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Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis.
- Source :
-
PLoS neglected tropical diseases [PLoS Negl Trop Dis] 2020 Oct 12; Vol. 14 (10), pp. e0008750. Date of Electronic Publication: 2020 Oct 12 (Print Publication: 2020). - Publication Year :
- 2020
-
Abstract
- Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.<br />Competing Interests: The authors have declared that no competing interests exist.
- Subjects :
- DNA, Protozoan analysis
DNA, Protozoan genetics
DNA, Ribosomal analysis
HSP70 Heat-Shock Proteins analysis
Humans
Leishmaniasis, Cutaneous pathology
Sensitivity and Specificity
Skin parasitology
DNA, Ribosomal genetics
HSP70 Heat-Shock Proteins genetics
Leishmania isolation & purification
Leishmaniasis, Cutaneous parasitology
Parasite Load methods
Real-Time Polymerase Chain Reaction methods
Subjects
Details
- Language :
- English
- ISSN :
- 1935-2735
- Volume :
- 14
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- PLoS neglected tropical diseases
- Publication Type :
- Academic Journal
- Accession number :
- 33044986
- Full Text :
- https://doi.org/10.1371/journal.pntd.0008750