LT/TNF) can provide protection against New World cutaneous leishmaniasis.

Old World Leishmaniasis: An Emerging Infection among Deployed US Military and Civilian Workers

Many veterans of Operation Iraqi Freedom are now returning to the United States after potential exposure to leishmaniasis. In the past year, large numbers of leishmaniasis cases of a magnitude not encountered in the United States since World War II have challenged clinicians in both the military and the civilian sectors. Many Reserve and National Guard troops were deployed to Iraq and are now back in their communities. Hundreds of leishmaniasis cases, which were managed by a few practitioners initially, permitted further appreciation of the epidemiology and diagnostic and treatment options for Old World leishmaniasis. We describe the current situation, with on-the-ground experience, complimented by a literature review, and we provide a practical list of options for the clinician likely to encounter this parasitic infection in the coming months and years.

Treatment of cutaneous leishmaniasis among travellers.

Leishmaniasis is endemic in 88 countries on five continents. There are 1-1.5 million cases of cutaneous leishmaniasis reported yearly worldwide. There has been a sharp increase in recorded cases over the last 10 years. Based on geographical distribution, cutaneous leishmaniasis is divided into Old World and New World leishmaniasis. In the past, species could be inferred from geographical setting or determined by performing culture and isoenzyme analysis. The recently developed and now widely available PCR technology allows a rapid diagnosis with determination of most species, and thus enables a species-orientated treatment. While the Old World species mostly cause benign and often self-limiting cutaneous disease, the American species cause a broad spectrum of conditions from benign to severe manifestations, including mucosal involvement. The response to treatment varies according to the species. Therefore, a species-specific approach is proposed. Drugs for systemic and topical treatment are presented and discussed with regard to their application, use and adverse effects. Indications for local or systemic treatment are proposed. Drugs under investigation are also mentioned. An overview of published treatment options and a treatment recommendation is given for each of the most important species. The level of evidence of the studies leading to these recommendations is given.

Transient effect of topical treatment of cutaneous leishmaniasis with imiquimod.

Treatment of cutaneous leishmaniasis can be painful and protracted and cosmetic results are often unsatisfying. The immune modulator imiquimod has been reported to be suitable for the treatment of a variety of infectious skin diseases and neoplasias. We investigated the efficacy of topical application of imiquimod in the treatment of old world leishmaniasis in a placebo-controlled prospective study. Twelve patients were treated with imiquimod cream using a standard protocol, i.e. topical application three times a week, and a further three served as control group. Lesions of cutaneous leishmaniasis regressed within the first 2-4 weeks in 10 of the 12 patients, whereas in two patients no change was observed. However, after 8 weeks all lesions showed progression. Our results thus demonstrate that topical application of imiquimod alone is ineffective in treating old world cutaneous leishmaniasis. Further studies are required to demonstrate a possible benefit of imiquimod in combination with other, preferably orally administered medicines.

Perforin and gamma interferon are critical CD8+ T-cell-mediated responses in vaccine-induced immunity against Leishmania amazonensis infection.

Previous studies have demonstrated that protection against New World leishmaniasis caused by Leishmania amazonensis can be elicited by immunization with the developmentally regulated Leishmania amastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8(+) (as well as CD4(+)) effector responses is required for protection. Further, mice lacking beta(2)-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8(+) T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8(+) T cells, secreting gamma interferon (IFN-gamma) and expressing perforin, was observed at the site of infection; subsequently, activated CD4(+) T cells producing IFN-gamma were primarily found. As protection correlated with the ratio of total IFN-gamma-producing cells (CD4(+) and CD8(+) T cells) to macrophages found at the site of infection, a role for IFN-gamma was evident; in addition, vaccination of IFN-gamma-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8(+) T cell effector mechanisms (perforin, IFN-gamma) are clearly required in the protective immune response against L. amazonensis infection in vaccinated mice.

