Multiple evolutionary lineages for the main vector of Leishmania guyanensis, Lutzomyia umbratilis (Diptera: Psychodidae), in the Brazilian Amazon.

Lutzomyia umbratilis is the main vector of Leishmania guyanensis in the Brazilian Amazon and in neighboring countries. Previous biological and molecular investigations have revealed significant differences between L. umbratilis populations from the central Brazilian Amazon region. Here, a phylogeographic survey of L. umbratilis populations collected from nine localities in the Brazilian Amazon was conducted using two mitochondrial genes. Statistical analyses focused on population genetics, phylogenetic relationships and species delimitations. COI genetic diversity was very high, whereas Cytb diversity was moderate. COI genealogical haplotypes, population structure and phylogenetic analyses identified a deep genetic differentiation and three main genetic groups. Cytb showed a shallower genetic structure, two main haplogroups and poorly resolved phylogenetic trees. These findings, allied to absence of isolation by distance, support the hypothesis that the Amazon and Negro Rivers and interfluves are the main evolutionary forces driving L. umbratilis diversification. The main three genetic groups observed represent three evolutionary lineages, possibly species. The first lineage occurs north of the Amazon River and east of Negro River, where Le. guyanensis transmission is intense, implying that L. umbratilis is an important vector there. The second lineage is in the interfluve between north of Amazon River and west of Negro River, an area reported to be free of Le. guyanensis transmission. The third lineage, first recorded in this study, is in the interfluve between south of Amazonas River and west of Madeira River, and its involvement in the transmission of this parasite remains to be elucidated.

Diagnosis and identification of Leishmania species in patients with cutaneous leishmaniasis in the state of Roraima, Brazil's Amazon Region.

BACKGROUND: Cutaneous leishmaniasis (CL) is an endemic disease in Brazil that is highly prevalent in the northern region of the country. Although there is a continuous and growing number of cases registered in the state of Roraima, there is limited information regarding the species of Leishmania that affect the human population. In this study, we aimed to characterize which Leishmania species cause human disease in those presenting with cutaneous leishmaniasis in endemic areas of the State of Roraima. METHODS: We conducted a prospective surveillance study between 2016 to 2018 in health centers located in the State of Roraima, Brazil. Participants with clinical suspicion of CL were enrolled and provided lesion samples for parasitological detection by microscopy. A subset of the samples was tested by polymerase chain reaction and sequencing of the internal transcribed spacer 1 (ITS-1 PCR) for molecular species identification. RESULTS: A total of 262 participants were enrolled in this study. Of those, 129 (49.27%) were positive by parasitological examination. Most positive subjects (81.58%) were male, and most cases presented a single lesion (80.26%). ITS-1 PCR and sequencing on a subset of 76 samples allowed us to detect nine different species of Leishmania: L. (V.) braziliensis, L (V.) panamensis, L. (V.) guyanensis, L. (V.) naiffi, L. (V.) shawi, L.(V.) utingensis, L. (V.) lindenbergi, L. (L.) amazonensis and L. (L.) mexicana. CONCLUSIONS: Our study provides the first assessment of circulating species of Leishmania in the State of Roraima, Brazil, and shows the high diversity in this region. This study opens the path for further research on the transmission of leishmaniasis in the northernmost Brazilian State including vector and reservoir surveillance as well as for intensification of investigation and control activities against CL in the region.

Antileishmanial activity of synthetic analogs of the naturally occurring quinolone alkaloid N-methyl-8-methoxyflindersin.

