The dermal leishmaniases of Brazil, with special reference to the eco-epidemiology of the disease in Amazonia

Six species of Leishmania are at present known to cause cutaneous and/or mucocutaneous leishamniasis in Brazil, and they are all to be found in the Amazon region of this country. The eco-epidemiology of each is discussed, with the observation that the Amazonian leishmaniases are all zoonoses, with their source in silvatic mammals and phlebotomine sandfly vectors. With man's destruction of the natural forest in southern Brazil, some sandfly species have survived by adapting to a peridomestic or domiciliary habitat in rural areas. Some domestic animals, such as dogs and equines are seemingly now involved in the epidemiology of the disease. No such process has yet been reported in the Amazon region, but may well take place with the continuing devastation of its forest.

Phase 1 Study of an Inactivated Vaccine against American Tegumentary Leishmaniasis in Normal Volunteers in Brazil

A Phase 1 double-blind placebo-controlled study was performed to evaluate a vaccine against American tegumentary leishmaniasis in 61 healthy male volunteers. Side effects and the immune response to the vaccine were evaluated, with 1- and 2- dose schemes, with intervals of 7 or 21 days, each dose containing 1440 mg of protein N antigen of a single strain of Leishmania amazonensis (PH 8) diluted in merthiolated saline (1:10,000). Merthiolated saline and an inert substance were used as placebos. No significant clinical alterations were found following the respective injections in the vaccinated individuals as compared to the placebos, except for local pain, which was associated significantly with injection of the vaccine. The laboratory alterations we observed bore no association with the clinical findings and were unimportant. We observed no differences between the groups with regard to seroconversion or the Montenegro skin test. However, the group that received a single dose of the vaccine and the one that received two doses with a 21-day interval displayed cutaneous induration significantly larger than in the control group, with 100%, 100%, and 66% conversion in the skin test, respectively. We concluded that the vaccine does not present any major side effect that would contraindicate its use in healthy individuals.

Zinc Sulphate in the Treatment of Cutaneous Leishmaniasis: an in Vitro and Animal Study

This study was designed to evaluate the effectiveness of zinc sulphate both in vitro and in an animal model against both strains of old world cutaneous leishmaniasis. The in vitro sensitivities of promastigotes and axenic amastigotes of both Leishmania major and L. tropica to zinc sulphate was determined, the LD50 calculated and compared to the standard treatment for cutaneous leishmaniasis pentavalent antimony compounds. The results show that the two forms of both strains were sensitive to zinc sulphate and their respective LD50 were lower compared to the pentavalent antimony compound. Furthermore the sensitivities of the forms of both strains were tested using a simple slide method and compared to results of the standard method. To confirm this result, zinc sulphate was administered orally to mice infected with cutaneous leishmaniasis both therapeutically and prophylactically. Results showed that oral zinc sulphate was effective in both treatment and prophylaxis for cutaneous leishmaniasis. These results encourage the use of oral zinc sulphate in the treatment of cutaneous leishmaniasis clinically.

Genomic rearrangements in trypanosomatids: an alternative to the "one gene" evolutionary hypotheses?

Most molecular trees of trypanosomatids are based on point mutations within DNA sequences. In contrast, there are very few evolutionary studies considering DNA (re) arrangement as genetic characters. Waiting for the completion of the various parasite genome projects, first information may already be obtained from chromosome size-polymorphism, using the appropriate algorithms for data processing. Three illustrative models are presented here. First, the case of Leishmania (Viannia) braziliensis/L. (V.) peruviana is described. Thanks to a fast evolution rate (due essentially to amplification/deletion of tandemly repeated genes), molecular karyotyping seems particularly appropriate for studying recent evolutionary divergence, including eco-geographical diversification. Secondly, karyotype evolution is considered at the level of whole genus Leishmania. Despite the fast chromosome evolution rate, there is qualitative congruence with MLEE- and RAPD-based evolutionary hypotheses. Significant differences may be observed between major lineages, likely corresponding to major and less frequent rearrangements (fusion/fission, translocation). Thirdly, comparison is made with Trypanosoma cruzi. Again congruence is observed with other hypotheses and major lineages are delineated by significant chromosome rearrangements. The level of karyotype polymorphism within that "species" is similar to the one observed in "genus" Leishmania. The relativity of the species concept among these two groups of parasites is discussed.

Presence of American cutaneous leishmaniasis vectors surrounding the city of Medellín, Colombia

The presence of Lutzomyia (Helcocyrtomyia) hartmanni, as a vector of Leishmania colombiensis and L. columbiana (Verrucarum group), recently incriminated in the transmission of leishmaniasis, and L. pia (Verrucarum group) are reported for the first time in a periurban area of Medellín city. There is thus a risk of leishmaniasis transmission in this town.

