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Leishmania are intracellular protozoan parasites that cause the vectorborne disease leishmaniasis, which occurs in [approximately equal to] 88 countries (1). Human infection can result in 2 main forms of disease, [...]

Tegumentary leishmaniasis (TL) is endemic but neglected in southern Europe. Therefore, this study aimed to analyze the Leishmania strains causing TL cases in northeastern Italy, where an upsurge of TL cases has been observed in the last decade. Sections from 109 formalin-fixed and paraffin-embedded (FFPE) biopsies of skin and mucosal tissues were collected from TL cases in the selected area. Two DNA targets were amplified and sequenced: the ribosomal internal transcribed spacer 1 (ITS1) and the heat-shock protein 70 gene (hsp70). An in silico analysis was also performed on 149 genomes belonging to the Leishmania donovani complex. A total of 88 out of 109 (80.7%) samples from 83 TL cases were successfully typed by ITS1 and/or hsp70. ITS1 analysis identified L. infantum in 67 cases (91.8%), while L. major (n = 4, 5.5%) and L. tropica (n = 2, 2.7%) were detected in the remaining cases that were categorized as imported. Further, the hsp70 typing of 75 autochthonous cases showed the presence of eight distinct sequence variants belonging to the Leishmania donovani complex, with high genetic variability when compared to known L. infantum populations. In conclusion, our findings show that peculiar L. infantum variants are emerging in the novel focus on TL in northeastern Italy. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), the fatal form of a parasitic disease caused by Leishmania donovani complex, has been known to exist in southern Somalia since the 1930s, but its presence in [...]

Visceral leishmaniasis (VL), also known as kala-azar, is a fatal vector-borne parasitic disease. Worldwide incidence is 500,000 cases per year; ≅ 90% of cases occur in India, Nepal, Bangladesh, Sudan, [...]

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. Author summary: Cutaneous and muco-cutaneous leishmaniasis (CL and MCL) are disfiguring diseases caused by a worldwide distributed parasite called Leishmania and its 20 species. Clinical manifestations span a wide continuum from single nodular lesion to disseminated form with mucosal involvement. No randomized clinical trial has ever been done exclusively in travellers and medical management is poorly evidence-based or based very predominantly on data obtained in endemic countries. Articles and reviews almost invariably propose a dichotomic view, with Old World CL described as a benign disease in contrast to New World CL strongly associated with destructive mucosal lesions. Our study is the first prospective clinical study providing a detailed description of the clinical presentation and risk of mucosal involvement in CL in several hundreds of patients , with frequent formal identification of the infecting Leishmania species. The harmonized data collection in patients infected in many transmission foci worldwide enabled direct comparisons of clinical patterns induced by different Leishmania species, and on the outcome following treatment with either local or systemic regimens. The study is based on an international harmonized data collection that allowed a wide capture of parasitologically confirmed cases. In striking contrast with previous assumptions, the study shows that CL acquired in Europe displays unexpected high rates of mucosal involvement comparable to those of CL acquired in Latin America, especially in immunocompromised travellers. It also shows that when used as per recommendations, local therapy is associated with high cure rates. [ABSTRACT FROM AUTHOR]

Abstract Background Peru is one of the Latin American countries with the highest malaria burden, mainly due to Plasmodium vivax infections. However, little is known about P. vivax transmission dynamics in the Peruvian Amazon, where most malaria cases occur. The genetic diversity and population structure of P. vivax isolates collected in different communities around Iquitos city, the capital of the Peruvian Amazon, was determined. Methods Plasmodium vivax population structure was determined by multilocus genotyping with 16 microsatellites on 159 P. vivax infected blood samples (mono-infections) collected in four sites around Iquitos city. The population characteristics were assessed only in samples with monoclonal infections (n = 94), and the genetic diversity was determined by calculating the expected heterozygosity and allelic richness. Both linkage disequilibrium and the genetic differentiation (θ) were estimated. Results The proportion of polyclonal infections varied substantially by site (11% - 70%), with the expected heterozygosity ranging between 0.44 and 0.69; no haplotypes were shared between the different populations. Linkage disequilibrium was present in all populations (IAS 0.14 - 0.61) but was higher in those with fewer polyclonal infections, suggesting inbreeding and a clonal population structure. Strong population differentiation (θ = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies. Conclusion The P. vivax populations circulating in the Peruvian Amazon basin are genetically diverse, strongly differentiated and they have a low effective recombination rate. These results are in line with the low and clustered pattern of malaria transmission observed in the region around Iquitos city.

