Visceral Leishmaniosis caused by Leishmania (L.) mexicana in a Mexican patient with human immunodeficiency virus infection

A 36 year old male was admitted in December 1997 to hospital with afternoon fever, malaise and hepatosplenomegaly. He also had a dry cough, dyspnoea and anaemia. Pneumonia caused by Pneumocystis carinii and human immunodeficiency virus (HIV) infection were documented. The HIV infection was confirmed in 1997 with 290,000 virus copies. The patient had been in the Mexican State of Chiapas which is known to be endemic for visceral leishmaniosis (VL) and localized cutaneous leishmaniosis (LCL). The visceral symptoms were diagnosed as VL and the causal agent was identified as Leishmania (L.) mexicana. Identification of Leishmania was carried out by the analysis of amplified DNA with specific primers belonging to the Leishmania subgenus and by dot blot positive hybridisation of these polymerase chain reaction derived products with kDNA from the L. (L.) mexicana MC strain used as probe. This is the first case in Mexico of VL caused by a species of Leishmania that typically produces a cutaneous disease form.

Clinical picture of cutaneous leishmaniases due to Leishmania (Leishmania) mexicana in the Yucatan peninsula, Mexico

Localized cutaneous leishmaniasis (LCL), known as "chiclero's ulcer" in southeast Mexico, was described by Seidelin in 1912. Since then, the sylvatic region of the Yucatan peninsula has been identified as an endemic focus of LCL. The purpose of the present work was to describe the clinical picture of LCL caused by Leishmania (Leishmania) mexicana in the Yucatan peninsula. A total of 136 cases of LCL, based on isolation and characterization of L. (L.) mexicana by isoenzymes and/or monoclonal antibodies, were selected. Some variability of clinical features regarding number, type, size, form, location and time of evolution of the lesions was observed. The most frequently observed presentation was a single, ulcerated, rounded small lesion, located on the ear, with an evolution time of less than three months, with neither cutaneous metastases nor lymphatic nor mucosal involvement. This picture corresponds to previous studies carried out in the same endemic area where an organism of the L. mexicana complex has been incriminated as a major aetiological agent of classical "chiclero's ulcer", confirming that in the Yucatan peninsula LCL due to L. (L.) mexicana when located on the pinna of the ear is a remarkable characteristic.

Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major.

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.

Effect of a second bloodmeal on the oesophagus colonization by Leishmania mexicana complex in Lutzomyia evansi (Diptera: Psychodidae)

Migration and colonization of the oesophagus by Leishmania mexicana parasites were enhanced after digestion of a second bloodmeal intake in Lutzomyia evansi. This event has epidemiological significance since it affects the infection susceptibility of this sand fly species, which is a proven vector of L. chagasi in Colombian and Venezuelan visceral leishmaniasis foci. Also, it may explain the host seeking behaviour displayed by some partially bloodfed flies found inside houses.

Skin reactions to thimerosal and Leishmania in dogs from a leishmaniasis endemic area: it is better to keep them apart

Positive Montenegro's skin test is a delayed type hypersensitivity reaction widely used as indicative of previous infection with Leishmania in both humans and dogs. Montenegro's antigen consists of a crude Leishmania antigen solution, usually containing thimerosal as preserving agent. In this work it is shown that a large proportion of dogs (11 out of 56) examined in an endemic area of leishmaniasis presented induration at the site of injection of a diluent containing thimerosal alone. This clearly demonstrates that thimerosal leads to a high number of false positive skin reactions in dogs and that its use in Montenegro's skin test antigenic preparations should be avoided.

Leishmania (Leishmania) major-infected rhesus macaques (Macaca mulatta) develop varying levels of resistance against homologous re-infections

Seven rhesus macaques were infected intradermally with 10(7) promastigotes of Leishmania (Leishmania) major. All monkeys developed a localized, ulcerative, self-healing nodular skin lesion at the site of inoculation of the parasite. Non-specific chronic inflammation and/or tuberculoid-type granulomatous reaction were the main histopathological manifestations of the disease. Serum Leishmania-specific antibodies (IgG and IgG1) were detected by ELISA in all infected animals; immunoblot analyses indicated that numerous antigens were recognized. A very high degree of variability was observed in the parasite-specific cell-mediated immune responses [as detected by measuring delayed-type hypersensitivity (DTH) reaction, in vitro lymphocyte proliferation, and gamma interferon (IFN-gamma) production] for individuals over time post challenge. From all the recovered monkeys (which showed resolution of the lesions after 11 weeks of infection), 57.2% (4/7) and 28.6% (2/7) animals remained susceptible to secondary and tertiary infections, respectively, but the disease severity was altered (i.e. lesion size was smaller and healed faster than in the primary infection). The remaining monkeys exhibited complete resistance (i.e. no lesion) to each rechallenge. Despite the inability to consistently detect correlates of cell-mediated immunity to Leishmania or correlation between resistance to challenge and DTH, lymphocyte transformation or IFN-gamma production, partial or complete acquired resistance was conferred by experimental infection. This primate model should be useful for measuring vaccine effectiveness against the human disease.

