Visceral leishmaniasis in the hills of western Nepal: A transmission assessment.

In Nepal, visceral leishmaniasis (VL) has been targeted for elimination as a public health problem by 2026. Recently, increasing numbers of VL cases have been reported from districts of doubtful endemicity including hills and mountains, threatening the ongoing VL elimination program in Nepal. We conducted a multi-disciplinary, descriptive cross-sectional survey to assess the local transmission of Leishmania donovani in seven such districts situated at altitudes of up to 1,764 meters in western Nepal from March to December 2019. House-to-house surveys were performed for socio-demographic data and data on past and current VL cases. Venous blood was collected from all consenting individuals aged ≥2 years and tested with the rK39 RDT. Blood samples were also tested with direct agglutination test, and a titer of ≥1:1600 was taken as a marker of infection. A Leishmania donovani species-specific PCR (SSU-rDNA) was performed for parasite species confirmation. We also captured sand flies using CDC light traps and mouth aspirators. The house-to-house surveys documented 28 past and six new VL cases of which 82% (28/34) were without travel exposure. Overall, 4.1% (54/1320) of healthy participants tested positive for L. donovani on at least one serological or molecular test. Among asymptomatic individuals, 17% (9/54) were household contacts of past VL cases, compared to 0.5% (6/1266) among non-infected individuals. Phlebotomus argentipes, the vector of L. donovani, was found in all districts except in Bajura. L. donovani was confirmed in two asymptomatic individuals and one pool of sand flies of Phlebotomus (Adlerius) sp. We found epidemiological and entomological evidence for local transmission of L. donovani in areas previously considered as non-endemic for VL. The national VL elimination program should revise the endemicity status of these districts and extend surveillance and control activities to curb further transmission of the disease.

Immune response profiles from humans experimentally exposed to Phlebotomus duboscqi bites.

Introduction: Cutaneous leishmaniasis is a neglected vector-borne parasitic disease prevalent in 92 countries with approximately one million new infections annually. Interactions between vector saliva and the human host alter the response to infection and outcome of disease. Methods: To characterize the human immunological responses developed against saliva of Phlebotomus duboscqi, a Leishmania major (L. major) vector, we repeatedly exposed the arms of 14 healthy U.S volunteers to uninfected P. duboscqi bites. Blood was collected a week after each exposure and used to assess total IgG antibodies against the proteins of P. duboscqi salivary gland homogenate (SGH) and the levels of IFN-gamma and IL-10 from peripheral blood mononuclear cells (PBMCs) stimulated with SGH or recombinant sand fly proteins. We analyzed skin punch biopsies of the human volunteer arms from the insect bite site and control skin site after multiple P. duboscqi exposures (four volunteers) using immunohistochemical staining. Results: A variety of immediate insect bite skin reactions were observed. Late skin reactions to insect bites were characterized by macular hyperpigmentation and/or erythematous papules. Hematoxylin and eosin staining showed moderate mononuclear skin infiltrate with eosinophils in those challenged recently (within 2 months), eosinophils were not seen in biopsies with recall challenge (6 month post bites). An increase in plasma antigen-specific IgG responses to SGH was observed over time. Western Blot results showed strong plasma reactivity to five P. duboscqi salivary proteins. Importantly, volunteers developed a cellular immunity characterized by the secretion of IFN-gamma upon PBMC stimulation with P. duboscqi SGH and recombinant antigens. Discussion: Our results demonstrate that humans mounted a local and systemic immune response against P. duboscqi salivary proteins. Specifically, PduM02/SP15-like and PduM73/adenosine deaminase recombinant salivary proteins triggered a Th1 type immune response that might be considered in future development of a potential Leishmania vaccine.

Neutrophil extracellular traps formation: effect of Leishmania major promastigotes and salivary gland homogenates of Phlebotomus papatasi in human neutrophil culture.

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.

TORC1 is an essential regulator of nutrient-controlled proliferation and differentiation in Leishmania.

Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite proliferation and differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8, and identify new parasite-specific proteins that interact in this complex. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient-rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites are unable to respond to nutrients to differentiate into proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. We additionally show that RPTOR1-/- metacyclic promastigotes develop into amastigotes but do not proliferate in the mammalian host to cause pathology. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.

Geographical and Molecular Analysis of Haplotype Variations in Leishmania major Among Infected Iranian Phlebotomus papatasi.