Background: In the endgame of the elimination initiative of visceral leishmaniasis (VL) on the Indian subcontinent, one of the main questions remaining is whether asymptomatically infected individuals also contribute to transmission. We piloted a minimally invasive microbiopsy device that could help answer this question. While the potential of this device has been previously illustrated in Ethiopia, no such information is available for the setting of the Indian subcontinent. In this proof of concept study we aimed to assess 1) to what extent skin parasite load obtained with the new microbiopsy device correlates with disease status, 2) to what extent skin parasite load correlates with blood parasite load in the same subject, and 3) to what extent the skin parasite load obtained from different sampling sites on the body correlates with one another. Methods: We performed a pilot study in Bihar, India, including 29 VL patients, 28 PKDL patients, 94 asymptomatically infected individuals, 22 endemic controls (EC), and 28 non-endemic controls (NEC). Presence of infection with L. donovani in the blood was assessed using Direct Agglutination Test, rK39 ELISA, Whole Blood Analysis measuring IFN-γ and qPCR. A skin sample was collected with the microbiopsy device on two different locations on the body. PKDL patients provided a third skin sample from the edge of a PKDL lesion. Parasite load in the skin was measured by qPCR. Findings: We found a clear correlation between the skin parasite load obtained with the microbiopsy device and disease status, with both higher skin parasite loads and higher proportions of positive skin samples in VL and PKDL patients compared to asymptomatics, EC, and NEC. No clear correlation between skin parasite load and blood parasite load was found, but a moderate correlation was present between the skin parasite load in arm and neck samples. In addition, we found four positive skin samples among asymptomatic individuals, and 85% of PKDL lesions tested positive using this microbiopsy device. Conclusions: In line with previous pilot studies, our results from an Indian setting suggest that the microbiopsy device provides a promising tool to measure skin parasite load, and – if validated by xenodiagnosis studies – could facilitate much needed larger scale studies on infectiousness of human subgroups. In addition, we advocate further evaluation of this device as a diagnostic tool for PKDL. [ABSTRACT FROM AUTHOR]

Background: In endemic regions, asymptomatic Leishmania infection is common. In HIV patients, detection of asymptomatic Leishmania infection could potentially identify those at risk of visceral leishmaniasis (VL). However, data on the prevalence, incidence, and determinants of asymptomatic infection, and the risk of VL are lacking. Methods: We conducted a cross-sectional survey at a single ART centre, followed by a prospective cohort study amongst HIV-infected adults in HIV care in a district hospital in a VL-endemic area in North-West Ethiopia (9/2015-8/2016). Asymptomatic Leishmania infection was detected using the direct agglutination test (DAT), rK39-rapid diagnostic test (RDT)), PCR on peripheral blood and the KAtex urine antigen test, and defined as positivity on any Leishmania marker. All individuals were followed longitudinally (irrespective of the Leishmania test results). Risk factors for asymptomatic Leishmania infection were determined using logistic regression. Results: A total of 534 HIV-infected individuals enrolled in HIV care were included in the study. After excluding 13 patients with a history of VL and an 10 patients with incomplete baseline Leishmania tests, 511 were included in analysis. The median age was 38 years (interquartile range (IQR) 30–45), 62.6% were male. The median follow-up time was 12 months (IQR 9–12). No deaths were reported during the study period. Most (95.5%) were on antiretroviral treatment at enrolment, for a median of 52 months (IQR 27–79). The median CD4 count at enrolment was 377 cells/mm3 (IQR 250–518). The baseline prevalence of Leishmania infection was 12.8% in males and 4.2% in females. Overall, 7.4% tested positive for rK39, 4.3% for DAT, 0.2% for PCR and 0.2% for KAtex. Independent risk factors for a prevalent infection were male sex (odds ratio (OR) 3.2; 95% confidence intervals (CI) 14–7.0) and concurrent malaria infection (OR 6.1; 95% CI 1.9–18.9). Amongst the 49 prevalent (baseline) infections with further follow-up, the cumulative incidence of losing the Leishmania markers by one year was 40.1%. There were 36 incident infections during the course of the study, with a cumulative one-year risk of 9.5%. Only one case of VL was detected during follow-up. Conclusions: We found a high prevalence of asymptomatic Leishmania infection, persisting in most cases. The incidence was more modest and overt VL was rare. Larger and longer studies with more complete follow-up may help to decide whether a test and treat strategy would be justified in this context. Trial registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. Methods and findings: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. Conclusions: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated. [ABSTRACT FROM AUTHOR]

