Dados do Trabalho

Título

Quantitative polymerase chain reaction (qPCR) protocol predictive of treatment outcome in cutaneous leishmaniasis caused by Leishmania braziliensis.

Introdução

Leishmania braziliensis is the most prevalent agent causing cutaneous leishmaniasis (CL) in Brazil. Despite the heterogeneous distribution of parasites within lesions, a more precise quantification strategy could be valuable to identify parasite load as a biomarker for disease severity and treatment failure. 

Objetivo (s)

To quantify parasite load using a qPCR assay in cutaneous lesion biopsies from patients with cutaneous leishmaniasis residing in an endemic area of Southeastern Bahia, Brazil. 

Material e Métodos

Employing L. braziliensis kDNA and human 18S rRNA endogenous control as targets, we developed a TaqMan qPCR assay to assess 38 randomly selected samples from a DNA bank of patients previously diagnosed with CL, and from 17 patients in the early stage of infection (ESI) prior to ulceration. Parasite load was calculated from a standard curve generated from 2×10⁵ parasites extracted from a culture of Leishmania promastigotes, followed by a 10-fold serial dilution to obtain a DNA quantity of 2 parasites. Target specificity was confirmed using biopsies from six healthy, non-endemic controls. This study was approved by the Research Ethics Committee and the National Research Ethics Commission (CONEP: 81315517.1.0000.5577), and all patients provided informed consent. Data analysis was performed using GraphPad Prism 8. Statistical comparisons and survival analysis were conducted to evaluate parasite load, clinical outcomes, and identify potential prognostic factors. 

Resultados e Conclusão

Our study demonstrated a strong association between parasite load, as determined by qPCR, and treatment failure in CL patients. Patients in the early stages of infection exhibited higher parasite burdens. ROC curve analysis identified a cut-off value of 14,718 parasites that effectively discriminated between patients who responded to treatment and those who failed,with a sensitivity of 58.82% and specificity of 95.45%. These findings highlight the potential of qPCR as a prognostic tool for guiding treatment decisions. qPCR may overestimate parasite load.   Our findings suggest that parasite load, as determined by qPCR, can serve as a valuable prognostic tool to aid in assessing disease severity and treatment outcomes in cutaneous leishmaniasis.       

Palavras Chave

Cutaneous leishmaniasis; Parasite load; qPCR; Clinical outcome