Leishmaniasis is a neglected, parasitic tropical disease caused by an intracellular protozoan from the genus Leishmania. Quinoline alkaloids, secondary metabolites found in plants from the Rutaceae family, have antiparasitic activity against Leishmania sp. N-methyl-8-methoxyflindersin (1), isolated from the leaves of Raputia heptaphylla and also known as 7-methoxy-2,2-dimethyl-2H,5H,6H-pyran[3,2-c]quinolin-5-one, shows antiparasitic activity against Leishmania promastigotes and amastigotes. This study used in silico tools to identify synthetic quinoline alkaloids having structure similar to that of compound 1 and then tested these quinoline alkaloids for their in vitro antiparasitic activity against Leishmania (Viannia) panamensis, in vivo therapeutic response in hamsters suffering from experimental cutaneous leishmaniasis (CL), and ex vivo immunomodulatory potential in healthy donors' human peripheral blood (monocyte)-derived macrophages (hMDMs). Compounds 1 (natural), 2 (synthetic), and 8 (synthetic) were effective against intracellular promastigotes (9.9, 3.4, and 1.6 µg/mL medial effective concentration [EC50], respectively) and amastigotes (5.07, 7.94, and 1.91 µg/mL EC50, respectively). Compound 1 increased nitric oxide production in infected hMDMs and triggered necrosis-related ultrastructural alterations in intracellular amastigotes, while compound 2 stimulated oxidative breakdown in hMDMs and caused ultrastructural alterations in the parasite 4 h posttreatment, and compound 8 failed to induce macrophage modulation but selectively induced apoptosis of infected hMDMs and alterations in the intracellular parasite ultrastructure. In addition, synthetic compounds 2 and 8 improved the health of hamsters suffering from experimental CL, without evidence of treatment-associated adverse toxic effects. Therefore, synthetic compounds 2 and 8 are potential therapeutic candidates for topical treatment of CL.

Eligibility for Local Therapies in Adolescents and Adults with Cutaneous Leishmaniasis from Southwestern Colombia: A Cross-Sectional Study.

Local therapies have been proposed as safe and effective alternatives to systemic drugs in cutaneous leishmaniasis (CL), especially among less severe cases. However, they are not widely available and used in endemic places, including Colombia, which has a high burden of disease. Further complicating the uptake of local therapies is that different treatment guidelines have been established by the World Health Organization (WHO) and Pan American Health Organization (PAHO). Using data from a large referral center in Colombia, we determined the proportion of patients who would be eligible for and potentially benefit from local therapies according to both international guidelines. The sample included 1,891 confirmed cases of CL aged ≥ 12 years, mostly infected with Leishmania Viannia panamensis (91%, n = 601/660), between 2004 and 2014. Overall, 57% of the sample had one lesion, whereas another 31% had two to three lesions. For 74% of patients, all lesions were in an area other than head or neck. The maximum lesion size was ≤ 3 cm for 58% and < 5 cm for 88% of the sample. Based on our data, up to 56% of patients could have been eligible for local therapies according to the WHO criteria. By contrast, only 23% were eligible according to the more restrictive PAHO criteria. Regardless, these data suggest that a substantial proportion of CL patients in Colombia may benefit from local therapies given their relatively mild presentation of disease and low risk of complications. Individualized risk-benefit assessment and guideline adjustments may increase local therapy eligibility and benefit a large number of patients.

Evaluation of point of care tests for the diagnosis of cutaneous leishmaniasis in Suriname.

BACKGROUND: Cutaneous leishmaniasis (CL) is a serious health problem in Suriname. To expand the diagnostic options, two newly developed diagnostic tests, i.e. the rapid diagnostic test CL Detect™ Rapid Test (CL Detect) and the Loopamp™ Leishmania Detection Kit (Loopamp) were evaluated. METHODS: Diagnostic test performance was compared to the routine diagnostic approach in place, i.e. clinical symptoms combined with microscopy, and to polymerase chain reaction (PCR), which was used as a reference standard. The study population (n = 93) was a typical representation of the CL affected population in Suriname and mainly infected with Leishmania guyanensis. RESULTS: CL Detect had a very low sensitivity compared to microscopy (36.7%) or PCR (35.8%), due to a high number of false negative results. The specificity of the CL Detect compared to microscopy and PCR was 85.7 and 83.3% respectively. Loopamp sensitivity was 84.8% compared to microscopy and 91.4% compared to PCR. The Loopamp test had a moderate specificity (42.9%) compared to microscopy, but a good specificity compared to PCR (91.7%). CONCLUSION: The CL Detect is not likely to be a good replacement for the routine diagnostic procedure for CL in Suriname. The high sensitivity of the easy to perform Loopamp enables the implementation of sensitive molecular diagnosis in resource limited settings.