Background: Leishmaniasis is a protozoan disease caused by parasites of the genus Leishmania, transmitted to humans by sandflies. The diagnosis of leishmaniasis is often challenging as it mimics many other infectious or malignant diseases. The disease can present in three ways: cutaneous, mucocutaneous, or visceral leishmaniasis, which rarely occur together or consecutively.Case Presentation: The patient was a 52 years old immunosuppressed Belgian woman with a long history of severe rheumatoid arthritis. She underwent bone marrow biopsy to explore thrombocytopenia. Diagnosis of visceral leishmaniasis was made by identification of Leishman Donovan (LD) bodies in macrophages. Treatment with liposomal amphotericin B was successful. She later developed cutaneous leishmaniasis treated with amphotericin B lipid complex. She next presented with relapsing cutaneous lesions followed by rapidly progressing lymphadenopathies. Biopsy confirmed the diagnosis of leishmaniasis. Treatments by miltefosine, amphotericin B, N-methyl-glucamine antimoniate were subsequently initiated. She later presented a recurrent bone marrow involvement treated with intramuscular paromomycin and miltefosine. She died two years later from leukemia. At the time of death, she presented with a mucosal destruction of the nose. A Leishmania-specific PCR (Polymerase Chain Reaction) identified L. infantum as etiological agent.Conclusions: Clinicians should be aware of the potential concomitant or sequential involvement of multiple anatomic localizations of Leishmania in immunosuppressed patients. [ABSTRACT FROM AUTHOR]

Background: We designed a straightforward method for discriminating circulating Leishmania populations in the Indian subcontinent (ISC). Research on transmission dynamics of visceral leishmaniasis (VL, or Kala-azar) was recently identified as one of the key research priorities for elimination of the disease in the ISC. VL in Bangladesh, India, and Nepal is caused by genetically homogeneous populations of Leishmania donovani parasites, transmitted by female sandflies. Classical methods to study diversity of these protozoa in other regions of the world, such as microsatellite typing, have proven of little use in the area, as they are not able to discriminate most genotypes. Recently, whole genome sequencing (WGS) so far identified 10 different populations termed ISC001-ISC010. Methodology / Principle findings: As an alternative to WGS for epidemiological or clinical studies, we designed assays based on PCR amplification followed by dideoxynucleotide sequencing for identification of the non-recombinant genotypes ISC001 up to ISC007. These assays were applied on 106 parasite isolates collected in Nepal between 2011 and 2014. Combined with data from WGS on strains collected in the period 2002–2011, we provide a proof-of-principle for the application of genotyping to study treatment outcome, and differential geographic distribution. Conclusions / Significance: Our method can aid in epidemiological follow-up of visceral leishmaniasis in the Indian subcontinent, a necessity in the frame of the Kala-azar elimination initiative in the region. [ABSTRACT FROM AUTHOR]

