Clinical and parasitological features of Leishmania infection among gold miners in the Oiapoque basin, an international Brazil-French Guiana border.

Gold miners working illegally in mines live in poor health conditions related to their strenuous work and precarious housing. Therefore, they are at higher risk for infectious diseases. American tegumentary leishmaniasis (ATL) appears to be of great concern to the population living in the Guiana Shield region. Our aim was to describe their demographic characteristics, the clinical features of cutaneous leishmaniasis (CL), and the frequency of Leishmania infection in people working in illegal gold mines in French Guiana. A cross-sectional study was carried out from October to December 2019 in Oiapoque city, Amapá, Brazil. Indeed, many gold miners working in French Guiana are originally from Brazil, and from Oiapoque in particular. A total of 105 participants from 31 different mining sites in French Guiana were recruited. Suspected Leishmania infection was confirmed by the following: detection of kDNA in blood or the lesion site; detection of specific antibodies; or detection of IFN-γ release after blood incubation with leishmanial antigens (IGRA-Leish). Nine active CL cases, 38 healed ATL (hATL) and 58 cases with no history of ATL (noATL), were identified. Only half of the treated hATL (50.0%; n = 14) reported having been assisted by a health care unit and the others treated themselves. PCR-kDNA for Leishmania was positive in the blood of 100% of CL cases. Curiously, blood PCR-kDNA was positive in 13% of hATL patients and in 15.5% of noATL patients. The IGRA-Leish was positive in 60.5% of hATL and in 37.9% of noATL. In addition to scars suggestive of CL, 71% of hATL had laboratory evidence of Leishmania infection. Restriction fragment polymorphism (RFLP) of the hsp70 gene identified a sympatric circulation of L. (V.) guyanensis (n = 4), L. (V.) braziliensis (n = 1), L. (L.) amazonensis (n = 2), L. (V.) shawi (n = 1) and L. (V.) naiffi/shawi (n = 1). Taking the laboratory techniques and the clinical evaluations together, 76% (n = 80) of the 105 participants had evidence of Leishmania infection. These results suggests that illegal gold miners working in French Guiana are at high risk for infection with different species of Leishmania, but their illegal condition and remoteness make it difficult for them to access health services.

Molecular investigation of Leishmania in sandflies (Diptera: Psychodidae) and rodents (Mammalia: Rodentia) in Nahavand, west of Iran.

Cutaneous leishmaniasis caused by different species of Leishmania is transmitted by Phlebotominae sandflies. This disease remains a public health concern in Iran. Therefore, the present study aimed to examine Leishmania infection in sandflies and reservoir rodents in six rural regions of Nahavand, located in western Iran. From May to October 2022, sandflies and rodents were collected and identified at the species level. Additionally, rodents' skin lesions and earlobe specimens were collected separately for microscopic and molecular examination. All specimens were tested for Leishmania DNA by PCRs targeting the parasite's ITS-2 and 18S rRNA gene and positive were Sanger sequenced. A total of 3396 sandflies belonging to seven subgenera and 11 species, i.e., Phlebotomus papatasi (42.7%), P. major (20.6%), P. mascitti (0.3%), P. neglectus (0.2%), P. alexandri (0.2%), P. turanicus (0.3%), Sergentomyia murgabiensis (18.1%), S. dentata (10.5%), S. theodori (5.8%), S. antennata (1.1%), and S. pawlowski (0.1%) were identified. Based on the species population, 29 pools of sandflies were examined for the presence of Leishmania DNA using conventional PCR (cPCR), and individual DNAs were tested when positive. Leishmania major DNA was detected in two P. papatasi and Leishmania sp. in one P. major individual sandfly. This is the first report of Leishmania infection in sandflies from Hamadan province. The captured rodents (n = 61) belonged to four families and seven species, i.e., Arvicola amphibius (37.7%), Mus musculus (29.5%), Microtus socialis (13.1%), Apodemus sylvaticus (11.5%), Talpa davidiana (4.9%), Apodemus witherbyi (1.6%), and Rattus norvegicus (1.6%). Microscopic and molecular examinations of the rodent lesions and earlobes scored negative results. The presence of Leishmania in the Phlebotominae sandflies in Nahavand indicates a potential threat to humans and animals in the region. Regular monitoring and examination of the sandflies' population and timely diagnosis and treatment of new patients are strongly recommended.

