Research Article: Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

Date Published: July 23, 2015

Publisher: Public Library of Science

Author(s): Milagros Suárez, Braulio M. Valencia, Marlene Jara, Milena Alba, Andrea K. Boggild, Jean-Claude Dujardin, Alejandro Llanos-Cuentas, Jorge Arevalo, Vanessa Adaui, Alain Debrabant.

Abstract: BackgroundCutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion.Methodology/Principal FindingsWe applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4).Conclusion/SignificanceOur results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is recommended instead of the more invasive biopsy.

Partial Text: Cutaneous leishmaniasis (CL) is a parasitic disease of significant public health problem in at least 18 countries of Latin America; about 67,000 CL cases were reported to occur annually in the last decade [1]. The disease is caused by protozoan parasites of the subgenera Leishmania (Viannia) and L. (Leishmania), with the former being responsible for most cases. The clinical phenotypes of CL are diverse and range from a single or few cutaneous ulcerative lesions at the site of infection that may heal spontaneously, diffuse and disseminated CL with multiple non-ulcerative lesions, to disfiguring mucocutaneous leishmaniasis that can be life-threatening [2,3]. The severity and outcome of the disease are dependent among others on the immune responses evoked by the host and the infecting Leishmania species [4,5].

Here we assessed whether the Leishmania (Viannia) parasite load differs by sampling site within CL ulcers and sampling method by means of qPCR. We observed that a significantly lower amount of parasites was quantified from the ulcer border as compared to the ulcer base and center. The fact that this finding was similarly observed with lesion biopsy and scraping specimens points out its robustness. This finding called our attention because most of available studies in the literature on skin lesions caused by Neotropical Leishmania parasites are based on biopsies collected from the border of the ulcer, in accordance with WHO recommendations [20], as this lesion site is regarded to likely concentrate a greater amount of parasites and viable infected mononuclear phagocytes compared to the necrotic center of the ulcer [21]. Nevertheless, consistent with our findings, in a recent study that evaluated the use of swab sampling over the ulcer coupled to qPCR for diagnosis of CL, Adams et al. [16] found indications of a greater quantity of parasite DNA in the ulcerated zone of the lesion (as compared to that found in the ulcer border using aspirate samples). Furthermore, Ramírez et al. [23] reported a significant increase in the sensitivities of microscopy and conventional PCR of dermal scrapings when samples were collected from the central region of the bottom of the ulcer rather than from the margin of the lesion. This appeared to be related to a higher parasite load and easily detectable amastigotes in that area [23].



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