Immunologically, active visceral leishmaniasis (VL) is characterized by profound immunosuppression, severe systemic inflammatory responses, and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication, and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis; however, their role in human VL is poorly understood. In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase, and elastase, all contained in neutrophils' granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analyzed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species, and phagocytose bacterial particles, but not Leishmania parasites. Our results suggest that impaired effector functions, increased activation, and immaturity of neutrophils play a key role in the pathogenesis of VL. [ABSTRACT FROM AUTHOR]

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Background: In the Indian subcontinent, Visceral leishmaniasis is endemic in a geographical area coinciding with the Lower Gangetic Plain, at low altitude. VL occurring in residents of hill districts is therefore often considered the result of Leishmania donovani infection during travel. Early 2014 we conducted an outbreak investigation in Okhaldhunga and Bhojpur districts in the Nepal hills where increasing number of VL cases have been reported. Methodology/Principal Findings: A house-to-house survey in six villages documented retrospectively 35 cases of Visceral Leishmaniasis (VL). Anti-Leishmania antibodies were found in 22/23 past-VL cases, in 40/416 (9.6%) persons without VL and in 12/155 (7.7%) domestic animals. An age- and sex- matched case-control study showed that exposure to known VL-endemic regions was no risk factor for VL, but having a VL case in the neighbourhood was. SSU-rDNA PCR for Leishmania sp. was positive in 24 (5%) of the human, in 18 (12%) of the animal samples and in 16 (14%) bloodfed female Phlebotomus argentipes sand flies. L. donovani was confirmed in two asymptomatic individuals and in one sand fly through hsp70-based sequencing. Conclusions/Significance: This is epidemiological and entomological evidence for ongoing local transmission of L. donovani in villages at an altitude above 600 meters in Nepal, in districts considered hitherto non-endemic for VL. The VL Elimination Initiative in Nepal should therefore consider extending its surveillance and control activities in order to assure VL elimination, and the risk map for VL should be redesigned. [ABSTRACT FROM AUTHOR]

Background: Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management. Methodology: We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania. Principal Findings: Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level. Conclusions/Significance: Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis. Author Summary: Cutaneous leishmaniasis is a disease due to a small parasite called Leishmania. This parasite causes disfiguring skin lesions that last for months or years. There are many different subtypes of Leishmania, each giving rise to lesions of different severity and responding to therapies in its own way. Treating physicians must know as soon as possible which subtype of Leishmania is involved to propose the best treatment. Because it is impossible to differentiate the Leishmania subtypes microscopically, the identification of the culprit subtype currently requires complex and expensive typing methods, the results of which are generally obtained several weeks after the diagnosis. Here, we have evaluated the ability of a new method using mass spectrometry to differentiate Leishmania subtypes. Our results were consistent with those provided by reference typing methods and were obtained rapidly after the parasite had been cultured in vitro. This new method may help physicians know very soon which Leishmania subtype is involved thereby facilitating treatment choice. [ABSTRACT FROM AUTHOR]

Background: Cutaneous leishmaniasis is caused by several Leishmania species that are associated with variable outcomes before and after therapy. Optimal treatment decision is based on an accurate identification of the infecting species but current methods to type Leishmania isolates are relatively complex and/or slow. Therefore, the initial treatment decision is generally presumptive, the infecting species being suspected on epidemiological and clinical grounds. A simple method to type cultured isolates would facilitate disease management. Methodology: We analyzed MALDI-TOF spectra of promastigote pellets from 46 strains cultured in monophasic medium, including 20 short-term cultured isolates from French travelers (19 with CL, 1 with VL). As per routine procedure, clinical isolates were analyzed in parallel with Multilocus Sequence Typing (MLST) at the National Reference Center for Leishmania. Principal Findings: Automatic dendrogram analysis generated a classification of isolates consistent with reference determination of species based on MLST or hsp70 sequencing. A minute analysis of spectra based on a very simple, database-independent analysis of spectra based on the algorithm showed that the mutually exclusive presence of two pairs of peaks discriminated isolates considered by reference methods to belong either to the Viannia or Leishmania subgenus, and that within each subgenus presence or absence of a few peaks allowed discrimination to species complexes level. Conclusions/Significance: Analysis of cultured Leishmania isolates using mass spectrometry allows a rapid and simple classification to the species complex level consistent with reference methods, a potentially useful method to guide treatment decision in patients with cutaneous leishmaniasis. [ABSTRACT FROM AUTHOR]

