Quantitative and ultrastructural studies on the uptake of drug loaded liposomes by mononuclear phagocytes infected with Leishmania donovani.

This study compared splenic and hepatic uptake of free and liposome-entrapped sodium antimony gluconate after i.v. administration to mice infected with Leishmania donovani. It was demonstrated that entrapment within liposomes greatly altered the kinetics of uptake of the drug. We were also able to show that liposomes composed of sphingomyelin, stearylamine and cholesterol were marginally better than any other preparation in delivering entrapped drug to liver and spleen. X-ray microanalytical studies on the uptake of liposomes by Kupffer cells infected with L. donovani have indicated that internalised liposomes probably fuse with parasitophorous vacuoles, transferring their contents into the immediate locality of the leishmanial parasites. It is proposed that this is the way in which liposome entrapped antileishmanial agents have an enhanced therapeutic effect over free drug therapy.

Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes.

This study describes the characterisation of externally oriented surface peptides of both morphological forms of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar). Using 125I surface labelling techniques and peptide extraction in the detergents Triton X-100 and Triton X-114, a major iodinable promastigote peptide at 63 kDa or 65 kDa (depending on detergent used) was identified. This peptide was demonstrated to be the immunodominant membrane peptide of L. donovani and was strongly recognised by human sera from parasitologically confirmed cases of kala-azar. This peptide was not demonstrated on the surface of tissue amastigotes, although in vitro translations of poly(A+) RNA from both promastigotes and amastigotes demonstrated that both forms possessed mRNA that directs the synthesis of a 63 kDa peptide. It is suggested therefore that in amastigotes this peptide may be a processed antigen. We also report the isolation of a recombinant cDNA clone in the bacteriophage vector lambda gt10 which encodes a 63 kDa polypeptide that is recognised by human kala-azar sera. It is proposed that this surface peptide could be used in a specific immunodiagnostic test for leishmaniasis.

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.

Synergism in vitro of lovastatin and miconazole as anti-leishmanial agents.

The antifungal drug miconazole and the cholesterol-lowering agent lovastatin (mevinolin) were used in combination to assess their potency as anti-leishmanial agents. The drug combination was synergistic, being more potent in terms of inhibition of promastigote proliferation, macrophage infection and amastigote numbers. In promastigote cultures the effect was more marked in Leishmania amazonensis than L. donovani. Analysis of the sterol compositions of both promastigote and amastigote cultures revealed the inhibition of sterol 14 alpha-demethylation by miconazole and showed some apparent evidence of inhibition of sterol biosynthesis by lovastatin.

A PEG-ELISA for the detection of Leishmania donovani antigen in circulating immune complexes.

Leishmanial antigen in circulating immune complexes (CIC) from sera of cotton-rats experimentally infected with Leishmania donovani and visceral leishmaniasis patients (VLP) was detected using a polyethylene glycol (PEG) enzyme-linked immunosorbent assay (PEG-ELISA). The immune complexes were precipitated in the cold with 12% PEG (average M(r) 6000) and then dissociated with glycine-HCl buffer. The dissociated antigen bound to the plate was then detected by peroxidase-labelled rabbit antibody raised to either amastigotes or to CIC. Serum samples from either controls or patients infected with heterologous organisms were used to define the sensitivity and specificity of the test. Leishmanial antigen was detected in the CIC from all experimentally infected animals (100% sensitivity) and in 22 of 25 of the CIC from VLP (88% sensitivity), using either conjugate. Immunoblotting of PEG-precipitated CIC from infected animals with both rabbit antisera revealed multiple antigen components. Antigens of 40, 42 and 45 kDa appeared to be specifically recognized by both antibodies; the components of 40 and 42 kDa were common to amastigote extracts and CIC from infected animals.

Antigenic analysis of Leishmania isolates from Tachira state, Venezuela.

Studies of cutaneous leishmaniasis in 3 endemic foci in Tachira state, western Venezuela have revealed sympatric populations of parasites causing both cutaneous and mucocutaneous disease. Immunological techniques and measurement of protease/acid phosphatase activities have been used to detect species-specific parasite antigens from 3 isolates from Tachira. Identified antigens of particular interest had molecular masses of 100, 82, 66, 50 and 27 kDa, but there was a high degree of heterogeneity between the antigens of the Tachira isolates and other Venezuelan strains of Leishmania braziliensis and L. mexicana. This heterogeneity has implications concerning the selection of antigens for use in serodiagnosis of leishmaniasis.

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).

Molecular approaches applied to the analysis of sympatric sandfly populations in endemic areas of western Venezuela.

To understand the epidemiology of cutaneous and mucocutaneous leishmaniasis in three distinct endemic foci of Tachira state, Western Venezuela, we aim to improve vector identification methods by developing species-specific sandfly DNA probes. These probes will be able to distinguish between sympatric sandfly populations thereby providing epidemiological data for determining the significance of individual sandfly groups related to their vectorial capacity.

The axenic cultivation of Leishmania donovani amastigotes.

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

[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.

New developments in the chemotherapy of leishmaniasis.

Several significant advances in the chemotherapy of leishmaniasis have occurred in the last 10 years. Some of these advances have arisen due to the greater awareness of the pharmacokinetic properties of drugs, such as the pentavalent antimonials, while others have resulted from the introduction of drugs new to the treatment of leishmaniasis, such as aminosidine which can be used both systemically and topically against cutaneous leishmaniasis. The most encouraging advance is the use of lipid-associated amphotericin B; very short treatments with these preparations have been shown to be effective. Other studies have shown the usefulness of combination therapy and the use of immune modulators. A number of biochemical pathways in Leishmania, such as those associated with purine and sterol metabolism, are known to be distinct from those of the mammalian hosts. These have been exploited in the search for the rational choice of anti-leishmanial agents.

Studies on the Phlebotomus fauna of Mount Elgon, Kenya.

In a study of sandflies in 229 of 237 caves found on the slopes of Mount Elgon, Kenya, the Phlebotomus (Larroussius) species P. pedifer and P. elgonensis were found distributed at different altitudes. The females of these two species, which were the only ones of the genus Phlebotomus encountered in the study, were morphologically indistinguishable using existing techniques at the time. On the basis of the morphological identification of the males, however, P. pedifer occurred mainly at the lower altitudes (1750-1900 m) while P. elgonensis predominated at higher altitudes (2300-2600 m). The man-biting activities and the presence of promastigotes in the guts of female sandflies were restricted to lower altitudes (below 1900 m), i.e. the area of maximum distribution of P. pedifer and of cases of cutaneous leishmaniasis. This supports the suggestion that P. pedifer is a vector of cutaneous leishmaniasis on Mount Elgon.

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.