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. 2011 Jun;55(6):2916-21.
doi: 10.1128/AAC.00812-10. Epub 2011 Apr 4.

Antimony-resistant clinical isolates of Leishmania donovani are susceptible to paromomycin and sitamaquine

Arpita Kulshrestha  1 , Ruchi Singh, Dhiraj Kumar, Narender Singh Negi, Poonam Salotra
Affiliations

Antimony-resistant clinical isolates of Leishmania donovani are susceptible to paromomycin and sitamaquine

Arpita Kulshrestha et al. Antimicrob Agents Chemother. 2011 Jun.

Abstract

Widespread antimonial resistance in anthroponotic visceral leishmaniasis (VL) makes it critical to monitor the susceptibility of prevailing field isolates to upcoming antileishmanials in order to frame the right treatment policies to protect these drugs against development of resistance. We aimed to generate the baseline data on natural in vitro susceptibility to paromomycin and sitamaquine in Leishmania donovani field isolates from VL patients (n = 20) coming from zones of varying sodium antimony gluconate (SAG) resistance. We further monitored nitric oxide (NO) release in infected macrophages treated with these drugs. Field isolates exhibited variable sensitivity to paromomycin and sitamaquine with respective mean 50% effective dose (ED₅₀) values ± standard error of the mean (SEM) of 3.9 ± 0.3 μM and 2.1 ± 0.2 μM at the intracellular amastigote stage and 29.8 ± 2.5 μM and 17.7 ± 1.0 μM at the promastigote stage. Susceptibilities at the two parasite stages did not correlate for either drug. Isolates from high SAG resistance zones exhibited significantly lower susceptibility to sitamaquine than those from low SAG resistance zones, while isolates from different zones showed similar susceptibilities to paromomycin. NO release was promoted in L. donovani-infected macrophages upon treatment with paromomycin/sitamaquine. NO inhibitors significantly compromised amastigote killing by sitamaquine, but not by paromomycin. In conclusion, SAG-resistant/sensitive VL isolates were susceptible to both paromomycin and sitamaquine. Paromomycin, exhibiting higher efficacy against SAG-resistant parasites and having a distinct mechanism of action, appears to be a promising drug for combination therapy.

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Figures

Fig. 1.
Fig. 1.
Map of Bihar and adjoining states showing origins of parasite isolates from areas of high and low endemicity. Clinical isolates of L. donovani were prepared from patients originating from the states of Bihar, West Bengal, and Uttar Pradesh, India, from zones of low and high endemicity for visceral leishmaniasis, which represent, respectively, zones of LR and HR to antimony. Modified from the district map of Bihar available at http://www.biharonline.gov.in.
Fig. 2.
Fig. 2.
Representative plots of susceptibilities of parasite isolates from high- and low-resistance zones to SAG, paromomycin, and sitamaquine. The ED50s of L. donovani isolates at the intracellular amastigote stage were determined by infection in the murine macrophage cell line J774A.1 as described in Materials and Methods. The ED50s of VL isolates from HR and LR zones to these drugs are represented by circles and squares, respectively. The horizontal bars represent the means of HR/LR, and the vertical bars represent the standard errors. Each individual value represents the mean ED50 of the results from three separate assays. *, P < 0.05; **, P < 0.001, as determined by the Mann Whitney U test.
Fig. 3.
Fig. 3.
Effects of paromomycin and sitamaquine on production of nitric oxide by macrophages and parasite survival. (A) Production of nitric oxide in parasite-infected macrophages upon treatment with paromomycin or sitamaquine. J774.A.1 murine macrophages that were uninfected (UI) or infected with L. donovani LdAG83 (1:10 infection ratio) (Inf) or infected macrophages treated with L-NMMA 1 h prior to drug exposure (inf+Inh) were incubated with medium alone (control) or medium containing increasing concentrations of paromomycin (P1, P3, P10, and P30, denoting 1, 3, 10, and 30 μM, respectively) or sitamaquine (S0.5, S1, S3, and S10, denoting 0.5, 1, 3, and 10 μM, respectively) for 48 h at 37°C. LPS (1 μg/ml) from E. coli was used as a positive control, while SAG was taken as the reference drug (SAG3 and SAG10, denoting 3 and 10 μg/ml, respectively). Nitrite concentrations were measured in supernatants by the Griess method. The graphs represent the means of triplicates from two independent experiments ± standard error of the mean (SEM). *, P < 0.05; **, P < 0.001, as determined by a t test. (B) Effects of nitric oxide inhibitor on parasite survival in L. donovani-infected macrophages treated with paromomycin/sitamaquine. The percent parasite survival in infected macrophages upon treatment with different drugs in the presence (L-NMM+) or absence (L-NMMA−) of inhibitor is shown. Other details are as in panel A.
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