Assessment of pan-Leishmania detection by recombinase polymerase amplification assay.

The spread of vector habitats along with increasing human mobility can introduce atypical Leishmania species and hence can challenge existing diagnostic practices for rapid detection of active infection with species outside the narrow target range. Here we assessed the pan-Leishmania detection ability of isothermal recombinase polymerase amplification (RPA) assays targeting 18S rRNA gene, cathepsin L-like cysteine proteinase B (Cpb) gene, and kinetoplast minicircle DNA (kDNA) regions. While the lowest limit of detection of the 18S rRNA-RPA and Cpb-RPA assays were estimated as 12 and 17 standard DNA molecules, respectively, both assays could amplify genomic DNA of 7 pathogenic Leishmania species. Evaluation of 18S rRNA-RPA and our previously developed kDNA-RPA assays on 70 real-time PCR-positive leishmaniasis samples of varying pathologies resulted in sensitivity rates of 35.71% and 88.57%, respectively, while the combined sensitivity was 98.57%. Combinatorial application of 18S rRNA-RPA and kDNA-RPA assays can be recommended for further diagnostic assessments.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia.

BACKGROUND: Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. METHODS: Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. RESULTS: The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. CONCLUSIONS: The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.

Phlebotominae Fauna (Diptera: Psychodidae) and Molecular Detection of Leishmania (Kinetoplastida: Trypanosomatidae) in Urban Caves of Belo Horizonte, Minas Gerais, Brazil.

Sand flies are often collected in urban areas, which has several implications for the risk of transmission of Leishmania Ross, 1903, to humans and other mammals. Given this scenario, we describe the sand fly fauna of caves and their surroundings in Mangabeiras Municipal Park (MMP) and Paredão Serra do Curral Park (PSCP), both located in the urban area of Belo Horizonte, Minas Gerais, Brazil, an endemic focus of visceral and cutaneous leishmaniasis. Collections were conducted monthly from November 2011 to October 2012, using CDC light traps exposed for two consecutive nights in four caves and their surroundings. Nonsystematized collections using Shannon traps and active searches were also performed around the caves. The presence of Leishmania DNA in collected female sand flies was evaluated by ITS1-PCR. A total of 857 sand flies representing fourteen species were collected in MMP, of which Evandromyia edwardsi (Mangabeira, 1941) was the most abundant. Leishmania amazonensis was detected in Brumptomyia nitzulescui (Costa Lima, 1932) and Ev. edwardsi, with the latter also having Leishmania braziliensis, Leishmania infantum, and Leishmania sp. A total of 228 sand flies representing four species were collected in PSCP, of which Sciopemyia microps (Mangabeira, 1942) was the most abundant. No females from PSCP were positive for Leishmania-DNA. Studies aimed at describing sand fly faunas of cave environments and detecting Leishmania are essential to understanding the relationship between these insects and this ecotope and assessing and monitoring areas that may pose risks to the health of visitors and employees.

Culicoides Latreille (Diptera: Ceratopogonidae) as potential vectors for Leishmania martiniquensis and Trypanosoma sp. in northern Thailand.

Biting midges of genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several pathogenic arboviruses and parasites of humans and animals. Several reports have suggested that biting midges might be a potential vector of Leishmania parasites. In this study, we screened for Leishmania and Trypanosoma DNA in biting midges collected from near the home of a leishmaniasis patient in Lamphun province, northern Thailand by using UV-CDC light traps. The identification of biting midge species was based on morphological characters and confirmed using the Cytochrome C oxidase subunit I (COI) gene. The detection of Leishmania and Trypanosoma DNA was performed by amplifying the internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rRNA) genes, respectively. All the amplified PCR amplicons were cloned and sequenced. The collected 223 biting midges belonged to seven species (Culicoides mahasarakhamense, C. guttifer, C. innoxius, C. sumatrae, C. huffi, C. oxystoma, and C. palpifer). The dominant species found in this study was C. mahasarakhamense (47.53%). Leishmania martiniquensis DNA was detected in three samples of 106 specimens of C. mahasarakhamense tested indicating a field infection rate of 2.83%, which is comparable to reported rates in local phlebotomines. Moreover, we also detected Trypanosoma sp. DNA in one sample of C. huffi. To our knowledge, this is the first molecular detection of L. martiniquensis in C. mahasarakhamense as well as the first detection of avian Trypanosoma in C. huffi. Blood meal analysis of engorged specimens of C. mahasarakhamense, C. guttifer, and C. huffi revealed that all specimens had fed on avian, however, further studies of the host ranges of Culicoides are needed to gain a better insight of potential vectors of emerging leishmaniasis. Clarification of the vectors of these parasites is also important to provide tools to establish effective disease prevention and control programs in Thailand.