PURPOSE: Leishmania major is main causative agent and Phlebotomus papatasi is only proven vector of Zoonotic Cutaneous Leishmaniasis (ZCL) in Iran. Human leishmaniasis is mostly susceptible to climatic conditions and molecular variations of Leishmania parasites within sandflies. METHODS: L. major was analyzed based on geographical, environmental, climatic changes and haplotype variations within P. papatasi. Molecular tools and different geographical aspects were employed using Arc-GIS software for mapping the geographic distribution of samples and other statistics tests. Fragments of ITS-rDNA, k-DNA, and microsatellite genes of Leishmania were used for PCR, RFLP, sequencing, and phylogenetic analyses. RESULTS: Totally 81 out of 1083 female P. papatasi were detected with Leishmania parasites: 70 and five were L. major and L. turanica, respectively. Golestan and Fars provinces had the highest (13.64%) and lowest (4.55%) infection rates, respectively. The infection rate among female P. papatasi collected from gerbil burrows was significantly higher (15.15%) than animal shelters, yards, and inside houses (4.48%) (P < 0.0%). Microsatellite was more sensitive (22.72%) than k-DNA (18.8%) and ITS-rDNA (7.48%). More molecular variations of L. major were found in Isfahan province. CONCLUSIONS: Arc-GIS software and other statistics tests were employed to find Leishmania positive and haplotype variations among sand flies. Geographical situations, altitude, climate, precipitation, humidity, temperature, urbanization, migrations, regional divergences, deforestation, global warming, genome instability, ecology, and biology of the sand flies intrinsically, and the reservoir hosts and neighboring infected locations could be reasons for increasing or decreasing the rate of Leishmania infection and haplotype variations.

Epidemiological survey, molecular profiling and phylogenetic analysis of cutaneous leishmaniasis in Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: Cutaneous leishmaniasis (CL), an emerging vector-borne ailment in Khyber Pakhtunkhwa (KPK), Pakistan, exhibits diverse spread patterns and outbreaks. METHODS: To comprehend its epidemiology and identify parasite species, we conducted an active survey on suspected CL cases (n=8845) in KPK. RESULTS: Microscopy and internal transcribed spacer-1 PCR-restriction fragment length polymorphism (RFLP) molecular techniques detected Leishmania spp. in blood samples. Phylogenetic analysis gauged genetic affinities with other areas. District Bannu displayed the highest CL impact (14.58%), while Swat had the lowest impact (4.33%) among cases. Annual blood examination rate, parasite incidence and slide positivity rate were 4.96 per 1000 people, 0.0233 and 0.047%, respectively. CL infections were prevalent in 1- to 20-y-olds, with males (57.17%) more vulnerable than females (42.82%). Single lesions occurred in 43.73% of patients, while 31.2% people had two lesions, 17.31% had three lesions and 7.74% had more than three lesions. Most had sand-fly exposure but lacked preventive measures like repellents and bed nets. Leishmania tropica was confirmed via RFLP analysis in amplified samples. Phylogenetic analysis unveiled genetic parallels between L. tropica of KPK and isolates from China, Iran, Afghanistan, India, Syria and Morocco. CONCLUSIONS: Urgent comprehensive control measures are imperative. Early detection, targeted interventions and raising awareness of CL and sand-fly vectors are vital for reducing the disease's impact. International collaboration and monitoring are crucial to tackle Leishmania spp.'s genetic diversity and curtail its cross-border spread.

Entomological, parasitological and molecular investigations in a new focus of cutaneous leishmaniasis in Youssoufia region, Morocco.