Identification of a differentially expressed mRNA in axenic Leishmania panamensis amastigotes

Differential display technique was applied in order to identify transcripts which are present in axenic amastigotes but not in promastigotes of the Leishmania panamensis parasites. One of them was cloned and the sequence reveals an open reading frame of 364 amino acids (aprox. 40 kDa). The deduced protein is homologous to the serine/threonine protein kinases and specially to the mitogen activates protein kinases from eukaryotic species. Southern blot analysis suggest that this transcript, named lpmkh, is present in the genome of the parasite as a single copy gene. These results could imply that lpmkh could be involved in the differentiation process or the preservation of amastigotes in axenic conditions.

Isolation of Leishmania infantum, zymodeme MON-1 from canine and human visceral leishmaniasis on Margarita Island, Venezuela

An increase in the incidence of human visceral leishmaniasis (HVL) has been detected in recent years on Margarita Island, located off the NE coast of Venezuela. Recent studies have revealed reactivity to rK39 antigen (Leishmania chagasi) in 20% of 541 sera from domestic dogs in endemic communities; PCR reactions were positive using primers for the L. donovani complex. Here we report that isolates from human and canine infection, identified by isoenzyme analysis, correspond to L. infantum, zymodeme MON-1. This appears to be the first isolation and identification of an isolate from HVL on Margarita Island and demonstrates the presence of this zymodeme in the canine population.

Dissociation between vasodilation and Leishmania infection-enhancing effects of sand fly saliva and maxadilan

In this study, the ability of maxadilan and Lutzomyia longipalpis salivary gland lysate to enhance the infection of CBA mice by Leishmania major and of BALB/c mice by L. braziliensis was tested. No difference was observed between sizes of lesion in CBA mice infected with L. major and treated or not with salivary gland lysate or maxadilan, although they were injected in concentrations that induced cutaneous vasodilation. Although parasites were more frequently observed in foot pads and spleens of animals treated with maxadilan than in the animals treated with salivary gland lysate or saline, the differences were small and not statistically significant. The lesions in BALB/c mice infected with L. braziliensis and treated with maxadilan were slightly larger than in animals that received Leishmania alone. Such differences disappeared 14 weeks after infection, and were statistically significant only in one of two experiments.

Leishmania cysteine proteinases: from gene to subunit vaccine.

Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.

Cotton Rats (Sigmodon hispidus) and Black Rats (Rattus rattus) as Possible Reservoirs of Leishmania spp. in Lara State, Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.

Frequency of Infection of Lutzomyia Phlebotomines with Leishmania braziliensis in a Brazilian Endemic Area as Assessed by Pinpoint Capture and Polymerase Chain Reaction

Leishmania infected of Lutzomyia spp. are rare in endemic areas. We tested the hypothesis that there is clustering of infected vectors by combining pinpoint capture with sensitive L. braziliensis kDNA minicircle specific PCR/dot blot in an endemic area in the State of Bahia. Thirty out of 335 samples (10 to 20 sand flies/sample; total of 4,027 female sand flies) were positive by PCR analysis and dot blot leading to a underestimated overall rate of 0.4% positive phlebotomines. However, 83.3% of the positive samples were contributed by a single sector out of four sectors of the whole studied area. This resulted in a rate of 1.5% Leishmania positive phlebotomines for this sector, far above rates of other sectors. Incidence of American cutaneous leishmaniasis cases for this sector was about twice that for other sectors. Our results show that there is a non-homogeneous distribution of Leishmania-infected vectors. Such a clustering may have implications in control strategies against leishmaniasis, and reinforces the necessity of understanding the ecological and geographical factors involved in leishmanial transmission.

Molecular Modeling Approaches for Determining Gene Function: application to a Putative Poly-A Binding Protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 aminoacids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.

Charaterization of Leishmania major Friedlin Telomeric Terminus

Here we have characterized Leishmania major (Friedlin) telomeric terminus (the very end) using recombinants obtained by a vector-adaptor cloning protocol. As in L. donovani, the last nine nucleotides of L. major terminus are 5'-GGTTAGGGT-OH 3', differing from Trypanosoma cruzi and T. brucei terminus 5'GGGTTAGGG-OH 3', thus indicating that these sequences are genus specific. We have also made a comparative analysis between L. major and L. donovani telomere-associated sequences, and described a novel non-repeated telomeric associated sequence common to L. major low molecular weight chromosomal bands.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.