Genomic Analysis of Colombian Leishmania panamensis strains with different level of virulence.

The establishment of Leishmania infection in mammalian hosts and the subsequent manifestation of clinical symptoms require internalization into macrophages, immune evasion and parasite survival and replication. Although many of the genes involved in these processes have been described, the genetic and genomic variability associated to differences in virulence is largely unknown. Here we present the genomic variation of four Leishmania (Viannia) panamensis strains exhibiting different levels of virulence in BALB/c mice and its application to predict novel genes related to virulence. De novo DNA sequencing and assembly of the most virulent strain allowed comparative genomics analysis with sequenced L. (Viannia) panamensis and L. (Viannia) braziliensis strains, and showed important variations at intra and interspecific levels. Moreover, the mutation detection and a CNV search revealed both base and structural genomic variation within the species. Interestingly, we found differences in the copy number and protein diversity of some genes previously related to virulence. Several machine-learning approaches were applied to combine previous knowledge with features derived from genomic variation and predict a curated set of 66 novel genes related to virulence. These genes can be prioritized for validation experiments and could potentially become promising drug and immune targets for the development of novel prophylactic and therapeutic interventions.

Leishmania (Viannia) guyanensis in tegumentary leishmaniasis.

Leishmania (Viannia) guyanensis is a causal agent of American tegumentary leishmaniasis (ATL). This protozoan has been poorly investigated; however, it can cause different clinical forms of ATL, ranging from a single cutaneous lesion to severe lesions that can lead to destruction of the nasopharyngeal mucosa. L. (V.) guyanensis and the disease caused by this species can present unique aspects revealing the need to better characterize this parasite species to improve our knowledge of the immunopathological mechanisms and treatment options for ATL. The mechanisms by which some patients develop a more severe form of ATL remain unclear. It is known that the host immune profile and parasite factors may influence the clinical manifestations of the disease. Besides intrinsic parasite factors, Leishmaniavirus RNA 1 (LRV1) infecting L. guyanensis can contribute to ATL immunopathogenesis. In this review, general aspects of L. guyanensis infection in humans and mouse models are presented.

Failure of fluconazole in treating cutaneous leishmaniasis caused by Leishmania guyanensis in the Brazilian Amazon: An open, nonrandomized phase 2 trial.

BACKGROUND: The treatment of Leishmaniasis caused by Leishmania (Viannia) guyanensis is based on a weak strength of evidence from very few clinical trials and some case series reports. Current treatment guidelines recommend pentamidine isethionate or meglumine antimoniate (Glucantime) as the first-line choices. Both are parenteral drugs with a low therapeutic indexes leading to a high risk of undesired effects. Imidazole derivatives interfere with the production of leishmanial ergosterol, an essential component of their membrane structure. One drug that has been studied in different clinical presentations of Leishmania is fluconazole, a hydrophilic bis-triazole, which is easily absorbed through the oral route with a low toxicity profile and is considered safe for children. This drug is readily available in poor countries with a reasonable cost making it a potential option for treating leishmaniasis. METHODS AND FINDINGS: An adaptive nonrandomized clinical trial with sequential groups with dose escalation of oral fluconazole was designed to treat adult men with localized cutaneous leishmaniasis (LCL) in Manaus, Brazil. Eligible participants were patients with LCL with confirmed Leishmania guyanensis infection. RESULTS: Twenty adult male patients were treated with 450 mg of fluconazole daily for 30 days. One patient (5%) was cured within 30 days of treatment. Of the 19 failures (95%), 13 developed a worsening of ulcers and six evolved lymphatic spreading of the disease. Planned dose escalation was suspended after the disappointing failure rate during the first stage of the trial. CONCLUSION/SIGNIFICANCE: Oral fluconazole, at the dose of 450mg per day, was not efficacious against LCL caused by Leishmania guyanensis in adult men. TRIAL REGISTRATION: Brazilian Clinical Trial Registration (ReBec)-RBR-8w292w; UTN number-1158-2421.

