The perfect storm: Cutaneous leishmaniasis in troops deployed in the Atlantic forest of Argentina.

American cutaneous leishmaniasis (ACL) is the most prevalent form of leishmaniasis, associated with an ulcerative and stigmatizing mucocutaneous pathology. This study assessed the incidence of Leishmania (Viannia) braziliensis in members of the Argentine Army who were exposed to sandfly bites in Iguazú National Park (INP), northeastern Argentina, during an outbreak of ACL in 2019, and the presence of Leishmania in rodents, opossums and phlebotomine sandflies collected in the area of exposure. Samples from military personnel, wild animals and phlebotomine sandflies were analysed. A total of 20 (40%) patients among the Army personnel and two Akodon montensis rodents (11%) were positive for the presence of Leishmania sp. genes by PCR, while Nyssomyia whitmani and Migonemyia migonei, competent vectors of Leishmania, were also found at the same site. Sequences of hsp70 DNA fragments obtained from human samples confirmed the identity of L. (V.) braziliensis. The risk to which military personnel carrying out activities in the forest are exposed is highlighted, and this risk extends to any worker and visitor who circulates without protection in the INP, coming into contact with transmission "hot spots" due to the concentration of vectors, reservoirs and/or parasites.

Leishmania (Leishmania) infantum DNA detection in Nyssomyia neivai in Vale do Ribeira, Paraná, Brazil.

BACKGROUND: The incidence of visceral leishmaniasis (VL) has increased in the Southern region of Brazil in recent years, especially in the State of Paraná. New species have been suggested with potential to act as vector in VL endemic areas. OBJECTIVES: Identify the Leishmania species in sand fly specimens collected from 2016 to 2018 in the municipality of Itaperuçu, Vale do Ribeira, Paraná, Brazil. METHODS: Light traps were used for collections and for the analysis of sand fly were used the multiplex polymerase chain reaction (PCR) methodology and subsequent sequencing. FINDINGS: Among the collected specimens, 88.62% were attributed to the species Nyssomyia neivai, which were grouped into 176 pools. Three positive pools were detected: two with Leishmania (Viannia) braziliensis and one with L. (Leishmania) infantum. The positivity rate for the parasite was 0.25% based on the presence of at least one infected insect in the pool. MAIN CONCLUSIONS: The detection of L. infantum in Ny. neivai draws attention due to its abundance and anthropophily in the State of Paraná. Moreover, this finding is considered as an alert and suggests that the vector competence of Ny. neivai and the criteria for its incrimination should be carried out, given its wide distribution in southern of Brazil.

Big is not better: Comparing two alpha-Gal-bearing glycotopes in neoglycoproteins as biomarkers for Leishmania (Viannia) braziliensis infection.

The protozoan parasite Leishmania (Viannia) braziliensis is among Latin America's most widespread Leishmania species and is responsible for tegumentary leishmaniasis (TL). This disease has multiple clinical presentations, with cutaneous leishmaniasis (CL) being the most frequent. It manifests as one or a few localized skin ulcers, which can spread to other body areas. Hence, early diagnosis and treatment, typically with pentavalent antimonials, is critical. Traditional diagnostic methods, like parasite culture, microscopy, or the polymerase chain reaction (PCR) for detection of the parasite DNA, have limitations due to the uneven distribution of parasites in biopsy samples. Nonetheless, studies have revealed high levels of parasite-specific anti-α-Gal antibodies in L. (V.) braziliensis-infected patients. Previously, we demonstrated that the neoglycoprotein NGP28b, consisting of the L. (Leishmania) major type-2 glycoinositolphospholipid (GIPL)-3-derived trisaccharide Galpα1,6Galpα1,3Galfß conjugated to bovine serum albumin (BSA) via a linker, acts as a reliable serological biomarker (BMK) for L. (V.) braziliensis infection in Brazil. This indicates the presence of GIPL-3 or a similar structure in this parasite, and its terminal trisaccharide either functions as or is part of an immunodominant glycotope. Here, we explored whether extending the trisaccharide with a mannose unit would enhance its efficacy as a biomarker for the serological detection of L. (V.) braziliensis. We synthesized the tetrasaccharide Galpα1,6Galpα1,3Galfß1,3Manpα(CH2)3SH (G31SH) and conjugated it to maleimide-functionalized BSA to afford NGP31b. When we assessed the efficacy of NGP28b and NGP31b by chemiluminescent enzyme-linked immunosorbent assay on a cohort of CL patients with L. (V.) braziliensis infection from Bolivia and Argentina against a healthy control group, both NGPs exhibited similar or identical sensitivity, specificity, and accuracy. This finding implies that the mannose moiety at the reducing end is not part of the glycotope recognized by the parasite-specific anti-α-Gal antibodies in patients' sera, nor does it exert a relevant influence on the terminal trisaccharide's conformation. Moreover, the mannose does not seem to inhibit glycan-antibody interactions. Therefore, NGP31b is a viable and dependable BMK for the serodiagnosis of CL caused by L. (V.) braziliensis.

