Clinical and parasitological factors in parasite persistence after treatment and clinical cure of cutaneous leishmaniasis

Abstract

Background: The determinants of parasite persistence or elimination after treatment and clinical resolution of cutaneous leishmaniasis (CL) are unknown. We investigated clinical and parasitological parameters associated with the presence and viability of Leishmania after treatment and resolution of CL caused by L. Viannia.

Methods: Seventy patients who were treated with meglumine antimoniate (n = 38) or miltefosine (n = 32) and cured, were included in this study. Leishmania persistence and viability were determined by detection of kDNA and 7SLRNA transcripts, respectively, before, at the end of treatment (EoT), and 13 weeks after initiation of treatment in lesions and swabs of nasal and tonsillar mucosa.

Results: Sixty percent of patients (42/70) had evidence of Leishmania persistence at EoT and 30% (9/30) 13 weeks after treatment initiation. A previous episode of CL was found to be a protective factor for detectable Leishmania persistence (OR: 0.16, 95%CI: 0.03-0.92). kDNA genotyping could not discern differences between parasite populations that persisted and those isolated at diagnosis.

Conclusions: Leishmania persist in skin and mucosal tissues in a high proportion of patients who achieved therapeutic cure of CL. This finding prompts assessment of the contribution of persistent infection in transmission and endemicity of CL, and in disease reactivation and protective immunity.

Fig 1. Schematic representation of study participants and samples obtained at different time points during treatment and clinical follow-up.

Fig 2. Frequency of detection of Leishmania in samples from CL patients before and after treatment.

Presence of Leishmania was determined by detection of Leishmania kDNA or 7SLRNA in lesion and mucosal samples (A) and parasite viability (B) was determined by detection of 7SLRNA transcripts. Graphs represent frequency of positivity in at least one sample (left panels), or independently in lesions (center panels) and in any mucosal tissue (right panels). Data are shown as relative frequencies based on the total number of patients at each sampling time. Differences were analyzed by the McNemar’s test.

Fig 3. Comparative analysis of pre-treatment and post-treatment clinical strains by kDNA genotyping and multilocus microsatellite typing.

UPGMA trees of distances calculated from MLMT data (A) and conserved block minicircle kDNA genotyping (B) from three pairs of L. V. panamensis and three pairs of L. V. braziliensis strains isolated before treatment (BT; black triangles) and at treatment failure (TF; black squares), alongside 34 L. V. panamensis clinical strains (open circles) and the reference L. V. panamensis strain LS94. Shadowed codes denote pairs of strains isolated from the same patient. (C) Pairwise sequence alignment of pre and post-treatment strains indicating % base-pair identity.

Fig 4. Analysis of Leishmania genetic diversity in clinical samples obtained from patients before and at the end of treatment.

Maximum Likelihood tree of distances calculated from sequences of the conserved block of minicircle kDNA from lesion (-L), tonsillar (-T) and nasal (-N) mucosa samples obtained before treatment (BT) and at the end of treatment (ET), alongside a panel of L. V. panamensis (Lp; n = 48), L. infantum (Li; n = 5), and L. mexicana complex (Lm; n = 4) sequences. Color blocks denote subpopulations defined by population structure analysis and include the estimated fixation index (F_ST). LP1-3: L. V. panamensis subpopulations. The bottom panel is the visual representation of the STRUCTURE analysis. Each strain/sequence (n = 83) is represented by a single vertical line divided into K colors, where K is the number of populations assumed (K = 5). Details of strains and kDNA sequences retrieved from NCBI are shown in S1 Table.