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.
Highlights
Visceral leishmaniasis (VL) is a neglected and poverty-related disease that causes significant morbidity and mortality
Leishmaniasis is caused by protozoan parasites of the genus Leishmania and is transmitted by inoculation of infective promastigotes by the female sand fly
Since ‘resistant’ patient isolates are not yet available, we artificially selected for PMM resistance using two different in vitro protocols with drug pressure on either the extracellular promastigote or on the intracellular amastigote stage
Summary
Visceral leishmaniasis (VL) is a neglected and poverty-related disease that causes significant morbidity and mortality. Treatment options are quite limited and the development of resistance to antimonials (Sb) has added to the problem [1]. To counter this evolution, the Kala-Azar Elimination programme was officially launched in India and Nepal in 2005 [2,3] and advocates the use of miltefosine (MIL) as first-line alternative to Sb. other drugs are still required for treating treatment failures. It was shown that PMM is not hampered by Sb-resistance [7], but appropriate measures should certainly be taken to assure its long-term effectiveness. Resistance in the field has not been reported yet, but this issue needs to be proactively addressed in laboratory studies to help steer decisions on future treatment policies, diagnosis and epidemiological resistance monitoring
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