Amphotericin B resistance in Leishmania amazonensis: In vitro and in vivo characterization of a Brazilian clinical isolate.

In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L. amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L. amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.

Steppe lemmings and Chinese hamsters as new potential animal models for the study of the Leishmania subgenus Mundinia (Kinetoplastida: Trypanosomatidae).

Leishmania, the dixenous trypanosomatid parasites, are the causative agents of leishmaniasis currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania, and the recently described Mundinia, consisting of six species distributed sporadically all over the world infecting humans and/or animals. These parasites infect various mammalian species and also cause serious human diseases, but their reservoirs are unknown. Thus, adequate laboratory models are needed to enable proper research of Mundinia parasites. In this complex study, we compared experimental infections of five Mundinia species (L. enriettii, L. macropodum, L. chancei, L. orientalis, and four strains of L. martiniquensis) in three rodent species: BALB/c mouse, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus). Culture-derived parasites were inoculated intradermally into the ear pinnae and progress of infection was monitored for 20 weeks, when the tissues and organs of animals were screened for the presence and quantity of Leishmania. Xenodiagnoses with Phlebotomus duboscqi were performed at weeks 5, 10, 15 and 20 post-infection to test the infectiousness of the animals throughout the experiment. BALB/c mice showed no signs of infection and were not infectious to sand flies, while Chinese hamsters and steppe lemmings proved susceptible to all five species of Mundinia tested, showing a wide spectrum of disease signs ranging from asymptomatic to visceral. Mundinia induced significantly higher infection rates in steppe lemmings compared to Chinese hamsters, and consequently steppe lemmings were more infectious to sand flies: In all groups tested, they were infectious from the 5th to the 20th week post infection. In conclusion, we identified two rodent species, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus), as candidates for laboratory models for Mundinia allowing detailed studies of these enigmatic parasites. Furthermore, the long-term survival of all Mundinia species in steppe lemmings and their infectiousness to vectors support the hypothesis that some rodents have the potential to serve as reservoir hosts for Mundinia.

Ticks analysis for molecular detection and phylogenetic evaluation of stray dogs infecting protozoa from Alborz, Iran.

Ticks play an important role in the transmission of parasitic diseases, especially pathogenic protozoa in canine hosts, and it is very important to determine the role and extent of their infection with these pathogens in order to determine important control strategies. This study assessed the molecular prevalence of three protozoan pathogens including Hepatozoon canis, Leishmania spp. and Babesia spp., in ticks using PCR. A total 300 stray dogs were investigated and 691 ticks (171 male, 377 female and 143 nymph) were detected directly from 45 infested dogs. Species, stage of growth, and gender were determined for each tick. DNA extracted from 224 ticks (26 male, 165 female and 33 nymph). The molecular presence of three protozoan pathogens including Hepatozoon spp. (18S rRNA gene), Leishmania infantum (kinetoplastid minicircle DNA) and Babesia spp. (ssrRNA gene) were investigated using PCR method. One species of ticks, Rhipicephalus sanguineus was identified. Two of the target pathogens, Hepatozoon spp. (7/83; 8.43 %) and Babesia spp. (1/83; 1.2 %), were detected by PCR method. Sequence analysis of the ssrRNA gene of detected Babesia spp. showed a close relationship to the deposited strains of Babesia vulpis in the gene bank. To the best of our knowledge, this is the first study to undertake a phylogenetic analysis of H. canis and Babesia spp. in stray dogs in Alborz province, Iran and the first report about molecular detection of Babesia vulpis from tick infesting dogs in Iran. According to the above results, it seems necessary to implement tick control programs in dogs.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

A Geomedical Survey: Is There an Association Between Climatic Conditions and Leishmania Species Distribution in Iran During the Years 1999-2021?