Background: Most of the Leishmania genome is reported to be constitutively expressed during the life cycle of the parasite, with a few regulated genes. Inter-species comparative transcriptomics evidenced a low number of species-specific differences related to differentially distributed genes or the differential regulation of conserved genes. It is of uppermost importance to ensure that the observed differences are indeed species-specific and not simply specific of the strains selected for representing the species. The relevance of this concern is illustrated by current study. Methodology/Principal Findings: We selected 5 clinical isolates of L. braziliensis characterized by their diversity of clinical and in vitro phenotypes. Real-time quantitative PCR was performed on promastigote and amastigote life stages to assess gene expression profiles at seven time points covering the whole life cycle. We tested 12 genes encoding proteins with roles in transport, thiol-based redox metabolism, cellular reduction, RNA poly(A)-tail metabolism, cytoskeleton function and ribosomal function. The general trend of expression profiles showed that regulation of gene expression essentially occurs around the stationary phase of promastigotes. However, the genes involved in this phenomenon appeared to vary significantly among the isolates considered. Conclusion/Significance: Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics. Results obtained on an individual strain are not necessarily representative of a given species. Therefore, extreme care should be taken when comparing the profiles of different species and extrapolating functional differences between them. Author Summary: Leishmania is a group of parasites (Protozoa, Trypanosomatidae) responsible for a wide spectrum of clinical forms. Among the factors explaining this phenotypic polymorphism, parasite features are important contributors. One approach to identify them consists in characterizing the gene expression profiles throughout the life cycle. In a recent study, the transcriptome of 3 Leishmania species was compared and this revealed species-specific differences, albeit in a low number. A key issue, however, is to ensure that the observed differences are indeed species-specific and not specific of the strains selected for representing the species. In order to illustrate the relevance of this concern, we analyzed here the gene expression profiles of 5 clinical isolates of L. braziliensis at seven time points of the life cycle. Our results clearly illustrate the unique character of each isolate in terms of gene expression dynamics: one Leishmania strain is not necessarily representative of a given species. [ABSTRACT FROM AUTHOR]

Abstract: Leishmania panamensis and Leishmania guyanensis are two species of the subgenus Viannia that are genetically very similar. Both parasites are usually associated with cutaneous leishmaniasis, but also have the potential to cause the mucocutaneous form of the disease. In addition, the study of foci and consequently the identification of vectors and probable reservoirs involved in transmission require a correct differentiation between both species, which is important at epidemiological level. We explored the possibility of identifying these species by using restriction fragment length polymorphisms (RFLP) in the gene coding for heat-shock protein 70 (hsp70). Previously, an hsp70 PCR-RFLP assay proved to be very effective in differentiating other Leishmania species when HaeIII is used as restriction enzyme. Based on hsp70 sequences analysis, BccI was found to generate species-specific fragments that can easily be recognized by agarose gel electrophoresis. Using the analysis of biopsies, scrapings, and parasite isolates previously grouped in a cluster comprising both L. panamensis and L. guyanensis, we showed that our approach allowed differentiation of both entities. This offers the possibility not only for identification of parasites in biological samples, but also to apply molecular epidemiology in certain countries of the New World, where several Leishmania species could coexist. [Copyright &y& Elsevier]