A Case of Cutaneous Leishmaniasis guyanensis Mimicking Otitis Externa.

Cutaneous leishmaniasis typically presents as a painless papule progressing to an ulcer or plaque. In this case study of the ear, the disease manifested as a small painful bump progressing into redness and swelling about the ear with purulent drainage. After multiple oral/intravenous antipseudomonal, antistaphylococcal, and antifungal treatments, there was no improvement. The skin progressed to an erythematous plaque and hemorrhagic ulcer; punch biopsy and speciation revealed Leishmaniasis guyanensis. The patient was switched to a seven-dose course of intravenous L-amphotericin B (visceral leishmaniasis protocol). Within 21 days, pain and edema resolved and the ulcers healed. Three-month follow-up demonstrated no recurrence. Further studies are needed to evaluate the use of L-amphotericin B in Leishmaniasis guyanensis.

Tilting the balance between RNA interference and replication eradicates Leishmania RNA virus 1 and mitigates the inflammatory response.

Many Leishmania (Viannia) parasites harbor the double-stranded RNA virus Leishmania RNA virus 1 (LRV1), which has been associated with increased disease severity in animal models and humans and with drug treatment failures in humans. Remarkably, LRV1 survives in the presence of an active RNAi pathway, which in many organisms controls RNA viruses. We found significant levels (0.4 to 2.5%) of small RNAs derived from LRV1 in both Leishmania braziliensis and Leishmania guyanensis, mapping across both strands and with properties consistent with Dicer-mediated cleavage of the dsRNA genome. LRV1 lacks cis- or trans-acting RNAi inhibitory activities, suggesting that virus retention must be maintained by a balance between RNAi activity and LRV1 replication. To tilt this balance toward elimination, we targeted LRV1 using long-hairpin/stem-loop constructs similar to those effective against chromosomal genes. LRV1 was completely eliminated, at high efficiency, accompanied by a massive overproduction of LRV1-specific siRNAs, representing as much as 87% of the total. For both L. braziliensis and L. guyanensis, RNAi-derived LRV1-negative lines were no longer able to induce a Toll-like receptor 3-dependent hyperinflammatory cytokine response in infected macrophages. We demonstrate in vitro a role for LRV1 in virulence of L. braziliensis, the Leishmania species responsible for the vast majority of mucocutaneous leishmaniasis cases. These findings establish a targeted method for elimination of LRV1, and potentially of other Leishmania viruses, which will facilitate mechanistic dissection of the role of LRV1-mediated virulence. Moreover, our data establish a third paradigm for RNAi-viral relationships in evolution: one of balance rather than elimination.

Mammalian Innate Immune Response to a Leishmania-Resident RNA Virus Increases Macrophage Survival to Promote Parasite Persistence.

Some strains of the protozoan parasite Leishmania guyanensis (L.g) harbor a viral endosymbiont called Leishmania RNA virus 1 (LRV1). LRV1 recognition by TLR-3 increases parasite burden and lesion swelling in vivo. However, the mechanisms by which anti-viral innate immune responses affect parasitic infection are largely unknown. Upon investigating the mammalian host's response to LRV1, we found that miR-155 was singularly and strongly upregulated in macrophages infected with LRV1+ L.g when compared to LRV1- L.g. LRV1-driven miR-155 expression was dependent on TLR-3/TRIF signaling. Furthermore, LRV1-induced TLR-3 activation promoted parasite persistence by enhancing macrophage survival through Akt activation in a manner partially dependent on miR-155. Pharmacological inhibition of Akt resulted in a decrease in LRV1-mediated macrophage survival and consequently decreased parasite persistence. Consistent with these data, miR-155-deficient mice showed a drastic decrease in LRV1-induced disease severity, and lesional macrophages from these mice displayed reduced levels of Akt phosphorylation.

