Transcriptomics analysis highlights potential ways in human pathogenesis in Leishmania braziliensis infected with the viral endosymbiont LRV1.

The parasite Leishmania (Viannia) braziliensis is widely distributed in Brazil and is one of the main species associated with human cases of different forms of tegumentary leishmaniasis (TL) such as cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). The mechanisms underlying the pathogenesis of TL are still not fully understood, but it is known that factors related to the host and the parasite act in a synergistic and relevant way to direct the response to the infection. In the host, macrophages have a central connection with the parasite and play a fundamental role in the defense of the organism due to their ability to destroy intracellular parasites and present antigens. In the parasite, some intrinsic factors related to the species or even the strain analyzed are fundamental for the outcome of the disease. One of them is the presence of Leishmania RNA Virus 1 (LRV1), an endosymbiont virus that parasitizes some species of Leishmania that triggers a cascade of signals leading to a more severe TL phenotype, such as ML. One of the strategies for understanding factors associated with the immune response generated after Leishmania/host interaction is through the analysis of molecular patterns after infection. Thus, the gene expression profile in human monocyte-derived macrophages obtained from healthy donors infected in vitro with L. braziliensis positive (LbLRV1+) and negative (LbLRV1-) for LRV1 was evaluated. For this, the microarray assay was used and 162 differentially expressed genes were identified in the comparison LbLRV1+ vs. LbLRV1-, 126 upregulated genes for the type I and II interferons (IFN) signaling pathway, oligoadenylate synthase OAS/RNAse L, non-genomic actions of vitamin D3 and RIG-I type receptors, and 36 down-regulated. The top 10 downregulated genes along with the top 10 upregulated genes were considered for analysis. Type I interferon (IFNI)- and OAS-related pathways results were validated by RT-qPCR and Th1/Th2/Th17 cytokines were analyzed by Cytometric Bead Array (CBA) and enzyme-linked immunosorbent assay (ELISA). The microarray results validated by RT-qPCR showed differential expression of genes related to IFNI-mediated pathways with overexpression of different genes in cells infected with LbLRV1+ compared to LbLRV1- and to the control. No significant differences were found in cytokine levels between LbLRV1+ vs. LbLRV1- and control. The data suggest the activation of gene signaling pathways associated with the presence of LRV1 has not yet been reported so far. This study demonstrates, for the first time, the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 when comparing infections with LbLRV1+ versus LbLRV1- and the control. This finding emphasizes the role of LRV1 in directing the host's immune response after infection, underlining the importance of identifying LRV1 in patients with TL to assess disease progression.

HRM Accuracy and Limitations as a Species Typing Tool for Leishmania Parasites.

High Resolution Melting Analysis (HRM) has been pointed out as a suitable alternative method to detect and identify Leishmania species. Herein, we aimed to evaluate the sensitivity, specificity, accuracy, and limitations of a HSP70-HRM protocol both as a diagnostic scheme applied in clinical samples and as a species typing tool for laboratory research and reference services. Our data reveal the pronounced species-typing potential of the HSP70-HRM in DNA from cultured parasites. For clinical samples, however, we advise caution due to parasite load-dependent accuracy. In light of these findings and considering the importance of parasite load determination for clinical and research purposes, we recommend the integration of the presented typing scheme and the previously published Leishmania quantifying approach as combined tools for clinicians, surveillance, and research.

Exploring Host-Specificity: Untangling the Relationship between Leishmania (Viannia) Species and Its Endosymbiont Leishmania RNA Virus 1.

A relevant aspect in the epidemiology of Tegumentary Leishmaniasis (TL) are the Leishmania parasites carrying a viral endosymbiont, Leishmania RNA Virus 1 (LRV1), a dsRNA virus. Leishmania parasites carrying LRV1 are prone to causing more severe TL symptoms, increasing the likelihood of unfavorable clinical outcomes. LRV1 has been observed in the cultured strains of five L. (Viannia) species, and host specificity was suggested when studying the LRV1 from L. braziliensis and L. guyanensis strains. The coevolution hypothesis of LRV1 and Leishmania was based on phylogenetic analyses, implying an association between LRV1 genotypes, Leishmania species, and their geographic origins. This study aimed to investigate LRV1 specificity relative to Leishmania (Viannia) species hosts by analyzing LRV1 from L. (Viannia) species. To this end, LRV1 was screened in L. (Viannia) species other than L. braziliensis or L. guyanensis, and it was detected in 11 out of 15 L. naiffi and two out of four L. shawi. Phylogenetic analyses based on partial LRV1 genomic sequencing supported the hypothesis of host specificity, as LRV1 clustered according to their respective Leishmania species' hosts. These findings underscore the importance of investigating Leishmania and LRV1 coevolution and its impact on Leishmania (Viannia) species dispersion and pathogenesis in the American Continent.

