Identification of the agent causing visceral leishmaniasis in Uzbeki and Tajiki foci by analysing parasite DNA extracted from patients' Giemsa-stained tissue preparations.

Our present study is the first attempt to characterize Leishmania parasites from foci in Uzbekistan and Tajikistan endemic for visceral leishmaniasis (VL). PCR-sequencing of the ribosomal internal transcribed spacer 1 and multilocus microsatellite typing (MLMT) were applied to DNA extracted from preparations of Giemsa-stained bone marrow aspirates from 13 cases of VL. L. infantum was shown to cause VL currently occurring in this area. MLMT applying 14 microsatellite markers, previously shown to be polymorphic for strains of the L. donovani complex, revealed that microsatellite profiles of parasites causing human VL in the Namangan and Jizzakh regions in Uzbekistan, and Penjikent region in Tajikistan, basically coincide with those of strains of L. infantum MON-1. Furthermore, these parasites were assigned to a distinct cluster genetically clearly separated from the populations of L. infantum MON-1 from Europe, the Middle East and North Africa. The existence of a genetically homogeneous but distinct group of L. infantum MON-1 indicates that the parasites circulating in the Uzbeki and Tajiki foci studied have been restricted there for a long time rather than having been recently introduced from elsewhere by human or animal reservoir migration.

The recent emergence of Leishmania tropica in Jericho (A'riha) and its environs, a classical focus of L. major.

Between 1997 and 2002, 49 strains of Leishmania were isolated from the cutaneous lesions of Palestinians living in and around Jericho. A polymerase chain reaction (PCR) amplifying the ribosomal internal transcribed spacer 1 (ITS1-PCR) was applied to their cultured promastigotes and to 207 individuals' skin scrapings spotted on filter-papers, 107 of which proved positive for leishmanial DNA. Species identification was performed by restricting the ITS1-PCR amplification products from the cultured promastigotes and the amastigotes in the scrapings with the endonuclease HaeIII. Of the 49 cultures, 28 (57%) were L. major and 21 (43%) were L. tropica. Of the 107 dermal samples tested directly, 53 (49.5%) were infected with L. major, 52 (48.5%) with L. tropica and two remained unidentified. This is the first time L. tropica has been exposed in the population of the Jericho area and on such a large scale. The itinerant behaviour of some of this population precludes categorically declaring that L. tropica has recently become established in this classical focus of L. major. For this and although 88.2% of the cases of L. tropica claimed not to have travelled out of the vicinity of Jericho, local infected sand fly vectors of L. tropica must be caught, identified and, if possible, shown to harbour infections, and, if one exists, an animal reservoir host should also be exposed to endorse whether the cases caused by L. tropica were imported or autochthonous.

Epidemiology of cutaneous leishmaniasis in the endemic area of Jericho, Palestine.

This study of cutaneous leishmaniasis in Jericho city and the adjacent Aqbat-Jaber refugee camp investigated the seroprevalance of Leishmania major and the risk factors associated with acquiring the disease. Clinical and parasitology identification of cases showed children and young men were more affected, with the head most affected in children. Enzyme-linked immunosorbent assay (ELISA) was used to test sera from 190 individuals. The overall seroprevalence of cutaneous leishmaniasis was 26.3%. A case-control study of 247 individual in 37 households showed that a higher level of education of the head of the household and having children sleep under bed nets were significantly related to a lower incidence of cutaneous leishmaniasis.

Epidemiology of cutaneous leishmaniasis in the endemic area of Jericho, Palestine

This study of cutaneous leishmaniasis in Jericho city and the adjacent Aqbat-Jaber refugee camp investigated the seroprevalance of Leishmania major and the risk factors associated with acquiring the disease. Clinical and parasitology identification of cases showed children and young men were more affected, with the head most affected in children. Enzyme-linked immunosorbent assay [ELISA] was used to test sera from 190 individuals. The overall seroprevalence of cutaneous leishmaniasis was 26.3%. A case-control study of 247 individual in 37 households showed that a higher level of education of the head of the household and having children sleep under bed nets were significantly related to a lower incidence of cutaneous leishmaniasis

Cutaneous involvement in a rare case of adult visceral leishmaniasis acquired in Israel.

Skin lesions are rare in visceral leishmaniasis, especially in Mediterranean countries. We describe an unusual case of visceral leishmaniasis in a 41-year-old man that began with a skin lesion. The parasites isolated from both the skin lesion and the bone marrow were typed as Leishmania donovani sensu stricto. This pathogen is not endemic in Israel or neighboring countries; its contribution to adult visceral leishmaniasis in Israel is summarized.

Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis.