Insecticide-treated net (ITN) use, factors associated with non-use of ITNs, and occurrence of sand flies in three communities with reported cases of cutaneous leishmaniasis in Ghana.

BACKGROUND: The insecticide treated bed net (ITN) has been proven for malaria control. Evidence from systematic review also suggests benefits of ITN roll out in reducing the incidence of cutaneous leishmaniasis (CL) and other vector borne diseases. METHODS: Using a community-based cross-sectional study design, ITN use, factors associated with non-use of ITNs, and occurrence of sand flies were investigated in three communities with reported cases of CL in the Oti region of Ghana. RESULTS: A total of 587 households comprising 189 (32.2%), 200 (34.1%), and 198 (33.7%) households from Ashiabre, Keri, and Sibi Hilltop communities with de facto population of 3639 participated in this study. The proportion of households that owned at least one ITN was 97.1%. The number of households having at least one ITN for every two members was 386 (65.8%) and 3159 (86.8%) household population had access to ITN. The household population that slept in ITN the night before this survey was 2370 (65.1%). Lack of household access to ITN (AOR = 1.80; CI: 1.31, 2.47), having a family size of more than 10 members (AOR = 2.53; CI: 1.20, 4.24), having more than 10 rooms for sleeping in a household (AOR = 10.18; CI: 1.28, 81.00), having 2-4 screened windows (AOR = 1.49; CI: 1.00, 2.20), and having 8-10 screened windows (AOR = 3.57; CI: 1.25, 10.17) were significantly associated with increased odds of not sleeping in ITN the night before the survey. A total of 193 female sand flies were trapped from various locations within the study communities. CONCLUSIONS: Factors associated with ITN non-use such as lack of household access to ITN should be incorporated into future efforts to improve ITN use. Species of sand flies and their potential vectorial role in the study communities should also be investigated.

Outbreak of Cutaneous Leishmaniasis among military personnel in French Guiana, 2020: Clinical, phylogenetic, individual and environmental aspects.

BACKGROUND: Cutaneous Leishmaniasis (CL) is endemic in French Guiana but cases are usually sporadic. An outbreak signal was issued on May 15th 2020 with 15 suspected cases after a military training course in the rainforest. An outbreak investigation was carried out. METHODOLOGY/PRINCIPAL FINDINGS: Thirty cases were confirmed. Leishmania guyanensis was the most frequent species (90%). The most frequent presentation was ulcerative (90%). Lesions on the face and hands were frequent (40% each). Eight cases (26%) presented a poor outcome after treatment with pentamidine and required a second line with amphotericin B. Three of them required further treatments with meglumine antimoniate or miltefosine. Two spots within the training area were deemed as likely sites of contamination, due to illegal logging. The isolated Leishmania strains did not form a separate cluster. Participation in Week 13 of year 2020 was associated with infection (OR = 4.59 [1.10-19.83]; p = 0.016) while undergoing only the "Fighting" exercise was protective (OR = 0.1 [0-0.74]; p = 0.021). There was no association between infection and other risk factors at the individual level. The attack rate of Regiment B (14/105 = 13.3%) was significantly higher (OR = 4.22 [1.84-9.53], p = 0.0001) compared to Regiment A (16/507 = 3.2%). The attack rate during this training course (30/858 = 3.5%) was significantly higher (OR 2.29 [1.28-4.13]; p = 0.002) than for other missions in French Guiana during the same period (22/1427 = 1.5%). CONCLUSIONS: This outbreak could be explained by a combination of factors: climatic conditions around week 13, at-risk activities including night trainings, absence of impregnation, a lesser experience of rainforest duties in Regiment B and illegal logging attracting sandflies on military training grounds.