Treatment of Old World Cutaneous Leishmaniasis by Pentamidine Isethionate

Background: Pentavalent antimonial derivatives (PAD), especially meglumine antimoniate (Glucantime^®), are usually considered as the first-line drugs for Old World leishmaniasis, but their potential toxicity and the number of required injection, either intralesional or intramuscular, prompt to search for alternative treatments. Objective: To evaluate the efficiency and tolerance of pentamidine isethionate in Old World leishmaniasis. Methods: An open pilot study included 11 patients from two regional academic centers, with varied parasitological forms of Old World leishmaniasis, treated with three strictly intramuscular injections of 4 mg/kg of base-pentamidine every other day. Results: Tolerance was good overal, and 8/11 (73%) of patients responded well with a quick healing of their lesions, little scarring and no relapse. Conclusion: Pentamidine isethionate is a safe and effective first-line treatment for Old World leishmaniasis. Larger-scale prospective studies comparing several dosage regimens of pentamidine and pentamidine isethionate to PAD are warranted.

Intra and inter-specific microsatellite variation in the Leishmania subgenus Viannia

Leishmania species of the subgenus Viannia are responsible for a large proportion of New World leishmaniasis. Here we report the development of a set of microsatellite markers which are able to discriminate between all species within the subgenus Viannia, including the closely related species pairs: Leishmania (V.) braziliensis and Leishmania (V.) peruviana; Leishmania (V.) panamensis and Leishmania (V.) guyanensis. Potential species hybrids were uncovered in the analysis. These markers are sufficiently polymorphic such that within-species epidemiological, population and genetic studies are theoretically possible for all species analyzed.

3 サシチョウバエの分類学 : 新大陸リーシュマニア症の伝播疫学に果す重要性(分類カレント・トピックス)

3 サシチョウバエの分類学 : 新大陸リーシュマニア症の伝播疫学に果す重要性(分類カレント・トピックス)

Treatment of cutaneous leishmaniasis.

The World Health Organization estimates that approximately 400,000 new cases of leishmaniasis occur worldwide each year. Cutaneous leishmaniasis is being encountered more frequently in the United States because of increasing travel and immigration from endemic areas. The indications for treatment and recommended treatment regimens reported in the infectious disease and dermatology literature vary widely. We examine both classic and newly developed therapeutic agents and modalities for cutaneous leishmaniasis. Proper therapy depends on species identification. New World leishmaniasis, in general, requires more aggressive therapy; parenteral antimonials are the drugs of choice. Physical modalities may suffice in most cases of Old World leishmaniasis because of its strong tendency toward spontaneous resolution.

Leishmaniasis of the tongue treated with liposomal amphotericin B

A 66-year-old man, immunosuppressed because of a lymphoma and with severe ischaemic heart disease and proteinuria, presented with a swelling on the tongue due to leishmaniasis. His travel history suggested that he had acquired the infection in the Mediterranean area some years earlier. He was treated with liposomal amphotericin B for 21 days and made a good recovery despite a temporary deterioration in renal function. Oral lesions are a rare manifestation of Old World leishmaniasis. Liposomal amphotericin B is a novel treatment that may be well tolerated when there is concern about the toxicity of more established drugs.

Diagnosis of New World leishmaniasis: Specific detection of species of the Leishmania braziliensis complex by amplification of kinetoplast DNA

We have sequenced single kinetoplast DNA minicircles from three species and part of a minicircle from the fourth major species within the Leishmania braziliensis complex. Alignment of these sequences with each other and with those of other kinetoplastids allowed the selection of a pair of oligonucleotides suitable as primers in a polymerase chain reaction which is highly specific for the Leishmania braziliensis complex. The reaction is capable of detecting less than one femtogramme of kinetoplast DNA. It has been tested with crude specimens from South American leishmaniasis patients, potential wild animal reservoirs and sandfly vectors. The tests indicate that these primers are suitable for diagnosis of leishmaniasis and potentially useful in epidemiological surveys.

Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test

The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L.braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria. Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1% of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidioidomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L.major-like antigen was better than L.b.braziliensis' to disclose cross-reacting antibodies.

The relationship of Leishmania braziliensis subspecies and immune response to disease expression in New World leishmaniasis.

