Detection of Leishmania RNA Virus in Clinical Samples from Cutaneous Leishmaniasis Patients Varies according to the Type of Sample.

Leishmania RNA virus (LRV) is a double-stranded RNA virus belonging to the Totiviridae family detected as cytoplasmic inclusions in some strains of the human parasite Leishmania spp. Experimental evidence supports the hypothesis that human coinfection with Leishmania spp.-LRV triggers an exacerbated immune response in the host that can be responsible for the observed complicated outcomes in cutaneous leishmaniasis (CL), such as mucosal leishmaniasis (ML) and treatment failure of CL. However, the reported frequencies of LRV associated with complicated outcomes in patient's series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. To assess whether or not the inconsistent information about the frequency of LRV associated with CL complicated outcomes could be related to the virus detection approach, the present study evaluated the LRV presence in clinical samples using a diagnostic algorithm according to the type of the sample. In 36 samples with diagnosis of complicated forms of CL (15 of ML and 21 of CL antimony treatment failure) and six samples with non-Leishmania spp. infection, the LRV presence was assessed by RT-PCR, RT-qPCR, and nested RT-PCR. Viral load was estimated in parasite clinical isolates. By combining the methods, LRV1 presence was confirmed in 45% (9/20) of isolates and 37.5% (6/16) of the incisional biopsies. Remarkably, in some cases (4/8), LRV1 was undetectable in the isolates but present in their respective biopsies, and less frequently, the opposite was observed (1/8), suggesting the possibility of loss of parasites harboring LRV1 during the in vitro growth.

[Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study].

INTRODUCTION: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. OBJECTIVE: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. MATERIALS AND METHODS: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. RESULTS: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. CONCLUSION: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Molecular typing of Leishmania (Leishmania) amazonensis and species of the subgenus Viannia associated with cutaneous and mucosal leishmaniasis in Colombia: A concordance study / Tipificación molecular de Leishmania (Leishmania) amazonensis y especies del subgénero Viannia asociadas a la leishmaniasis cutánea y la mucosa en Colombia: un estudio de concordancia

Resumen Introduction: Multilocus enzyme electrophoresis (MLEE) is the reference standard for the characterization of Leishmania species. The test is restricted to specialized laboratories due to its technical complexity, cost, and time required to obtain results. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is used to identify Leishmania species. Objective: To establish the concordance between the two tests as identifying methods for circulating species in Colombia. Materials and methods: A total of 96 isolates from patients with cutaneous or mucosal leishmaniasis were selected and identified by MLEE and PCR-RFLP with miniexon and hsp70 as the molecular targets, which were used sequentially. Restriction enzymes HaeIII and BccI were similarly applied. Cohen's kappa coefficient and the 95% confidence interval (CI) were calculated. Results: The kappa coefficient and the 95% CI between MLEE and PCR-RFLP displayed "very good" concordance with a coefficient of 0.98 (CI95%: 0.98 to 1.00). The identified species were Leishmania Viannia braziliensis, Leishmania Viannia panamensis, Leishmania Viannia guyanensis and Leishmania Leishmania amazonensis. A total of 80 of the 96 isolates were sequenced and the results obtained by PCR-RFLP were confirmed. Conclusion: Due to the concordance obtained between tests results with the amplification of the genes miniexon and hsp70, PCR-RFLP is proposed as an alternative for identifying circulating Leishmania species in Colombia.

Abstract Introducción. La electroforesis de enzimas multilocus (Multilocus Enzyme Electrophoresis, MLEE) es el estándar de referencia para la tipificación de las especies de Leishmania. La prueba está restringida a laboratorios especializados por su complejidad técnica, sus costos y el tiempo necesario para obtener resultados. La PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) se utiliza para tipificar especies de Leishmania. Objetivo. Establecer la concordancia entre las dos pruebas como métodos de tipificación de las especies circulantes en Colombia. Materiales y métodos. Se seleccionaron 96 aislamientos de pacientes con leishmaniasis cutánea o mucocutánea y se tipificaron mediante MLEE y PCR-RFLP con los blancos moleculares miniexon y hsp70 usados en serie. Las enzimas de restricción aplicadas fueron la HaeIII y la BccI, respectivamente. Se calculó el coeficiente kappa y un intervalo de confianza (IC) de 95 %. Resultados. Se determinó que la concordancia fue "muy buena" al obtener un coeficiente de 0,98 (IC95%: 0,98-1,00). Las especies identificadas fueron: Leishmania Viannia braziliensis, L. (V.) panamensis, L. (V.) guyanensis y L. (L,) amazonensis. De los 96 aislamientos, 80 se enviaron a secuenciación y se confirmaron los resultados obtenidos mediante PCR-RFLP. Conclusión. Dada la concordancia obtenida con la PCR-RFLP amplificando los genes miniexon y hsp70, se propone esta prueba como alternativa para la tipificación de especies de Leishmania circulantes en Colombia.