Leishmania donovani is the only cause of visceral leishmaniasis in East Africa; previous descriptions of L. infantum and "L. archibaldi" from this region are a consequence of convergent evolution in the isoenzyme data.

Isoenzyme-based studies have identified 3 taxa/species/'phylogenetic complexes' as agents of visceral leishmaniasis in Sudan: L. donovani, L. infantum and "L. archibaldi". However, these observations remain controversial. A new chitinase gene phylogeny was constructed in which stocks of all 3 putative species isolated in Sudan formed a monophyletic clade. In order to construct a more robust classification of the L. donovani complex, a panel of 16 microsatellite markers was used to describe 39 stocks of these 3 species. All "L. donovani complex" stocks from Sudan were again found to form a single monophyletic clade. L. donovani ss stocks from India and Kenya were found to form 2 region-specific clades. The partial sequence of the glutamate oxaloacetate transaminase (GOT) gene of 17 L. donovani complex stocks was obtained. A single nucleotide polymorphism in the GOT gene appeared to underlie the isoenzyme classification. It was concluded that isoenzyme-based identification is unsafe for stocks isolated in L. donovani endemic areas and identified as L. infantum. It was also concluded that the name L. archibaldi is invalid and that only a single visceralizing species, Leishmania donovani, is found in East Africa.

Evaluation of a urinary antigen-based latex agglutination test in the diagnosis of kala-azar in eastern Nepal.

BACKGROUND: We evaluated the diagnostic accuracy as well as the reproducibility of the urine latex agglutination test 'KAtex' in the diagnosis of kala-azar in patients recruited at a tertiary care centre in Dharan, Nepal, between November 2000 and January 2002. METHODS: All patients presenting with fever of 2 weeks or more and splenomegaly were consecutively enrolled. Bone marrow and--if negative--spleen aspirates were examined for Leishmania donovani. Serum and urine samples were taken in duplicate for the Direct Agglutination Test (DAT) and KAtex. The reference laboratory determined sensitivity and specificity of KAtex. Reproducibility between both laboratories was assessed. RESULTS: KAtex was performed on urine from 155 parasitologically confirmed kala-azar and 77 non-kala-azar cases (parasitology and DAT-negative). KAtex showed a sensitivity of 47.7% (74/155, 95% CI: 39.7-55.9) and a specificity of 98.7% (76/77, 95% CI: 93.0-100.0). Reproducibility of KAtex showed a kappa of 0.684 (P < 0.001, n = 232). CONCLUSION: KAtex evaluation showed high specificity, low sensitivity and moderate reproducibility. A urine test for kala-azar could become a real breakthrough in kala-azar management if its reproducibility and sensitivity could be further improved.

Detection of antileishmanial IgG antibodies and Leishmania-specific antigens in sera from Yemeni patients with visceral leishmaniasis.

As part of an attempt to develop an effective method for the serodiagnosis of visceral leishmaniasis (VL), the sera from 43 Yemeni cases of the disease were screened both for antileishmanial IgG antibodies and for Leishmania-specific antigens. Indirect ELISA and capture ELISA were used to test each serum for the antibodies and antigens, respectively. Sera from patients with diseases other than leishmaniasis (29 for the antibody-detection assays and 42 for the antigen-detection) and from apparently healthy volunteers were also tested. For each type of assay, the threshold for seropositivity was set three standard deviations above the mean absorbance value for the sera from the healthy volunteers. Thirty-seven of the 43 VL sera were found positive for Leishmania-specific IgG antibodies and 37 were found positive for Leishmania-specific antigens. The sensitivities of the antibody- and antigen-detection assays were both therefore 86%. The overall specificity of the antibody-detection ELISA (67%) was, however, slightly higher than that of the capture-ELISA (64%).

Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa.

Current treatments for diseases caused by apicomplexan and trypanosomatid parasites are inadequate due to toxicity, the development of drug resistance and an inability to eliminate all life cycle stages of these parasites from the host. New therapeutics agents are urgently required. It has recently been demonstrated that type II fatty acid biosynthesis occurs in the plastid of Plasmodium falciparum and Toxoplasma gondii and inhibitors of this pathway such as triclosan and thiolactomycin restrict their growth. Furthermore, Trypanosoma brucei has recently been demonstrated to use type II fatty acid biosynthesis for myristate synthesis and to be susceptible to thiolactomycin. As this pathway is absent from mammals, it may provide an excellent target for novel antimicrobial agents to combat these diverse parasites. Leishmania and Trypanosoma parasites produce ergosterol-related sterols by a biosynthetic pathway similar to that operating in pathogenic fungi and their growth is susceptible to sterol biosynthesis inhibitors. Thus, inhibition of squalene 2,3-epoxidase by terbinafine, 14alpha-methylsterol 14-demethylase by azole and triazole compounds and delta(24)-sterol methyl transferase by azasterols all cause a depletion of normal sterols and an accumulation of abnormal amounts of sterol precursors with cytostatic or cytoxic consequences. However, Leishmania parasites can survive with greatly altered sterol profiles induced by continuous treatment with low concentrations of some inhibitors and they also have some ability to utilise and metabolise host sterol. These properties may permit the parasites to evade treatment with sterol biosynthesis inhibitors in some clinical situations and need to be taken into account in the design of future drugs.