Leishmania (Viannia) naiffi Lainson & Shaw 1989.

Just over 30 years ago, a new species of Leishmania of the subgenus Leishmania (Viannia) was described infecting the armadillo Dasypus novemcinctus; then, a report of human infection followed. From the Brazilian Amazon and apparently restricted to this region and its close borders, Leishmania (Viannia) naiffi has been characterized as a species that grows easily in axenic culture medium and causes few to no lesions after inoculation in experimental animal models. Results in the last decade indicate the occurrence of L. naiffi in vectors and human infections, including a report of therapeutic failure possibly associated with Leishmania RNA virus 1. Overall, such accounts suggest that the parasite is more dispersed and the disease less self-healing than previously expected.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare.

BACKGROUND: Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES: Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS: Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS: The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS: The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Raising the suspicion of a non-autochthonous infection: identification of Leishmania guyanensis from Costa Rica exhibits a Leishmaniavirus related to Brazilian north-east and French Guiana viral genotypes.

BACKGROUND: Costa Rica has a history of neglecting prevention, control and research of leishmaniasis, including limited understanding on Leishmania species causing human disease across the country and a complete lack of knowledge on the Leishmania RNA virus, described as a factor linked to the worsening and metastasis of leishmanial lesions. OBJECTIVES: The aim of this work was to describe a case of cutaneous leishmaniasis by Leishmania (Viannia) guyanensis, bearing infection with Leishmaniavirus 1 (LRV1) in Costa Rica, raising the suspicion of imported parasites in the region. METHODS: The Leishmania strain was previously identified by routine hsp70 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in Costa Rica and subsequently characterised by isoenzyme electrophoresis and Sanger sequencing in Brazil. Screening for LRV1 was conducted with a dual RT-PCR approach and sequencing of the fragment obtained. FINDINGS: Since 2016 Costa Rica performs Leishmania isolation and typing as part of its epidemiological surveillance activities. Amongst 113 strains typed until 2019, only one was characterised as a L. (V.) guyanensis, corresponding to the first confirmed report of this species in the country. Interestingly, the same strain tested positive for LRV1. Sequencing of the viral orf1 and 2, clustered this sample with other LRV1 genotypes of South American origin, from the Northeast of Brazil and French Guiana. MAIN CONCLUSION: The unique characteristics of this finding raised the suspicion that it was not an autochthonous strain. Notwithstanding its presumed origin, this report points to the occurrence of said endosymbiont in Central American Leishmania strains. The possibility of its local dispersion represents one more challenge faced by regional health authorities in preventing and controlling leishmaniasis.

First report of Leishmania (Mundinia) martiniquensis in South American territory and confirmation of Leishbunyavirus infecting this parasite in a mare

BACKGROUND Epidemiological data related to leishmaniases or Leishmania infection in horses are scarce. However, studies carried out in different regions in the world showed equids parasitised by Leishmania braziliensis, L. infantum and L. martiniquensis. OBJECTIVES Identify the Leishmania species causing cutaneous leishmaniasis in a mare, living in Rio de Janeiro State (Brazil), and search the presence of Leishmania viruses in the isolated parasite. METHODS Isoenzymes and polymerase chain reaction (PCR) targeting ITSrDNA region followed by sequencing were conducted for typing the isolated parasite. A search for Leishmania virus infection was also performed. FINDINGS The mare presented skin nodules and ulcers in the left pinna caused by Leishmania spp. that was detected by culture and PCR. The parasite was identified as Leishmania (Mundinia) martiniquensis, infected by Leishbunyavirus (LBV), representing the first description of this species in South America. The animal travelled to different Brazilian regions, but not to outside the country. MAIN CONCLUSIONS The worldwide distribution of L. martiniquensis and its infection by LBV were confirmed in this study, indicating the autochthonous transmission cycle in Brazil. The clinical profile of the disease in the mare, showing fast spontaneous healing of cutaneous lesions, may indicate that skin lesions related to L. martiniquensis infection in horses might be underdiagnosed.

Interspecies and Intrastrain Interplay among Leishmania spp. Parasites.