BACKGROUND AND AIMS: Leishmaniasis is a neglected tropical infection caused by Leishmania parasite that affect human and animal. In Morocco, the cutaneous leishmaniasis has spread substantially to the new areas. The surveillance limited to active foci may underestimate the occurrence of cutaneous leishmaniasis (CL). This study aims to investigate the local transmission of CL in rural districts of Youssoufia province, central Morocco, as a potential focus of CL. METHODS: For this purpose, parasitological, molecular and entomological investigations were carried out in this area. Data collection concerns potential vectors and human cases. Thus, 402 patients were examined for suspected leishmaniasis lesions in three localities of the province of Youssoufia. In these same localities, 983 sand flies were collected by CDC light traps and sticky paper during one-night per month during 6 months. These sand flies were all identified morphologically using the Moroccan identification key. RESULTS: The results showed that among the 25 skin lesions detected in a population of 402 individuals, 18 were confirmed by kDNA nested PCR as CL positive patients, of which only 25% were positive by direct examination. Leishmania tropica and Leishmania major were identified as causative agents of CL in the study area. Direct parasitological examination showed a low sensitivity (27.78%), especially for L. major, although its specificity was evaluated at 100%. Regarding entomological results, both genera of the Moroccan sand fly were collected in the study area: Genus/Phlebotomus (75.28%) and Sergentomyia (24.72%). Phlebotomus (P) papatasi, the proven vector of L. major, was the most abundant species (33.98%), followed by Paralongicollum sergenti (22.58%), the confirmed vector of L. tropica; while Sergentomyia (S) minuta, P. longicuspis, S. fallax and P. kazeruni were collected with, respectively, 17.60%, 16.99%, 7.12% and 1.73%. CONCLUSION: This study constitutes the first report of CL in the study areas, as well as the coexistence of L. tropica and L. major in these rural localities. Local transmission of CL is highly probable, as indicated by the prevalence of the two proven vectors of L. major and L. tropica. To control the spread of this disease, our results suggest the use of highly sensitive molecular methods to detect CL cases in potential leishmaniasis foci, which will improve surveillance.

Cutaneous and Visceral Leishmaniasis Caused by the Same Zymodeme of Leishmania donovani in Kerala, India.

The tribal population in and around the Western Ghats region of India is affected by both cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) with typical clinical symptoms. In this study, we recorded and analyzed seven CL and three VL cases from this emerging belt. All the cases were found as autochthonous transmission. Multiple genetic markers (minicircle kinetoplast DNA polymerase chain reaction and restriction fragment length polymorphism of 3'untranslated region heat shock protein (HSP) 70, a larger segment of HSP 70, and 6-phosphogluconate dehydrogenase [PGDH] gene sequences) were used to identify and characterize the parasite. It was found that both clinical manifestations are caused by zymodeme MON-37 of Leishmania donovani. We have investigated the detailed entomological and epidemiological aspects of disease transmission. An abundant population of the proven vector Phlebotomus argentipes was observed in the study villages.

Species identification of phlebotomine sandflies using deep learning and wing interferential pattern (WIP).

Sandflies (Diptera; Psychodidae) are medical and veterinary vectors that transmit diverse parasitic, viral, and bacterial pathogens. Their identification has always been challenging, particularly at the specific and sub-specific levels, because it relies on examining minute and mostly internal structures. Here, to circumvent such limitations, we have evaluated the accuracy and reliability of Wing Interferential Patterns (WIPs) generated on the surface of sandfly wings in conjunction with deep learning (DL) procedures to assign specimens at various taxonomic levels. Our dataset proves that the method can accurately identify sandflies over other dipteran insects at the family, genus, subgenus, and species level with an accuracy higher than 77.0%, regardless of the taxonomic level challenged. This approach does not require inspection of internal organs to address identification, does not rely on identification keys, and can be implemented under field or near-field conditions, showing promise for sandfly pro-active and passive entomological surveys in an era of scarcity in medical entomologists.

Interactions between Leishmania parasite and sandfly: a review.

Leishmaniasis transmission cycles are maintained and sustained in nature by the complex crosstalk of the Leishmania parasite, sandfly vector, and the mammalian hosts (human, as well as zoonotic reservoirs). Regardless of the vast research on human host-parasite interaction, there persists a substantial knowledge gap on the parasite's development and modulation in the vector component. This review focuses on some of the intriguing aspects of the Leishmania-sandfly interface, beginning with the uptake of the intracellular amastigotes from an infected host to the development of the parasite within the sandfly's alimentary canal, followed by the transmission of infective metacyclic stages to another potential host. Upon ingestion of the parasite, the sandfly hosts an intricate repertoire of immune barriers, either to evade the parasite or to ensure its homeostatic coexistence with the vector gut microbiome. Sandfly salivary polypeptides and Leishmania exosomes are co-egested with the parasite inoculum during the infected vector bite. This has been attributed to the modulation of the parasite infection and subsequent clinical manifestation in the host. While human host-based studies strive to develop effective therapeutics, a greater understanding of the vector-parasite-microbiome and human host interactions could help us to identify the targets and to develop strategies for effectively preventing the transmission of leishmaniasis.

