Abstract 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 co-infection 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 cutaneous leishmaniasis. However, the reported frequencies of LRV associated to complicated outcomes in patient¿s series are highly variable, diminishing the relevance on the virus presence in the pathogenesis of the disease. In order to assess whether or not the inconsistent information about the frequency of LRV associated to CL complicated outcomes could be related with the virus detection approach, the present study evaluated LRV presence in clinical samples using a diagnostic algorithm according with the type of sample. In 36 samples with diagnosis of complicated forms of CL (15 of ML and 21 of CL antimony treatment failure) and 6 samples with non-Leishmania spp. infection, LRV presence was assessed by RT-PCR, RT-qPCR and nested RT-PCR. Viral load was estimated in parasites clinical isolates. By combining 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.