PURPOSE: Iran is among the high-risk leishmaniasis regions in the world. WHO recommends the use of GIS as an ideal tool for healthcare authorities to predict the evolution of a disease, delimit the risk of outbreaks and identify critical areas. The aim of this research is to find the association between the main species of Leishmania (L. major, L. tropica, L. infantum) dispersion and climatic variables in Iran. METHODS: All molecular-based reports of leishmaniasis from Iran between 1999 and 2021 were gathered from reliable medical sources. Meteorological data (air and soil temperatures, annual rainfall and humidity) of the country along the study period were obtained from the Iranian Climatological Research Centre. The data concerning species distribution and climatic conditions during this period were moved to a base-map through raster layers using ArcGIS 10.4.1 software. The relationship between parasitological and climatic models was examined using ANOVA. RESULTS: High risk area maps, based on the cut-off thresholds, were generated for Leishmania major, L. tropica and L. infantum. According to the molecular-based reports, the L. major distribution was significantly related to all climatic variables, while L. tropica was merely related to rainfall and humidity, and the L. infantum distribution was significantly associated with rainfall, soil and air temperatures. CONCLUSION: The association between climatic conditions and Leishmania species distribution in Iran has been confirmed. Consequently, both, the relationship between climatic conditions and the geographical distribution of Leishmania species, and the use of GIS to better understand the spatial epidemiology of leishmaniasis, have been reaffirmed.

Chagas Disease and Leishmaniasis, Sympatric Zoonoses Present in Human from Rural Communities of Venezuela.

PURPOSE: Trypanosoma cruzi and Leishmania spp. coexist in several endemic areas, and there are few studies of Chagas disease and leishmaniasis coinfection worldwide; for this reason, the objective of this work was to determine the Chagas disease and leishmaniasis coinfection in several rural communities co-endemic for these diseases. METHODS: A total of 1107 human samples from six co-endemic rural communities of Cojedes state, Venezuela, were analyzed. Serum samples were evaluated by ELISA, indirect hemagglutination, and indirect immunofluorescence for Chagas disease diagnosis, and individuals were evaluated for leishmaniasis by leishmanin skin test (LST). Approximately, 30% of the individuals were also analyzed by PCR (blood clot samples) for T. cruzi and for Leishmania spp. RESULTS: The 14.7% of the individuals were positive to Trypanosoma cruzi infection by serology, and 25.8% were positive to Leishmania spp. current or past infection by LST. Among the group with PCR results, 7.8% were positive for T. cruzi, and 9.4% for Leishmania spp. The coinfection T. cruzi/Leishmania spp. was 6.5%. The T. cruzi DTUs of the positive blood clot samples were TcI, revealed using the molecular markers: (i) intergenic region of the miniexon, (ii) D7 divergent domain of the 24Sα rDNA, (iii) size-variable domain of the 18S rDNA, and (iv) hsp60-PCR-RFLP (EcoRV). The Leishmania species identified were L. (Leishmania) mexicana and L. (Viannia) braziliensis. CONCLUSION: A high prevalence was found for T. cruzi and Leishmania spp. single and coinfections in almost all communities studied, being these results of relevance for the implementation of control programs in co-endemic areas.

Evaluation of the CL Detect Rapid Test in Ethiopian patients suspected for Cutaneous Leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is common in Ethiopia, mainly affecting impoverished populations in rural areas with poor access to health care. CL is routinely diagnosed using skin slit smear microscopy, which requires skilled staff and appropriately equipped laboratories. We evaluated the CL Detect Rapid Test (InBios, Washington, USA), which is supplied with a dental broach sampling device, as a diagnostic alternative which could be used in field settings. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the diagnostic accuracy of the CL Detect Rapid Test on skin slit and dental broach samples from suspected CL patients at the Leishmaniasis Research and Treatment Center in Gondar, Ethiopia. A combined reference test of microscopy and PCR on the skin slit sample was used, which was considered positive if one of the two tests was positive. We recruited 165 patients consecutively, of which 128 (77.6%) were confirmed as CL. All microscopy-positive results (n = 71) were also PCR-positive, and 57 patients were only positive for PCR. Sensitivity of the CL Detect Rapid Test on the skin slit was 31.3% (95% confidence interval (CI) 23.9-39.7), which was significantly higher (p = 0.010) than for the dental broach (22.7%, 95% CI 16.3-30.6). Sensitivity for both methods was significantly lower than for the routinely used microscopy, which had a sensitivity of 55.5% (IQR 46.8-63.8) compared to PCR as a reference. CONCLUSIONS/SIGNIFICANCE: The diagnostic accuracy of the CL Detect Rapid Test was low for skin slit and dental broach samples. Therefore, we do not recommend its use neither in hospital nor field settings. TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov as NCT03837431.

