In Vitro Drug Susceptibility of a Leishmania (Leishmania) infantum Isolate from a Visceral Leishmaniasis Pediatric Patient after Multiple Relapses

Abstract

The parasitic protozoan Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis in South America, an infectious disease associated with malnutrition, anemia, and hepatosplenomegaly. In Brazil alone, around 2700 cases are reported each year. Treatment failure can occur as a result of drug, host, and/or parasite-related factors. Here, we isolated a Leishmania species from a pediatric patient with visceral leishmaniasis that did not respond to chemotherapy, experiencing a total of nine therapeutic relapses and undergoing a splenectomy. The parasite was confirmed as L. (L.) infantum after sequencing of the ribosomal DNA internal transcribed spacer, and the clinical isolate, in both promastigote and amastigote forms, was submitted to in vitro susceptibility assays with all the drugs currently used in the chemotherapy of leishmaniasis. The isolate was susceptible to meglumine antimoniate, amphotericin B, pentamidine, miltefosine, and paromomycin, similarly to another strain of this species that had previously been characterized. These findings indicate that the multiples relapses observed in this pediatric patient were not due to a decrease in the drug susceptibility of this isolate; therefore, immunophysiological aspects of the patient should be further investigated to understand the basis of treatment failure in this case.

Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate

“…The susceptibility of antileishmanial drugs against promastigotes of the M2269 strain, the AAB clinical isolate, and an AmB-resistant clonal line was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoline bromide) colorimetric assay, as previously described [31]. Briefly, 2×10 6 log-phase parasites were incubated in presence of the following drugs serially diluted (1:2): AmB (800 to 3.12 nM), SbIII (1,000 to 1.56 μM), PEN (100 to 1.56 μM), MF (200 to 3.12 μM), or PM (1,000 to 6.25 μM) for 24 h. Three independent experiments were performed in triplicate and the 50% effective concentration (EC 50 ) was determined by sigmoidal regression curves generated using the GraphPad Prism 8 software.…”

“…The drug susceptibility assays for intracellular amastigotes were performed as previously described [31]. Briefly, bone marrow-derived macrophages (BMDMs) obtained from BALB/c mice were incubated in a 5% CO 2 atmosphere at 37˚C [32].…”

“…c EC 50 mean values ± standard deviation of three independent experiments d 50% cytotoxic concentration (CC 50 ) of each drug that reduces bone marrow-derived macrophage viability by 50%, as determined previously[31] (-) Data not determined. https://doi.org/10.1371/journal.pntd.0012175.t001…”