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.

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.

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.

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.

Prevalence and risk factors of Toxoplasma gondii and Leishmania spp. infections in apparently healthy dogs in west Shewa zone, Oromia, Ethiopia.

BACKGROUND: In urban settings, the presence of a high density of the human population and contact with domestic and/or stray animals such as dogs and cats can be risk factors for the transmission of zoonotic protozoa parasites. Toxoplasma gondii (T. gondii) and Leishmania spp. are zoonotic protozoon parasites with significant health burdens worldwide. METHODS: A cross-sectional study was used to investigate the antibody prevalence and risk factors of T. gondii and Leishmania spp. infections in 385 randomly selected dogs of Ambo, Bako, and Gojo towns of West Shewa Zone, Oromia regional state, Ethiopia. A questionnaire survey was administered to households to collect data on potential risk factors. Dog sera samples were assayed for T. gondii IgG antibodies using the direct agglutination test while Leishmania spp. specific antibodies tested using an indirect enzyme-linked immunosorbent assay (ELISA). Logistic regression was used for data analysis. RESULTS: Overall, T. gondii and Leishmania spp. infection seroprevalence was found to be 82.86% (95% confidence interval (CI): 78.71-86.49%) and 92.47% (95% CI: 89.36-94.90%), respectively. Seropositivity for both T. gondii and Leishmania spp. was found in 82.58% of the dogs. None of the investigated factors were associated with Leishmania spp. seropositivity (p > 0.05). The seroprevalence of T. gondii was significantly different among the study towns (p = 0.003). The risk of T. gondii infection was 2.71 times higher in adult dogs than juvenile dogs (p = 0.043). Dogs kept simultaneously with other domestic animals had increased odds of T. gondii seropositivity compared to those with no other domestic animals (Adjusted Odds ratio: 1.96, p = 0.021). However, altitude, sex, breed, housing, feeding, educational level of head of the household, and dog's living area were not significantly associated with T. gondii seropositivity (p > 0.05). CONCLUSIONS: The high seropositivity and the simultaneous presence of antibodies of T. gondii and Leishmania spp. in dogs suggest the widespread nature of these parasites in the environment and the high potential of transmission to other animals and humans. Further epidemiological studies, isolation and molecular characterization of the parasites, and educational campaigns are suggested.

Remarkable diversity, new records and Leishmania detection in the sand fly fauna of an area of high endemicity for cutaneous leishmaniasis in Acre state, Brazilian Amazonian Forest.

The species richness of Amazonian phlebotomines is considered to be one of the highest in the world. In the present study, we investigated the richness and diversity of phlebotomine fauna in Xapuri city, Acre state, Western Brazilian Amazonia, which is an area that is highly endemic for cutaneous leishmaniasis. Sand fly collections were performed monthly from August 2013 to July 2015 (288 h total of sampling effort) in intradomiciliary, peridomiciliary, and forested environments of two localities. Collected females were dissected, microscopically examined for flagellates in their guts, and preserved in ethanol. A total of 21,197 specimens comprising 14 genera and 57 species were collected, and the majority of these were Nyssomyia, Psychodopygus, and Trichophoromyia genera. Three new records of phlebotomine species for Acre are presented here, including Brumptomyia brumpti, Psathyromyia pradobarrientosi, and for the first time in Brazil, Th. omagua. In Xapuri, the phlebotomine fauna of different ecotopes was varied in regard to abundance, diversity, and frequency, and they included proven and permissive vectors of Leishmania spp. The fauna discovered in the forested areas (57 species) was richer and more diverse than was that (33 species) identified in the peri­ and intra-domiciles. The identification of Leishmania subgenera that were present in sand fly guts according to SSU rRNA sequences revealed ten and three species harboring Leishmania of subgenera Viannia and Leishmania (most likely Leishmania amazonensis), respectively. The presence of Leishmania (Leishmania) in sand flies are reported here for the first time in Acre. The presence of L. (Viannia) spp. in Brumptomyia sp. and Lutzomyia sherlocki. and the occurrence of mixed infections with Leishmania of both subgenera in Ps. lainsoni have been reported for the first time in Brazil. Taken together, data from previous studies and from the present study highlight the remarkable complexity of phlebotomine fauna that is possibly due to the well-preserved Xapuri forested areas sustaining vital economic activities of plant extraction and ecological tourism. Our findings also provide new insights into the ongoing adaptation of Trichophoromyia and Psychodopygus species to human habitats.

