Experimental Induction of Paromomycin Resistance in Antimony-Resistant Strains of L. donovani: Outcome Dependent on In Vitro Selection Protocol

Abstract

Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 µM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 µM inclusion level that did not affect normal growth. Comparison of the IC50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC50 = 199 µM) compared to the parent strain (IC50 = 45 µM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field.

In Vivo Selection of Paromomycin and Miltefosine Resistance in Leishmania donovani and L. infantum in a Syrian Hamster Model

“…Since the first MIL-resistant isolate was reported in 2012, a second naturally resistant strain was found by our research group (3, 4). As for PMM resistance, this was already reported to develop fairly readily (4,7,9,20). Moreover, numerous in vitro studies demonstrated quick selection of MIL and PMM resistance on promastigotes (6)(7)(8).…”

“…Moreover, numerous in vitro studies demonstrated quick selection of MIL and PMM resistance on promastigotes (6)(7)(8). To target the more relevant amastigote stage in mammalian infection, our research group previously developed an in vitro resistance selection protocol on intracellular amastigotes (9). For PMM, several strains with PMM-resistant amastigotes but still fully PMMsusceptible promastigotes were obtained.…”

“…Conversely, laboratory studies already demonstrated that MIL and PMM resistance can be selected in vitro using axenic promastigotes (6)(7)(8). Considering the debatable relevance of promastigote-based studies, our group developed an in vitro resistance selection protocol on intracellular amastigotes, revealing a process-dependent outcome (4,9). Rapid generation of PMM-resistant amastigotes for several Leishmania donovani and Leishmania infantum strains was obtained, while the derived promastigotes remained fully PMM susceptible.…”

Elucidation of Cellular Mechanisms Involved in Experimental Paromomycin Resistance in Leishmania donovani

“…There are limited studies on PMM resistance in Leishmania, which evince the involvement of mitochondrial membrane potential and decreases in drug uptake (14)(15)(16). In our previous report, we compared the outcomes and stability of experimental PMM resistance induction in promastigotes and intracellular amastigotes (17). Here, we exploited laboratory-generated PMM-resistant (PMM-R) parasites to examine changes in membrane fluidity, drug accumulation, mutations in rRNA genes, and mRNA expression of ATP-binding cassette (ABC) transporter genes (MDR1 and MRPA) and protein phosphatase 2A (PP2A).…”

Lesleishmaniosesvues au travers du réseau international des Instituts Pasteur

“…Elle est actuellement en phase III des essais cliniques réalisés en Afrique de l'Est. Cependant, des études de laboratoire ont montré que ces deux molécules pouvaient sélectionner rapidement des parasites résistants [24][25][26][27], ce qui repré-sente un risque lors de traitements, même de courtes durées. Pour la miltéfosine, sa disponibilité sur le marché des médicaments de comptoir et son utilisation incontrôlée peuvent grandement augmenter ce risque de développement de résistances.…”