Multivariate analyses of clinical presentation, subspecies identity of the causal organism, and the Leishmania-specific immune response parameters (indirect fluorescent antibody test [IFAT], cutaneous delayed type hypersensitivity [DTH], and in vitro lymphocyte transformation [LT]) of 441 patients with tegumentary leishmaniasis were used to examine the human host-parasite interaction in L. braziliensis infection. Mucocutaneous disease (P less than .002) and L. braziliensis braziliensis infection (P less than .001) were independently associated with significantly higher IFAT titers and cutaneous DTH than were cutaneous disease or L. braziliensis panamensis infection. Lesion size was also correlated with IFAT titer (P. less than .001). Although time of lesion evolution was highly correlated with all parameters, differences associated with subspecies and disease form were independent of lesion duration (three-way analysis of variance). In contrast with the cutaneous DTH response, the in vitro lymphocyte proliferative response to Leishmania antigen did not correlate with disease form and only weakly with infecting subspecies when time of evolution and subspecies were controlled. The association of mucosal disease presentation with a particular subspecies and the independent correlation of both variables with heightened IFAT titers and cutaneous DTH to Leishmania antigen supports the possibility of immune mechanisms of pathogenesis in human tegumentary leishmaniasis.

Indirect immunofluorescence test in New World leishmaniasis: serological and clinical relationship.

The indirect immunofluorescence test (IF) for anti-Leishmania antibodies (IgG and IgM) was performed with sera from the following groups of individuals: 214 cutaneous leishmaniasis patients, 28 healthy subjects with positive Montenegro's skin test (MST), 29 healthy subjects with negative MST and 16 visceral leishmaniasis patients. The first four groups came from a suburban area of Rio de Janeiro (Jacarepaguá) where cutaneous leishmaniasis caused by Leishmania braziliensis braziliensis is endemic. It was observed that IF-IgM titers were significantly higher amongst the cutaneous leishmaniasis patients with less than four months of disease as compared to those with longer periods and that IF-IgG titers were significantly higher in patients with multiple lesions as compared to those with single lesions. The visceral leishmaniasis patients had IF-IgG titers significantly higher than those from cutaneous leishmaniasis patients. A group of 28 individuals selected amongst the 214 cutaneous leishmaniasis patients had their IF-titers (IgG and IgM) compared to those of the two control groups of healthy subjects from the endemic area, respectively with positive and negative MST. Significantly higher titers of IF-IgG and IF-IgM were found in the group with active disease. The same group of patients showed IF-IgG titers significantly lower at the end of the antimonial therapy than those observed during this treatment.

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.

Serodiagnosis of New World leishmaniasis by using a genus-specific antigen in enzyme linked immunosorbent assays.

A genus-specific monoclonal antibody (83L-5G9), generated against promastigotes of Leishmania braziliensis panamensis (WRAIR-470), has been used as a ligand in immunochromatography columns for the recovery and purification of the genus-specific antigen. When the purified polypeptide, actually a doublet comprised of a 58 kd and a 31 kd moiety, was used as the antigen in enzyme linked immunosorbent assays (ELISA), it was reactive with 36 of 85 sera from Leishmania patients and with 1 of 49 sera from confirmed cases of Chagas' disease. An additional 25 sera from an unexposed population and five specimens from individuals seropositive for Toxoplasma gondii were nonreactive.

The ribosomal gene spacer as a tool for the taxonomy of Leishmania

A recombinant DNA ribosomal gene spacer of Leishmania braziliensis Y was used as probe to test different Leishmania species. Based on the similarity of their restriction patterns, three groups were distinguished with respect to international Leishmania references: first a group with a similar restriction pattern to L. braziliensis Y and the reference organism L. mexicana garnhami JAP78; a second group with restriction patterns similar to the reference organism L. mexicana mexicana M379; and finally a group where all the restriction patterns were related to the reference organism L. braziliensis braziliensis M2903. These results support the existence of L. garnhami as an independent Leishmania species; they confirm previous studies on L. mexicana and L. braziliensis and open the way for the more exact diagnosis of New World Leishmaniasis.

