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1.
AAPS PharmSciTech ; 21(7): 275, 2020 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033847

RESUMO

In the published manuscript, co-author Sarah Hendrickx name was misspelled and co-author Guy Caljon's last and first names were inadvertently switched.

3.
Trends Parasitol ; 36(9): 785-795, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32713762

RESUMO

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes.


Assuntos
Resistência a Medicamentos , Insetos Vetores/parasitologia , Leishmaniose/transmissão , Psychodidae/parasitologia , Animais , Antiparasitários/farmacologia , Humanos , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Estágios do Ciclo de Vida/fisiologia
4.
Parasit Vectors ; 13(1): 276, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487217

RESUMO

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

5.
Front Immunol ; 11: 1113, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32582193

RESUMO

Type I interferons (IFNs) induced by an endogenous Leishmania RNA virus or exogenous viral infections have been shown to exacerbate infections with New World Cutaneous Leishmania parasites, however, the impact of type I IFNs in visceral Leishmania infections and implicated mechanisms remain to be unraveled. This study assessed the impact of type I IFN on macrophage infection with L. infantum and L. donovani and the implication of sialoadhesin (Siglec-1/CD169, Sn) as an IFN-inducible surface receptor. Stimulation of bone marrow-derived macrophages with type I IFN (IFN-α) significantly enhanced susceptibility to infection of reference laboratory strains and a set of recent clinical isolates. IFN-α particularly enhanced promastigote uptake. Enhanced macrophage susceptibility was linked to upregulated Sn surface expression as a major contributing factor to the infection exacerbating effect of IFN-α. Stimulation experiments in Sn-deficient macrophages, macrophage pretreatment with a monoclonal anti-Sn antibody or a novel bivalent anti-Sn nanobody and blocking of parasites with soluble Sn restored normal susceptibility levels. Infection of Sn-deficient mice with bioluminescent L. infantum promastigotes revealed a moderate, strain-dependent role for Sn during visceral infection under the used experimental conditions. These data indicate that IFN-responsive Sn expression can enhance the susceptibility of macrophages to infection with visceral Leishmania promastigotes and that targeting of Sn may have some protective effects in early infection.

6.
J Microbiol Methods ; 173: 105935, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376283

RESUMO

BACKGROUND: Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. METHODOLOGY: The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. PRINCIPAL FINDINGS: Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. CONCLUSIONS/SIGNIFICANCE: The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

7.
Artigo em Inglês | MEDLINE | ID: mdl-32388220

RESUMO

OBJECTIVES: This study evaluated the implications of clinically acquired miltefosine resistance (MIL-R) by assessing virulence in mice and sand flies to reveal the potential of MIL-R strains to circulate. METHODS: Experimental infections with the MIL-R clinical Leishmania infantum isolate MHOM/FR/2005/LEM5159, having a defect in the LiROS3 subunit of the MIL-transporter, and its syngeneic experimentally reconstituted MIL-S counterpart (LEM5159LiROS3) were performed in BALB/c mice and Lutzomyia longipalpis and Phlebotomus perniciosus sand flies. In mice, the amastigote burdens in liver and spleen were compared microscopically using Giemsa smears and by bioluminescent imaging. During the sand fly infections, the percentage of infected flies, parasite load, colonization of the stomodeal valve and metacyclogenesis were evaluated. The stability of the MIL-R phenotype after sand fly and mouse passage was determined as well. RESULTS: The fitness of the MIL-R strain differed between the mouse and sand fly infection model. In mice, a clear fitness loss was observed compared to the LiROS3-reconstituted susceptible strain. This defect could be rescued by episomal reconstitution with a wildtype LiROS3 copy. However, this fitness loss was not apparent in the sand fly vector, resulting in metacyclogenesis and efficient colonization of the stomodeal valve. Resistance was stable after passage in both sand fly and mouse. CONCLUSION: The natural MIL-R strain is significantly hampered in its ability to multiply and cause a typical visceral infection pattern in BALB/c mice. However, this LiROS3-deficient strain efficiently produced mature infections and metacyclic promastigotes in the sand fly vector highlighting the transmission potential of this particular MIL-R clinical Leishmania strain.