Anti-Leishmania compounds can be screened using Leishmania spp. expressing red fluorescence (tdTomato).

The main challenges associated with leishmaniasis chemotherapy are drug toxicity, the possible emergence of resistant parasites, and a limited choice of therapeutic agents. Therefore, new drugs and assays to screen and detect novel active compounds against leishmaniasis are urgently needed. We thus validated Leishmania braziliensis (Lb) and Leishmania infantum (Li) that constitutively express the tandem tomato red fluorescent protein (tdTomato) as a model for large-scale screens of anti-Leishmania compounds. Confocal microscopy of Lb and Li::tdTomato revealed red fluorescence distributed throughout the entire parasite, including the flagellum, and flow cytometry confirmed that the parasites emitted intense fluorescence. We evaluated the infectivity of cloned promastigotes and amastigotes constitutively expressing tdTomato, their growth profiles in THP-1 macrophages, and susceptibility to trivalent antimony, amphotericin, and miltefosine in vitro. The phenotypes of mutant and wild-type parasites were similar, indicating that the constitutive expression of tdTomato did not interfere with the evaluated parameters. We applied our validated model to a repositioning strategy and assessed the susceptibility of the parasites to eight commercially available drugs. We also screened 32 natural plant and fungal extracts and 10 pure substances to reveal new active compounds. The infectivity and Glucantime treatment efficacy of BALB/c mice and golden hamsters infected with Lb and Li::tdTomato mutant lines, respectively, were very similar compared to animals infected with wild-type parasites. Standardizing our methodology would offer more rapid, less expensive, and easier assays to screen of compounds against L. braziliensis and L. infantum in vitro and in vivo. Our method could also enhance the discovery of active compounds for treating leishmaniasis.

Novel detection of Leishmania RNA virus-1 (LRV-1) in clinical isolates of Leishmania Viannia panamensis.

American tegumentary leishmaniasis comprises a discrete set of clinical presentations endemic to Latin America. Leishmania RNA virus-1 (LRV-1) is a double-stranded RNA virus identified in 20­25% of the Leishmania Viannia braziliensis and L. V. guyanensis, however not in L. V. panamensis. This is the first report of LRV-1 in L. V. panamensis and its associations with clinical phenotypes of ATL. Unique surplus discard clinical isolates of L. V. panamensis were identified from the Public Health Ontario Laboratory (PHOL) and the Leishmania Clinic of the Instituto de Medicina Tropical 'Alexander von Humboldt' between 2012 and 2019 and screened for LRV-1 by real-time polymerase chain reaction. Patient isolates were stratified according to clinical phenotype. Of 30 patients with L. V. panamensis, 14 (47%) and 16 (53%) patients had severe and non-severe ATL, respectively. Five (36%) of 14 severe cases and 2 (12%) of 16 non-severe cases were positive for LRV-1, respectively. No differences in sex were observed for clinical phenotype and LRV-1 status. Although an association between LRV-1 status and clinical phenotype was not demonstrated, this is the first description of the novel detection of LRV-1 in L. V. panamensis, a species that has been documented predominantly in Central America.