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.

Chemical study of Adenocalymma axillarum crude leaf extract and isolated compounds

Abstract Adenocalymma axillarum (K.Schum.) L.G. Lohmann is a liana belonging to the family Bignoniaceae. In traditional medicine, the genus Adenocalymma is used to treat fever, skin ailments, and body, joint, and facial muscle pains, and it is also applied as cosmetic. Biological assays conducted with the A. axillarum crude leaf ethanol extract have indicated leishmanicidal activity and absence of cytotoxicity. This study aimed to analyze the A. axillarum leaf ethanol crude extract by high-performance liquid chromatography-high-resolution mass spectrometry- diode array detector (HPLC-HRMS-DAD) and to evaluate the leishmanicidal and cytotoxic activities of this crude extract, its fractions, and isolated compounds. HPLC-HRMS-DAD analysis of this extract revealed that it consisted mainly of flavonoids, with nine major compounds. Extract purification yielded 4-hydroxy-N-methylproline, 6-β-hydroxyipolamiide, quercetin-3-O-robinobioside, hyperin, isorhamnetin-3-O-robinobioside, and 3'-O-methylhyperin, which were identified by Nuclear Magnetic Resonance. The isolated compounds were inactive against Leishmania amazonensis promastigotes and human lung fibroblast cells.

Species identification and phylogenetic analysis of Leishmania isolated from patients, vectors and hares in the Xinjiang Autonomous Region, The People's Republic of China.

BACKGROUND: Visceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics. METHODOLOGY/PRINCIPAL FINDINGS: Here, an eleven-year survey (2004-2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship. CONCLUSIONS/SIGNIFICANCE: The above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir.

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.

BACKGROUND: Leishmaniasis 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 FINDINGS: Clinical, 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/SIGNIFICANCE: An 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.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Molecular identification of two newly identified human pathogens causing leishmaniasis using PCR-based methods on the 3' untranslated region of the heat shock protein 70 (type I) gene.

PCR-based methods to amplify the 3' untranslated region (3'-UTR) of the heat shock protein 70 (type I) gene (HSP70-I) have previously been used for typing of Leishmania but not with Leishmania (Mundinia) martiniquensis and L. (Mundinia) orientalis, newly identified human pathogens. Here, the 3'-UTRs of HSP70-I of L. martiniquensis, L. orientalis, and 10 other species were sequenced and analyzed. PCR-Restriction Fragment Length Polymorphism (RFLP) analysis targeting the 3'-UTR of HSP70-I was developed. Also, the detection limit of HSP70-I-3'-UTR PCR methods was compared with two other commonly used targets: the 18S small subunit ribosomal RNA (SSU-rRNA) gene and the internal transcribed spacer 1 region of the rRNA (ITS1-rRNA) gene. Results showed that HSP70-I-3'-UTR PCR methods could be used to identify and differentiate between L. martiniquensis (480-2 bp) and L. orientalis (674 bp) and distinguished them from parasites of the subgenus Viannia and of the subgenus Leishmania. PCR-RFLP patterns of the 3'-UTR of HSP70-I fragments digested with BsuRI restriction enzyme successfully differentiated L. martiniquensis, L. orientalis, L. braziliensis, L. guyanensis = L. panamensis, L. mexicana = L. aethiopica = L. tropica, L. amazonensis, L. major, and L. donovani = L. infantum. For the detection limit, the HSP70-I-3'-UTR PCR method could detect the DNA of L. martiniquensis and L. orientalis at the same concentration, 1 pg/µL, at a similar level to the SSU-rRNA PCR. The PCR that amplified ITS1-rRNA was more sensitive (0.01 pg/µL) than that of the HSP70-I-3'-UTR PCR. However, the sizes of both SSU-rRNA and ITS1-rRNA PCR amplicons could not differentiate between L. martiniquensis and L. orientalis. This is the first report of using HSP70-I-3'-UTR PCR based methods to identify the parasites causing leishmaniasis in Thailand. Also, the BsuRI-PCR-RFLP method can be used for differentiating some species within other subgenera.

Development of an Amplicon-Based Next-Generation Sequencing Protocol to Identify Leishmania Species and Other Trypanosomatids in Leishmaniasis Endemic Areas.

