RESUMO
OBJECTIVES: The outcome of American tegumentary leishmaniasis (ATL) may depend on the presence of the Leishmania RNA virus (LRV). This virus may be involved in treatment failure. We aimed to determine whether genetic clusters of LRV1 are involved in this therapeutic outcome. METHODS: The presence of LRV1 was assessed in 129 Leishmania guyanensis isolates from patients treated with pentamidine in French Guiana. Among the 115 (89%) isolates found to carry LRV1, 96 were successfully genotyped. Patient clinical data were linked to the LRV data. RESULTS: The rate of treatment failure for LRV1-positive isolates was 37% (15/41) versus 40% (2/5) among LRV1-negative isolates (p 0.88). Concerning LRV1 genotypes, two predominant LRV1 groups emerged, groups A (23% (22/96)) and B (70% (67/96)). The treatment failure rate was 37% (3/8) for group A and 45% (9/20) for group B (p 0.31). DISCUSSION: Neither the presence nor genotype of LRV1 in patients with L. guyanensis seemed to correlate with pentamidine treatment failure.
Assuntos
Leishmania guyanensis/virologia , Leishmaniose Cutânea/tratamento farmacológico , Leishmaniavirus/classificação , Pentamidina/uso terapêutico , Adulto , Feminino , Guiana Francesa , Variação Genética , Técnicas de Genotipagem , Humanos , Leishmaniavirus/genética , Leishmaniavirus/isolamento & purificação , Masculino , Filogenia , Estudos Retrospectivos , Análise de Sequência de RNA , Falha de Tratamento , Adulto JovemRESUMO
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.
Assuntos
Leishmania/virologia , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/virologia , Leishmaniavirus/genética , RNA Viral/isolamento & purificação , Humanos , Leishmania/classificação , Leishmaniavirus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Carga ViralRESUMO
RNA virus 1-1 (LRV-1-1) is a dsRNA virus identified in isolates of Leishmania (Viannia) braziliensis and thought to advance localized cutaneous leishmaniasis (LCL) to mucocutaneous or mucosal leishmaniasis (MCL/ML). We examined the prevalence of LRV-1 and its correlation to phenotypes of American tegumentary leishmaniasis caused by L. (V.) braziliensis from Peru to better understand its epidemiology. Clinical isolates of L. (V.) braziliensis were screened for LRV-1 by real-time polymerase chain reaction (PCR) and stratified according to the phenotype: LCL (< 4 ulcers in number) MCL/ML; inflammatory ulcers (erythematous, purulent, painful ulcers with or without lymphatic involvement) or multifocal ulcers (≥ 4 in ≥ 2 anatomic sites). Proportionate LRV-1 positivity was compared across phenotypes. Of 78 L. (V.) braziliensis isolates, 26 (54.2%) had an inflammatory phenotype, 22 (28%) had the MCL/ML phenotype, whereas 30 (38.5%) had LCL. Mucocutaneous or mucosal leishmaniasis was found exclusively in adult male enrollees. Leishmania RNA virus 1 positivity by phenotype was as follows: 9/22 (41%) with MCL/ML; 5/26 (19%) with an inflammatory/multifocal cutaneous leishmaniasis phenotype; and 7/30 (23%) with LCL (P = 0.19). Leishmania RNA virus 1 positivity was not associated with age (P = 0.55) or gender (P = 0.49). Relative LRV-1 copy number was greater in those with MCL/ML than those with inflammatory/multifocal CL (P = 0.02). A direct association between LRV-1 status and clinical phenotype was not demonstrated; however, relative LRV-1 copy number was highest in those with MCL/ML. Future analyses to understand the relationship between viral burden and pathogenesis are required to determine if LRV-1 is truly a contributor to the MCL/ML phenotype.
Assuntos
Leishmania braziliensis/virologia , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Leishmaniavirus/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peru/epidemiologia , Adulto JovemRESUMO
INTRODUCTION: Leishmania RNA virus type 1 (LRV1) is an endosymbiont of some Leishmania (Vianna) species in South America. Presence of LRV1 in parasites exacerbates disease severity in animal models and humans, related to a disproportioned innate immune response, and is correlated with drug treatment failures in humans. Although the virus was identified decades ago, its genomic diversity has been overlooked until now. METHODOLOGY/PRINCIPLES FINDINGS: We subjected LRV1 strains from 19 L. (V.) guyanensis and one L. (V.) braziliensis isolates obtained from cutaneous leishmaniasis samples identified throughout French Guiana with next-generation sequencing and de novo sequence assembly. We generated and analyzed 24 unique LRV1 sequences over their full-length coding regions. Multiple alignment of these new sequences revealed variability (0.5%-23.5%) across the entire sequence except for highly conserved motifs within the 5' untranslated region. Phylogenetic analyses showed that viral genomes of L. (V.) guyanensis grouped into five distinct clusters. They further showed a species-dependent clustering between viral genomes of L. (V.) guyanensis and L. (V.) braziliensis, confirming a long-term co-evolutionary history. Noteworthy, we identified cases of multiple LRV1 infections in three of the 20 Leishmania isolates. CONCLUSIONS/SIGNIFICANCE: Here, we present the first-ever estimate of LRV1 genomic diversity that exists in Leishmania (V.) guyanensis parasites. Genetic characterization and phylogenetic analyses of these viruses has shed light on their evolutionary relationships. To our knowledge, this study is also the first to report cases of multiple LRV1 infections in some parasites. Finally, this work has made it possible to develop molecular tools for adequate identification and genotyping of LRV1 strains for diagnostic purposes. Given the suspected worsening role of LRV1 infection in the pathogenesis of human leishmaniasis, these data have a major impact from a clinical viewpoint and for the management of Leishmania-infected patients.
