Quantitative Nucleic Acid Sequence-Based Assay as a New Molecular Tool for Detection and Quantification of Leishmania Parasites in Skin Biopsy Samples

Abstract

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.

Cutaneous Leishmaniasis (Leishmania major Infection) in Dutch Troops Deployed in Northern Afghanistan: Epidemiology, Clinical Aspects, and Treatment

“…Parasitologic methods were microscopy (Giemsa-stained smear), culture in Novy-MacNeal-Nicolle medium, polymerase chain reaction with molecular characterization of the parasites by sequence analysis of the polymerase chain reaction amplicon, 6 and quantitative nucleic acid sequence-based amplification. 7 Treatment consisted of three ilSb v injections with intervals of 1-3 days with cryotherapy preceding the first injection. Cryotherapy was applied with a liquid nitrogen spray apparatus (Cry-Ac; Brymill, CT) within rubber cones matching the diameter of the lesions.…”

Molecular approaches for a better understanding of the epidemiology and population genetics ofLeishmania

“…Assays using alternative amplification technologies such as quantitative nucleic acid sequence-based amplification (QT-NASBA) based on amplification of 18S RNA and loop-mediated isothermal amplification (LAMP) of kinetoplast minicircle DNA do not allow discrimination of different Leishmania species (Van der Meide et al 2005;Takagi et al 2009). Commercialized assays that have been developed for direct detection of Leishmania are not able to identify the infecting species.…”

First Case of Cutaneous Leishmaniasis Caused by Leishmania (Viannia) braziliensis in Suriname

“…Nucleic acid amplification tests (NAAT) are the standard techniques for species identification and have a high sensitivity and specificity. 5,6 This report describes a patient who presented with ulcerative lesions after a hunting trip to West Suriname. With NAAT analysis, the patient proved to be infected with Leishmania braziliensis, a species of Leishmania not reported in Suriname until now.…”