Trypanosomatid infections are an important public health threat affecting many low-income countries across the tropics, particularly in the Americas. Trypanosomatids can infect many vertebrate, invertebrate, and plant species and play an important role as human pathogens. Among these clinically relevant pathogens are species from the genera Leishmania and Trypanosoma. Mixed trypanosomatid infections remain a largely unexplored phenomenon. Herein, we describe the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatid species in mammalian reservoirs, human patients, and sand fly vectors throughout regions of Leishmania endemicity. Sixty-five samples from different departments of Colombia, including two samples from Venezuela, were analyzed: 49 samples from cutaneous leishmaniasis (CL) patients, 8 from sand flies, 2 from domestic reservoirs (Canis familiaris), and 6 from wild reservoirs (Phyllostomus hastatus). DNA from each sample served to identify the presence of trypanosomatids through conventional PCR using heat shock protein 70 (HSP70) gene as the target. PCR products underwent sequencing by Sanger sequencing and NGS, and trypanosomatid species were identified by using BLASTn against a reference database built from trypanosomatid-derived HSP70 sequences. The alpha and beta diversity indexes of amplicon sequence variants were calculated for each group. The results revealed the presence of mixed infections with more than two Leishmania species in 34% of CL samples analyzed. Trypanosoma cruzi was identified in samples from wild reservoirs, as well as in sand fly vectors. Coinfection events with three different Leishmania species were identified in domestic reservoirs. These findings depose the traditional paradigm of leishmaniasis as being a single-species-driven infection and redraw the choreography of host-pathogen interaction in the context of multiparasitism. Further research is needed to decipher how coinfections may influence disease progression. This knowledge is key to developing an integrated approach for diagnosis and treatment. IMPORTANCE Traditionally, there has been a frequent, yet incorrect assumption that phlebotomine vectors, animal reservoirs, and human hosts are susceptible to Leishmania infection by a single parasite species. However, current evidence supports that these new vector-parasite-reservoir associations lend vectors and reservoirs greater permissiveness to certain Leishmania species, thus promoting the appearance of coinfection events, particularly in disease-endemic regions. The present study describes the application of an amplicon-based next-generation sequencing (NGS) assay to detect and identify trypanosomatid species in mammalian reservoirs, human patients, and sand fly vectors from regions of endemicity for leishmaniasis. This changes our understanding of the clinical course of leishmaniasis in areas of endemicity.

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

BACKGROUND: Cutaneous leishmaniasis (CL) is frequent in travellers and can involve oro-nasal mucosae. Clinical presentation impacts therapeutic management. METHODOLOGY: Demographic 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 FINDINGS: Compared 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/SIGNIFICANCE: CL 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.

Chromosome-scale genome sequencing, assembly and annotation of six genomes from subfamily Leishmaniinae.

We provide the raw and processed data produced during the genome sequencing of isolates from six species of parasites from the sub-family Leishmaniinae: Leishmania martiniquensis (Thailand), Leishmania orientalis (Thailand), Leishmania enriettii (Brazil), Leishmania sp. Ghana, Leishmania sp. Namibia and Porcisia hertigi (Panama). De novo assembly was performed using Nanopore long reads to construct chromosome backbone scaffolds. We then corrected erroneous base calling by mapping short Illumina paired-end reads onto the initial assembly. Data has been deposited at NCBI as follows: raw sequencing output in the Sequence Read Archive, finished genomes in GenBank, and ancillary data in BioSample and BioProject. Derived data such as quality scoring, SAM files, genome annotations and repeat sequence lists have been deposited in Lancaster University's electronic data archive with DOIs provided for each item. Our coding workflow has been deposited in GitHub and Zenodo repositories. This data constitutes a resource for the comparative genomics of parasites and for further applications in general and clinical parasitology.

In Vivo Structure-Function Analysis and Redox Interactomes of Leishmania tarentolae Erv.