8.
Methods Mol Biol ; 2116: 791-800, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32221956

RESUMO

In vitro growth (inhibition) assays have a dual application, either supporting the discovery of novel drugs or as a monitoring tool of drug resistance in patient isolates. From an experimental design point of view, both are quite different with regard to the infecting Leishmania species and strain, the wide variety of permissive host cells (primary cells versus cell lines), drug exposure times, detection methods and endpoint criteria. Recognizing the need for enhanced assay standardization to decrease interlaboratory variation and improve proper interpretation of results, a detailed description is given of the basic fundamental procedures and requirements for routine in vitro growth of Leishmania spp. with specific focus on the intracellular amastigote susceptibility assay. Although the described experimental procedures focus on visceral Leishmania species, the same assay principles may apply for the cutaneous species as well.

9.
Parasit Vectors ; 13(1): 96, 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32087758

RESUMO

BACKGROUND: Since the introduction of miltefosine (MIL) as first-line therapy in the kala-azar elimination programme in the Indian subcontinent, treatment failure rates have been increasing. Since parasite infectivity and virulence may become altered upon treatment relapse, this laboratory study assessed the phenotypic effects of repeated in vitro and in vivo MIL exposure. METHODS: Syngeneic Leishmania donovani lines either or not exposed to MIL were compared for drug susceptibility, rate of promastigote multiplication and metacyclogenesis, macrophage infectivity and behaviour in the sand fly vector, Lutzomyia longipalpis. RESULTS: Promastigotes of both in vitro and in vivo MIL-selected strains displayed a slightly reduced drug susceptibility that was associated with a reduced MIL-accumulation linked to a lower copy number (disomic state) of chromosome 13 harboring the miltefosine transporter (LdMT) gene. In vitro selected promastigotes showed a lower rate of metacyclogenesis whereas the in vivo derived promastigotes displayed a moderately increased growth rate. Repeated MIL exposure did neither influence the parasite load nor metacyclogenesis in the sand fly vector. CONCLUSIONS: Recurrent in vitro and in vivo MIL exposure evokes a number of very subtle phenotypic and genotypic changes which could make promastigotes less susceptible to MIL without attaining full resistance. These changes did not significantly impact on infection in the sand fly vector.


Assuntos
Antiprotozoários/farmacologia , Insetos Vetores/parasitologia , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Fosforilcolina/análogos & derivados , Psychodidae/parasitologia , Aclimatação , Animais , Resistência a Medicamentos , Humanos , Leishmania donovani/patogenicidade , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/transmissão , Testes de Sensibilidade Parasitária , Fenótipo , Fosforilcolina/farmacologia , Virulência
10.
PLoS Negl Trop Dis ; 13(12): e0007885, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31790397

RESUMO

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Testes de Sensibilidade Parasitária/métodos , Animais , Células Cultivadas , Feminino , Humanos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/parasitologia , Camundongos , Testes de Sensibilidade Parasitária/normas
11.
Artigo em Inglês | MEDLINE | ID: mdl-31525614

RESUMO

OBJECTIVES: To gain insight into the propagation of miltefosine (MIL) resistance in visceral leishmaniasis, this laboratory study explored development of resistant parasites with a defective miltefosine transporter (MT) in sand flies. METHODS: Infectivity, colonization of stomodeal valve and metacyclogenesis of a MIL-resistant (MIL-R) Leishmania infantum LEM3323 line with a defective MT were assessed in the natural sand fly vectors Phlebotomus perniciosus and Lutzomyia longipalpis. Given our recent description of partial drug dependency of the MT-deficient line, the impact of MIL pre-exposure on sand fly infectivity was explored as well. RESULTS: A significant reduction in sand fly infection, stomodeal valve colonization and differentiation into metacyclics (determined by a lower flagellum/cell body length ratio) was observed in both vectors for MIL-R as compared to the isogenic parent MIL-susceptible line. Re-introduction of the wildtype MT gene into MIL-R tended to partially rescue the capacity to infect sand flies. Pre-exposure to MIL did not alter infectivity of the MIL-R line. CONCLUSION: The MIL resistant L. infantum LEM3323 line is significantly hampered in its development and transmissibility potential in two sand fly vector species. Additional studies are warranted to evaluate whether this applies to other visceral Leishmania parasites with acquired MIL-resistance.