A cross-sectional study of Leishmania spp. in draft horses from the Distrito Federal, Brazil: Seroprevalence, spatial distribution, and associated factors.

Equine leishmaniasis is a neglected tropical disease caused by the protozoan of the Leishmania genus, and it has been reported in several countries around the world, especially Brazil. Therefore, the present investigation aims to conduct a cross-sectional study to determine the prevalence, spatial distribution, and associated factors with seropositivity for Leishmania spp. in draft horses from the Distrito Federal, Brazil. The serological survey was conducted on 411 animals, employing the Indirect Immunofluorescence Test (IFA) and Enzyme-Linked Immunosorbent Assay (ELISA). The Kappa (κ) and gross agreement indexes evaluated the Leishmania spp. seropositivity by IFA and ELISA test. The statistical analysis was performed using the Chi-square test and logistic regression. The spatial analysis showed the areas with the highest number of seropositive and the Moran autocorrelation analyses between the spatial distribution and the epidemiological model's explanatory variables. A 27.01 % co-positivity was observed with a κ index of 52.64 %. The final model considered the variables: access to water bodies (p-value = 0.008, Odds Ratio (OR) = 2.26, Confidence Interval (CI) = 1.24-4.13), the absence of the use of ectoparasiticide (p-value = 0.008, OR = 1.93 CI = 1.18-3.15) and traveling animal (p-value = 0.059, OR = 1.54, CI = 0.98-2.41). The Kernel map showed hot areas with a high concentration of nine positive animals per area and some lighter areas ranging from five to seven positive animals per area where control measures should be performed. The Moran autocorrelation analysis was significant for the variables: traveling animal (Moran's I = 0.540 and pseudo-p-value = 0.001) and the absence of use ectoparasiticide (Moran's I = 0.259 and pseudo-p-value = 0.005). The current study exposes a high seroprevalence of Leishmania spp. in horses in the Distrito Federal, Brazil. Moreover, it proposes that traveling animal, the access to water bodies and the absence of the use of ectoparasiticide are significantly associated with seropositivity for Leishmania spp. in draft horses, which may contribute to the implementation of prophylactic and controls measures where leishmaniasis is already stalled.

Spatial and Temporal Variability of Visceral Leishmaniasis in Colombia, 2007 to 2018.

Visceral leishmaniasis (VL) is a neglected tropical disease associated with poverty and is endemic in 56 countries worldwide. Brazil, Venezuela, and Colombia are the most affected countries in South America. In Colombia, the National Public Health Surveillance System (SIVIGILA) consolidates epidemiological information and monitors all VL cases nationwide. However, to date, no studies have investigated the occurrence of VL in Colombia using metadata analysis. We studied the demographic data, the spatial and temporal distribution of VL cases, and the association with vector distribution of Leishmania species in Colombia from 2007 to 2018. We found 306 VL cases reported to SIVIGILA for this period, with a coverage of 25.5 cases/year, and a mortality of 2.28% (seven deaths). The highest number of confirmed cases (N = 52) occurred in 2007; the lowest (N = 9) occurred in 2012. The cases were reported mainly in children (< 7 years) affiliated with the subsidized health regimen. Regarding the geographic distribution, the cases were reported by 42 municipalities distributed in 10 departments. The occurrence of VL cases toward the northeast of Colombia, and the distribution of vectors, such as Lutzomyia longipalpis and Lu. evansi, may be changing the panorama of VL in the country. We conclude that VL, mainly in recent years, shows a temporal and spatial variability associated with the occurrence of cases in new settings. Our findings increase our understanding and knowledge of this disease, and suggest the need to monitor and prioritize areas with changes in geographic expansion to improve prevention and control actions in the country.

Sand fly identification and screening for Leishmania spp. in six provinces of Thailand.