Detection of Internal Transcribed Spacer 1 and hsp70 Genetic Markers Using Restriction Fragment Length Polymorphisms and Sequencing in Identification of Leishmania Species Causing Tegumentary Leishmaniasis in Brazil.

The identification of Leishmania species that cause tegumentary leishmaniasis (TL) is important for taxonomic and prognostic purposes. Molecular analysis using different Leishmania genomic targets is the most useful method for identifying Leishmania species. Therefore, we evaluated the performance of ribosomal RNA internal transcribed spacer 1 (ITS1) and heat shock protein (hsp70) genetic markers by polymerase chain reaction (PCR), followed by restriction fragment length polymorphism analysis (RFLP) and sequencing, for identification of Leishmania species. Samples from 84 Brazilian patients were amplified. Internal transcribed spacer 1 PCR followed by RFLP (HaeIII) [ITS1-RFLP (HaeIII)] identified 46.4% (39/84) of the samples as compatible with the Viannia subgenus. Internal transcribed spacer 1 PCR followed by sequencing (ITS1-sequencing) identified Leishmania (Viannia) braziliensis in 91.7% (77/84) of the TL samples, Leishmania (Leishmania) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 2.4% (2/84), and L. (L.) infantum in 1.2% (1/84). One of the samples showed the same proportion of similarity with L. (V.) guyanensis and L. (V.) panamensis. hsp70 nested PCR followed by RFLP (HaeIII) [nested hsp70-RFLP (HaeIII)] identified 91.7% (77/84) of the samples as compatible with L. (V.) braziliensis/L. (V.) naiffi, 3.6% (3/84) with L. (L.) amazonensis, 1.2% (1/84) with L. (L.) infantum, and 3.6% (3/84) with L. (V.) guyanensis. hsp70 PCR followed by sequencing (hsp70-sequencing) identified L. (V.) braziliensis in 91.7% (77/84) of the TL samples, L. (L.) amazonensis in 3.6% (3/84), L. (V.) guyanensis in 3.6% (3/84), and L. (L.) infantum in 1.2% (1/84). Our findings clearly showed that nested hsp70-RFLP (HaeIII) is better than ITS1-RFLP (HaeIII) and that ITS1 or hsp70 PCR followed by sequencing was adequate for identifying Leishmania species. We also found that Leishmania (Viannia) braziliensis is the most common species causing TL in Brazil. Therefore, sequencing multiple target genes such as ITS1 and hsp 70 is more accurate than RFLP for identifying Leishmania species.

Assessing reservoir host status in leishmaniasis with special reference to the infectiousness of Leishmania (Viannia) braziliensis infections in wild rodents.

Molecular methods have been responsible for a notable increase in the detection of Leishmaniinae infections in wild animals. Determining their infectiousness is of paramount importance in evaluating their epidemiological significance. One of the most efficient ways of determining infectiousness for vector borne diseases is xenodiagnosis with the appropriate vector. However, this is logistically very difficult to accomplish in the field, and an ideal solution is to find a molecular surrogate for xenodiagnosis. In this review we discuss different approaches to the problem by focusing on the infectiousness of Leishmania (Viannia) braziliensis in rodents under laboratory and field conditions. Comparisons with similar studies for other Leishmania species emphasizes that there are pivotal differences in the infectiousness and the importance of asymptomatic infections in different hosts. Potentially the most promising surrogate is the real time quantitative PCR (qPCR). However, its success depends on choosing a tissue that relates to the vector's feeding location and the parasite's tissue tropism. This requires detailed knowledge of the infection of each species in its wild hosts. We conclude that for L. (V.) braziliensis infections in wild rodents the tissue of choice for a molecular xenodiagnostic test, based on the qPCR is blood, providing that a significant number of samples must be examined.