The origin and dispersion of human parasitic diseases in the Old World (Africa, Europe and Madagascar)

The ancestors of present-day man (Homo sapiens sapiens) appeared in East Africa some three and a half million years ago (Australopithecs), and then migrated to Europe, Asia, and later to the Americas, thus beginning the differentiation process. The passage from nomadic to sedentary life took place in the Middle East in around 8000 BC. Wars, spontaneous migrations and forced migrations (slave trade) led to enormous mixtures of populations in Europe and Africa and favoured the spread of numerous parasitic diseases with specific strains according to geographic area. The three human plasmodia (Plasmodium falciparum, P. vivax, and P. malariae) were imported from Africa into the Mediterranean region with the first human migrations, but it was the Neolithic revolution (sedentarisation, irrigation, population increase) which brought about actual foci for malaria. The reservoir for Leishmania infantum and L. donovani - the dog - has been domesticated for thousands of years. Wild rodents as reservoirs of L. major have also long been in contact with man and probably were imported from tropical Africa across the Sahara. L. tropica, by contrast, followed the migrations of man, its only reservoir. L. infantum and L. donovani spread with man and his dogs from West Africa. Likewise, for thousands of years, the dog has played an important role in the spread and the endemic character of hydatidosis through sheep (in Europe and North Africa) and dromadary (in the Sahara and North Africa). Schistosoma haematobium and S. mansoni have existed since prehistoric times in populations living in or passing through the Sahara. These populations then transported them to countries of Northern Africa where the specific, intermediary hosts were already present. Madagascar was inhabited by populations of Indonesian origin who imported lymphatic filariosis across the Indian Ocean (possibly of African origin since the Indonesian sailors had spent time on the African coast before reaching Madagascar). Migrants coming from Africa and Arabia brought with them the two African forms of bilharziosis: S. haematobium and S. mansoni.

Anti-leishmanial and structure-activity relationship of ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives

A series of ring substituted 3-phenyl-1-(1,4-di-N-oxide quinoxalin-2-yl)-2-propen-1-one derivatives were synthesized and tested for in vitro leishmanicidal activity against amastigotes of Leishmania amazonensis in axenical cultures and murine infected macrophages. Structure-activity relationships demonstrated the importance of a radical methoxy at position R3', R4' and R5'. (2E)-3-(3,4,5-trimethoxy-phenyl)-1-(3,6,7-trimethyl-1,4-dioxy-quinoxalin-2-yl)-propenone was the most active. Cytotoxicity on macrophages revealed that this product was almost six times more active than toxic.

Comparative evaluation of phenol and thimerosal as preservatives for a candidate vaccine against American cutaneous leishmaniasis

For decades thimerosal has been used as a preservative in the candidate vaccine for cutaneous leishmaniasis, which was developed by Mayrink et al. The use of thimerosal in humans has been banned due to its mercury content. This study addresses the standardization of phenol as a new candidate vaccine preservative. We have found that the proteolytic activity was abolished when the test was conducted using the candidate vaccine added to merthiolate (MtVac) as well as to phenol (PhVac). The Montenegro's skin test conversion rates induced by MtVac and by PhVac was 68.06% and 85.9%, respectively, and these values were statistically significant (p < 0.05). The proliferative response of peripheral mononuclear blood cells shows that the stimulation index of mice immunized with both candidate vaccines was higher than the one in control animals (p < 0.05). The ability of the candidate vaccines to induce protection in C57BL/10 mice against a challenge with infective Leishmania amazonensis promastigotes was tested and the mice immunized with PhVac developed smaller lesions than the mice immunized with MtVac. Electrophoresis of phenol-preserved antigen revealed a number of proteins, which were better preserved in PhVac. These results do in fact encourage the use of phenol for preserving the immunogenic and biochemical properties of the candidate vaccine for cutaneous leishmaniasis.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii(SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Estudo de flebotomíneos (Diptera, Psychodidae) em área urbana do município de Bonito, Mato Grosso do Sul, Brasil

O objetivo do trabalho foi identificar a fauna flebotomínea em áreas do perímetro urbano do município de Bonito, Mato Grosso do Sul, Brasil. O estudo foi desenvolvido de março de 2005 a fevereiro de 2006, em 17 ecótopos distribuídos em 12 locais, três no Centro e nove em diferentes bairros. As capturas foram realizadas quinzenalmente com armadilhas automáticas luminosas. Capturou-se 2.680 espécimes, 2.283 machos e 397 fêmeas, de 12 espécies, Brumptomyia avellari, Brumptomyia brumpti, Bichromomyia flaviscutellata, Evandromyia corumbaensis, Evandromyia sallesi, Lutzomyia longipalpis, Micropygomyia acanthopharynx, Micropygomyia quinquefer, Nyssomyia whitmani, Psathyromyia aragaoi, Psathyromyia punctigeniculata e Psathyromyia shannoni. Lutzomyia longipalpis, vetora do agente da leishmaniose visceral americana, foi a espécie mais freqüente e a mais abundante, representando 93,5% dos flebotomíneos capturados e índice de abundância padronizado de 0,85. Com freqüência mais expressiva nos ecótopos próximos de galinheiro e de pocilga, esta espécie foi capturada em todos os meses do ano, com picos no verão, inverno e primavera. As demais espécies foram pouco freqüentes. Ressalta-se que a captura de Bichromomyia flaviscutellata, no intradomicilio e peridomicílio, nas proximidade de mata remanescente, tem grande significado epidemiológico uma vez que essa espécie é a principal vetora da Leishmania (Leishmania) amazonensis, agente etiológico da leishmaniose cutânea difusa anérgica. Portanto, na área urbana de Bonito foram encontradas duas espécies que comprovadamente participam da transmissão de leishmanioses, Lutzomyia longipalpis e Bichromomyia flaviscutellata, ambas encontradas naturalmente infectadas pelos respectivos agentes.