The ongoing risk of Leishmania donovani transmission in eastern Nepal: an entomological investigation during the elimination era.

BACKGROUND: Visceral leishmaniasis (VL), a life-threatening neglected tropical disease, is targeted for elimination from Nepal by the year 2026. The national VL elimination program is still confronted with many challenges including the increasingly widespread distribution of the disease over the country, local resurgence and the questionable efficacy of the key vector control activities. In this study, we assessed the status and risk of Leishmania donovani transmission based on entomological indicators including seasonality, natural Leishmania infection rate and feeding behavior of vector sand flies, Phlebotomus argentipes, in three districts that had received disease control interventions in the past several years in the context of the disease elimination effort. METHODS: We selected two epidemiologically contrasting settings in each survey district, one village with and one without reported VL cases in recent years. Adult sand flies were collected using CDC light traps and mouth aspirators in each village for 12 consecutive months from July 2017 to June 2018. Leishmania infection was assessed in gravid sand flies targeting the small-subunit ribosomal RNA gene of the parasite (SSU-rRNA) and further sequenced for species identification. A segment (~ 350 bp) of the vertebrate cytochrome b (cytb) gene was amplified from blood-fed P. argentipes from dwellings shared by both humans and cattle and sequenced to identify the preferred host. RESULTS: Vector abundance varied among districts and village types and peaks were observed in June, July and September to November. The estimated Leishmania infection rate in vector sand flies was 2.2% (1.1%-3.7% at 95% credible interval) and 0.6% (0.2%-1.3% at 95% credible interval) in VL and non-VL villages respectively. The common source of blood meal was humans in both VL (52.7%) and non-VL (74.2%) villages followed by cattle. CONCLUSIONS: Our findings highlight the risk of ongoing L. donovani transmission not only in villages with VL cases but also in villages not reporting the presence of the disease over the past several years within the districts having disease elimination efforts, emphasize the remaining threats of VL re-emergence and inform the national program for critical evaluation of disease elimination strategies in Nepal.

Blood meal analysis and molecular detection of mammalian Leishmania DNA in wild-caught Sergentomyia spp. from Tunisia and Saudi Arabia.

Phlebotomine sand flies (Diptera: Phlebotominae) belonging to the genus Phlebotomus are vectors of pathogens such as arboviruses, bacteria, and parasites (Leishmania). Species of the genus Sergentomyia (Se.) transmit Sauroleishmania (Reptile Leishmania) and feed on cold-blooded vertebrates; recently, they have been incriminated in mammalian Leishmania transmission. In addition, they have been reported to feed on warm-blooded vertebrates. This study aimed to (i) screen wild-caught Sergentomyia species for the detection of mammalian Leishmania and (ii) identify the blood meal origin of engorged females. The sand flies were collected using centers for disease control and prevention (CDC) traps, mounted and identified morphologically. Only females of the genus Sergentomyia were screened for Leishmania infection using PCR targeting the 18S ribosomal DNA locus. For positive specimens, Leishmania parasites were typed using nested PCR targeting ribosomal internal transcribed spacer 1 followed by digestion with HaeIII. The PCR-RFLP results were confirmed through sequencing. Blood meal identification was performed through PCR amplification of the vertebrate cytochrome b gene using degenerate primers followed by sequencing. In total, 6026 sand fly specimens were collected between 2009 and 2018. Among these, 511 belonged to five species of Sergentomyia genus: Se. minuta (58.51%), Se. fallax (18.01%), Se. clydei (14.68%), Se. dreyfussi (6.26%), and Se. antennata (2.54%). A total of 256 female Sergentomyia sp. specimens were screened for Leishmania infection. Seventeen (17) were positive (6.64%). Two Leishmania species were identified. Leishmania major DNA was detected in five specimens; this included three Se. fallax, one Se. minuta, and one Se. dreyfussi collected from Tunisia. Leishmania infantum/L. donovani complex was detected in four Se. minuta and three Se. dreyfussi specimens collected from Tunisia. In addition, we identified the blood meal origin of five engorged Se. minuta specimens collected from Tunisia. Sequencing results revealed two blood sources: humans (n = 4) and reptiles (n = 1) indicating possible role of Sergentomyia species in the transmission of human Leishmania. In addition, these species could be involved in the life cycle of L. infantum/L. donovani complex and L. major. The results of the blood meal origin showed that Sergentomyia fed on both cold- and warm-blooded vertebrates. These findings enable a better understanding of the behavior of this sand fly genus. Further studies should focus on the role of Sergentomyia in human Leishmania transmission and possible control of this disease.