Leishmania parasites present astonishing adaptative abilities that represent a matter of life or death within disparate environments during the heteroxenous parasite life cycle. From an evolutionary perspective, organisms develop methods of overcoming such challenges. Strategies that extend beyond the genetic diversity have been discussed and include variability between parasite cells during the infections of their hosts. The occurrence of Leishmania subpopulation fluctuations with variable structural genomic contents demonstrates that a single strain might shelter the variability required to overcome inconsistent environments. Such intrastrain variability provides parasites with an extraordinary ability to adapt and thus survive and propagate. However, different perspectives on this evolution have been proposed. Strains or species living in the same environment can cooperate but also compete. These interactions might increase the replication rate of some parasites but cause the loss of more aggressive competitors for others. Adaptive responses to intra- and interspecific competition can evolve as a fixed strategy (replication is adapted to the average genetic complexity of infections) or an optional strategy (replication varies according to the genetic complexity of the current infection). This review highlights the complexity of interspecies and intrastrain interactions among Leishmania parasites as well as the different factors that influence this interplay.

Raising the suspicion of a non-autochthonous infection: identification of Leishmania guyanensis from Costa Rica exhibits a Leishmaniavirus related to Brazilian north-east and French Guiana viral genotypes

BACKGROUND Costa Rica has a history of neglecting prevention, control and research of leishmaniasis, including limited understanding on Leishmania species causing human disease across the country and a complete lack of knowledge on the Leishmania RNA virus, described as a factor linked to the worsening and metastasis of leishmanial lesions. OBJECTIVES The aim of this work was to describe a case of cutaneous leishmaniasis by Leishmania (Viannia) guyanensis, bearing infection with Leishmaniavirus 1 (LRV1) in Costa Rica, raising the suspicion of imported parasites in the region. METHODS The Leishmania strain was previously identified by routine hsp70 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in Costa Rica and subsequently characterised by isoenzyme electrophoresis and Sanger sequencing in Brazil. Screening for LRV1 was conducted with a dual RT-PCR approach and sequencing of the fragment obtained. FINDINGS Since 2016 Costa Rica performs Leishmania isolation and typing as part of its epidemiological surveillance activities. Amongst 113 strains typed until 2019, only one was characterised as a L. (V.) guyanensis, corresponding to the first confirmed report of this species in the country. Interestingly, the same strain tested positive for LRV1. Sequencing of the viral orf1 and 2, clustered this sample with other LRV1 genotypes of South American origin, from the Northeast of Brazil and French Guiana. MAIN CONCLUSION The unique characteristics of this finding raised the suspicion that it was not an autochthonous strain. Notwithstanding its presumed origin, this report points to the occurrence of said endosymbiont in Central American Leishmania strains. The possibility of its local dispersion represents one more challenge faced by regional health authorities in preventing and controlling leishmaniasis.

Overcoming the Negligence in Laboratory Diagnosis of Mucosal Leishmaniasis.

The northern region of Brazil, which has the largest number of cases of tegumentary leishmaniasis (TL) in the country, is also the region that has the highest diversity of species of vectors and Leishmania parasites. In this region, cases of mucosal leishmaniasis (ML), a clinical form of TL, exceed the national average of cases, reaching up to 12% of the total annual TL notifications. ML is associated with multiple factors, such as the parasite species and the viral endosymbiont Leishmania RNA virus 1 (LRV1). Being a chronic parasitological disease, laboratory diagnosis of ML poses a challenge for health services. Here, we evaluated more than 700 clinical samples from patients with clinical suspicion of TL, including patients with cutaneous leishmaniasis (CL) and mucosal leishmaniasis, comparing the results of parasitological tests-direct parasitological examination by microscopy (DP) and conventional PCR (cPCR) targeting of both kDNA and hsp70. The DP was performed by collecting material from lesions through biopsies (mucosal lesions) or scarification (cutaneous lesions); for PCR, a cervical brush was used for sample collection. Blood samples were tested employing standardized real-time PCR (qPCR) protocol targeting the HSP70 gene. PCR tests showed higher sensitivity than DP for both CL and ML samples. Considering ML samples only (N = 89), DP showed a sensitivity of 49.4% (N = 44) against 98.8% (N = 88) for kDNA PCR. The qPCR hsp70 for blood samples from patients with ML (N = 14) resulted in superior sensitivity (50%; N = 7) compared to DP (21.4%; N = 3) for samples from the same patients. Our results reinforced the need to implement a molecular test for the diagnosis of ML, in addition to proposing methods less invasive for collecting material from TL patients. Sample collection using a cervical brush in lesions observed in CL and ML patients is easy to perform and less invasive, compared to scarification and biopsies. Blood samples could be a good source for qPCR diagnosis for ML patients. Thus, we propose here a standardized method for collection and for performing of molecular diagnosis of clinical samples from suspicious ML patients that can be applied in reference services for improving ML diagnosis.