Assuntos
Antiprotozoários/farmacologia , Insetos Vetores/parasitologia , Leishmania infantum/crescimento & desenvolvimento , Phlebotomus/parasitologia , Fosforilcolina/análogos & derivados , Psychodidae/parasitologia , Análise de Variância , Animais , Resistência a Medicamentos , Feminino , Concentração Inibidora 50 , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/transmissão , Camundongos , Camundongos Endogâmicos BALB C , Fosforilcolina/farmacologia , Coelhos
12.
Parasitol Res ; 118(10): 2743-2752, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31473855

RESUMO

Leishmaniasis is a neglected parasitic disease for which the current antileishmania therapeutics are hampered by drug toxicity, high cost, need for parenteral administration, increasing treatment failure rates, and emergence of drug resistance. The R&D pipeline had run fairly dry for several years, but fortunately some new drug candidates are now under (pre)clinical development. Identification of novel drugs will nevertheless remain essential to adequately sustain and improve effective disease control in the future. In this review, a package of standard and accessible R&D approaches is discussed with expansion to some alternative strategies focusing on parasite-host and vector-host interactions.


Assuntos
Antiprotozoários/farmacologia , Descoberta de Drogas , Leishmania/efeitos dos fármacos , Animais , Resistência a Medicamentos , Humanos , Leishmania/crescimento & desenvolvimento , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia
13.
J Antimicrob Chemother ; 74(2): 395-406, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30412253

RESUMO

Objectives: Miltefosine is currently the only oral drug for visceral leishmaniasis, and although deficiency in an aminophospholipid/miltefosine transporter (MT) is sufficient to elicit drug resistance, very few naturally miltefosine-resistant (MIL-R) strains have yet been isolated. This study aimed to make a detailed analysis of the impact of acquired miltefosine resistance and miltefosine treatment on in vivo infection. Methods: Bioluminescent versions of a MIL-R strain and its syngeneic parental line were generated by integration of the red-shifted firefly luciferase PpyRE9. The fitness of both lines was compared in vitro (growth rate, metacyclogenesis and macrophage infectivity) and in BALB/c mice through non-invasive bioluminescence imaging under conditions with and without drug pressure. Results: This study demonstrated a severe fitness loss of MT-deficient parasites, resulting in a complete inability to multiply and cause a typical visceral leishmaniasis infection pattern in BALB/c mice. The observed fitness loss could not be rescued by host immune suppression with cyclophosphamide, whereas episomal reconstitution with a wild-type MT restored parasite virulence, hence linking parasite fitness to MT mutation. Remarkably, in vivo miltefosine treatment or in vitro miltefosine pre-exposure significantly rescued MIL-R parasite virulence. The in vitro pre-exposed MIL-R promastigotes showed a longer and more slender morphology, suggesting an altered membrane composition. Conclusions: The profound fitness loss of MT-deficient parasites most likely explains the low frequency of MIL-R clinical isolates. The observation that miltefosine can reverse this phenotype indicates a drug dependency of the MT-deficient parasites and emphasizes the importance of resistance profiling prior to miltefosine administration.