Morphology does not allow differentiating the species of the Phlebotomus perniciosus complex: Molecular characterization and investigation of their natural infection by Leishmania infantum in Morocco.

Morphological and DNA-based complemented approaches were applied for characterization of sympatric populations of Phlebotomus longicuspis and Phlebotomus perniciosus in Morocco. Both sand fly species are generally recorded in sympatry in North Africa but on few occasions have been molecularly characterized. The diagnostic confusion of these species has led to errors in their geographical distribution and probably, in the assignment of their role in the transmission of L. infantum. Sand flies were caught inside households in El Borouj, central Morocco, in 2014-2015. For female sand flies, detection of L. infantum natural infection and blood meal identification were carried out. According to morphological identification, Phlebotomus longicuspis s.l. (34.7%) was the second most abundant Phlebotomus species after P. sergenti, followed by atypical Phlebotomus perniciosus (7.1%); 11.6% of the male specimens of P. longicuspis s.l. were identified as P. longicuspis LCx according to the number of coxite setae. The density of Larroussius species was very high (31 Larroussius/light trap/night) in the peripheral neighbourhood of Oulad Bouchair (p = 0.001) where the first case of cutaneous leishmaniasis due to Leishmania infantum was detected in 2017. Phylogenetic trees based on three independent genes highlighted three well-supported clusters within P. perniciosus complex that could be interpreted as corresponding to P. perniciosus, P. longicuspis s.s. and an undescribed species, all coexisting in sympatry. Some females with typical morphology of P. longicuspis were genetically homologous to P. perniciosus. The taxa cannot be differentiated by morphological methods but characterized by a distinctive genetic lineage for which the synapomorphic characters are described. Leishmania infantum was detected in females of all clusters with a low parasite load. Population genetics will help to assess the threat of the geographical spread of L. infantum in Morocco by determining the density, abundance and vector role of the species of the P. perniciosus complex identified correctly.

First evidence of experimental genetic hybridization between cutaneous and visceral strains of Leishmania donovani within its natural vector Phlebotomus argentipes.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites of genus Leishmania, and transmitted by different species of Phlebotomine sand flies. More than 20 species of Leishmania are known to cause disease in humans and other animals. Leishmania donovani species complex is known to have a vast diversity of clinical manifestations in humans, but underlying mechanisms for such diversity are yet unknown. Long believed to be strictly asexual, Leishmania have been shown to undergo a cryptic sexual cycle inside its sandfly vector. Natural populations of hybrid parasites have been associated with the rise of atypical clinical outcomes in the Indian subcontinent (ISC). However, formal demonstration of genetic crossing in the major endemic sandfly species in the ISC remain unexplored. Here, we investigated the ability of two distinct variants of L. donovani associated with strikingly different forms of the disease to undergo genetic exchange inside its natural vector, Phlebotomus argentipes. Clinical isolates of L. donovani either from a Sri Lankan cutaneous leishmaniasis (CL) patient or an Indian visceral leishmaniasis (VL) patient were genetically engineered to express different fluorescent proteins and drug-resistance markers and subsequently used as parental strains in experimental sandfly co-infection. After 8 days of infection, sand flies were dissected and midgut promastigotes were transferred into double drug-selective media. Two double drug-resistant, dual fluorescent hybrid cell lines were recovered, which after cloning and whole genome sequencing, were shown to be full genomic hybrids. This study provides the first evidence of L. donovani hybridization within its natural vector Ph. argentipes.

Entomological Exploration of Sand Flies in Human Communities Affected by Cutaneous and Visceral Leishmaniasis in El Hajeb Province, Morocco.