The Maze Pathway of Coevolution: A Critical Review over the Leishmania and Its Endosymbiotic History.

The description of the genus Leishmania as the causative agent of leishmaniasis occurred in the modern age. However, evolutionary studies suggest that the origin of Leishmania can be traced back to the Mesozoic era. Subsequently, during its evolutionary process, it achieved worldwide dispersion predating the breakup of the Gondwana supercontinent. It is assumed that this parasite evolved from monoxenic Trypanosomatidae. Phylogenetic studies locate dixenous Leishmania in a well-supported clade, in the recently named subfamily Leishmaniinae, which also includes monoxenous trypanosomatids. Virus-like particles have been reported in many species of this family. To date, several Leishmania species have been reported to be infected by Leishmania RNA virus (LRV) and Leishbunyavirus (LBV). Since the first descriptions of LRVs decades ago, differences in their genomic structures have been highlighted, leading to the designation of LRV1 in L. (Viannia) species and LRV2 in L. (Leishmania) species. There are strong indications that viruses that infect Leishmania spp. have the ability to enhance parasitic survival in humans as well as in experimental infections, through highly complex and specialized mechanisms. Phylogenetic analyses of these viruses have shown that their genomic differences correlate with the parasite species infected, suggesting a coevolutionary process. Herein, we will explore what has been described in the literature regarding the relationship between Leishmania and endosymbiotic Leishmania viruses and what is known about this association that could contribute to discussions about the worldwide dispersion of Leishmania.

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis.

Leishmaniasis is a worldwide neglected disease, encompassing asymptomatic infections and different clinical forms, such as American Tegumentary Leishmaniasis (ATL) which is part of the complex of diseases caused by protozoan parasites from Leishmania genus, transmitted by sand fly vectors. As a neglected disease, much effort is still needed in treatment and diagnosis. Currently, ATL diagnosis is mainly made by parasite detection by microscopy. The sensitivity of the method varies, and factors such as collection procedures interfere. Molecular approaches, specially based on Real Time PCR (qPCR) technique, has been widely used to detect Leishmania infection and to quantify parasite load, once it is a simple, rapid and sensitive methodology, capable to detect low parasite concentrations and less prone to variability. Although many studies have been already published addressing the use of this technique, an improvement on these methodologies, including an analytical validation, standardization and data association is demanded. Moreover, a proper validation by the assay by the use of clinical samples is still required. In this sense, the purpose of the present work is to compare the performance of qPCR using two commonly used targets (18S rDNA and HSP70) with an internal control (RNAse P) in multiplex reactions. Additionally, we validated reactions by assaying 88 samples from patients presenting different clinical forms of leishmaniasis (cutaneous, mucosal, recent and old lesions), representing the diversity found in Brazil's Amazon Region. Following the methodology proposed herein, the results indicate the use of both qPCR assays, 18S rDNA and HSP70, to achieve a very good net sensitivity (98.5%) and specificity (100%), performing simultaneous or sequential testing, respectively. With this approach, our main goal is to conclude the first step of a further multicenter study to propose the standardization of detection and quantification of Leishmania.

First report of Leishmania (Viannia) lindenbergi causing tegumentary leishmaniasis in the Brazilian western Amazon region.

Tegumentary Leishmaniasis (TL) in the Brazilian Amazon region is associated with several Leishmania species. In this report, we describe two cases of TL related to Leishmania lindenbergi occurring in different locations of Rondônia state. After clinical diagnosis, lesion samples were collected for parasitological diagnoses via direct microscopic visualization, parasite isolation, and PCR. PCR reactions were positive in both clinical samples. Parasite isolation was possible for both patients, and isolates were submitted to species identification by isoenzyme electrophoresis and DNA sequencing. This report is the first to describe human infections caused by L. lindenbergi since the initial description and record of human infection by this species in 2002.

Draft Whole-Genome Sequence of Leishmania (Viannia) braziliensis Presenting Leishmania RNA Virus 1, from Western Amazon, Brazil.