Background: Peru is one of the Latin American countries with the highest malaria burden, mainly due to Plasmodium vivax infections. However, little is known about P. vivax transmission dynamics in the Peruvian Amazon, where most malaria cases occur. The genetic diversity and population structure of P. vivax isolates collected in different communities around Iquitos city, the capital of the Peruvian Amazon, was determined. Methods: Plasmodium vivax population structure was determined by multilocus genotyping with 16 microsatellites on 159 P. vivax infected blood samples (mono-infections) collected in four sites around Iquitos city. The population characteristics were assessed only in samples with monoclonal infections (n = 94), and the genetic diversity was determined by calculating the expected heterozygosity and allelic richness. Both linkage disequilibrium and the genetic differentiation (θ) were estimated. Results: The proportion of polyclonal infections varied substantially by site (11% - 70%), with the expected heterozygosity ranging between 0.44 and 0.69; no haplotypes were shared between the different populations. Linkage disequilibrium was present in all populations (IA S 0.14 - 0.61) but was higher in those with fewer polyclonal infections, suggesting inbreeding and a clonal population structure. Strong population differentiation (θ = 0.45) was found and the Bayesian inference cluster analysis identified six clusters based on distinctive allele frequencies. Conclusion: The P. vivax populations circulating in the Peruvian Amazon basin are genetically diverse, strongly differentiated and they have a low effective recombination rate. These results are in line with the low and clustered pattern of malaria transmission observed in the region around Iquitos city. [ABSTRACT FROM AUTHOR]

Summary: Monitoring Leishmania infection in sand flies is important for understanding the eco-epidemiology of kala-azar and assessing the impact of the recently launched kala-azar control programme in the Indian subcontinent. We applied a PCR technique that targets rRNA genes to estimate the natural incidence of Leishmania infection in sand flies sampled in six villages of the Terai region of Nepal. Amplifications were made on 135 pools of sand flies and confirmed by sequencing. Seven pools were found to be PCR positive: in five of them we identified the rDNA signature found in Leishmania spp., whereas two other pools revealed a sequence compatible with other trypanosomatids. Different methodologies were applied to evaluate the infection rate from pools of unequal size and estimated the infection rate to range from 0.468% to 0.578% for the Leishmania group and from 0.185% to 0.279% for the non-Leishmania group. Our results highlight the diversity of flagellate infections likely to be encountered in Phlebotomus argentipes populations. Our methodology allows clear discrimination of Leishmania from other trypanosomatids and should be applied on larger insect samples or in longitudinal studies. [Copyright &y& Elsevier]

Abstract: We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was further simplified by using an oligochromatography dipstick to detect L. infantum and L. donovani products. Because the analytical sensitivity is lower than that of certain other species- and genus-specific PCRs, our assays are especially valuable for use on cultured isolates or directly on cryostabilates. As such, they can be implemented by research and health centers having access to culturing, DNA isolation, and PCR. [Copyright &y& Elsevier]

Reference 1 Raguenaud M-E, Jansson A, Vanlerberghe V, Van der Auwera G, Deborggraeve S, Dujardin J-C, et al. (2007) Epidemiology and Clinical Features of Patients with Visceral Leishmaniasis Treated by an MSF Clinic in Bakool Region, Somalia, 2004-2006.PLoS Negl Trop Dis1(1): e85. https://doi.org/10.1371/journal.pntd.0000085, 17989791 The correct name is: Ioannis Orfanos. Correction: Epidemiology and Clinical Features of Patients with Visceral Leishmaniasis Treated by an MSF Clinic in Bakool Region, Somalia, 2004-2006. [Extracted from the article]

Species-directed therapy of leishmaniasis has been recommended for travelers since 2014, but little is known about species distribution and treatment practices in non-endemic countries. We aimed to describe leishmaniasis cases in Belgium since species typing became available and evaluate its impact on patient management. Retrospective analysis of all patients diagnosed by PCR at our national reference laboratory from 2010 to 2018. Species were typed by Hsp-70 sequencing. We identified 18 visceral leishmaniasis (VL) and 147 (muco)cutaneous leishmaniasis ((M)CL) cases. VL was exclusively due to L. infantum and consistently treated with liposomal amphotericin B, with four observed failures. (M)CL was caused by ten different species. Of 62 cases diagnosed and species typed after 2014 with timing information, 28 (45.2%) were treated before the species result was available. Therapy was not species-directed in 10/32(28.1%) of those treated after species identification. Patients treated according to the guidelines tended to have a favorable outcome more often than those who were not (36/44, 81.8% versus 8/19, 57.9%; p = 0.045). In contrast to VL, various species caused (M)CL in our setting and species result was often not considered for treatment. Outcome tended to be better however when therapy was species-directed. [ABSTRACT FROM AUTHOR]