The role of promastigote secretory gel in the origin and transmission of the infective stage of Leishmania mexicana by the sandfly Lutzomyia longipalpis.

Transmission of leishmaniasis is effected by a specific developmental stage, the metacyclic promastigote. The precursors of metacyclic promastigotes were a distinct subpopulation of parasites, identified for the first time as a new stage in the life-cycle and named leptomonad promastigotes. Microdissection of infected sandflies into 4 midgut regions and foregut allowed precursor-product relationships to be established for amastigote-procyclic promastigote, procyclic-nectomonad promastigote, nectomonad-leptomonad promastigote and leptomonad-metacyclic promastigote developmental switches. Metacyclic promastigotes occurred mainly in the thoracic midgut and cardia, coincident with the accumulation of a promastigote secretory gel (PSG) plug in these anterior regions. The gel-like plug was isolated from flies with mature infections and found to contain predominantly leptomonad promastigotes. The PSG plug also contained the majority (75%) of the total metacyclic promastigote population in the sandflies, which were concentrated at the anterior pole. The PSG plug was found to be the main site of metacyclogenesis, and acted as a reservoir of leptomonad promastigotes from which metacyclic forms differentiated and migrated forward to promote the infective potential of the fly. The PSG plug occluded and distorted the midgut, forcing the stomodeal valve open and affecting the feeding success of the sandflies, such that they experienced difficulty in taking a full meal. Collectively, these data support the role of the PSG in the transmission of leishmaniasis, by conditioning the midgut environment for metacyclogenesis and altering the feeding ability of infected sandflies.

Latex agglutination test for the detection of urinary antigens in visceral leishmaniasis.

This paper describes a new latex agglutination test ('KATEX') for the detection of leishmanial antigen in the urine of patients with visceral leishmaniasis. In preliminary laboratory trials, using urine collected from well-defined cases and controls from Brazil, Yemen and Nepal, the test had 100% specificity and a sensitivity between 68 and 100%. When used in a time-course experiment in cotton rats infected with Leishmania donovani, the test became positive 1 week after inoculation and antigen levels in urine declined rapidly after chemotherapy (the test was negative before the end of the course of treatment). Finally, in an integrated study performed in Sudan, KATEX was compared to microscopy and four different serological tests in a group of 73 patients having presented with clinical manifestations suggestive of visceral leishmaniasis. Compared to microscopy, KATEX performed better than any single serological test in predicting positivity and a particularly good result was obtained by combining KATEX and the direct agglutination test (DAT).

The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana.

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.

[Katex for the diagnosis of human visceral leishmaniasis]. / Le Katex pour le diagnostic de la leishmaniose viscérale humaine.

Katex is a latex agglutination test allowing highly specific detection Leishmania antigen in urine of patients with visceral leishmaniasis. A multicentric study of this new diagnostic tool which is also effective in patients co-infected by leishmaniasis and HIV is currently in progress in Sudan, India, Nepal, Brazil and Spain. The authors describe the utility of this technique in comparison with other routine diagnostic procedures such as microscopic examination and serological tests. Preliminary results suggest that it could be used to confirm infection in the field and to monitor treatment efficacy.

Evidence for a neotropical origin of Leishmania.

Contradictory biogeographic hypotheses for either a Neotropical or a Palaearctic origin of the genus Leishmania have been proposed. Hypotheses constructed on the basis of biogeographic data must be tested against an independent dataset and cannot be supported by biogeographic data alone. In the absence of a fossil record for the Leishmania these two hypotheses were tested against a combined dataset of sequences from the DNA polymerase A catalytic subunit and the RNA polymerase II largest subunit. The phylogeny obtained provided considerable support for a Neotropical origin of the genus Leishmania and leads us to reject the hypothesis for a Palaearctic origin.

Utilization of leucine and acetate as carbon sources for sterol and fatty acid biosynthesis by Old and New World Leishmania species, Endotrypanum monterogeii and Trypanosoma cruzi.

The relative roles of acetate and leucine in the provision of a carbon source for fatty acid and sterol biosynthesis in several trypanosomatid species were investigated using 14C- and 13C-labelled acetate, glucose and leucine as substrates. Promastigotes of Leishmania species synthesized a large proportion of their sterol from leucine. L. major (LV39), L. amazonensis and L. mexicana were the most efficient utilizers of leucine, producing at least 70-77% of their sterol from leucine; L. braziliensis, L. donovani and L. tropica apparently produced less sterol from leucine (23-36%) and L. major (LV561), L. adleri and L. panamamensis were intermediate, utilizing leucine to provide 51-58% of their sterol. In all the cases the balance of the sterol produced was apparently synthesized from carbon arising from acetate. The related trypanosomatid Endotrypanum monterogeii also produced a large amount (77%) of its sterol from leucine rather than acetate. By contrast Trypanosoma cruzi elaborated only 8% of its sterol from leucine and used acetate far more effectively than the Leishmania species for sterol biosynthesis. The fatty acid moieties of the triacylglycerols and phospholipids were produced from acetate. Leucine was also incorporated into the fatty acids to varying extents in the different organisms showing that leucine can also be metabolized in trypanosomatids to generate acetyl-CoA.

