80 research outputs found
Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014
Leishmaniasis is a vector-borne disease which is endemic in 98 countries
worldwide [1]. It is caused by protozoan parasites of the genus Leishmania,
which are transmitted by female sand flies of the genera Lutzomyia and
Phlebotomus. Many infected individuals never develop symptoms, but those who
do can exhibit various disease manifestations [2]. Visceral leishmaniasis (VL)
or kala-azar is the severe form, whereby parasites infect internal organs and
the bone marrow, a lethal condition if left untreated. Other disease types are
restricted to the skin (cutaneous leishmaniasis, CL) or the mucosae of the
nose and mouth (mucosal leishmaniasis, ML). Finally, a particular cutaneous
disease sometimes develops in cured VL patients: post kala-azar dermal
leishmaniasis (PKDL). Typically, VL is caused by two species: Leishmania
donovani and Leishmania infantum. The latter can also cause CL, as can all
other pathogenic species. Some particular species (e.g. L. braziliensis and L.
aethiopica) can lead to overt ML. As many as 20 different Leishmania species
are able to infect humans, and globally there are over 1 million new disease
cases per annum [1,3]. Leishmaniasis is endemic in southern Europe, and in
other European countries cases are diagnosed in travellers who have visited
affected areas both within the continent and beyond. Although treatment in
practice is often guided only by clinical presentation and patient history, in
some cases determination of the aetiological subgenus, species complex or
species is recommended for providing optimal treatment [2,4,5]. For example, a
patient returning from South America with CL might be infected with Leishmania
braziliensis, which necessitates systemic drug therapy and counselling about
the risk of developing mucosal leishmaniasis in the future. The same patient
could also be infected with Leishmania mexicana, which is managed by less
intensive treatment and which is not associated with mucosal disease [6].
Determining the infecting species and its probable source permits selection of
the correct drug, route of administration (intralesional, oral systemic, or
parenteral) and duration [7]. Unfortunately, for CL it is impossible to
predict the species responsible for an ulcerating lesion clinically, and the
morphology of amastigotes does not differ between species. When the
geographical origin of infection is known, for instance when a patient in an
endemic region is treated at a local hospital, the species can be guessed
often from the known local epidemiology, as species distribution follows a
geographical pattern [8]. However, especially in infectious disease clinics
that treat patients who have stayed in various endemic countries, the
geographic origin of infections may be unknown. For instance, people residing
in Europe who have travelled outside Europe may come from, or have also
visited, Leishmania-endemic areas within Europe, especially the Mediterranean
basin. Even when the location of infection is known, several species can co-
circulate in a given endemic area, in which case the species can only be
determined by laboratory tests. Culture and subsequent isoenzyme analysis is
time consuming and available in very few specialised centres, so it is
impractical as a front-line diagnostic test in clinical laboratories. Hence,
well-performed reliable molecular methods are necessary for species
identification. Several Leishmania typing methods have been published
(reviewed in [9]), and as a result each laboratory uses its own preferred
assay. The most popular assays nowadays are those that can be applied directly
to clinical samples, thereby circumventing the need for parasite isolation and
culture. However, few tests have been standardised, and no commercial kits are
currently available. As a result, clinical and epidemiological studies make
use of various techniques, and in patient management other methods are often
deployed. In this study we compare the typing performance in 16 clinical
laboratories across Europe, which use a variety of methods for species
discrimination
Ongoing outbreak of visceral leishmaniasis in Bologna Province, Italy, November 2012 to May 2013
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Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples
Background: Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods: The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results: The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3%, 58.6%, 40.5% and 10.8% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97% for both methods. Conclusion: This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China
A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition
Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin–trypanothione reductase–NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis
ASPETTI NORMATIVI ED ANALITICI IN RELAZIONE AI LIMITI MASSIMI DEI RESIDUI DI PESTICIDI NEGLI ALIMENTI
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