BACKGROUND: Phlebotomine sand flies are vectors of Leishmania spp. At least 27 species of sand flies have been recorded in Thailand. Although human leishmaniasis cases in Thailand are mainly imported, autochthonous leishmaniasis has been increasingly reported in several regions of the country since 1999. Few studies have detected Leishmania infection in wild-caught sand flies, although these studies were carried out only in those areas reporting human leishmaniasis cases. The aim of this study was therefore to identity sand fly species and to investigate Leishmania infection across six provinces of Thailand. METHODS: Species of wild-caught sand flies were initially identified based on morphological characters. However, problems identifying cryptic species complexes necessitated molecular identification using DNA barcoding in parallel with identification based on morphological characters. The wild-caught sand flies were pooled and the DNA isolated prior to the detection of Leishmania infection by a TaqMan real-time PCR assay. RESULTS: A total of 4498 sand flies (1158 males and 3340 females) were caught by trapping in six provinces in four regions of Thailand. The sand flies were morphologically classified into eight species belonging to three genera (Sergentomyia, Phlebotomus and Idiophlebotomus). Sergentomyia iyengari was found at all collection sites and was the dominant species at most of these, followed in frequency by Sergentomyia barraudi and Phlebotomus stantoni, respectively. DNA barcodes generated from 68 sand flies allowed sorting into 14 distinct species with 25 operational taxonomic units, indicating a higher diversity (by 75%) than that based on morphological identification. Twelve barcoding sequences could not be assigned to any species for which cytochrome c oxidase subunit I sequences are available. All tested sand flies were negative for Leishmania DNA. CONCLUSIONS: Our results confirm the presence of several sand fly species in different provinces of Thailand, highlighting the importance of using DNA barcoding as a tool to study sand fly species diversity. While all female sand flies tested in this study were negative for Leishmania, the circulation of Leishmania spp. in the investigated areas cannot be ruled out.

Prevalence and associated risk factors of Leishmania infection among immunocompetent hosts, a community-based study in Chiang Rai, Thailand.

BACKGROUND: Leishmaniasis is an emerging infectious disease reported in the north and south of Thailand of which patients with HIV/AIDS are a high risk group for acquiring the infection. A lack of information regarding prevalence, and the risk association of Leishmania infection among asymptomatic immunocompetent hosts needs further investigation. Information on potential vectors and animal reservoirs in the affected areas is also important to control disease transmission. METHODS: An outbreak investigation and a cross-sectional study were conducted following one index case of cutaneous leishmaniasis (CL) caused by L. martiniquensis in an immunocompetent male patient reported in August 2015, Chiang Rai Province, Thailand. From September to November 2015, a total of 392 participants at two study areas who were related to the index case, 130 students at a semi-boarding vocational school and 262 hill tribe villagers in the patient's hometown, were recruited in this study. The nested internal transcribed spacer 1-PCR (ITS1-PCR) was performed to detect Leishmania DNA in buffy coat, and nucleotide sequencing was used to identify species. Antibody screening in plasma was performed using the Direct Agglutination Test (DAT), and associated risk factors were analyzed using a standardized questionnaire. Captured sandflies within the study areas were identified and detected for Leishmania DNA using nested ITS1-PCR. Moreover, the animal reservoirs in the study areas were also explored for Leishmania infection. RESULTS: Of 392 participants, 28 (7.1%) were positive for Leishmania infection of which 1 (4.8%) was L. martiniquensis, 12 (57.1%) were L. orientalis and 8 (38.1%) were Leishmania spp. Of 28, 15 (53.6%) were DAT positive. None showed any symptoms of CL or visceral leishmaniasis. Risk factors were associated with being female (adjusted odds ratio, AOR 2.52, 95%CI 1.01-6.26), increasing age (AOR 1.05, 95%CI 1.02-1.08), having an animal enclosure in a housing area (AOR 3.04, 95%CI 1.13-8.22), being exposed to termite mounds (AOR 3.74, 95%CI 1.11-12.58) and having domestic animals in a housing area (AOR 7.11, 95%CI 2.08-24.37). At the semi-boarding vocational school, six Sergentomyia gemmea samples were PCR positive for DNA of L. orientalis and one S. gemmea was PCR positive for DNA of L. donovani/L. infantum. Additionally, one Phlebotomus stantoni was PCR positive for DNA of L. martiniquensis, and one black rat (Rattus rattus) was PCR positive for DNA of L. martiniquensis. CONCLUSION: This information could be useful for monitoring Leishmania infection among immunocompetent hosts in affected areas and also setting up strategies for prevention and control. A follow-up study of asymptomatic individuals with seropositive results as well as those with positive PCR results is recommended.