Elucidation of carbon sources used for the biosynthesis of fatty acids and sterols in the trypanosomatid Leishmania mexicana.

Sterols are necessary for the growth of trypanosomatid protozoans; sterol biosynthesis is a potential target for the use and development of drugs to treat the diseases caused by these organisms. This study has used (14)C-labelled substrates to investigate the carbon sources utilized by promastigotes and amastigotes of Leishmania mexicana for the production of sterol [mainly ergosta-5,7,24(24(1))-trien-3beta-ol] and the fatty acid moieties of the triacylglycerol (TAG) and phospholipid (PL) of the organism. The isoprenoid precursor mevalonic acid (MVA) was incorporated into the sterols, and the sterol precursor squalene, by the promastigotes of L. mexicana. However, acetate (the precursor to MVA in most organisms) was a very poor substrate for sterol production but was readily incorporated into the fatty acids of TAG and PL. Other substrates (glucose, palmitic acid, alanine, serine and isoleucine), which are metabolized to acetyl-CoA, were also very poor precursors to sterol but were incorporated into TAG and PL and gave labelling patterns of the lipids similar to those of acetate. In contrast, the amino acid leucine was the only substrate to be incorporated efficiently into the squalene and sterol of L. mexicana promastigotes. Quantitative measurements revealed that at least 70-80% of the sterol synthesized by the promastigotes of L. mexicana is produced from carbon provided by leucine metabolism. Studies with the amastigote form of L. mexicana showed that in this case leucine was again the major sterol precursor, whereas acetate was utilized for fatty acid production.

The axenic cultivation of Leishmania donovani amastigotes.

Full text is available as a scanned copy of the original print version.

In vitro stimulation of metacyclogenesis in Leishmania braziliensis, L. donovani, L. major and L. mexicana.

Promastigotes of Leishmania braziliensis, L. donovani, L. major and L. mexicana recently derived from tissue amastigotes were cultured in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum and 25 micrograms gentamicin sulfate/ml at pH 5.5. These cultures produced more metacyclic promastigotes in their stationary-phase populations than others cultured at pH 7.0. Metacyclic promastigotes possessed a short (< or = 8 microns) and narrow (< or = 1.5 microns) cell body with a flagellum twice or more the length of the cell body. Promastigotes from acidic cultures were more resistant to complement-mediated lysis and more infective in vivo than those grown at neutral pH. These results demonstrate that induction of metacyclogenesis by acidic pH is a response conserved across a variety of species of Leishmania.

The Leishmania hertigi (Kinetoplastida; Trypanosomatidae) complex and the lizard Leishmania: their classification and evidence for a neotropical origin of the Leishmania-Endotrypanum clade.

The relationships of the Leishmania hertigi complex and the lizard Leishmania species to the main groups of mammalian Leishmania and Endotrypanum parasites were examined. Restriction fragment length polymorphisms and sequences of small subunit ribosomal RNA genes and hybridization studies of kinetoplast DNA indicated that the L. hertigi complex was more closely related to the genus Endotrypanum than to the genus Leishmania. The lizard Leishmania species were found to be at the crown of the Leishmania tree. The data provides strong evidence for a Neotropical origin of the Endotrypanum/Leishmania clade since the parasites closest to the root of the tree are all found exclusively in the Neotropics. The evolution of the Leishmania/Endotrypanum clade in relation to the evolution of the known hosts of these parasites is discussed.

A time-course study of circulating antigen and parasite-specific antibody in cotton rats infected with Leishmania donovani.

Levels of circulating antigen in a group of cotton rats infected with Leishmania donovani were followed over a 28-week period, using a modified, polyethylene-glycol (PEG) ELISA. Circulating antigen could be detected from 1 week post-infection and gradually increased over time. In infected cotton rats treated with a curative dose of Pentostam at 12 weeks post-infection, antigen levels peaked and then declined. Antigen was still detected in some of the treated animals at 28 weeks post-infection. The antibodies used in the PEG-ELISA were also used in a capture ELISA to detect parasite antigens in urine. Urine samples which were positive by capture ELISA were also analysed by western blotting, in an attempt to identify the parasite antigens present. Three components, of 45, 47 and 58 kDa, were detected.

The direct agglutination test: a non-specific test specific for the diagnosis of visceral leishmaniasis?

Serology has an important role to play in the diagnosis of the severe clinical syndrome of visceral leishmaniasis (VL). The direct agglutination test (DAT), a simple agglutination test which requires no laboratory facilities, has become the preferred test, particularly for field studies. The nature of the antigens responsible for the agglutination of leishmanial promastigotes by the serum of VL patients is not known. A series of experiments which provide some clues to the molecular basis for the test and which indicate that there might be more in DAT than meets the eye is reported.