Assuntos
Variação Genética , Leishmania/virologia , Leishmaniavirus/classificação , Leishmaniavirus/isolamento & purificação , Filogenia , Adulto , Idoso , Análise por Conglomerados , Feminino , Guiana Francesa , Genoma Viral , Humanos , Leishmania/isolamento & purificação , Leishmaniose/parasitologia , Leishmaniavirus/genética , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Adulto JovemRESUMO
Tegumentary Leishmaniasis (TL) is endemic in Latin America, and Brazil contributes approximately 20 thousand cases per year. The pathogenesis of TL, however, is still not fully understood. Clinical manifestations vary from cutaneous leishmaniasis (CL) to more severe outcomes, such as disseminated leishmaniasis (DL), mucosal leishmaniasis (ML) and diffuse cutaneous leishmaniasis (DCL). Many factors have been associated with the severity of the disease and the development of lesions. Recent studies have reported that the presence of Leishmania RNA virus 1 infecting Leishmania (Leishmania RNA virus 1, LRV1) is an important factor associated with the severity of ML in experimental animal models. In the present study, 156 patients who attended Rondonia's Hospital of Tropical Medicine with both leishmaniasis clinical diagnoses (109 CL; 38 ML; 5 CL+ML; 3 DL and 1 DCL) and molecular diagnoses were investigated. The clinical diagnosis were confirmed by PCR by targeting hsp70 and kDNA DNA sequences and the species causing the infection were determined by HSP70 PCR-RFPL. The presence of LVR1 was tested by RT-PCR. Five Leishmania species were detected: 121 (77.6%) samples were positive for Leishmania (Viannia) braziliensis, 18 (11.5%) were positive for Leishmania (V.) guyanensis, 3 (1.8%) for Leishmania (V.) lainsoni, 2 (1.3%) for Leishmania (Leishmania) amazonensis and 2 (1.3%) for Leishmania (V.) shawi. Six (3.9%) samples were positive for Leishmania sp. but the species could not be determined, and 4 (2.6%) samples were suggestive of mixed infection by L. (V.) braziliensis and L. (V.) guyanensis. The virus was detected in L. braziliensis (N = 54), L. guyanensis (N = 5), L. amazonensis (N = 2), L. lainsoni (N = 1) and inconclusive samples (N = 6). Patients presenting with CL+ML, DL and DCL were excluded from further analysis. Association between the presence of the virus and the disease outcome were tested among the remaining 147 patients (CL = 109 and ML = 38). Of them, 71.1% (n = 27) mucosal lesions were positive for LRV1, and 28.9% (n = 11) were negative. In cutaneous lesions, 36.7% (n = 40) were positive and 63.3% (n = 69) were negative for LRV1. The ratio P(ML|LRV1+)/P(ML|LRV1-) was 2.93 (CI95% 1.57...5.46; p<0.001), thus corroborating the hypothesis of the association between LRV1 and the occurrence of mucosal leishmaniasis, as previously described in animal models; it also indicates that LRV1 is not the only factor contributing to the disease outcome.
Assuntos
Leishmania/patogenicidade , Leishmania/virologia , Leishmaniose Mucocutânea/patologia , Leishmaniose Mucocutânea/parasitologia , Leishmaniavirus/isolamento & purificação , Mucosa/patologia , RNA Viral/isolamento & purificação , Brasil , Estudos de Casos e Controles , Humanos , Leishmania/classificação , Leishmania/genética , Leishmaniavirus/genética , Dados de Sequência Molecular , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.
Assuntos
Leishmaniavirus/isolamento & purificação , Vírion/isolamento & purificação , Virologia/métodos , Leishmaniavirus/ultraestrutura , Microscopia Eletrônica de Transmissão , Coloração e Rotulagem/métodos , Vírion/ultraestruturaRESUMO
Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.
Assuntos
Leishmaniavirus/isolamento & purificação , Vírion/isolamento & purificação , Virologia/métodos , Leishmaniavirus/ultraestrutura , Microscopia Eletrônica de Transmissão , Coloração e Rotulagem/métodos , Vírion/ultraestruturaRESUMO
Leishmania RNA Virus (LRV, Totiviridae) infect Leishmania cells and subvert mice immune response, probably promoting parasite persistence, suggesting significant roles for LRV in host-parasite interaction. Here we describe a new LRV1-4 purification protocol, enabling capsid visualization by negatively stained electron microscopy representing a significant contribution to future LRV investigations.
Assuntos
Leishmaniavirus/isolamento & purificação , Vírion/isolamento & purificação , Virologia/métodos , Leishmaniavirus/ultraestrutura , Microscopia Eletrônica de Transmissão , Coloração e Rotulagem/métodos , Vírion/ultraestruturaRESUMO
Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.
Assuntos
Leishmania braziliensis/virologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Leishmaniavirus/genética , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Adolescente , Adulto , Idoso , Animais , Brasil/epidemiologia , Criança , Pré-Escolar , Cicatriz/patologia , Feminino , Humanos , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Leishmaniavirus/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Método Simples-CegoRESUMO
Leishmaniavirus is a double-stranded RNA virus that persistently infects some strains of the protozoan parasite Leishmania. There is considerable interest in the possibility that the presence of this virus alters parasite phenotype and may affect disease pathogenesis. If so, the virus marker could provide a valuable prognostic indicator for human leishmaniasis, particularly in those cases caused by New World parasite strains. The virus has been detected in cultured L. braziliensis, L. b. guyanensis, and L. major. To date there has been no information as to the extent of infection in samples prior to culturing in the laboratory. This study demonstrates, through the reverse transcription-polymerase chain reaction, that Leishmaniavirus exists in human biopsy samples of leishmaniasis prior to manipulation in culture.