Por favor, use este identificador para citar o enlazar este Item:http://hdl.handle.net/20.500.12105/16277
Título
Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp
Autor(es)
Chicharro, Carmen ISCIII | Nieto Martinez, Francisco Javier ISCIII | Miguelañez, Silvia ISCIII | Garcia, Emilia ISCIII | Ortega, Sheila ISCIII | Peña Gallego, Ana ISCIII | Rubio Muñoz, Jose Miguel ISCIII | Flores-Chavez, Maria ISCIII
Fecha de publicación
2023-06-15
Cita
Microbiol Spectr. 2023 Jun 15;11(3):e0335422.
Idioma
Inglés
Tipo de documento
research article
Resumen
Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.
Palabras clave
Leishmaniasis | Molecular diagnosis | Sensitivity | Specificity | Nested PCR | qPCR | Parasite load | Gel system
MESH
Leishmania | Leishmaniasis | Animals | Humans | Real-Time Polymerase Chain Reaction | Spain | Reproducibility of Results | DNA, Protozoan | Sensitivity and Specificity | Mammals
Versión en línea
DOI
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