Highlights: [•] Hsp20 is a suitable molecular marker for Leishmania typing. [•] The same pair of oligonucleotides allows hsp20 amplification from different Leishmania species. [•] Nine species and two subspecies are recognized among the Leishmania pathogenic species. [•] A reliable taxonomy for Leishmania is possible based on molecular phylogenetic analyses. [ABSTRACT FROM AUTHOR]

Trypanosome evolution was so far essentially studied on the basis of phylogenetic analyses of small subunit ribosomal RNA (SSU-rRNA) and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes. We used for the first time the 70 kDa heat-shock protein gene ( hsp 70) to investigate the phylogenetic relationships among 11 Trypanosoma species on the basis of 1380 nucleotides from 76 sequences corresponding to 65 strains. We also constructed a phylogeny based on combined datasets of SSU-rDNA, gGAPDH and hsp 70 sequences. The obtained clusters can be correlated with the sections and subgenus classifications of mammal-infecting trypanosomes except for Trypanosoma theileri and Trypanosoma rangeli . Our analysis supports the classification of Trypanosoma species into clades rather than in sections and subgenera, some of which being polyphyletic. Nine clades were recognized: Trypanosoma carassi , Trypanosoma congolense , Trypanosoma cruzi , Trypanosoma grayi , Trypanosoma lewisi , T. rangeli , T. theileri , Trypanosoma vivax and Trypanozoon . These results are consistent with existing knowledge of the genus' phylogeny. Within the T. cruzi clade, three groups of T. cruzi discrete typing units could be clearly distinguished, corresponding to TcI, TcIII, and TcII + V + VI, while support for TcIV was lacking. Phylogenetic analyses based on hsp 70 demonstrated that this molecular marker can be applied for discriminating most of the Trypanosoma species and clades. [ABSTRACT FROM AUTHOR]

Objectives. Explore a new target for molecular diagnosis of Leishmania. Materials and methods. We evaluated the utility of the gene that encodes the heat shock protein 20-kDa (Hsp20) for detecting Leishmania by polymerase chain reaction (PCR). PCR was normalized and analytical parameters were determined, as well as the validity and diagnostic accuracy, and concordance with the PCR - 18S. PCR-Hsp20 with DNA was obtained from a group of clinical samples from different sources. Results. The analytical parameters were adequate. The sensitivity obtained was 86% and the specificity was 100%. The concordance with the reference method was good (K = 0.731), which supports its potential use for diagnosis. The possibility of subsequent identification of the species by sequencing the amplified product gives an additional advantage. Conclusions. The usefulness of this gene asa new target for the detection of Leishmania was demonstrated. Because of its potential, it is recommended to improve the sensitivity of the method and to evaluate it in different endemic regions. [ABSTRACT FROM AUTHOR]

Abstract: In order to get more insight into its evolution and geographical distribution, we investigated the Leishmania (Viannia) braziliensis species complex using amplified fragment length polymorphisms and sequencing of a heat-shock protein 70 gene fragment. Previously, several assays had alluded to the high genetic diversity of the group, and single-locus assays typically identified two species, i.e. L. braziliensis and Leishmania peruviana, with occasional genetic signatures of both in the same strain. By analysis of 53 parasite isolates from Peru, and eight additional ones from other countries, we identified an atypical L. braziliensis cluster, and confirmed the origin of L. peruviana from the L. braziliensis cluster during the colonization of the western Andean coastal valleys. We discuss the clinical and taxonomical implications of our findings in relation to currently used species typing assays. [Copyright &y& Elsevier]