Diversity and dissemination of viruses in pathogenic protozoa.

Viruses are the most abundant biological entities on Earth and play a significant role in the evolution of many organisms and ecosystems. In pathogenic protozoa, the presence of viruses has been linked to an increased risk of treatment failure and severe clinical outcome. Here, we studied the molecular epidemiology of the zoonotic disease cutaneous leishmaniasis in Peru and Bolivia through a joint evolutionary analysis of Leishmania braziliensis and their dsRNA Leishmania virus 1. We show that parasite populations circulate in tropical rainforests and are associated with single viral lineages that appear in low prevalence. In contrast, groups of hybrid parasites are geographically and ecologically more dispersed and associated with an increased prevalence, diversity and spread of viruses. Our results suggest that parasite gene flow and hybridization increased the frequency of parasite-virus symbioses, a process that may change the epidemiology of leishmaniasis in the region.

Unveiling drug-tolerant and persister-like cells in Leishmania braziliensis lines derived from patients with cutaneous leishmaniasis.

Introduction: Resistance against anti-Leishmania drugs (DR) has been studied for years, giving important insights into long-term adaptations of these parasites to drugs, through genetic modifications. However, microorganisms can also survive lethal drug exposure by entering into temporary quiescence, a phenomenon called drug tolerance (DT), which is rather unexplored in Leishmania. Methods: We studied a panel of nine Leishmania braziliensis strains highly susceptible to potassium antimonyl tartrate (PAT), exposed promastigotes to lethal PAT pressure, and compared several cellular and molecular parameters distinguishing DT from DR. Results and discussion: We demonstrated in vitro that a variable proportion of cells remained viable, showing all the criteria of DT and not of DR: i) signatures of quiescence, under drug pressure: reduced proliferation and significant decrease of rDNA transcription; ii) reversibility of the phenotype: return to low IC50 after removal of drug pressure; and iii) absence of significant genetic differences between exposed and unexposed lineages of each strain and absence of reported markers of DR. We found different levels of quiescence and DT among the different L. braziliensis strains. We provide here a new in-vitro model of drug-induced quiescence and DT in Leishmania. Research should be extended in vivo, but the current model could be further exploited to support R&D, for instance, to guide the screening of compounds to overcome the quiescence resilience of the parasite, thereby improving the therapy of leishmaniasis.

Co-expression analysis of lncRNA and mRNA suggests a role for ncRNA-mediated regulation of host-parasite interactions in primary skin lesions of patients with American tegumentary leishmaniasis.

Leishmaniasis, caused by different Leishmania species, manifests as cutaneous or visceral forms. In the American continent, the cutaneous form is called American tegumentary leishmaniasis (ATL) and is primarily caused by Leishmania (Viannia) braziliensis. Mucosal leishmaniasis (ML), the most severe form of ATL, arises in approximately 20% of patients from a primary cutaneous lesion. Evidence indicates changes in overall expression patterns of mRNAs and lncRNAs of the host in response to Leishmania infection, with the parasite capable of modulating host immune response, which may contribute to disease progression. We evaluated whether the co-expression of lncRNAs and their putative target mRNAs in primary cutaneous lesions of patients with ATL could be associated with the development of ML. Previously available public RNA-Seq data from primary skin lesions of patients infected with L. braziliensis was employed. We identified 579 mRNAs and 46 lncRNAs differentially expressed in the primary lesion that subsequently progressed to mucosal disease. Co-expression analysis revealed 1324 significantly correlated lncRNA-mRNA pairs. Among these, we highlight the positive correlation and trans-action between lncRNA SNHG29 and mRNA S100A8, both upregulated in the ML group. S100A8 and its heterodimeric partner S100A9 form a pro-inflammatory complex expressed by immune cells and seems to participate in host innate immune response processes of infection. These findings expand the knowledge of the Leishmania-host interaction and indicate that the expression of lncRNAs in the primary cutaneous lesion could regulate mRNAs and play roles in disease progression.

Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Diversity and geographical distribution of Leishmania species and the emergence of Leishmania (Leishmania) infantum and L. (Viannia) panamensis in Central-Western Venezuela.

Transmission of cutaneous leishmaniasis in Venezuela reveals diverse and changing epidemiological landscapes, as well as a spectrum of clinical phenotypes presumed to be linked to a variety of Leishmania species. Central-western Venezuela constitutes one of the highest endemic epicenters in the country, and updated molecular epidemiological information is still lacking. Therefore, in this study we aimed to characterize the landscape of circulating Leishmania species across central-western Venezuela through the last two decades, performed comparisons of haplotype and nucleotide diversity, and built a geospatial map of parasite species distribution. A total of 120 clinical samples were collected from patients across the cutaneous disease spectrum, retrieving parasitic DNA, and further characterizing by PCR and sequencing of the HSP70 gene fragment. This data was later collated with further genetic, geospatial and epidemiological analyses. A peculiar pattern of species occurrence including Leishmania (Leishmania) amazonensis (77.63% N=59), Leishmania (Leishmania) infantum (14.47% N=11), Leishmania (Viannia) panamensis (5.26% N=4) and Leishmania (Viannia) braziliensis (2.63% N=2) was revealed, also highlighting a very low genetic diversity amongst all analyzed sequences. Geographical distribution showed that most cases are widely distributed across the greater urban-sub urban area of the Irribaren municipality. L.(L.) amazonensis appears to be widely dispersed throughout Lara state. Statistical analyses failed to reveal significance for any comparisons, leading to conclude a lack of association between the infective Leishmania species and clinical phenotypes. To the best of our knowledge, this is an unprecedented study which addresses comprehensively the geographical distribution of Leishmania species in central-western Venezuela throughout the last two decades, and the first to incriminate L. (L.) infantum as an etiologic agent of cutaneous leishmaniasis in this region. Our findings support that Leishmania endemism in central-western Venezuela is caused mainly by L.(L.) amazonensis. Future studies are needed to unveil additional details on the ecological intricacies and transmission aspects of leishmaniasis (i.e. sampling phlebotomines and mammals) and to adopt adequate public health prevention and control strategies and mitigate disease impact in this endemic region.

Polyunsaturated fatty acids alter the formation of lipid droplets and eicosanoid production in Leishmania promastigotes.

BACKGROUND: The knowledge about eicosanoid metabolism and lipid droplet (LD) formation in the Leishmania is very limited and new approaches are needed to identify which bioactive molecules are produced of them. OBJECTIVES: Herein, we compared LDs and eicosanoids biogenesis in distinct Leishmania species which are etiologic agents of different clinical forms of leishmaniasis. METHODS: For this, promastigotes of Leishmania amazonensis, L. braziliensis and L. infantum were stimulated with polyunsaturated fatty acids (PUFA) and LD and eicosanoid production was evaluated. We also compared mutations in structural models of human-like cyclooxygenase-2 (GP63) and prostaglandin F synthase (PGFS) proteins, as well as the levels of these enzymes in parasite cell extracts. FINDINGS: PUFAs modulate the LD formation in L. braziliensis and L. infantum. Leishmania spp with equivalent tissue tropism had same protein mutations in GP63 and PGFS. No differences in GP63 production were observed among Leishmania spp, however PGFS production increased during the parasite differentiation. Stimulation with arachidonic acid resulted in elevated production of hydroxyeicosatetraenoic acids compared to prostaglandins. MAIN CONCLUSIONS: Our data suggest LD formation and eicosanoid production are distinctly modulated by PUFAS dependent of Leishmania species. In addition, eicosanoid-enzyme mutations are more similar between Leishmania species with same host tropism.

