Leishmaniasis in the major endemic region of Plurinational State of Bolivia: Species identification, phylogeography and drug susceptibility implications

- Leishmaniasis is highly endemic in the Department of La Paz (Bolivia).
- L. (V.) braziliensis, L. (L) mexicana-amazonensis and L. (V.) lainsoni are the major circulating species.
- L. (V.) braziliensis is the most prevalent monophyletic clade.
- Intracellular amastigotes show high variation in susceptibility to conventional drugs.
- L. (L.) mexicana-amazonensis is more susceptibility to miltefosine than L. (V.) braziliensis.

The Plurinational State of Bolivia is one of the Latin American countries with the highest prevalence of leishmaniasis, highlighting the lowlands of the Department of La Paz where about 50% of the total cases were reported. The control of the disease can be seriously compromised by the intrinsic variability of the circulating species that may limit the efficacy of treatment while favoring the emergence of resistance. Fifty-five isolates of Leishmania from cutaneous and mucocutaneous lesions from patients living in different provinces of the Department of La Paz were tested. Molecular characterization of isolates was carried out by 3 classical markers: the rRNA internal transcribed spacer 1 (ITS-1), the heat shock protein 70 (HSP70) and the mitochondrial cytochrome b (Cyt-b). These markers were amplified by PCR and their products digested by the restriction endonuclease enzymes AseI and HaeIII followed by subsequent sequencing of Cyt-b gene and ITS-1 region for subsequent phylogenetic analysis. The combined use of these 3 markers allowed us to assign 36 isolates (65.5%) to the complex Leishmania (Viannia) braziliensis, 4 isolates (7, 27%) to L. (Viannia) lainsoni. and the remaining 15 isolates (23.7%) to a local variant of L. (Leishmania) mexicana. Concerning in vitro drug susceptibility the amastigotes from all isolates where highly sensitive to Fungizone® (mean IC50 between 0.23 and 0.5 μg/mL) whereas against Glucantime® the sensitivity was moderate (mean IC50 ranging from 50.84 μg/mL for L. (V.) braziliensis to 18.23 μg/mL for L. (L.) mexicana. L. (V.) lainsoni was not sensitive to Glucantime®. The susceptibility to miltefosine was highly variable among species isolates, being L. (L.) mexicana the most sensitive, followed by L. (V.) braziliensis and L. (V.) lainsoni (mean IC50 of 8.24 μg/mL, 17.85 μg/mL and 23.28 μg/mL, respectively).

Fifty five human isolates of Leishmania from Bolivia were identified by PCR amplification and sequencing of ITS-1 and Cytochrome B gene followed by in vitro evaluation of drug susceptibility.166