Identification and Biological Characterization of Leishmania (Viannia) guyanensis Isolated from a Patient with Tegumentary Leishmaniasis in Goiás, a Nonendemic Area for This Species in Brazil.

The aim of this study was to characterize clinical field isolates of Leishmania spp. obtained from patients with American Tegumentary Leishmaniasis (ATL) who live in Goiás state, Brazil. The presumed areas of infection were in Goiás, Tocantins, and Pará states. Three isolates of parasites were identified as L. (Viannia) braziliensis and one as L. (V.) guyanensis. The in vitro growth profiles were found to be similar for all parasites. Nevertheless, in C57BL/6 mice, L. (V.) guyanensis infection was better controlled than L. (V.) braziliensis. Yet in C57BL/6 mice deficient in interferon gamma, L. (V.) guyanensis lesions developed faster than those caused by L. (V.) braziliensis isolates. In BALB/c mice, the development of lesions was similar for isolates from both species; however, on the 11th week of infection, amastigotes could not be observed in macrophages from L. (V.) guyanensis-infected mice. Thus, L. (V.) guyanensis can be circulating in Goiás, a state where autochthonous cases of this species had not yet been reported. Considering the difficulties to differentiate L. (V.) guyanensis from L. (V.) braziliensis at the molecular, morphological, and clinical (human and murine models) levels, the presence of L. (V.) guyanensis infections is possibly underestimated in several regions of Brazil.

Functional variations in MBL2 gene are associated with cutaneous leishmaniasis in the Amazonas state of Brazil.

Functional variations in the mannose-binding lectin (MBL2) gene causing low levels of serum MBL are associated with susceptibility to numerous infectious diseases. We investigated whether there is genetic association of MBL2 variant alleles with cutaneous leishmaniasis (CL) caused by Leishmania guyanensis. We used PCR-restriction fragment length polymorphism to genotype six MBL2 variants, three in the promoter region and three in the exon 1. An association was noted between the single nucleotide polymorphism -221X/Y of the MBL2 gene and CL (P=2.9 × 10(-6); odds ratio (OR)=1.9 (1.4-2.5) consistent with the hypothesis that the -221X allele confers high risk to development of CL among L. guyanensis-infected individuals. Furthermore, L. guyanensis-infected individuals bearing the codon 57 allele C had a higher risk of developing CL (P=5 × 10(-5); OR=1.9 (1.4-2.6)). The low MBL expressor haplotype LXPB was also associated to CL (P=6 × 10(-4)). This study raises the possibility that functional polymorphisms in MBL2 gene play a role in clinical outcome of Leishmania infection.

An epidemic outbreak of canine cutaneous leishmaniasis in Colombia caused by Leishmania braziliensis and Leishmania panamensis.

The largest recorded outbreak of cutaneous leishmaniasis in Colombia's history occurred during 2005-2009 in soldiers of the Colombian Army, with ~40,000 cases. This outbreak was caused by the influx of military personnel into the jungle with the mission of combat illicit crops and the guerrilla. The soldiers remain for long periods within the rainforest and are exposed to the bite of infected sand flies. During the military activities, soldiers work with dogs specially trained to detect landmines, and therefore, dogs are also exposed to the infected sand flies and show high incidence of cutaneous leishmaniasis (CL). This work describes an epidemic outbreak of canine CL caused by Leishmania braziliensis and Leishmania panamensis in Colombia, South America. The clinical features of the disease and the response to treatment with pentavalent antimonials observed in 72 guard dogs from the Colombian Army are described. A program for prevention and control of canine CL is also discussed.

First case of cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Suriname.

The main causative agent of cutaneous leishmaniasis (CL) in Suriname is Leishmania (Viannia) guyanensis. This case report presents a patient infected with Leishmania (Viannia) braziliensis, a species never reported before in Suriname. This finding has clinical implications, because L. braziliensis has a distinct clinical phenotype characterized by mucocutaneous leishmaniasis, a more extensive and destructive form of CL that requires different treatment. Clinicians should be aware that chronic cutaneous ulcers in patients from the Guyana region could be caused by L. braziliensis.