Leishmania aethiopica infections in man result in a spectrum of diseases from LCL to DCL. These clinical manifestations have been attributed to genetic differences within the host or the parasites. In this study two different PCR-based methods were used to elucidate genetic variation within the species L. aethiopica. Inter- and intra-specific variations were detected in the ITS of the ribosomal operon in different strains and species of Leishmania, using a PCR-RFLP approach, and by a PCR fingerprinting technique that used single non-specific primers to amplify polymorphic regions of the genomic DNA. Both methods revealed genetic heterogeneity among ten L. aethiopica isolates examined. Unrooted distance trees separated the ten strains into two different genetic groups. This subdivision was correlated to the geographical origin of the isolates rather than to the clinical manifestation of the disease.

Emergence of visceral leishmaniasis in central Israel.

In 1994-1995, a child and five dogs from villages located between Jerusalem and Tel-Aviv, Israel were diagnosed with visceral leishmaniasis (VL). Based on these findings, the distribution of VL in domestic and wild canids in central Israel was examined. In the two villages where canine index cases were identified, a substantial proportion (11.5%, 14 of 122) of the dogs examined were seropositive. However, the rate of infection in five neighboring villages was only 1% (1 of 99). Parasites were cultured from 92% (12 of 13) of the seropositive dogs biopsied and the strains were characterized as Leishmania infantum by a clamped polymorphic-polymerase chain reaction, monoclonal antibodies, and/or excreted factor serology. The discovery of VL close to major urban centers is an important public health issue. The disease appears to have emerged recently in this area, and it is unclear whether the parasite was re-introduced or was continuously present at low levels in this region. The presence of seropositive wild canids, jackals (7.6%, 4 of 53) and red foxes (5%, 1 of 20), in central Israel, and the reappearance of the jackal population after near extinction suggests that wild canids may play a role in spreading this disease.

Structure of Leishmania lipophosphoglycan: inter- and intra-specific polymorphism in Old World species.

The most abundant surface macromolecule on the promastigote stage of leishmanial parasites is a polymorphic lipophosphoglycan (LPG). We have elucidated the structures of two new LPGs, from Leishmania tropica (LRC-L36) and L. aethiopica (LRC-L495), and investigated the nature of intra-specific polymorphism in the previously characterized LPG of L. major (LRC-L456 and -L580). These molecules contain a phosphoglycan chain, made up of repeating PO4-6Gal beta 1-4Man units and a conserved hexaglycosyl-phosphatidylinositol membrane anchor. Extensive polymorphism occurs in the extent to which the LPG repeat units are substituted with different glycan side chains. The L. tropica LPG is the most complex LPG characterized to date, as most of the repeat units are substituted with more than 19 different glycan side chains. All of these side chains, including the novel major glycans, Arap beta 1-3Glc beta 1- and +/- Arap beta 1-2Glc beta 1-4[+/- Arap beta 1-2]Glc beta 1-, are linked to the C-3 position of the backbone disaccharide galactose. In contrast, the L. aethiopica LPG repeat units are partially substituted (35%) with single alpha-mannose residues that are linked, unusually, to the C-2 position of the mannose in the backbone disaccharide. Polymorphism is also evident in the spectrum of alpha-mannose-containing oligosaccharides that cap the non-reducing terminus of the phosphoglycan chains of these LPGs. Finally, analysis of the L. major LPGs showed that, while some strains contain LPGs which are highly substituted with side chains of beta Gal, Gal beta 1-3Gal beta 1- and Arap beta 1-2Gal beta 1-3Gal beta 1-, the LPGs of other strains (i.e L. major LRC-L456) are essentially unsubstituted. Recent studies have shown that the LPG side chains and cap structures can mediate promastigote attachment to a number of different receptors along the midgut of the sandfly vector. The possible significance of LPG polymorphism on the ability of these parasites to infect a number of different sandfly vectors is discussed.

Glycoinositol-phospholipid profiles of four serotypically distinct Old World Leishmania strains.