Comparison of Sampling Procedures for the Molecular Diagnosis of Leishmaniases.

The present work evaluates sampling protocols, storage procedures, and DNA purification methods for Leishmania spp. detection and quantification in different biological samples. The efficiency of three preservation solutions, a phosphate buffer solution, an ethylenediaminetetraacetic acid (EDTA) buffer solution, and 70% ethanol, was compared in combination with three DNA extraction protocols: a commercial silica column kit, salting-out protein precipitation, and organic extraction with phenol-chloroform. Tissue samples from BALB/c mice experimentally infected with Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, or Leishmania (Leishmania) infantum were stored in the three preservation solutions and subsequently subjected to the three different DNA extraction methods. The extracted DNA was then used in real-time polymerase chain reaction (PCR) assays for the detection and quantification of parasite ribosomal small subunit DNA targets as well as mammalian glyceraldehyde-3-phosphate dehydrogenase (gapdh) targets. The results of the optimized protocols showed that the DNA extraction method did not influence test quality, but DNA from samples preserved with the EDTA buffer solution produced higher amounts of target amplicons. Based on these results, we concluded that samples from suspected cases of leishmaniasis for submission to molecular diagnostic procedures should be preferentially preserved in EDTA, followed by any one of the DNA purification methods evaluated.

Study on the zoonotic cycle of tegumentary leishmaniasis in an endemic area of a metropolitan region in the Northeastern region of Brazil.

This study was conducted to characterize the transmission cycle of the tegumentary leishmaniasis (TL) in an old colonization area at Pernambuco State, Brazil. The aims were to identify autochthonous cases, sandflies fauna, domestic animals as possible reservoir hosts and the Leishmania species involved in this endemic area. A total of 168 suspected human cases of TL and 272 domestic animals (canine, feline, equine, goat, and sheep) were included. The sandflies were captured and identified by species. Patients were predominantly male and the average age was 37+18.1 years old. Of 85 patients who had skin lesions, 25.6% of them had direct positive smears for TL and 34 isolates were identified as Leishmania (Viannia) braziliensis. The confirmation for TL diagnosed by molecular detection (PCR) was almost three times more sensitive than the direct test [p < 0.001; PR = 2.72] associated with clinical examination. The Kappa test on PCR between two different specimens, biopsy, and skin lesion swab was 60.8% (p < 0.001). More than 200 specimens of sandflies (80 males and 159 females) were captured and identified as Lutzomyia whitmani (99.6%) and Lu. evandroi (0.4%). The detection of L. (V.) braziliensis by Real-Time PCR in the blood of a captured fed female was positive in 59.3% of Lu. whitmani. Of the 272 domestic animals included, 61.76% were male (n = 168). Thirty-six animals (13.2%) had lesions compatible with TL (34 dogs, 1 cat and 1 sheep) and 3 of them, all dogs, had lesions on the snout, showing destruction of cartilage and mucosa. The study suggests the participation of domestic animals as possible reservoirs. However, further studies are necessary to better understand the transmission cycle and take recommended measures in order to control the disease.

Leishmania V. braziliensis infection in asymptomatic domestic animals within an endemic region in the Northeast of Brazil.

BACKGROUND: American cutaneous leishmaniasis is a commonly neglected, vector-borne tropical parasitic disease that is a major public health concern in Brazil. Leishmania (Viannia) braziliensis is the main species associated with the disease. Accurate diagnosis is based on epidemiological surveillance, clinical assessment, and laboratory testing. Leishmania (V.) braziliensis has been detected in several wild and synanthropic mammals. Their epidemiological role has not been entirely elucidated. This study aimed to assess potential L. braziliensis infections in asymptomatic domestic animals, by molecular and serological testing in endemic areas, in the metropolitan region of Recife. METHODS: Blood samples and conjunctival fluids were collected from 232 animals (canids, felids, equines, and caprines) for the detection of L. braziliensis using molecular tests (conventional and real-time polymerase chain reaction [PCR and qPCR]). For immunological detection, blood samples from 115 dogs were assessed using enzyme-linked immunosorbent assay. RESULTS: Real-time quantitative PCR showed positive results for blood and conjunctival samples in all investigated species. The results of the blood and conjunctival samples were 68.2% and 26.9% in Canis familiaris, 100% and 41.7% in Felis catus, 77.3% and 30.8% in Equus caballus/Equus asinus, and 50% and 33.3% in Capra hircus samples, respectively. CONCLUSIONS: Results from this study adds valuable information to our understanding of the role of asymptomatic domestic animals, L. braziliensis life cycle, and American cutaneous leishmaniasis in Northeast Brazil.