Wild, synanthropic and domestic hosts of Leishmania in an endemic area of cutaneous leishmaniasis in Minas Gerais State, Brazil.

Domestic, synanthropic and wild hosts of Leishmania spp. parasites were studied in an area endemic for American tegumentary leishmaniasis (ATL), specifically in northern Minas Gerais State, Brazil. Domestic dogs and small forest mammals are reservoir hosts for L. (Leishmania) infantum. However, the role that these animals play in the transmission cycle of the Leishmania spp. that cause cutaneous leishmaniasis is not well known. This study evaluated 72 rodents, 25 marsupials and 98 domestic dogs found in two villages of the Xakriabá Indigenous Territory, an area of intense ATL transmission. A total of 23 dogs (23.47%) were shown to be positive according to at least one test; 8 dogs (8.16%) tested positive in a single serological test and 15 dogs (15.31%) tested positive by IFAT and ELISA. Eleven dogs were euthanised to allow for molecular diagnosis, of which nine (81.8%) tested positive by PCR for Leishmania in at least one tissue. Seven animals were infected only with L. (L.) infantum, whilst two displayed a mixed infection of L. (L.) infantum and L. (V.) braziliensis. Isoenzymatic characterisation identified L. (L.) infantum parasites isolated from the bone marrow of two dogs. Of the 97 small mammals captured, 24 tested positive for Leishmania by PCR. The results showed that L. (V.) braziliensis, L. (L.) infantum and L. (V.) guyanensis are circulating among wild and synanthropic mammals present in the Xakriabá Reserve, highlighting the epidemiological diversity of ATL in this region.

Leishmania RNA virus controls the severity of mucocutaneous leishmaniasis.

Mucocutaneous leishmaniasis is caused by infections with intracellular parasites of the Leishmania Viannia subgenus, including Leishmania guyanensis. The pathology develops after parasite dissemination to nasopharyngeal tissues, where destructive metastatic lesions form with chronic inflammation. Currently, the mechanisms involved in lesion development are poorly understood. Here we show that metastasizing parasites have a high Leishmania RNA virus-1 (LRV1) burden that is recognized by the host Toll-like receptor 3 (TLR3) to induce proinflammatory cytokines and chemokines. Paradoxically, these TLR3-mediated immune responses rendered mice more susceptible to infection, and the animals developed an increased footpad swelling and parasitemia. Thus, LRV1 in the metastasizing parasites subverted the host immune response to Leishmania and promoted parasite persistence.

Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis.

The levels of regulatory T cells (Treg cells), analyzed by Foxp3 mRNA expression, were determined in lesions from patients with acute cutaneous leishmaniasis (ACL) and chronic cutaneous leishmaniasis (CCL). We demonstrated that Treg cells preferentially accumulate in lesions from ACL patients during the early phase of infection (lesion duration of less than 1 month). In addition, levels of Foxp3 mRNA transcripts were significantly higher in specimens from patients with CCL than in those from patients with ACL, suggesting a critical role of intralesional Treg cells in CCL. Intralesional Treg cells from both ACL and CCL patients were shown to have suppressive functions in vitro, since they inhibited the gamma interferon (IFN-gamma) produced by CD4(+) CD25(-) T cells purified from peripheral blood mononuclear cells from the same patient in response to Leishmania guyanensis stimulation. Intralesional 2,3-indoleamine dioxygenase (IDO) mRNA expression was associated with that of Foxp3, suggesting a role for IDO in the suppressive activity of intralesional Treg cells. In addition, a role, albeit minor, of interleukin-10 (IL-10) was also demonstrated, since neutralization of IL-10 produced by intralesional T cells increased IFN-gamma production by effector cells in an in vitro suppressive assay. These results confirm the role of intralesional Treg cells in the immunopathogenesis of human Leishmania infection, particularly in CCL patients.