Glycoinositol-phospholipids (GIPLs) are the major glycolipid class and prominant surface antigens of leishmanial parasites. The GIPLs from four serologically distinct Old World strains of Leishmania were characterized to determine inter- and intra-specific differences in these glycolipids. These studies showed that: (1) the major GIPLs of Leishmania topica (LRC-L36) and Leishmania aethiopica (LRC-L495) belong to the alpha-mannose-terminating GIPL series (iM2, iM3 and iM4) that are structurally related to the glycosyl-phosphatidylinositol anchors of both the surface proteins and the abundant lipophosphoglycan (LPG). In contrast, the GIPLs from two Leishmania major strains (LRC-L456 and LRC-L580) belong to the alpha-galactose-terminating GIPL series (GIPL-1, -2 and -3) that are more structurally related to the LPG anchor; (2) the GIPL profiles of the L. major strains differed in that a significant proportion of the GIPL-2 and -3 species (approximately 40% and 80%, respectively) in LRC-L580 are substituted with a glucose-1-PO4 residue, while this type of substitution was not detected in LRC-L456; and (3) all the GIPLs contained either an alkylacyl- or a lysoalkyl-phosphatidylinositol lipid moiety. However, the alkyl chain compositions of different GIPLs within the same strain was variable. In L. major, the major GIPL species contained alkylacylglycerols with predominantly C18:0 and C24:0 alkyl chains, whereas the glucose-1-PO4-substituted GIPLs contained exclusively lysoalkylglycerols with C24:0 alkyl chains. In L. tropica, the major GIPL, iM2, contained predominantly C24:0 alkyl chains whereas the structurally related iM3 and iM4 GIPLs in this strain contained predominantly C18:0 alkyl chains. In L. aethiopica all the GIPLs (iM2, iM3, iM4) contained C18:0 alkyl chains. These data suggest that the synthesis of the GIPLs may occur in more than one subcellular compartment. The possibility that species-specific differences in the predominantly surface glycan structures may modulate the interaction of the parasite with the insect and mammalian hosts is discussed.

The kinetoplastid membrane protein 11 of Leishmania donovani and African trypanosomes is a potent stimulator of T-lymphocyte proliferation.

Kinetoplastid membrane protein 11 (KMP-11) from Leishmania donovani is an abundant 11-kDa surface membrane glycoprotein. Lymph node cells from mice of six different H-2 haplotypes immunized with KMP-11 or with L. donovani promastigotes were stimulated to proliferate in vitro KMP-11. Primed purified T cells required antigen presentation since they were not stimulated unless KMP-11-pulsed or L. donovani-infected macrophages were added. Promastigotes of a wide variety of Leishmania species and procyclic forms of African trypanosomes stimulated proliferation of KMP-11-primed or L. donovani promastigote-primed lymph node cells. All of the Leishmania promastigotes and African trypanosomes tested contained an 11-kDa protein, as detected by immunoblotting with KMP-11-specific monoclonal antibodies. The widespread distribution of the 11-kDa (KMP-11) molecules and their ability to stimulate strong T-lymphocyte proliferation in a non-H-restricted fashion suggest that they may be important molecules for induction of cell-mediated immune responses.

Distribution of lipophosphoglycan-associated epitopes in different Leishmania species and in African trypanosomes.

Monoclonal antibody (mAb) CA7AE binds specifically to the phosphorylated Gal-beta 1,4-Man disaccharide repeat epitope of Leishmania donovani lipophosphoglycan (LPG). This mAb detected the repeat epitope in most but not all of a wide variety of Leishmania species and strains examined. MAb CA7AE also bound to both glycoprotein and carbohydrate antigens in medium from L. donovani promastigote cultures. Specifically, mAb CA7AE bound the delipidated form of LPG, the phosphoglycan, and a glycoprotein both of which are released into the medium by the parasite indicating that both share a specific phosphorylated carbohydrate epitope. The epitope was detected in sera from L. donovani-infected (kala-azar positive) patients when mAb CA7AE was used in an antigen-capture enzyme-linked immunosorbent assay (ELISA). MAb L157 is specific for a protein that is found associated with L. donovani LPG, the lipophosphoglycan-associated protein (LPGAP). This mAb bound to molecules in all 19 strains (representing 9 species) of Leishmania promastigotes and to molecules in 2 species of Trypanosoma procyclic culture forms. This wide distribution of the LPGAP epitope implies that it may have a conserved function, for example, in the biochemistry or arrangement of parasite surface molecules. In addition, since the LPGAP is involved in the stimulation of T lymphocyte proliferation, its wide distribution amongst different Leishmania species suggests that it may be an ideal molecule for testing as a vaccine for leishmaniasis.

Visceral leishmaniasis: a difficult diagnosis and unusual causative agent.

In a young man who had a prolonged fever of unknown origin, hepatosplenomegaly, and progressive pancytopenia, stained smears, blood-agar cultures of bone marrow, and serologic testing for antileishmanial antibodies were negative. Biopsies from liver and bone marrow were uninformative. Visceral leishmaniasis was diagnosed only after splenectomy, when amastigotes were finally cultured from the spleen. The parasite was shown to be an unusual leishmanial parasite, possessing a mixture of intrinsic biochemical and serologic characteristics displayed independently by Leishmania tropica and Leishmania donovani sensu lato, the latter being the usual cause of visceral leishmaniasis. After splenectomy, parasites were also demonstrated in stained bone marrow aspirate smears. Recovery was uneventful after treatment with antimony for 28 days. Visceral leishmaniasis can be a cause of fever of unknown origin and should be considered in its differential diagnosis in endemic areas.

Serodiagnosis of canine visceral leishmaniasis in Portugal: comparison of three methods.

