Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol

Sarah Hendrickx, Raquel Andrea Inocêncio da Luz, V. Bhandari, K. Kuypers, Craig Shaw, Julien Lonchamp, P. Salotra, Katharine Carter, Katharine Carter, S. Sundar, S. Rijal, Jean-Claude Dujardin, Paul Cos, Louis Maes

Research output: Contribution to journalArticle

28 Citations (Scopus)

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.
LanguageEnglish
Article numbere1664
Number of pages7
JournalPLOS Neglected Tropical Diseases
Volume6
Issue number5
DOIs
Publication statusPublished - 29 May 2012

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Paromomycin
Antimony
Inhibitory Concentration 50
Phenotype
India
Drug Stability
Nepal
Visceral Leishmaniasis
Mesocricetus
Drug Resistance
Cricetinae
In Vitro Techniques
Clone Cells
Outcome Assessment (Health Care)
Pressure
Growth
Pharmaceutical Preparations

Keywords

  • paromomycin
  • antimony
  • L. donovani
  • in vitro selection protocol

Cite this

Hendrickx, Sarah ; Inocêncio da Luz, Raquel Andrea ; Bhandari, V. ; Kuypers, K. ; Shaw, Craig ; Lonchamp, Julien ; Salotra, P. ; Carter, Katharine ; Carter, Katharine ; Sundar, S. ; Rijal, S. ; Dujardin, Jean-Claude ; Cos, Paul ; Maes, Louis. / Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani : outcome dependent on in vitro selection protocol. In: PLOS Neglected Tropical Diseases. 2012 ; Vol. 6, No. 5.
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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.",
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Hendrickx, S, Inocêncio da Luz, RA, Bhandari, V, Kuypers, K, Shaw, C, Lonchamp, J, Salotra, P, Carter, K, Carter, K, Sundar, S, Rijal, S, Dujardin, J-C, Cos, P & Maes, L 2012, 'Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: outcome dependent on in vitro selection protocol' PLOS Neglected Tropical Diseases, vol. 6, no. 5, e1664. https://doi.org/10.1371/journal.pntd.0001664

Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani : outcome dependent on in vitro selection protocol. / Hendrickx, Sarah; Inocêncio da Luz, Raquel Andrea; Bhandari, V.; Kuypers, K.; Shaw, Craig; Lonchamp, Julien; Salotra, P.; Carter, Katharine; Carter, Katharine; Sundar, S.; Rijal, S.; Dujardin, Jean-Claude; Cos, Paul; Maes, Louis.

In: PLOS Neglected Tropical Diseases, Vol. 6, No. 5, e1664, 29.05.2012.

Research output: Contribution to journalArticle

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T1 - Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani

T2 - PLOS Neglected Tropical Diseases

AU - Hendrickx, Sarah

AU - Inocêncio da Luz, Raquel Andrea

AU - Bhandari, V.

AU - Kuypers, K.

AU - Shaw, Craig

AU - Lonchamp, Julien

AU - Salotra, P.

AU - Carter, Katharine

AU - Carter, Katharine

AU - Sundar, S.

AU - Rijal, S.

AU - Dujardin, Jean-Claude

AU - Cos, Paul

AU - Maes, Louis

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N2 - 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.

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KW - paromomycin

KW - antimony

KW - L. donovani

KW - in vitro selection protocol

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DO - 10.1371/journal.pntd.0001664

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