Sand flies are the exclusive vectors of leishmaniasis. This group of parasitic diseases is a serious public health problem in Morocco. The aim of this study was to investigate the sand fly fauna, mainly the species composition, biodiversity, and seasonal activity of sand flies in El Hajeb in central Morocco. A total of six stations (Aït Naaman, Aït Rbaa, Aït Brahim, Ain Taoujdate, Sidi Mbarek, and Aït Oufella) were studied, five of which had recently recorded cases of leishmaniasis. Sand fly bimonthly captures were carried out using a sticky paper trap in different biotopes from March to December 2019. A total of 14590 adult sand flies were collected. The activity of the sand fly started in April and declined in November. The periods of high abundance were July, September, and November. Morphological identification of sand flies shows the presence of twelve species: Phlebotomus papatasi, P. longicuspis, P. perniciosus, P. sergenti, P. bergeroti, P. alexandri, P. dreyfussi, P. ariasi, Sergentomyia fallax, S. minuta, S. schwetzi, and S. antennata. The analysis showed that species belonging to the genus Phlebotomus were the most dominant (93.3%) and the risk periods were spread during the summer and autumn seasons. The present study provides for the first time information on the species of sand flies in El Hajeb. It, therefore, provides decision makers with an important tool to conduct vector control actions during peak periods in order to limit the transmission of leishmaniasis. A preprint was made available by the research square in the following link: "https://assets.researchsquare.com/files/rs-1409330/v1/dfef7013-0327-4a54-897f-214924a2d950.pdf?c=1646838874."

Phylogenetic and biogeographical traits predict unrecognized hosts of zoonotic leishmaniasis.

The spatio-temporal distribution of leishmaniasis, a parasitic vector-borne zoonotic disease, is significantly impacted by land-use change and climate warming in the Americas. However, predicting and containing outbreaks is challenging as the zoonotic Leishmania system is highly complex: leishmaniasis (visceral, cutaneous and muco-cutaneous) in humans is caused by up to 14 different Leishmania species, and the parasite is transmitted by dozens of sandfly species and is known to infect almost twice as many wildlife species. Despite the already broad known host range, new hosts are discovered almost annually and Leishmania transmission to humans occurs in absence of a known host. As such, the full range of Leishmania hosts is undetermined, inhibiting the use of ecological interventions to limit pathogen spread and the ability to accurately predict the impact of global change on disease risk. Here, we employed a machine learning approach to generate trait profiles of known zoonotic Leishmania wildlife hosts (mammals that are naturally exposed and susceptible to infection) and used trait-profiles of known hosts to identify potentially unrecognized hosts. We found that biogeography, phylogenetic distance, and study effort best predicted Leishmania host status. Traits associated with global change, such as agricultural land-cover, urban land-cover, and climate, were among the top predictors of host status. Most notably, our analysis suggested that zoonotic Leishmania hosts are significantly undersampled, as our model predicted just as many unrecognized hosts as unknown hosts. Overall, our analysis facilitates targeted surveillance strategies and improved understanding of the impact of environmental change on local transmission cycles.

Molecular detection of Leishmania (Sauroleishmania) adleri (Trypanosomatida: Trypanosomatidae) in Sergentomyia sp. sand flies (Diptera: Psychodidae) in Mali and Niger.

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies.

Species diversity of phlebotomine sand flies and sympatric occurrence of Leishmania (Mundinia) martiniquensis, Leishmania (Leishmania) donovani complex, and Trypanosoma spp. in the visceral leishmaniasis focus of southern Thailand.

Autochthonous leishmaniasis in Thailand has recently been a public health concern due to an increasing number of new clinical cases. Most indigenous cases were diagnosed with Leishmania (Mundinia) martiniquensis, and Leishmania (Mundinia) orientalis. However, some doubts regarding vector misidentification have arisen and need to be elucidated. Accordingly, we aimed to assess the species composition of sand flies and determine the molecular prevalence of trypanosomatids in the transmission area of leishmaniasis in southern Thailand. In the present study, a total of 569 sand flies were caught from the vicinity of a visceral leishmaniasis patient's house in Na Thawi District, Songkhla Province. Of these, 229 parous and gravid females consisted of Sergentomyia khawi, Se. barraudi, Phlebotomus stantoni, Grassomyia indica, and Se. hivernus, accounting for 31.4%, 30.6%, 29.7%, 7.9%, and 0.4%, respectively. However, Se. gemmea, which has previously been proposed as the most abundant species and putative vector of visceral leishmaniasis, was not found in the present study. Based on ITS1-PCR and sequence analysis, two specimens of Gr. indica and Ph. stantoni showed positive amplification of L. martiniquensis and L. donovani complex, respectively, the first one being presumed indigenous and the second one being not. Anuran Trypanosoma was also molecularly detected using SSU rRNA-PCR and ubiquitously found in 16 specimens of four dominant sand fly species except for Se. hivernus. The obtained sequences could be phylogenetically categorized into the two major amphibian clades (An04/Frog1 and An01+An02/Frog2). The existence of the monophyletic subgroup and distinct lineage suggests them as novel Trypanosoma species. The TCS network analysis of these anuran Trypanosoma sequences also revealed high haplotype diversity (Hd = 0.925 ± 0.050), but low nucleotide diversity (π = 0.019 ± 0.009). Furthermore, the living anuran trypanosomes were microscopically demonstrated in a single specimen of Gr. indica, supporting the vectorial capacity. Importantly, our data confirmed the scarcity of Se. gemmea and also uncovered, for the first time, the co-circulation of L. martiniquensis, L. donovani complex, and suspected novel anuran Trypanosoma spp. in phlebotomine sand files, implicating their potential role as vectors of trypanosomatid parasites. Therefore, the novel data from this study would greatly facilitate the comprehension of the complexity of trypanosomatid transmission and the establishment of prevention and control measures for this neglected disease more effectively.