Clinical-therapeutic follow-up of patients with American cutaneous leishmaniasis caused by different Leishmania spp. in Brazil.

American cutaneous leishmaniasis (ACL) may present different clinical manifestations, immune and therapeutic responses, depending on the Leishmania species, as well as inoculum size and factors inherent to the affected individual. Thus, the aim of this study was to carry out clinical-therapeutic follow-up of Brazilian patients with ACL caused by different Leishmania species. Between 2015 and 2018, patients with ACL from Amazonas and Pernambuco states (Brazil) were submitted to blood collection before and after treatment. The qPCR technique was used to quantify the parasite load. To identify the Leishmania species, one of the following techniques was employed: a conventional PCR performed from biopsy or blood DNA, followed by sequencing; or Multilocus Enzyme Electrophoresis from Leishmania isolated from biopsy/aspirated lesion. A total of 10.8% (23/213) of the patients included in positive cases were followed-up. All 23 patients were clinically and epidemiologically compatible with ACL and were also positive in parasitological tests (86.96%), molecular tests (73.91%) or both (60.87%). Seventeen samples collected before treatment and 11 collected after treatment were positive in the qPCR assay, with a mean parasite load (MPL) of 38.33 fg/µL and 11.81 fg/µL, respectively. Eight samples were positive in both collections. Thirteen patients (56.52%) were clinically cured (wound healing). Ten patients (43.47%) were not clinically cured at the time of return with the attending physician. Identification of Leishmania species was carried out in samples from nine patients, and six were identified as L. (Viannia) braziliensis, 2 as L (Viannia) guyanensis and 1 as L (Leishmania) amazonensis. One patient infected with L. guyanensis and other with L. braziliensis were not clinically cured and increased the mean parasite load after treatment. The data obtained from the followed-up patients and the relationship between clinical evolution and the infecting species demonstrate the need to understand its etiology to define the effective therapeutic protocol.

Multiparametric analysis of host and parasite elements in new world tegumentary leishmaniasis.

Tegumentary leishmaniasis is a tropical disease caused by protozoa of the genus Leishmania. Clinically, the disease presents a broad spectrum of symptoms, the mechanisms underlying the development of lesions remaining to be fully elucidated. In the present work, we performed a correlation and multiparametric analysis to evaluate how parasite- and host-related aspects associate with each other, and with the different clinical manifestations of tegumentary leishmaniasis. This cross-sectional study involved 75 individuals from endemic areas of Brazil, grouped according to their symptoms. Leishmania species were determined by DNA sequencing, and parasite load, antibody production, and cytokine profile were evaluated by kDNA qPCR, ELISA, and flow cytometry. Data were analyzed using the Chi-square test, principal component analysis, canonical discriminant analysis, and correlation analysis. Among the recruited patients, 23 (31%) were asymptomatic, 34 (45%) had primary cutaneous leishmaniasis, 10 (13%) presented recurrent cutaneous leishmaniasis, and eight (11%) had mucocutaneous leishmaniasis. Leishmania species identified included L. amazonensis, L. braziliensis, and L. guyanensis. Surprisingly, no Leishmania RNA virus infection was detected in any sample. In summary, our work showed that parasite load, antibody production, and cytokine levels alone are not determinants for tegumentary leishmaniasis symptoms. However, the correlation analysis allowed us to observe how these factors are correlated to each other within the groups, which revealed a unique network for each clinical manifestation. Our work reinforces the complexity of tegumentary leishmaniasis outcomes - which are associated with multiple host and parasite-related elements and provides a holistic model of the disease.