Síntese, caracterização e estudo da atividade inibitória de novas dialquilfosforilarilidrazonas sobre o crescimento de tripanossomatídeos

A new series of dialkylphosphorylhydrazones was synthesized through the condensation of aromatic aldehydes with different phosphorylhydrazines. All synthesized compounds were characterized by IR, ¹H-NMR, 13C-NMR and 31P-NMR spectroscopies. The in vitro investigation of the activity of these compounds against Leishmania amazonensis promastigotes and epimastigotes of T. cruzi, showed an efficient inhibition of proliferation, at non toxic concentrations to mammalian cells. The results have shown some derivatives as potential antiparasitic agents against trypanosomatids.

Leishmaniose cutânea experimental: II - aspectos evolutivos da infecção no primata Cebus apella (Cebidae) pela Leishmania (V.) Braziliensis e L. (L.) Amazonensis

Objetivando avaliar o potencial do primata C. apella como modelo experimental da leishmaniose cutânea, produzida pela L. (V.) braziliensis e L. (L.) Amazonensis , inocularam-se, via intradérmica, 3 X 10(6) de promastigotas dessas leishmanias, em 8 sítios da cauda de 10 espécimens desse primata, 5 deles com a L. (V.) braziliensis e outros 5 com a L. (L.) Amazonensis . Posteriormente, às inoculações, o exame semanal dos animais e biópsias mensais, revelaram os seguintes resultados relativos a cada parasita: a) L. (V.) braziliensis : o período de incubação foi de 15-20 dias; aos 30 dias evidenciaram-se lesões pápulo-eritematosas, que evoluíram para nódulos ao fim de 60 dias; no 3.° mês, notou-se ulceração espontânea destas lesões e, no 4° mês, deu-se o início da reparação das lesões ulceradas, culminando com a cura em um dos animais após 5 meses, em dois após 6 meses, noutro após 7 meses e, no último, após 10 meses. Quanto ao parasitismo nas lesões, foi demonstrado nos 5 animais, até 90 dias; depois disto, somente em 2 até 120 dias e, por fim, até 180 dias apenas naquele que curou depois de 10 meses, b) L. (L.) Amazonensis : o período de incubação foi de 20 dias; aos 30 dias notou- se lesões pápulo-eritematosas, que também evoluíram para nódulos ao fim de 60 dias, porém, a partir do 3.° mês, estas lesões regrediram rapidamente ao fim de 90 dias, quando não mais detectou-se o parasita na pele dos animais. Em relação aos testes de Montenegro, somente 2 dos 5 animais infectados com a L. (V.) braziliensis reagiram ao teste, 60 e 90 dias após as inoculações. Os resultados observados permitiram confirmar a infectividade do C. apella a estas leishmanias e, também, reforçar a indicação desse primata como modelo experimental da leishmaniose cutânea causada por estes parasitas.

Cell populations in lesions of cutaneous leishmaniasis of leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta

The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T-cells accounted for 44.7 ± 13.1% of the infiltrate in active lesions (versus CD2+= 40.3 ± 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 ± 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 ± 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis.

Glycosphingolipid antigens from Leishmania (L.) amazonensis amastigotes: Binding of anti-glycosphingolipid monoclonal antibodies in vitro and in vivo

Specific glycosphingolipid antigens of Leishmania (L.) amazonensis amastigotes reactive with the monoclonal antibodies (MoAbs) ST-3, ST-4 and ST-5 were isolated, and their structure was partially elucidated by negative ion fast atom bombardment mass spectrometry. The glycan moieties of five antigens presented linear sequences of hexoses and N-acetylhexosamines ranging from four to six sugar residues, and the ceramide moieties were found to be composed by a sphingosine d18:1 and fatty acids 24:1 or 16:0. Affinities of the three monoclonal antibodies to amastigote glycosphingolipid antigens were also analyzed by ELISA. MoAb ST-3 reacted equally well with all glycosphingolipid antigens tested, whereas ST-4 and ST-5 presented higher affinities to glycosphingolipids with longer carbohydrate chains, with five or more sugar units (slow migrating bands on HPTLC). Macrophages isolated from footpad lesions of BALB/c mice infected with Leishmania (L.) amazonensis were incubated with MoAb ST-3 and, by indirect immunofluorescence, labeling was only detected on the parasite, whereas no fluorescence was observed on the surface of the infected macrophages, indicating that these glycosphingolipid antigens are not acquired from the host cell but synthesized by the amastigote. Intravenous administration of 125I-labeled ST-3 antibody to infected BALB/c mice showed that MoAb ST-3 accumulated significantly in the footpad lesions in comparison to blood and other tissues