Sera collected in Portugal from 43 dogs were screened for specific antibodies to Leishmania donovani antigens. Three different techniques were compared: an indirect immunofluorescence assay (IFA), a direct enzyme-linked immunosorbent assay (ELISA) and a competitive-ELISA (C-ELISA) using two species-specific monoclonal antibodies, D2 and D13. By IFA, 22 of the sera examined showed positive reactions, compared with 26 by ELISA or 27 by C-ELISA. There was no direct correlation observed between the serum titre by IFA and the strength of the reaction in ELISA or inhibition in C-ELISA. However, a good correlation was observed between sera identified as positive (95.5%) by all three techniques. Western blotting on leishmanial membranes showed that common antigens with Mr of 26,000 and 70-84,000 were recognized by all infected dog sera, regardless of the serum titre. In large scale studies, ELISAs are preferred to IFA for the rapid diagnosis of canine visceral leishmaniasis because of their greater simplicity.

An experimental model for canine visceral leishmaniasis.

Seven mixed-breed dogs were challenged with either promastigotes or amastigotes of Leishmania donovani infantum strains recently isolated from naturally infected dogs. Different routes and numbers of parasites were utilized and each dog was monitored for at least 1 year post-infection. Anti-parasite specific antibody levels were measured by enzyme-linked immunosorbence, immunofluorescence, crossed-immune electrophoresis and Western blotting on crude antigen. Western blotting on two pure parasite proteins, dp72 and gp70-2, was also done. Mitogenic and antigen-specific stimulation of peripheral blood lymphocytes was monitored; and the haematological, clinical and parasitological parameters measured. Dogs challenged with amastigotes exhibited a more pronounced humoral response to leishmanial antigens. Only in one case was strong antigen-specific proliferation detected. Clinical signs of disease, including hypergammaglobulinaemia, enlarged lymph nodes and the presence of parasites, were also more apparent in the dogs challenged with amastigotes. None of the seven dogs died. Serum antibodies to leishmanial antigens were apparent between 1.5 to 3 months following challenge and correlated with the appearance of enlarged lymph nodes, hypergammaglobulinaemia and the presence of parasites in tissue biopsies. Serum antibodies remained chronically high in these dogs throughout the period of the study. Only one dog (1/3) challenged intravenously with promastigotes and the dog challenged intradermally with amastigotes produced transient antibody responses to leishmanial antigen.

Leishmania major: resistance of promastigotes to paromomycin, and susceptibility of amastigotes to paromomycin-methylbenzethonium chloride ointment.

Cutaneous lesions caused by Leishmania major in BALB/c mice were cured completely when treated topically with an ointment comprising 15% paromomycin sulphate and 1-2% methylbenzethonium chloride ointment in soft white paraffin twice daily for 10 days. No parasites were detected in tissue smears or in cultures from treated cutaneous lesions. Re-developing lesions, considered to be resulting from the migration of parasites from internal organs, showed almost the same response to topical treatment. Promastigotes of the virulent clone 121 of L. major LRC-L137 which were exposed to 100 micrograms ml-1 of paromomycin in RPMI medium at 28 degrees C developed resistance to the drug over 10 passages of exposure. Enzyme analysis of susceptible and resistant promastigotes of this clone showed no differences with regard to their profiles based on 11 enzymes.

Monoclonal antibodies against leishmanial membranes react with specific excreted factors (EF).

The interaction was examined between two reagents used for the speciation of Leishmania: spent culture-medium excreted factors (EF) and antileishmanial monoclonal antibodies (MCA). Thirty-three MCAs: seven against L. major, five against L. tropica, eight against L. aethiopica, 11 against L. donovani sensu lato and two against all Leishmania species were screened by double diffusion for reactions with EFs representing seven different sub-serotypes (A1,A2,A4,B1,B2,B3 and A3B2). Only five MCAs showed any reactivity. Three MCAs to L. major (T1, T2 and T7) precipitated only sub-serotype A1 and A4 EF, and two MCAs to L. tropica (T11 and T15) precipitated only sub-serotype A2 EF. Leishmania major and L. tropica are both serotype A species. None of the antiL. aethiopica and antiL. donovani MCAs reacted with any of the EF preparations. Both of these species belong to the serotype B. MCAs T11 and T15, the first recorded with a specificity for only sub-serotype A2 EF, were tested further against 28 sub-serotype A2 and three sub-serotype A2B2EFs from L. tropica strains. All 31 EFs formed visible bands with standard rabbit polyclonal antileishmanial serotype A serum. Only 18 of them precipitated with T11 and T15, even though parasite lysates prepared from promastigotes of several non-reactors tested positive with the MCAs. The lack of homogenous reactivity with T11 and T15 among the A2 EFs suggests that the EFs from these strains possess structural differences which affect EF-MCA precipitation.