A Leishmania infantum genetic marker associated with miltefosine treatment failure for visceral leishmaniasis

Abstract

Background: Miltefosine has been used successfully to treat visceral leishmaniasis (VL) in India, but it was unsuccessful for VL in a clinical trial in Brazil.

Methods: To identify molecular markers that predict VL treatment failure whole genome sequencing of 26 L. infantum isolates, from cured and relapsed patients allowed a GWAS analysis of SNPs, gene and chromosome copy number variations.

Findings: A strong association was identified (p = 0·0005) between the presence of a genetically stable L. infantumMiltefosine Sensitivity Locus (MSL), and a positive response to miltefosine treatment. The risk of treatment failure increased 9·4-fold (95% CI 2·11-53·54) when an isolate did not have the MSL. The complete absence of the MSL predicted miltefosine failure with 0·92 (95% CI 0·65-0·996) sensitivity and 0·78 (95% CI 0·52-0·92) specificity. A genotyping survey of L. infantum (n = 157) showed that the frequency of MSL varies in a cline from 95% in North East Brazil to <5% in the South East. The MSL was found in the genomes of all L. infantum and L. donovani sequenced isolates from the Old World (n = 671), where miltefosine can have a cure rate higher than 93%.

Interpretation: Knowledge on the presence or absence of the MSL in L. infantum will allow stratification of patients prior to treatment, helping to establish better therapeutic strategies for VL treatment. FUND: CNPq, FAPES, GCRF MRC and Wellcome Trust.

Keywords: Miltefosine Susceptibility Locus; Miltefosine treatment failure; Prognostic marker; Visceral leishmaniasis; Whole-genome sequencing.

Fig. 1

Flow charts of phase-two Brazilian miltefosine trial and L. infantum parasites recovered from enrolled patients (A), and the key steps used in the bioinformatics analysis (B). SNPs, Single Nucleotide Polymorphisms; InDels, Insertions and Deletions; CNV, Copy Number Variation; GWAS, Genome-Wide Association Study.

Fig. 2

Technical validation of bioinformatics data of structural variation. (A) PCR strategy to verify the presence of miltefosine sensitivity locus (MSL) and their genes individually: LinJ.31.2370, LinJ.31.2380, LinJ.31.2390 and LinJ.31.2400. (B) Results of PCR reaction presented in “A”. The * highlights the L. infantum isolates from cured patients that showed mixed genetic profile for the presence of MSL, and were subsequently cloned. The ^ highlights the L. infantum isolates used as controls for the cloning process. The presence of the complete MSL in the L. infantum genome is demonstrated by a PCR product of ~14 kb, whilst loss is demonstrated by a ~1·2 kb DNA fragment.

Fig. 3

Identification of repeated sequences flanking MSL in the Leishmania genome and possible mechanisms for MSL loss. (A) Position of repeats sequences on chromosome 31 of L. infantum JPCM5, identified by sequence of novel junction formed after MSL deletion in L. infantum isolates, (upper map). Repetitive elements with >90% identity to the L. infantum RAG337 can be found in equivalent positions on chromosome 31 of L. donovani BPK282A1 and flank a region that is syntenic with the MSL (lower map). (B) Model for loss of the MSL: Homologous recombination between two direct-repeat sequences flanking the MSL (RAG337–1′ and RAG337–2′), leading to formation of a circular extrachromosomal element.

Fig. 4

Geographical distribution of MSL in L. infantum circulating in different regions of Brazil. ES, Espírito Santo; MG, Minas Gerais; BA, Bahia; MA, Maranhão; PI, Piauí; RN, Rio Grande do Norte.