Mucosal Leishmaniasis caused by Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in the Brazilian Amazon

Background

Leishmania (Viannia) braziliensis is a parasite recognized as the most important etiologic agent of mucosal leishmaniasis (ML) in the New World. In Amazonia, seven different species of Leishmania, etiologic agents of human Cutaneous Leishmaniasis, have been described. Isolated cases of ML have been described for several different species of Leishmania: L. (V.) panamensis, L. (V.) guyanensis and L. (L.) amazonensis.

Methodology

Leishmania species were characterized by polymerase chain reaction (PCR) of tissues taken from mucosal biopsies of Amazonian patients who were diagnosed with ML and treated at the Tropical Medicine Foundation of Amazonas (FMTAM) in Manaus, Amazonas state, Brazil. Samples were obtained retrospectively from the pathology laboratory and prospectively from patients attending the aforementioned tertiary care unit.

Results

This study reports 46 cases of ML along with their geographical origin, 30 cases caused by L. (V.) braziliensis and 16 cases by L. (V.) guyanensis. This is the first record of ML cases in 16 different municipalities in the state of Amazonas and of simultaneous detection of both species in 4 municipalities of this state. It is also the first record of ML caused by L. (V.) guyanensis in the states of Pará, Acre, and Rondônia and cases of ML caused by L. (V.) braziliensis in the state of Rondônia.

Conclusions/Significance

L. (V.) braziliensis is the predominant species that causes ML in the Amazon region. However, contrary to previous studies, L. (V.) guyanensis is also a significant causative agent of ML within the region. The clinical and epidemiological expression of ML in the Manaus region is similar to the rest of the country, although the majority of ML cases are found south of the Amazon River.

Author Summary

Leishmaniasis is considered a neglected disease with 1.5 million new cases of cutaneous leishmaniasis (CL) occurring each year. In the Amazon region and in the Americas in general, ML is caused by Leishmania (Viannia) braziliensis, though in rare cases it has been related to other species. ML, which is associated with inadequate treatment of CL, normally manifests itself years after the occurrence of CL. Clinical features evolve slowly and most often affect the nasal cavity, in some cases causing perforation, or even destruction, of the septum. Diagnosis is made using the Montenegro skin test, serology and histopathology of the patients'' mucosal tissues, or by isolation of the parasites. PCR is the best way to identify the species of leishmaniasis and is therefore the diagnostic method of choice. This paper describes 46 cases of ML and their geographical origin, 30 cases associated with L. (V.) braziliensis and 16 with L. (V.) guyanensis. The species of leishmaniasis was identified using mucosal biopsies taken from Amazonian patients who were diagnosed and treated for ML in the tertiary care unit, in Manaus, Amazonas state, Brazil. This is the highest number of ML cases caused by L. (V.) guyanensis that has ever been reported.     

Population Structure and Evidence for Both Clonality and Recombination among Brazilian Strains of the Subgenus Leishmania (Viannia)

Background/Objectives: Parasites of the subgenus Leishmania (Viannia) cause varying clinical symptoms ranging from cutaneous leishmaniases (CL) with single or few lesions, disseminated CL (DL) with multiple lesions to disfiguring forms of mucocutaneous leishmaniasis (MCL). In this population genetics study, 37 strains of L. (V.) guyanensis, 63 of L. (V.) braziliensis, four of L. (V.) shawi, six of L. (V.) lainsoni, seven of L. (V.) naiffi, one each of L. (V.) utingensis and L. (V.) lindenbergi, and one L. (V.) lainsoni/L. naiffi hybrid from different endemic foci in Brazil were examined for variation at 15 hyper-variable microsatellite markers. Methodology/Principal findings: The multilocus microsatellite profiles obtained for the 120 strains were analysed using both model- and distance-based methods. Significant genetic diversity was observed for all L. (Viannia) strains studied. The two cluster analysis approaches identified two principal genetic groups or populations, one consisting of strains of L. (V.) guyanensis from the Amazon region and the other of strains of L. (V.) braziliensis isolated along the Atlantic coast of Brazil. A third group comprised a heterogeneous assembly of species, including other strains of L. braziliensis isolated from the north of Brazil, which were extremely polymorphic. The latter strains seemed to be more closely related to those of L. (V.) shawi, L. (V.) naiffi, and L. (V.) lainsoni, also isolated in northern Brazilian foci. The MLMT approach identified an epidemic clone consisting of 13 strains of L. braziliensis from Minas Gerais, but evidence for recombination was obtained for the populations of L. (V.) braziliensis from the Atlantic coast and for L. (V.) guyanensis. Conclusions/Significance: Different levels of recombination versus clonality seem to occur within the subgenus L. (Viannia). Though clearly departing from panmixia, sporadic, but long-term sustained recombination might explain the tremendous genetic diversity and limited population structure found for such L. (Viannia) strains.     