Assuntos
Aptidão Genética/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/genética , Macrófagos/parasitologia , Proteínas de Membrana Transportadoras/genética , Fosforilcolina/análogos & derivados , Animais , Feminino , Imunossupressão , Leishmania infantum/crescimento & desenvolvimento , Leishmaniose Visceral/parasitologia , Luciferases/metabolismo , Medições Luminescentes , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Testes de Sensibilidade Parasitária , Fosforilcolina/farmacologia , Virulência/efeitos dos fármacos
14.
Acta Clin Belg ; 74(5): 317-325, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30105946

RESUMO

Background: The importance of early cardiopulmonary resuscitation (CPR) during an out-of-hospital cardiac arrest (OHCA) cannot be emphasized enough and has a major impact on survival. Unfortunately, CPR education in schools is often inadequate or non-existing due to lack of educators and financial resources. The introduction of application-based teaching could facilitate education as no instructor is needed. The aim of present study is to compare app-based self-teaching (intervention group) with a traditional instructor-led course (control group). This trial is in line with the endorsement of training school children by the World Health Organization (WHO). Methods: This randomized controlled trial contains 165 participants between 16 and 18 years. The control group followed an instructor-led course, while the intervention group used a tablet application to complete a self-education course.Both groups were taught in separated classes during 40 min and used manikins and automated external defibrillator (AED) trainer kits. Before and after training, both groups were evaluated in a practical test to examine their skills (an overall score and sub-scores on different sections were obtained, following ERC 2015 guidelines). Results: No significant difference (p = 0.304) in overall teaching effectiveness was found between both groups. Although, in a sub-analysis, we found significantly better results for the instructor group for checking airway (p = 0.018), asking for an AED (p < 0.01) and shocking the patient (p = 0.002). Conclusions: App-based teaching can be a valuable alternative for classic instructor-based education. Yet new apps have to be evaluated before being implemented in CPR education.


Assuntos
Reanimação Cardiopulmonar/educação , Parada Cardíaca Extra-Hospitalar/terapia , Software , Adolescente , Instrução por Computador , Feminino , Humanos , Aprendizagem , Masculino , Manequins , Ensino
15.
Nat Prod Res ; 33(12): 1778-1782, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29424240

RESUMO

Here is reported the anti Leishmania infantum activity of 48 hexane, CH2Cl2 and MeOH extracts from 16 macroalgae collected on the Iberian Coast. Seven hexane and CH2Cl2 Cystoseira baccata, Cystoseira barbata, Cystoseira tamariscifolia, Cystoseira usneoides, Dictyota spiralis and Plocamium cartilagineum extracts were active towards promastigotes (IC50 29.8-101.8 µg/mL) inducing strong morphological alterations in the parasites. Hexane extracts of C. baccata and C. barbata were also active against intracellular amastigotes (IC50 5.1 and 6.8 µg/mL, respectively). Fatty acids, triacylglycerols, carotenoids, steroids and meroterpenoids were detected by nuclear magnetic resonance (NMR), and gas chromatography in the Cystoseira extracts. These results suggest that Cystoseira macroalgae contain compounds with antileishmanial activity, which could be explored as scaffolds to the development of novel sources of antiparasitic derivatives.


Assuntos
Antiprotozoários/farmacologia , Leishmania infantum/efeitos dos fármacos , Feófitas/química , Alga Marinha/química , Antiprotozoários/química , Carotenoides/análise , Cromatografia Gasosa , Avaliação Pré-Clínica de Medicamentos/métodos , Ácidos Graxos/análise , Ácidos Graxos/química , Espectroscopia de Ressonância Magnética , Esteroides/análise
16.
Parasitol Res ; 117(11): 3601-3612, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30141075