Leishmania braziliensis causing human disease in Northeast Brazil presents loci with genotypes in long-term equilibrium.

BACKGROUND: Leishmaniases are neglected tropical diseases that inflict great burden to poor areas of the globe. Intense research has aimed to identify parasite genetic signatures predictive of infection outcomes. Consistency of diagnostic tools based on these markers would greatly benefit from accurate understanding of Leishmania spp. population genetics. We explored two chromosomal loci to characterize a population of L. braziliensis causing human disease in Northeast Brazil. METHODOLOGY/PRINCIPAL FINDINGS: Two temporally distinct samples of L. braziliensis were obtained from patients attending the leishmaniasis clinic at the village of Corte de Pedra: (2008-2011) primary sample, N = 120; (1999-2001) validation sample, N = 35. Parasites were genotyped by Sanger's sequencing of two 600 base pairs loci starting at nucleotide positions 3,074 and 425,451 of chromosomes 24 and 28, respectively. Genotypes based on haplotypes of biallelic positions in each locus were tested for several population genetic parameters as well as for geographic clustering within the region. Ample geographic overlap of genotypes at the two loci was observed as indicated by non-significant Cusick and Edward's comparisons. No linkage disequilibrium was detected among combinations of haplotypes for both parasite samples. Homozygous and heterozygous genotypes displayed Hardy-Weinberg equilibrium (HWE) at both loci in the two samples when straight observed and expected counts were compared by Chi-square (p>0.5). However, Bayesian statistics using one million Monte-Carlo randomizations disclosed a less robust HWE for chromosome 24 genotypes, particularly in the primary sample (p = 0.04). Fixation indices (Fst) were consistently lower than 0.05 among individuals of the two samples at both tested loci, and no intra-populational structuralization could be detected using STRUCTURE software. CONCLUSIONS/SIGNIFICANCE: These findings suggest that L. braziliensis can maintain stable populations in foci of human leishmaniasis and are capable of robust genetic recombination possibly due to events of sexual reproduction during the parasite's lifecycle.

Genetic diversity and population structure of Leishmania (Viannia) braziliensis in the Peruvian jungle.

BACKGROUND: Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis is highly prevalent in the Peruvian jungle, where it affects military forces deployed to fight against drug trafficking and civilian people that migrate from the highland to the lowland jungle for economic activities such as mining, agriculture, construction, and chestnut harvest. We explored the genetic diversity and population structure of 124 L. (V.) braziliensis isolates collected from the highland (Junín, Cusco, and Ayacucho) and lowland Peruvian jungle (Loreto, Ucayali, and Madre de Dios). All samples were genotyped using Multilocus Microsatellite Typing (MLMT) of ten highly polymorphic markers. PRINCIPAL FINDINGS: High polymorphism and genetic diversity were found in Peruvian isolates of L. (V.) braziliensis. Most markers are not in Hardy-Weinberg equilibrium; this deviation is most likely caused by local inbreeding, as shown by the positive FIS values. Linkage Disequilibrium in subpopulations was not strong, suggesting the reproduction was not strictly clonal. Likewise, for the first time, two genetic clusters of this parasite were determined, distributed in both areas of the Peruvian jungle, which suggested a possible recent colonization event of the highland jungle from the lowland jungle. CONCLUSIONS: L. (V.) braziliensis exhibits considerable genetic diversity with two different clusters in the Peruvian jungle. Migration analysis suggested a colonization event between geographical areas of distribution. Although no human migration was observed at the time of sampling, earlier displacement of humans, reservoirs, or vectors could have been responsible for the parasite spread in both regions.