The Role of Nitric Oxide and Reactive Oxygen Species in the Killing of Leishmania braziliensis by Monocytes from Patients with Cutaneous Leishmaniasis

Human cutaneous leishmaniasis (CL) caused by Leishmania braziliensis, presents an exaggerated Th1 response that is associated with ulcer development. Macrophages are the primary cells infected by Leishmania parasites and both reactive oxygen species (ROS) and nitric oxide (NO) are important in the control of Leishmania by these cells. The mechanism involved in the killing of L. braziliensis is not well established. In this study, we evaluate the role of ROS and NO in the control of L. braziliensis infection by monocytes from CL patients. After in vitro infection with L. braziliensis, the oxidative burst by monocytes from CL patients was higher when compared to monocytes from healthy subjects (HS). Inhibition of the ROS pathway caused a significant decrease in the oxidative burst in L. braziliensis infected monocytes from both groups. In addition, we evaluated the intracellular expression of ROS and NO in L. braziliensis-infected monocytes. Monocytes from CL patients presented high expression of ROS after infection with L. braziliensis. The expression of NO was higher in monocytes from CL patients as compared to expression in monocytes from HS. A strong positive correlation between NO production and lesion size of CL patients was observed. The inhibition of ROS production in leishmania-infected monocytes from CL patients allowed the growth of viable promastigotes in culture supernatants. Thus, we demonstrate that while production of ROS is involved in L. braziliensis killing, NO alone is not sufficient to control infection and may contribute to the tissue damage observed in human CL.     

Detection of Leishmania RNA Virus in Leishmania Parasites

Background

Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.

Methodology/Principal Findings

This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.

Conclusions/Significance

We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.     

Leishmaniavirus-Dependent Metastatic Leishmaniasis Is Prevented by Blocking IL-17A

Cutaneous leishmaniasis has various outcomes, ranging from self-healing reddened papules to extensive open ulcerations that metastasise to secondary sites and are often resistant to standard therapies. In the case of L. guyanensis (L.g), about 5–10% of all infections result in metastatic complications. We recently showed that a cytoplasmic virus within L.g parasites (LRV1) is able to act as a potent innate immunogen, worsening disease outcome in a murine model. In this study, we investigated the immunophenotype of human patients infected by L.g and found a significant association between the inflammatory cytokine IL-17A, the presence of LRV1 and disease chronicity. Further, IL-17A was inversely correlated to the protective cytokine IFN-γ. These findings were experimentally corroborated in our murine model, where IL-17A produced in LRV1+ L.g infection contributed to parasite virulence and dissemination in the absence of IFN-γ. Additionally, IL-17A inhibition in mice using digoxin or SR1001, showed therapeutic promise in limiting parasite virulence. Thus, this murine model of LRV1-dependent infectious metastasis validated markers of disease chronicity in humans and elucidated the immunologic mechanism for the dissemination of Leishmania parasites to secondary sites. Moreover, it confirms the prognostic value of LRV1 and IL-17A detection to prevent metastatic leishmaniasis in human patients.     

Leishmania RNA virus-1 is similarly detected among metastatic and non-metastatic phenotypes in a prospective cohort of American Tegumentary Leishmaniasis

American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.     

High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species

Background

Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol.

Methods/Principal Findings

Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol.

Conclusions/Significance

HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA.     

Molecular tools confirm natural Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids causing cutaneous leishmaniasis in the Amazon region of Brazil

Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.     

