RESUMEN
The nitro-heterocyclic compound benznidazole (BZ) is the first-line drug for the treatment of Chagas disease, caused by the protozoan Trypanosoma cruzi. However, therapeutic failures are common for reasons that include the influences of parasite and host genetics, the effects of toxicity on adherence to treatment, and difficulties in demonstrating parasitological cure. To obtain information on the origin of the resistance to BZ and eliminate from the scenery the participation of the host, initially we mapped the susceptibility to the drug in thirteen species of seven genera of the family Trypanosomatidae. We verified that all Trypanosoma species are sensitive to low concentrations of the drug (IC50 2.7 to 25⯵M) while Non-Trypanosoma species are highly resistant to these concentrations. The two groups of parasites correspond to the major phylogenetic lineages of trypanosomatids. Next, we searched in the trypanosomatid genome databases homologs of two type-I nitroreductases (NTR-1 and OYE) and an ABC transporter (ABCG1) that have been associated with BZ resistance in T. cruzi. The predicted proteins were characterized regarding domains and used for phylogenetic analyses. Homologous NTR-1 genes were found in all trypanosomatids investigated and the structural characteristics of the enzyme suggest that it may be functional. OYE genes were absent in BZ-sensitive African trypanosomes, which excludes the participation of this enzyme in BZ bio-activation. Two copies of ABCG1 genes were observed in most BZ resistant species, while Trypanosoma species exhibit only one copy per haploid genome. Functional studies are required to verify the involvement of these genes in BZ resistance. In addition, since multiple mechanisms can contribute to BZ susceptibility, our study poses a range of organisms highly resistant to BZ in which these aspects can be investigated. Preliminary studies on BZ uptake indicate marked differences between BZ-sensitive and BZ-resistant species.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Resistencia a Medicamentos/genética , Nitroimidazoles/uso terapéutico , Filogenia , Tripanocidas/uso terapéutico , Trypanosoma/efectos de los fármacos , Trypanosoma/genética , Transportador de Casetes de Unión a ATP, Subfamilia G/genética , Transportadoras de Casetes de Unión a ATP/genética , Secuencia de Aminoácidos/genética , Animales , Geografía , Humanos , Proteínas de Transporte de Membrana/genética , Nitroimidazoles/toxicidad , Nitrorreductasas/genética , Tripanocidas/toxicidadRESUMEN
Treatment failure and parasite drug susceptibility in dermal leishmaniasis caused by Leishmania (Viannia) species are poorly understood. Prospective evaluation of drug susceptibility of strains isolated from individual patients before drug exposure and at clinical failure allows intrinsic and acquired differences in susceptibility to be discerned and analyzed. To determine whether intrinsic susceptibility or loss of susceptibility to miltefosine contributed to treatment failure, we evaluated the miltefosine susceptibility of intracellular amastigotes and promastigotes of six Leishmania (Viannia) braziliensis and six Leishmania (Viannia) panamensis strains isolated sequentially, at diagnosis and treatment failure, from two children and four adults ≥55 years old with concurrent conditions. Four patients presented only cutaneous lesions, one had mucosal disease, and one had disseminated mucocutaneous disease. Expression of the Leishmania drug transporter genes abca2, abca3, abcc2, abcc3, abcg4, abcg6, and LbMT was evaluated by quantitative reverse transcription-PCR (qRT-PCR). Intracellular amastigotes (median 50% effective concentration [EC50], 10.7 µmol/liter) were more susceptible to miltefosine than promastigotes (median EC50, 55.3 µmol/liter) (P < 0.0001). Loss of susceptibility at failure, demonstrated by a miltefosine EC50 of >32 µmol/liter (the upper limit of intracellular amastigote assay), occurred in L. panamensis infection in a child and in L. braziliensis infection in an adult and was accompanied by decreased expression of the miltefosine transporter LbMT (LbMT/ß-tubulin, 0.42- to 0.26-fold [P = 0.039] and 0.70- to 0.57-fold [P = 0.009], respectively). LbMT gene polymorphisms were not associated with susceptibility phenotype. Leishmania ABCA3 transporter expression was inversely correlated with miltefosine susceptibility (r = -0.605; P = 0.037). Loss of susceptibility is one of multiple factors involved in failure of miltefosine treatment in dermal leishmaniasis.