Schizodeme and Zymodeme Characterization of Leishmania in the Investigation of Foci of Visceral and Cutaneous Leishmaniasis

Leishmania parasites were isolated from humans and canines in foci of cutaneous and visceral leishmaniasis. After in vitro cultivation the parasites were examined by the following biochemical techniques: (i) restriction analysis of kinetoplast DNA (kDNA) also known as schizodeme analysis (Morel et al., 1980); (ii) zymodeme analysis (Barret et al., 1980); by agarose gel electrophoresis and (iii) isoelectricfocusing in polyacrylamide gels. The strains of cutaneous and visceralizing leishmanias studied could be differentiated by schizodeme analysis, using the endonuclease MspI, into three complexes agreeing with those accepted for human New World leishmaniasis. In the municipality of Rio de Janeiro, isolates from a focus of cutaneous leishmaniasis were identified as L. braziliensis braziliensis and from a focus of visceral leishmaniasis were identified as L. donovani by zymodeme characterization. Identical restriction enzyme profiles of kDNA from human and canine isolates indicated that in the cutaneous focus at Jacarepaguá, Rio de Janeiro, the same strain was probably circulating in both the canine and human populations. This suggests a possible role for dogs as a reservoir host for L. braziliensis braziliensis. In addition, our results confirm the importance of dogs as reservoirs in visceral leishmaniasis. The stability of the electrophoretic patterns of restriction digest ("fingerprints") of Leishmania kDNA as well as differences in the sensitivity of the techniques used were demonstrated. Strains from widely different geographical areas as well as strains maintained in vivo and in vitro showed identical kDNA restriction patterns, while strains showing similar banding patterns by enzyme electrophoresis could be differentiated by schizodeme analysis. These results demonstrate the usefulness of an integrated biochemical approach in the identification of Leishmania.

Micro enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis.

Of 21 confirmed cases of New World leishmaniasis, 16 exhibited antibody to antigens of the promastigote of Leishmania braziliensis panamensis by the enzyme-linked immunosorbent assay (ELISA). Comparison of antibody titers obtained by ELISA with titers obtained by indirect immunofluorescence using an amastigote substrate confirmed that the sensitivities of the two techniques were within the same range (r = 0.80). Although sera from patients with New World leishmaniasis failed to react with antigens extracted from epimastigotes of Trypanosoma cruzi, sera from 39 cases of Chagas' disease were reactive with promastigotes of L. braziliensis panamensis. This apparent unidirectional cross-reactivity has been attributed to differences in potency of the antigenic stimulus presented in the two diseases.

Aboriginal new world epidemiolgy and medical care, and the impact of Old World disease imports.

Various workers, including T. D. Stewart, claim that the aboriginal Americas were relatively disease-free because of the bering Strait cold-screen, eliminating many pathogens, and the paucity of zoonotic infections because of few domestic animals. Evidence of varying validity suggests that precontact Americns had their own strains of treponemic infections, bacillary and amoebic dysenteries, influenza and viral penumonia and other respiratory diseases, salmonellosis and perhaps other food poisoning, various arthritides, some endoparasites such as the ascarids, and several geographically circumscribed diseases such as the rickettsial verruca (Carrion's disease) and New World leishmaniasis and trypanosomiasis. Questionably aboriginal are tuberculosis and typhus. Accordingly, virtually all the "crowd-type" ecopathogenic diseases such as smallpox, yellow fever, typhoid, malaria, measles, pertussis, polio, etc., appear to have been absent from the New World, and were only brought in by White conquerors and their Black slaves. My hypothesis is that native American medical care systems--especially in the more culturally advanced areas--were sufficiently sophisticated to deal with native disease entities with reasonable competence. But native medical systems could not cope with the "crowd-type" disease imports that struck Indian and Eskimos as "virgin-field" populations. Reanalysis of native population losses through a genocidal combination of diease, war, slavery and attendant cultural disruption by Dobyns, Cook and others strongly suggest that traditiona estimates underplayed the death toll by a factor of the general order of ten. This would make for an immediately pre-contact Indian population of some 90-111 million instead of the tradition 8-11 million. Evidence is growing that Indians may have been no more susceptible to new pathogens that are other "virgin soil" populations, and thus their immune systems need not be considered less effective than those in other people. Present-day high mortality rates in Indians of both continents from infectious disease imports may be more socioeconomic than anything else.