Miltefosine and antimonial drug susceptibility of Leishmania Viannia species and populations in regions of high transmission in Colombia

Abstract

Background: Pentavalent antimonials have been the first line treatment for dermal leishmaniasis in Colombia for over 30 years. Miltefosine is administered as second line treatment since 2005. The susceptibility of circulating populations of Leishmania to these drugs is unknown despite clinical evidence supporting the emergence of resistance.

Methodology/principal findings: In vitro susceptibility was determined for intracellular amastigotes of 245 clinical strains of the most prevalent Leishmania Viannia species in Colombia to miltefosine (HePC) and/or meglumine antimoniate (Sb(V)); 163, (80%) were evaluated for both drugs. Additionally, susceptibility to Sb(V) was examined in two cohorts of 85 L. V. panamensis strains isolated between 1980-1989 and 2000-2009 in the municipality of Tumaco. Susceptibility to each drug differed among strains of the same species and between species. Whereas 68% of L. V. braziliensis strains presented in vitro resistance to HePC, 69% were sensitive to Sb(V). Resistance to HePC and Sb(V) occurred respectively, in 20% y 21% of L. panamensis strains. Only 3% of L. V. guyanensis were resistant to HePC, and none to Sb(V). Drug susceptibility differed between geographic regions and time periods. Subpopulations having disparate susceptibility to Sb(V) were discerned among L. V. panamensis strains isolated during 1980-1990 in Tumaco where resistant strains belonged to zymodeme 2.3, and sensitive strains to zymodeme 2.2.

Conclusions/significance: Large scale evaluation of clinical strains of Leishmania Viannia species demonstrated species, population, geographic, and epidemiologic differences in susceptibility to meglumine antimoniate and miltefosine, and provided baseline information for monitoring susceptibility to these drugs. Sensitive and resistant clinical strains within each species, and zymodeme as a proxy marker of antimony susceptibility for L. V. panamensis, will be useful in deciphering factors involved in susceptibility and the distribution of sensitive and resistant populations.

Figure 1. Susceptibility of L. Viannia species in Colombia to meglumine antimoniate and miltefosine.

Reduction of parasite burden at the discriminatory concentration of anti-leishmanial drugs for clinical strains of L. V. panamensis, L. V. braziliensis and L. V. guyanensis. The cutoff thresholds (dotted horizontal line) of <41% reduction of parasite burden by Sb^V (A) and <50% reduction of parasite burden by HePC (B), and indeterminate zones (gray region) were defined based on previously described ROC curves as reduction of parasite burden between 35% and 48% for Sb^V and 44% and 56% for HePC (Fernandez O, et al 2012). ^a Strains presenting in vitro resistance. Kruskal-Wallis (K-W) for Sb^V: P = 0.0009 and K-W for HePC: P<0.0001. * Dunn's multiple comparison test P<0.05: Compared with L. V. braziliensis.

Figure 2. Susceptibility of Leishmania Viannia species from major natural geographic regions to meglumine antimoniate and miltefosine.

Map of major natural geographic regions of Colombia (A). Susceptibility expressed as reduction of parasite burden at the discriminatory concentration of Sb^V (B) and HePC (C) in clinical strains of L. V. panamensis, L. V. braziliensis and L. V. guyanensis. Distributions of % reduction of parasite burden represent the median and the interquartile range. Qualitative classification of susceptibility is defined by the cutoff thresholds (dotted horizontal line) and indeterminate ranges are illustrated by gray shading. K-W for Sb^V: P = 0.012 and K-W for HePC: P<0.0001. * Dunn's multiple comparison test P<0.05.

Figure 3. Susceptibility of L. V. panamensis and L. V. braziliensis to meglumine antimoniate and miltefosine by geographic region.

Reduction of parasite burden at the discriminatory concentration of Sb^V (A and C) and HePC (B and D) for L. V. panamensis and L. V. braziliensis strains from Pacific, Andean, Orinoquía and Amazon regions. Because of the lower frequency of L. V. panamensis in regions east of the Andes mountain range, L. V. panamensis strains are combined for Orinoquía and Amazon regions. Qualitative classification of susceptibility is defined by the cutoff thresholds (dotted horizontal line) and indeterminate ranges are illustrated by gray shading. K-W for L. V. panamensis and HePC, P = 0.0012 and Dunn procedure, * P<0.05: Compared with Orinoquía and Amazon regions.

Figure 4. Susceptibility of L. V. panamensis strains to meglumine antimoniate in endemic foci of Tumaco and association of drug susceptibility with zymodeme.

Distribution and median susceptibility of clinical strains from the Rosario River and Mira River foci during the decades 1980–1989 and 2000–2009. K-W, P<0.0001 and Dunn procedure (A). Comparison of susceptibility of strains pertaining to zymodeme 2.3 and zymodeme 2.2 isolated during 1980–1989 from the Rosario River and Mira River foci to Sb^V (B and C). Qualitative classification of susceptibility is defined by the cutoff thresholds (dotted horizontal line) and indeterminate ranges are illustrated by gray shading. Mann-Whitney, P<0.0001.

Figure 5. Analysis of genetic diversity of L. V. panamensis and L. V. guyanensis strains.

The relationship between genetic diversity, zymodeme, drug susceptibility and geographic distribution was examined based on an unrooted neighbor joining tree generated from the distances calculated for microsatellite data. Strains included L. V. panamensis isolated from patients in the Municipality of Tumaco, Nariño pertaining to zymodemes 2.3 (n = 10) and 2.2 (n = 10), L. V. panamensis strains from the Pacific coast region not typed by isoenzyme analysis, and L. V. guyanensis strains isolated from patients from Andean (n = 9) and Amazon regions (n = 8). Range of susceptibility to antimony at 32 µg Sb^V/ml is shown for the genetically discriminated groups of strains. ND* Zymodeme not determined.

Figure 6. Association of zymodeme, age and occupation in the Municipality of Tumaco.

Children <7 years of age were more frequently infected with strains of L. V. panamensis pertaining to zymodeme 2.3, while farmers were more often infected by strains belonging to zymodeme 2.2. n = 280 (A), Chi-square test, P<0.05. Distribution of L. V. panamensis strains belonging to zymodeme 2.2 (n = 181) and 2.3 (n = 149) by age of patients with cutaneous leishmaniasis from the Municipality of Tumaco, 1980–1989 (B and C).