RESUMO

Primary mouse macrophages are frequently used to provide an in vitro intracellular model to evaluate antileishmanial drug efficacy. The present study compared the phenotypic characteristics of Swiss, BALB/c, and C57BL/6 mouse bone marrow-derived macrophages and peritoneal exudate cells using different stimulation and adherence protocols upon infection with a Leishmania infantum laboratory strain and two clinical isolates. Evaluation parameters were susceptibility to infection, permissiveness to amastigote multiplication, and impact on drug efficacy. Observed variations in infection of peritoneal exudate cells can mostly be linked to changes in the inflammatory cytokine profiles (IL-6, TNF-α, KC/GRO) rather than to differences in initial production of nitric oxide and reactive oxygen species. Optimization of the cell stimulation and adherence conditions resulted in comparable infection indices among peritoneal exudate cells and the various types of bone marrow-derived macrophages. BALB/c-derived bone marrow-derived macrophages were slightly more permissive to intracellular amastigote replication. Evaluation of antileishmanial drug potency in the various cell systems revealed minimal variation for antimonials and paromomycin, and no differences for miltefosine and amphotericin B. The study results allow to conclude that drug evaluation can be performed in all tested primary macrophages as only marginal differences are observed in terms of susceptibility to infection and impact of drug exposure. Combined with some practical considerations, the use of 24-h starch-stimulated, 48-h adhered, Swiss-derived peritoneal exudate cells can be advocated as an efficient, reliable, relatively quick, and cost-effective tool for routine drug susceptibility testing in vitro whenever the use of primary cells is feasible.


Assuntos
Antiprotozoários/uso terapêutico , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmaniose/tratamento farmacológico , Leishmaniose/imunologia , Macrófagos/imunologia , Anfotericina B/uso terapêutico , Animais , Antimônio/uso terapêutico , Células Cultivadas , Resistência a Medicamentos/genética , Feminino , Interleucina-6/sangue , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Testes de Sensibilidade Parasitária , Paromomicina/uso terapêutico , Fosforilcolina/análogos & derivados , Fosforilcolina/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/sangue
17.
Artigo em Inglês | MEDLINE | ID: mdl-30104270

RESUMO

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.


Assuntos
Leishmaniose/tratamento farmacológico , Leishmaniose/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Antiprotozoários/uso terapêutico , AMP Cíclico/metabolismo , Leishmania major/efeitos dos fármacos , Leishmania major/enzimologia , Leishmania major/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Inibidores de Fosfodiesterase/uso terapêutico
18.
J Mol Diagn ; 20(2): 253-263, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29355825

RESUMO

Several methods have been developed for the detection of Leishmania, mostly targeting the minicircle kinetoplast DNA (kDNA). A new RNA real-time quantitative PCR (qPCR) assay was developed targeting the conserved and highly expressed spliced-leader (SL) mini-exon sequence. This study compared the limits of detection of various real-time PCR assays in hamsters infected with Leishmania infantum, in spiked human blood, and in clinical blood samples from visceral leishmaniasis patients. The SL-RNA assay showed an excellent analytical sensitivity in tissues (0.005 and 0.002 parasites per mg liver and spleen, respectively) and was not prone to false-positive reactions. Evaluation of the SL-RNA assay on clinical samples demonstrated lower threshold cycle values than the kDNA qPCR, an excellent interrun stability of 97%, a 93% agreement with the kDNA assay, and an estimated sensitivity, specificity, and accuracy of 93.2%, 94.3%, and 93.8%, respectively. The SL-RNA qPCR assay was equally efficient for detecting Leishmania major, Leishmania tropica, Leishmania mexicana, Leishmania guayensis, Leishmania panamensis, Leishmania braziliensis, L. infantum, and Leishmania donovani and revealed similar SL-RNA levels in the different species and the occurrence of polycistronic SL-containing transcripts in Viannia species. Collectively, this single SL-RNA qPCR assay enables universal Leishmania detection and represents a particularly useful addition to the widely used kDNA assay in clinical studies in which the detection of viable parasites is pivotal to assess parasitological cure.


