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

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 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.

Figure 1.

Real-time PCR (RT-PCR) is useful for detection of low viral loads in Leishmania spp. isolates infected with Leishmania RNA virus 1 (LRV1). (A) Amplification of LRV RNA capsid fragment (∼485 bp) using modified RT-PCR. MM = molecular marker, Positive (POS): cDNA from (LRV1+)-MHOM/BR/75/M4147, Negative (NEG): non-template control (NTC). (B) Viral loads of the same samples presented in (A), estimated using two different modified RT-qPCRs, Ramos Pereira’s, and Itos.’ Labels on the top of the gel in (A) and on x axis in (B) correspond to isolate ID according to Table 1.