The role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis

In tegumentary leishmaniasis caused by Leishmania braziliensis, there is evidence that increased production of IFN-γ, TNF-α and absence of IL-10 is associated with strong inflammatory reaction and with tissue destruction and development of the lesions observed in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). We evaluate the role of regulatory cytokines and cytokine antagonists in the downregulation of immune response in L. braziliensis infection. Peripheral blood mononuclear cells from CL and ML were stimulated with soluble Leishmania antigen in the presence or absence of regulatory cytokines (IL-10, IL-27 and TGF-β) or antagonists of cytokines (α-TNF-α and α-IFN-γ). Cytokines production (IL-10, IL-17, TNF-α and IFN-γ) was measured by ELISA. IL-10 and TGF-β downmodulate TNF-α and IL-17 production, whereas IL-27 had no effect in the production of TNF-α, IFN-γ and IL-17 in these patients. Neutralization of TNF-α decreased IFN-γ level and the neutralization of IFN-γ decreased TNF-α level and increased IL-10 production. This study demonstrate that IL-10 and TGF-β are cytokines that appear to be more involved in modulation of immune response in CL and ML patients. IL-10 might have a protective role, since the neutralization of IFN-γ decreases the production of TNF-α in an IL-10-dependent manner.     

Differential Gene Expression and Infection Profiles of Cutaneous and Mucosal Leishmania braziliensis Isolates from the Same Patient

BackgroundLeishmaniasis is a complex disease in which clinical outcome depends on factors such as parasite species, host genetics and immunity and vector species. In Brazil, Leishmania (Viannia) braziliensis is a major etiological agent of cutaneous (CL) and mucosal leishmaniasis (MCL), a disfiguring form of the disease, which occurs in ~10% of L. braziliensis-infected patients. Thus, clinical isolates from patients with CL and MCL may be a relevant source of information to uncover parasite factors contributing to pathogenesis. In this study, we investigated two pairs of L. (V.) braziliensis isolates from mucosal (LbrM) and cutaneous (LbrC) sites of the same patient to identify factors distinguishing parasites that migrate from those that remain at the primary site of infection.Conclusions/SignificanceDespite sharing high similarity at the genome structure and ploidy levels, the parasites exhibited divergent expressed genomes. The proteome and metabolome results indicated differential profiles between the cutaneous and mucosal isolates, primarily related to inflammation and chemotaxis. BALB/c infection revealed that the cutaneous isolates were more virulent than the mucosal parasites. Furthermore, our data suggest that the LbrPGF2S protein is a candidate to contribute to parasite virulence profiles in the mammalian host.     

Clinical diversity and treatment results in Tegumentary Leishmaniasis: A European clinical report in 459 patients

BackgroundCutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management.MethodologyDemographic and clinical data from 459 travellers infected in 47 different countries were collected by members of the European LeishMan consortium. The infecting Leishmania species was identified in 198 patients.Principal findingsCompared to Old World CL, New World CL was more frequently ulcerative (75% vs 47%), larger (3 vs 2cm), less frequently facial (17% vs 38%) and less frequently associated with mucosal involvement (2.7% vs 5.3%). Patients with mucosal lesions were older (58 vs 30 years) and more frequently immunocompromised (37% vs 3.5%) compared to patients with only skin lesions. Young adults infected in Latin America with L. braziliensis or L. guyanensis complex typically had an ulcer of the lower limbs with mucosal involvement in 5.8% of cases. Typically, infections with L. major and L. tropica acquired in Africa or the Middle East were not associated with mucosal lesions, while infections with L. infantum, acquired in Southern Europe resulted in slowly evolving facial lesions with mucosal involvement in 22% of cases. Local or systemic treatments were used in patients with different clinical presentations but resulted in similarly high cure rates (89% vs 86%).Conclusion/SignificanceCL acquired in L. infantum-endemic European and Mediterranean areas displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised patients. When used as per recommendations, local therapy is associated with high cure rates.     

Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.     

