A Novel Bioimpedance-Based Detection of Miltefosine Susceptibility Among Clinical Leishmania donovani Isolates of the Indian Subcontinent Exhibiting Resistance to Multiple Drugs

Abstract

The extent of susceptibility towards miltefosine (Mil), amphotericin B (AmpB), and paromomycin (Paro) was measured among 19 clinical isolates of Leishmania donovani (LD). Thirteen of these clinical isolates were reported to exhibit low susceptibility towards sodium stibogluconate (SSG-R), while six of them were highly susceptible (SSG-S). The degree of clearance of amastigotes (EC50) for these predefined SSG-R- and SSG-S-infected macrophages was determined against Mil, AmpB, and Paro. Two out of the 13 SSG-R isolates (BHU575 and BHU814) showed low susceptibility towards all three drugs studied, while the rest of the 11 SSG-R isolates showed varying degrees of susceptibility either towards none or only towards individual drugs. Interestingly, all the SSG-S isolates showed high susceptibility towards Mil/AmpB/Paro. The total intracellular non-protein thiol content of the LD promastigotes, which have been previously reported to be positively co-related with EC50 towards SSG, was found to be independent from the degree of susceptibility towards Mil/AmpB/Paro. Impedance spectra analysis, which quantifies membrane resistance, revealed lower impedimetric values for all those isolates exhibiting low efficacy to Mil (Mil-R). Our analysis points out that while non-protein thiol content can be an attribute of SSG-R, lower impedimetric values can be linked with lower Mil susceptibility, although neither of these parameters seems to get influenced by the degree of susceptibility towards AmpB/Paro. Finally, a correlation analysis with established biological methods suggests that impedance spectral analysis can be used for the accurate determination of lower Mil susceptibility among LD isolates, which is further validated in the LD-infected in vivo hamster model.

Novel Pyrido[2′,1′:2,3]imidazo[4,5-c]quinoline Derivative Selectively Poisons Leishmania donovani Bisubunit Topoisomerase 1 to Inhibit the Antimony-Resistant Leishmania Infection in Vivo

“…Next, to propound C17 as a new drug candidate against drug-resistant leishmaniasis, we tested its efficacy toward the LdTop1 from the antimony-resistant field isolate of L. donovani , BHU575 (less susceptible to SSG, amphotericin B, and miltefosine), obtained from severely VL affected belt of India. , First, we confirmed the similar levels of endogenous LdTop1 activity from the two strains of L donovani AG83 (wild type) and BHU575 (antimony-resistant) in the plasmid DNA relaxation assays (Figure S3A). Figure A shows that indeed C17 inhibited the relaxation activity of LdTop1 from Leishmania BHU575 whole cell extracts in a dose-dependent manner (Figure A, lanes 4–8), with an IC 50 of 8.75 μM (Figure B) which was comparable with wild type parasites (Table ).…”

“…The structure−activity relationships (SAR) of the twenty compounds resulting from the parental structure of the PIQ core suggested that compound C17 is the most potent LdTop1 inhibitor both in the in vitro and ex vivo DNA relaxations assays (Table 2). C17 inhibits the growth of both the antimony-resistant field isolate of Leishmania (BHU575) which is also known to be less susceptible toward the clinically used antileishmanials like sodium stibo-gluconate (SSG), miltefosine, paromomycin, and amphotericin B, 34 as well as the wild-type drug-responsive strain (Ag83) by targeting LdTop1. C17 did not inhibit the activity of human monomeric Top1 which further exalts its promise as a selective antileishmanial drug candidate.…”

Transcriptome Analysis of Intracellular Amastigotes of Clinical Leishmania infantum Lines from Therapeutic Failure Patients after Infection of Human Macrophages

“…Knowledge of molecular changes associated with drug resistance and TF could help to increase the efficiency of new therapeutic strategies to be used in patients with leishmaniasis and TF. However, these studies have been undertaken principally in promastigote forms of Leishmania lines that are experimentally resistant to the common drugs, with very few studies including the use of clinical drug-resistant isolates and/or clinical isolates from patients with leishmaniasis and TF [ 19 , 20 , 21 ]. Molecular approaches to characterize the mechanisms of drug resistance or TF in these Leishmania lines include microarray studies [ 22 ], proteomic analysis [ 23 ], and, more recently, RNA-seq [ 24 , 25 , 26 , 27 ].…”