Sensitivity of a Vacuum Aspiratory Culture Technique for Diagnosis of Localized Cutaneous Leishmaniasis in an Endemic Area of Leishmania (Viannia) braziliensis Transmission

Sixty eight patients with localized cutaneous leishmaniasis from an area with Leishmania (Viannia) braziliensis transmission had cultures performed with a modified Marzochi´s vacuum aspiratory puncture technique to establish sensitivity and contamination rate with this new method. Overall sensitivity of three aspirates was 47.1%; (CI95% 39.4; 59.4) significantly greater than the sensitivity of a single one aspirate. Fungal contamination was observed in 6/204 (2.9%) inoculated culture tubes. We recommend that this useful technique should be adopted as routine for primary isolation of L. (V.) braziliensis from localized cutaneous ulcers.

Sensitivity of Lymph Node Aspiration in Localized Cutaneous Leishmaniasis Due to Leishmania (Viannia) braziliensis

Twenty nine patients with localized cutaneous leishmaniasis had lymph node and skin ulcer aspirations for culture of Leishmania with the modified Marzochi´s vacuum aspiratory technique. Sensitivity of lymph node aspiration was 58.6% and 34.5% for skin ulcer aspiration (P=0.06). Combined sensitivity of the two methods was 79.3%. There was no agreement between methods (Kappa Index = -0.084; CI95% -0,45; 0,28) showing the potential complementary roles in diagnostic approach.

Atypical Mucocutaneous Leishmaniasis Caused by Leishmania braziliensis in an Acquired Immunodeficiency Syndrome Patient: T-cell Responses and Remission of Lesions Associated with Antigen Immunotherapy

An atypical case of acquired immunodeficiency syndrome-associated mucocutaneous lesions due to Leishmania braziliensis is described. Many vacuolated macrophages laden with amastigote forms of the parasite were found in the lesions. Leishmanin skin test and serology for leishmaniasis were both negative. The patient was resistant to therapy with conventional drugs (antimonial and amphotericin B). Interestingly, remission of lesions was achieved after an alternative combined therapy of antimonial associated with immunotherapy (whole promastigote antigens). Peripheral blood mononuclear cells were separated and stimulated in vitro with Leishmania antigens to test the lymphoproliferative responses (LPR). Before the combined immunochemotherapy, the LPR to leishmanial antigens was negligible (stimulation index - SI=1.4). After the first course of combined therapy it became positive (SI=4.17). The antigen responding cells were predominantly T-cells (47.5%) most of them with CD8+ phenotype (33%). Very low CD4+ cells (2.2%) percentages were detected. The increased T-cell responsiveness to leishmanial antigens after combined therapy was accompanied by interferon-g (IFN-g) production as observed in the cell culture supernatants. In this patient, healing of the leishmaniasis lesions was associated with the induction of a specific T-cell immune response, characterized by the production of IFN-g and the predominance of the CD8+ phenotype among the Leishmania-reactive T-cells.

Contribution to the sand fly fauna (Diptera: Phlebotominae) of Rio Grande do Sul, Brazil and Leishmania (Viannia) infections

American cutaneous leishmaniasis (ACL) is endemic in the State of Rio Grande do Sul, Brazil. As reports of nearby hospitals suggest, the Parque Estadual do Turvo seems to serve as a source for the disease. During three months from November 1996 to February 1997 we collected, in this park, 2,228 sandflies (10 Lutzomyia species and 2 6species). We applied the polymerase chain reaction to 920 females which belong to the following species: Lutzomyia migonei, Lu. pessoai, Lu. fischeri, Lu. misionensis, Lu. lanei, Lu. neivai, Lu. shannoni, and Lu. monticola, in an attempt to verify natural infection by Leishmania (Viannia), the causative agent of ACL. Le. (Viannia) infections were demonstrated by DNA amplification from two Lu. pessoai and one Lu. misionensis female. Lu. pessoai have been found with leptomonas in the gut believed to be Le. (V.) braziliensis in other endemic areas of northeastern and southeastern Brazil. However, Lu. misionensis has never been found carrying a natural infection of Le. (Viannia).

Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains

A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35oC. The findings open the possibility of using canine peritoneal cells as a model for the screenning of leishmanicide drugs and to study the pathogenesis of these species.

The status of the Lutzomyia longipalpis species complex and possible implications for Leishmania transmission

The sand fly Lutzomyia longipalpis sensu latu has been identified as the principal vector of American visceral leishmaniasis, a potentially fatal disease that primarily affects children in several countries of South and Central America. Over the past several years increases have occurred both in the number of reported cases and the population at risk: approximately 1.6 million people reside in highly endemic areas with 16,000 cases reported annually. Several studies have attempted to relate the epidemiology of this disease to variability in Lu. longipalpis that is now recognized to be a complex of at least three sibling species. Morphological variation in this species was first noted by Mangabeira (1969). Since then physiological and biochemical differences have been reported by several investigators. Recent reports in Costa Rica of the presence of Lu. longipalpis in a focus of cutaneous leishmaniasis caused by Leishmania chagasi may be an additional indication of variability in this species. While existing evidence indicates that the morphospecies Lu. longipalpis may represent a complex of sibling species, genetic, epidemiological and ecological distinctions have not been fully resolved. Thus, delimitation of systematic boundaries within the complex and corresponding to geographic distributions and roles in transmission remain unresolved. The purpose of this review is to summarize from the literature observations of polymorphism in this morphospecies and consider what significance this reported variability may have to the epidemiology of visceral leishmaniasis.