Specificity of the rapid rK39 antigen-based immunochromatographic test Kalazar Detect(r) in patients with cutaneous leishmaniasis in Brazil

The aim of this study was to evaluate the specificity of a rapid immunochromatographic test that was developed to detect antibodies against the rK39 antigen for the diagnosis of visceral leishmaniasis (VL). This evaluation was performed using sera from patients with a confirmed diagnosis of active cutaneous leishmaniasis. The sera from 272 patients with a confirmed diagnosis of localised cutaneous leishmaniasis (CL) who resided in an area endemic for Leishmania braziliensis in Brazil were obtained before the initiation of antileishmanial treatment. Kalazar Detect^(r)(InBios, Seattle, WA) recombinant K39 antigen-based immunochromatographic strips were used according to the manufacturer''s instructions. The test results were evaluated independently by two examiners in sequential order. The positive controls for the test included five serum samples from five patients with parasitologically confirmed diagnosis of VL caused by Leishmania infantum in Brazil. Overall, 100% of the samples obtained from patients with CL were negative, confirming the absence of a serological cross-reaction for individuals with cutaneous disease when these patients were evaluated using the rapid test. The lack of a cross-reaction in patients who were infected by parasites of the same genus highlights the specificity of the rK39 antigen for the diagnosis of VL in areas with the sympatric circulation of L. braziliensis and L. infantum.     

Changes in the microbiological diagnosis and epidemiology of cutaneous leishmaniasis in real-time PCR era: A six-year experience in a referral center in Barcelona

BackgroundLeishmaniasis is a neglected disease caused by different species of the protozoa Leishmania spp. Cutaneous lesions are the most common clinical manifestation. This disease is prevalent in tropical and subtropical areas, including the Mediterranean basin. In Spain, Leishmania (L.) infantum is the only endemic species, but imported cases are often diagnosed. Different classical parasitological methods can be performed for cutaneous leishmaniasis (CL) diagnosis; but currently molecular techniques serve as a relevant tool for the detection and characterization of Leishmania parasites. We aimed to evaluate clinical and epidemiological characteristics of CL diagnosed patients by real-time PCR in a tertiary hospital over a six-year period.Methodology/Principal findingsClinical, epidemiological and microbiological data were retrospectively collected and analyzed. In our study, CL was confirmed in 59 (31.4%) out of 188 patients by real-time PCR, showing an increase over recent years: 11 cases of CL between 2014 and 2016 and 48 between 2017 and 2019. Real-time PCR was performed on skin swabs and/or biopsies samples, with a positivity of 38.5% and 26.5%, respectively. Results were 100% concordant when biopsy and skin swab were performed simultaneously. L. (L.) infantum was the most frequent species detected (50%), followed by L. (L.) major (45%) and Viannia subgenus (5%), which were detected only in imported cases. L. (L.) major was almost entirely detected in travelers/migrants from Morocco. Multiple and atypical skin lesions were more common in imported cases than in autochthonous cases (44.4% vs. 21.8%).Conclusions/SignificanceAn increase in both autochthonous and imported CL cases has been observed in past years in our hospital. Molecular techniques assist in improving CL diagnosis and characterization of the Leishmania species, mainly in imported cases.     

Down-Regulation of TLR and JAK/STAT Pathway Genes Is Associated with Diffuse Cutaneous Leishmaniasis: A Gene Expression Analysis in NK Cells from Patients Infected with Leishmania mexicana

An important NK-cell inhibition with reduced TNF-α, IFN-γ and TLR2 expression had previously been identified in patients with diffuse cutaneous leishmaniasis (DCL) infected with Leishmania mexicana. In an attempt to pinpoint alterations in the signaling pathways responsible for the NK-cell dysfunction in patients with DCL, this study aimed at identifying differences in the NK-cell response towards Leishmania mexicana lipophosphoglycan (LPG) between patients with localized and diffuse cutaneous leishmaniasis through gene expression profiling. Our results indicate that important genes involved in the innate immune response to Leishmania are down-regulated in NK cells from DCL patients, particularly TLR and JAK/STAT signaling pathways. This down-regulation showed to be independent of LPG stimulation. The study sheds new light for understanding the mechanisms that undermine the correct effector functions of NK cells in patients with diffuse cutaneous leishmaniasis contributing to a better understanding of the pathobiology of leishmaniasis.