Leishmania (Viannia) braziliensis is the main etiological agent of tegumentary leishmaniasis in the neotropics. Here, we report a draft genome sequence (31.2 Mb) of an L. braziliensis strain from the western Amazon region of Brazil. This genome sequence will complement those available for other Leishmania species and contribute to further studies focusing on this parasite and the neglected diseases associated with it.

First autochthonous case of canine visceral leishmaniasis in Rondônia, Brazil, a region with no history of visceral leishmaniasis.

This report describes the first autochthonous case of canine visceral leishmaniasis in Rondônia, northern Brazil. A canine resident of the municipality of Cacoal, with clinical signs and symptoms of visceral leishmaniasis, was treated by a veterinarian. Samples were analyzed by a reference laboratory. Dual-path platform (DPP) assay, indirect immunofluorescence technique (IIT), enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), isolation in a culture medium, and direct parasitological analysis were performed. DPP assay, IIT, and ELISA revealed positive results for Leishmania; PCR identified the species as Leishmania infantum. Based on the clinical presentation and test results, canine visceral leishmaniasis was diagnosed.

First autochthonous case of canine visceral leishmaniasis in Rondônia, Brazil, a region with no history of visceral leishmaniasis

Abstract This report describes the first autochthonous case of canine visceral leishmaniasis in Rondônia, northern Brazil. A canine resident of the municipality of Cacoal, with clinical signs and symptoms of visceral leishmaniasis, was treated by a veterinarian. Samples were analyzed by a reference laboratory. Dual-path platform (DPP) assay, indirect immunofluorescence technique (IIT), enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), isolation in a culture medium, and direct parasitological analysis were performed. DPP assay, IIT, and ELISA revealed positive results for Leishmania; PCR identified the species as Leishmania infantum. Based on the clinical presentation and test results, canine visceral leishmaniasis was diagnosed.

New insights into the genetic diversity of Leishmania RNA Virus 1 and its species-specific relationship with Leishmania parasites.

Cutaneous leishmaniasis is a neglected parasitic disease that manifests in infected individuals under different phenotypes, with a range of factors contributing to its broad clinical spectrum. One factor, Leishmania RNA Virus 1 (LRV1), has been described as an endosymbiont present in different species of Leishmania. LRV1 significantly worsens the lesion, exacerbating the immune response in both experimentally infected animals and infected individuals. Little is known about the composition and genetic diversity of these viruses. Here, we investigated the relationship between the genetic composition of LRV1 detected in strains of Leishmania (Viannia) braziliensis and L. (V.) guyanensis and the interaction between the endosymbiont and the parasitic species, analyzing an approximately 850 base pair region of the viral genome. We also included one LRV1 sequence detected in L. (V.) shawi, representing the first report of LRV1 in a species other than L. braziliensis and L. guyanensis. The results illustrate the genetic diversity of the LRV1 strains analyzed here, with smaller divergences detected among viral sequences from the same parasite species. Phylogenetic analyses showed that the LRV1 sequences are grouped according to the parasite species and possibly according to the population of the parasite in which the virus was detected, corroborating the hypothesis of joint evolution of the viruses with the speciation of Leishmania parasites.

MOLECULAR CHARACTERIZATION OF AMERICAN CUTANEOUS LEISHMANIASIS IN THE TRI­BORDER AREA OF ASSIS BRASIL, ACRE STATE, BRAZIL.

In this study, Leishmania species were identified by Polymerase Chain Reaction (PCR). The epidemiology of patients suspected of having American Cutaneous Leishmaniasis in the municipality of Assis Brasil, Acre State, located in the Brazil/Peru/Bolivia tri-border was also investigated. By PCR, the DNA of Leishmania was detected in 100% of the cases (37 samples) and a PCR-Restriction Fragment Length Polymorphism (RFLP) of the hsp 70 gene identified the species in 32 samples: Leishmania (Viannia) braziliensis (65.6%) , L. (V.) shawi (28.1%) , L. (V.) guyanensis (3.1%) and mixed infection L. (V.) guyanensis and L. (Leishmania) amazonensis(3.1%)This is the first report of L. (V.) shawi and L. (L.) amazonensis in Acre. The two predominant species were found in patients living in urban and rural areas. Most cases were found in males living in rural areas for at least three years and involved in rural work. This suggests, in most cases, a possible transmission of the disease from a rural/forest source, although some patients had not engaged in activities associated with permanence in forestall areas, which indicate a possible sandflies adaptation to the periurban setting.

Further Evidence of an Association between the Presence of Leishmania RNA Virus 1 and the Mucosal Manifestations in Tegumentary Leishmaniasis Patients.

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.