Assuntos
DNA de Cinetoplasto/análise , Leishmania infantum/genética , Leishmaniose Visceral/sangue , Leishmaniose Visceral/diagnóstico , Mesocricetus/parasitologia , RNA Líder para Processamento/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Animais , Criança , Pré-Escolar , Cricetinae , Confiabilidade dos Dados , Feminino , Humanos , Fígado/parasitologia , Sensibilidade e Especificidade , Baço/parasitologia
19.
J Antimicrob Chemother ; 73(2): 392-394, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29165590

RESUMO

Objectives: Increasing numbers of miltefosine treatment failures in visceral leishmaniasis therapy and reports of miltefosine resistance in the Indian subcontinent resulted in the recommendation to use liposomal amphotericin B as first-line therapy. Cross-resistance between miltefosine and amphotericin B has recently been documented, suggesting a role of mutations in the miltefosine transporter, a complex encoded by the MT and ROS3 genes. This study aimed to further explore the putative role of MT/ROS3 defects in the molecular basis of amphotericin B cross-resistance. Methods: The susceptibility profiles of different miltefosine-resistant Leishmania infantum strains with well-characterized mutations in the transporter complex and the corresponding episomally restored susceptible parasite lines were determined using both the routine extracellular promastigote assay and the intracellular amastigote assay. Results: In vitro amastigote and promastigote susceptibility testing of the two miltefosine-resistant and the episomally reconstituted L. infantum lines revealed full susceptibility to amphotericin B, despite the variable miltefosine susceptibility profile. Conclusions: Mutations present in either the MT and/or ROS3 gene are not sufficient to elicit higher tolerance to amphotericin B. Additional synergistic adaptations may be responsible for the miltefosine/amphotericin B cross-resistance described earlier.


Assuntos
Anfotericina B/farmacologia , Antiprotozoários/farmacologia , Resistência a Medicamentos , Leishmania infantum/efeitos dos fármacos , Proteínas de Membrana Transportadoras/deficiência , Fosforilcolina/análogos & derivados , Proteínas de Protozoários/genética , Humanos , Leishmaniose Visceral/parasitologia , Proteínas Mutantes/genética , Testes de Sensibilidade Parasitária , Fosforilcolina/farmacologia
20.
PLoS Negl Trop Dis ; 11(5): e0005620, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28505185

RESUMO

BACKGROUND: Since miltefosine monotherapy against visceral leishmaniasis (VL) caused by Leishmania donovani has been discontinued in the Indian subcontinent due to an increase in the number of treatment failures, single dose liposomal amphotericin B is now advocated as a treatment option of choice. Paromomycin-miltefosine combination therapy can be used as substitute first-line treatment in regions without cold-chain potential. Previous laboratory studies in the closely related species Leishmania infantum have demonstrated that paromomycin monotherapy fairly rapidly selects for resistance producing a phenotype with increased fitness. Given the possible clinical implications of these findings for the current field situation, the present study aimed to identify the potential hazards of paromomycin-miltefosine combination therapy. PRINCIPAL FINDINGS: Drug interaction studies using the fixed-ratio isobologram method revealed an indifferent interaction between paromomycin and miltefosine. In hamsters infected with L. infantum, the combination resulted in cumulative efficacy in reducing parasite burdens in the liver, spleen and bone marrow. Selected resistant lines against the single drugs did not display cross-resistance. When the intracellular amastigote stage was repeatedly exposed to the paromomycin-miltefosine combination, either in vitro or in vivo, no significant susceptibility decrease towards either drug was noted. CONCLUSIONS: These results suggest that implementation of paromomycin-miltefosine combination therapy indeed could represent a safe and affordable treatment option for L. donovani VL as miltefosine appears to overrule the anticipated rapid development of PMM resistance.


Assuntos
Antiprotozoários/administração & dosagem , Leishmania infantum/efeitos dos fármacos , Leishmaniose Visceral/tratamento farmacológico , Paromomicina/administração & dosagem , Fosforilcolina/análogos & derivados , Animais , Cricetinae , Resistência a Múltiplos Medicamentos , Quimioterapia Combinada , Feminino , Camundongos , Fosforilcolina/administração & dosagem
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