Dipeptidyl peptidase III as a DNA marker to investigate epidemiology and taxonomy of Old World Leishmania species.

BACKGROUND: Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. In Leishmania, DPPIII, was reported with other peptidases to play a significant role in parasites' growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop and evaluate a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene for Leishmania species identification and for phylogeny, and thus for diagnostic and molecular epidemiology studies of Old World Leishmania species. METHODOLOGY: Orthologous DDPIII genes were searched in all Leishmania genomes and aligned to design PCR primers and identify relevant restriction enzymes. A PCR assays was developed and seventy-two Leishmania fragment sequences were analyzed using MEGA X genetics software to infer evolution and phylogenetic relationships of studied species and strains. A PCR-RFLP scheme was also designed and tested on 58 OW Leishmania strains belonging to 8 Leishmania species and evaluated on 75 human clinical skin samples. FINDINGS: Sequence analysis showed 478 variable sites (302 being parsimony informative). Test of natural selection (dN-dS) (-0.164, SE = 0.013) inferred a negative selection, characteristic of essential genes, corroborating the DPPIII importance for parasite survival. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment. Sequence analyses and phylogenies confirmed occurrence of 6 clusters congruent to L. major, L. tropica, L. aethiopica, L. arabica, L. turanica, L. tarentolae species, and one to the L. infantum and L. donovani species complex. A PCR-RFLP algorithm for Leishmania species identification was designed using double digestions with HaeIII and KpnI and with SacI and PvuII endonucleases. Overall, this PCR-RFLP yielded distinct profiles for each of the species L. major, L. tropica, L. aethiopica, L. arabica and L. turanica and the L. (Sauroleishmania) L. tarentolae. The species L. donovani, and L. infantum shared the same profile except for strains of Indian origin. When tested on clinical samples, the DPPIII PCR showed sensitivities of 82.22% when compared to direct examination and was able to identify 84.78% of the positive samples. CONCLUSION: The study demonstrates that DPPIII gene is suitable to detect and identify Leishmania species and to complement other molecular methods for leishmaniases diagnosis and epidemiology. Thus, it can contribute to evidence-based disease control and surveillance.

A review of the leishmanin skin test: A neglected test for a neglected disease.

The leishmanin skin test (LST) has been used for decades to detect exposure and immunity to the parasite Leishmania, the causative agent of the neglected tropical disease leishmaniasis. In the LST, Leishmania antigen (leishmanin) is intradermally injected into the forearm. In an individual who has been previously infected, a delayed-type hypersensitivity (DTH) reaction results in a measurable induration at the site of the injection, indicating that previous exposure to Leishmania has resulted in the development of cell-mediated immunity. LST positivity is associated with long-lasting protective immunity against reinfection, most notably as reported for visceral leishmaniasis (VL). Despite efforts over the past few decades, leishmanin antigen is no longer produced under good manufacturing practice (GMP) conditions anywhere in the world. Consequently, the use of the LST in epidemiological studies has declined in favor of serological and molecular tests. In this review, we provide a historical overview of the LST and justification for the reintroduction of leishmanin. A GMP-grade leishmanin can be used to detect immunity in vivo by the LST and can be investigated for use in an interferon-γ release assay (IGRA), which may serve as an in vitro version of the LST. The LST will be a valuable tool for surveillance and epidemiological studies in support of the VL elimination programs and as a surrogate marker of immunity in vaccine clinical trials. METHODS: A review of the literature was conducted using PubMed as the primary database, with MeSH terms "leishmanin skin test" OR "Montenegro test" OR "Montenegro skin test." Articles written in English that describe the history or standardization of leishmanin, the use of leishmanin in an IGRA, or the use of the LST in epidemiological studies or vaccine trials were prioritized in our appraisal of the literature.