A neglected among the neglected: a review of cutaneous leishmaniasis in Guatemala.

Cutaneous leishmaniasis (CL) is a parasitic vector-borne disease affecting mostly low- and middle-income countries. CL is endemic in Guatemala, where an increase in the number of cases and incidence and a changing disease distribution in the past decade have been reported. Important research was conducted in Guatemala in the 1980s and 1990s to understand the epidemiology of CL and two Leishmania species were identified as the aetiologic agents. Several species of sand flies have been reported, five of which are naturally infected with Leishmania. Clinical trials conducted in the country evaluated different treatments against the disease and provided solid evidence for CL control strategies that are applicable worldwide. More recently, in the 2000s and 2010s, qualitative surveys were conducted to understand community perceptions of the disease and to highlight the challenges and enablers for disease control. However, limited recent data have been generated regarding the current CL situation in Guatemala, and key information necessary for effective disease control, such as incrimination of vectors and reservoirs, is still lacking. This review describes the current state of knowledge of CL in Guatemala, including the main parasite and sand fly species, disease reservoirs, diagnosis and control, as well as the perceptions of communities in endemic regions.

Experimental feeding of Sergentomyia minuta on reptiles and mammals: comparison with Phlebotomus papatasi.

BACKGROUND: Sergentomyia minuta (Diptera: Phlebotominae) is an abundant sand fly species in the Mediterranean basin and a proven vector of reptile parasite Leishmania (Sauroleishmania) tarentolae. Although it feeds preferentially on reptiles, blood meal analyses and detection of Leishmania (Leishmania) infantum DNA in wild-caught S. minuta suggest that occasional feeding may occur on mammals, including humans. Therefore, it is currently suspected as a potential vector of human pathogens. METHODS: A recently established S. minuta colony was allowed to feed on three reptile species (i.e. lizard Podarcis siculus and geckos Tarentola mauritanica and Hemidactylus turcicus) and three mammal species (i.e. mouse, rabbit and human). Sand fly mortality and fecundity were studied in blood-fed females, and the results were compared with Phlebotomus papatasi, vector of Leishmania (L.) major. Blood meal volumes were measured by haemoglobinometry. RESULTS: Sergentomyia minuta fed readily on three reptile species tested, neglected the mouse and the rabbit but took a blood meal on human. However, the percentage of females engorged on human volunteer was low in cage (3%) and feeding on human blood resulted in extended defecation times, higher post-feeding mortality and lower fecundity. The average volumes of blood ingested by females fed on human and gecko were 0.97 µl and 1.02 µl, respectively. Phlebotomus papatasi females readily fed on mouse, rabbit and human volunteer; a lower percentage of females (23%) took blood meal on the T. mauritanica gecko; reptilian blood increased mortality post-feeding but did not affect P. papatasi fecundity. CONCLUSIONS: Anthropophilic behaviour of S. minuta was experimentally demonstrated; although sand fly females prefer reptiles as hosts, they were attracted to the human volunteer and took a relatively high volume of blood. Their feeding times were longer than in sand fly species regularly feeding on mammals and their physiological parameters suggest that S. minuta is not adapted well for digestion of mammalian blood. Nevertheless, the ability to bite humans highlights the necessity of further studies on S. minuta vector competence to elucidate its potential role in circulation of Leishmania and phleboviruses pathogenic to humans.