Import and oxidative folding of proteins in the mitochondrial intermembrane space differ among eukaryotic lineages. While opisthokonts such as yeast rely on the receptor and oxidoreductase Mia40 in combination with the Mia40:cytochrome c oxidoreductase Erv, kinetoplastid parasites and other Excavata/Discoba lack Mia40 but have a functional Erv homologue. Whether excavate Erv homologues rely on a Mia40 replacement or directly interact with imported protein substrates remains controversial. Here, we used the CRISPR-Cas9 system to generate a set of tagged and untagged homozygous mutants of LTERV from the kinetoplastid model parasite Leishmania tarentolae. Modifications of the shuttle cysteine motif of LtErv were lethal, whereas replacement of clamp residue Cys17 or removal of the kinetoplastida-specific second (KISS) domain had no impact on parasite viability under standard growth conditions. However, removal of the KISS domain rendered parasites sensitive to heat stress and led to the accumulation of homodimeric and mixed LtErv disulfides. We therefore determined and compared the redox interactomes of tagged wild-type LtErv and LtErvΔKISS using stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry. While the Mia40-replacement candidate Mic20 and all but one typical substrate with twin Cx3/9C-motifs were absent in both redox interactomes, we identified a small set of alternative potential interaction partners with putative redox-active cysteine residues. In summary, our study reveals parasite-specific intracellular structure-function relationships and redox interactomes of LtErv with implications for current hypotheses on mitochondrial protein import in nonopisthokonts. IMPORTANCE The discovery of the redox proteins Mia40/CHCHD4 and Erv1/ALR, as well as the elucidation of their relevance for oxidative protein folding in the mitochondrial intermembrane space of yeast and mammals, founded a new research topic in redox biology and mitochondrial protein import. The lack of Mia40/CHCHD4 in protist lineages raises fundamental and controversial questions regarding the conservation and evolution of this essential pathway. Do protist Erv homologues act alone, or do they use the candidate Mic20 or another protein as a Mia40 replacement? Furthermore, we previously showed that Erv homologues in L. tarentolae and the human pathogen L. infantum are not only essential but also differ structurally and mechanistically from yeast and human Erv1/ALR. Here, we analyzed the relevance of such structural differences in vivo and determined the first redox interactomes of a nonopisthokont Erv homologue. Our data challenge recent hypotheses on mitochondrial protein import in nonopisthokonts.

Detection of Leishmania tarentolae in lizards, sand flies and dogs in southern Italy, where Leishmania infantum is endemic: hindrances and opportunities.

BACKGROUND: Leishmania tarentolae is a protozoan isolated from geckoes (Tarentola annularis, Tarentola mauritanica), which is considered non-pathogenic and is transmitted by herpetophilic Sergentomyia spp. sand flies. This species occurs in sympatry with Leishmania infantum in areas where canine leishmaniasis is endemic. In the present study, we investigated the circulation of L. tarentolae and L. infantum in sand flies, dogs and lizards in a dog shelter in southern Italy, where canine leishmaniasis by L. infantum is endemic. METHODS: Sheltered dogs (n = 100) negative for Leishmania spp. (March 2020) were screened by immunofluorescence antibody test (IFAT) using promastigotes of both species at two time points (June 2020 and March 2021). Whole blood from dogs, tissues of Podarcis siculus lizards (n = 28) and sand flies (n = 2306) were also sampled and tested by a duplex real-time PCR (dqPCR). Host blood meal was assessed in sand flies by PCR. RESULTS: Overall, 16 dogs became positive for L. infantum and/or L. tarentolae by IFAT at one or both sampling periods. One canine blood sample was positive for L. infantum, whilst two for L. tarentolae by dqPCR. At the cytology of lizard blood, Leishmania spp. amastigote-like forms were detected in erythrocytes. Twenty-two tissue samples, mostly lung (21.4%), scored molecularly positive for L. tarentolae, corresponding to 10 lizards (i.e., 35.7%). Of the female Sergentomyia minuta sampled (n = 1252), 158 scored positive for L. tarentolae, four for L. infantum, and one co-infected. Two Phlebotomus perniciosus (out of 29 females) were positive for L. tarentolae. Engorged S. minuta (n = 10) fed on humans, and one P. perniciosus, positive for L. tarentolae, on lagomorphs. CONCLUSIONS: Dogs and lacertid lizards (Podarcis siculus) were herein found for the first time infected by L. tarentolae. The detection of both L. tarentolae and L. infantum in S. minuta and P. perniciosus suggests their sympatric circulation, with a potential overlap in vertebrate hosts. The interactions between L. tarentolae and L. infantum should be further investigated in both vectors and vertebrate hosts to understand the potential implications for the diagnosis and control of canine leishmaniasis in endemic areas.