Studies on the effectiveness of diarylheptanoids derivatives against Leishmania amazonensis

In a previous work we demonstrated that diarylheptanoids extracted from Centrolobium sclerophyllum are very active against Leishmania amazonensis promastigotes. In order to continue our studies with these class of compounds, we decided to evaluate the activity of several diarylheptanoids derived from curcumin (diferuloyl methane) against the extracellular form (promastigotes) of L. amazonensis. Furthermore, an experiment against the intracellular form of the parasite (amastigotes) was carried out, comparing the most active compound among the curcumin derivatives (the methylcurcumin) with des-O-methylcentrolobine, the most active diarylheptanoid derived from C. sclerophyllum.

Establishment and characterization of a new continuous cell line from Lutzomyia longipalpis (Diptera: Psychodidae) and its susceptibility to infections with arboviruses and Leishmania chagasi

Embryonic tissue explants of the sand fly Lutzomyia longipalpis (Lutz & Neiva 1912) the main vector of Leishmania chagasi (Cunha and Chagas), were used to obtain a continuous cell line (Lulo). The tissues were seeded in MM/VP12 medium and these were incubated at 28ºC. The first subculture was obtained 45 days after explanting and 96 passages have been made to date. Lulo is composed of epithelioid cells, showed a 0.04 generations/hour exponential growth rate and population doubling time at 24.7 h. The cell line isoenzymatic profiles were determined by using PGI, PGM, MPI and 6-PGDH systems, coinciding with patterns obtained from the same species and colony's pupae and adults. The species karyotype characteristics were recognized (2n = 8), in which pair 1 is subtelocentric and pairs 2, 3 and 4 are metacentric. Lulo was free from bacterial, fungal, mycoplasmic and viral infection. Susceptibility to five arbovirus was determined, the same as Lulo interaction with Leishmania promastigotes.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocyted by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

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.

Action of pentoxifylline on experimental cutaneous leishmaniasis due to Leishmania (Leishmania) amazonensis

In the animal model of leishmaniasis caused by Leishmania (Leishmania) amazonensis there is a complex mechanism of the host-parasite interaction. The present study was performed to interfere with the inflammatory reaction to the parasites, through immune modulation. Female C5BL/6 isogenic mice were used, some of which were inoculated on the right ear and others on the right footpad with 3.10(6) stationary phase promastigotes of the MHOM/BR/PH8 strain of L. (L.) amazonensis, and were allocated in three groups: the first received pentoxifylline 8mg/kg every 12 h, since the first day; the second one received the same dose since the 40th day of infection and a control group that did not receive any treatment. All the ears excised were analyzed to determine the variation in weight between both ears and for histopathological analyses. A quantification of the parasites was done using the limiting dilution assay. A significant reduction of the number of parasites, was observed among the animals treated which had an accordingly significant reduction on the weight of the ears. Pentoxifylline reduced the macrophages propensity to vacuolation and induced a more effective destruction of the parasites by these cells. Moreover, the group that began the treatment later did not show the same effectiveness.

Sand fly evolution and its relationship to Leishmania transmission

The evolutionary relationships of sand flies and Leishmania are discussed in this report, which draws distinctions between co-association, co-evolution and co-speciation (or co-cladogenesis). Examples focus on Phlebotomus vectors of Le. infantum and Le. major in the Mediterranean subregion.

Acidic ribosomal proteins and histone H3 from Leishmania present a high rate of divergence

Another additional peculiarity in Leishmania will be discussed about of the amino acid divergence rate of three structural proteins: acidic ribosomal P1 and P2b proteins, and histone H3 by using multiple sequence alignment and dendrograms. These structural proteins present a high rate of divergence regarding to their homologous protein in Trypanosoma cruzi. At this regard, L. (V.) peruviana P1 and T. cruzi P1 showed 57.4% of divergence rate. Likewise, L. (V.) braziliensis histone H3 and acidic ribosomal P2 protein exhibited 31.8% and 41.7% respectively of rate of divergence in comparison with their homologous in T. cruzi.

Retention of Leishmania (Leishmania) Mexicana in naturally infected rodents from the State of Campeche, Mexico

In the State of Campeche, Mexico, zoonotic cutaneous leishmaniasis is mainly due to Leishmania (L.) mexicana. The parasite population is maintained in a mammalian species, a reservoir in which the ideal course of infection should be long and relatively nonpathogenic. The objective of the present study was to document the retention of L. (L.) mexicana in 29 naturally infected rodents. These cricetids lived in captivity for up to two years and were tested monthly for the presence of the parasite, by cultures of needle aspirates from the base of the tail. Peromyscus yucatanicus and Ototylomys phyllotis were incriminated as the primary reservoir hosts. The finding that the multiplication of parasites in P. yucatanicus might be triggered by temperature, suggests that this animal would be a good choice for further research on L. (L.) mexicana.