Diagnostic challenges in cutaneous leishmaniasis due to atypical Leishmania infantum: pathologists' insights from re-emergence zones.

Background: Leishmaniasis, a parasitic infection affecting both humans and animals, is increasingly spreading across Mediterranean and European regions, largely driven by human migration and environmental changes. In countries like Türkiye and across Europe, which have seen large influxes of migrants, the incidence of cutaneous leishmaniasis (CL) is rising, with cases now appearing in cities where the disease was previously undocumented. In these previously non-endemic areas, physicians unfamiliar with the characteristic lesions may misdiagnose CL, particularly in cases with only cutaneous manifestations. This study aims to evaluate the impact of re-emerging CL on the routine diagnostic practices of pathologists in Türkiye, by retrospectively reviewing cases. Methods: We conducted a retrospective analysis of CL cases diagnosed between 2013 and 2022 at a single pathology center in Türkiye, covering multiple provinces. Twelve cases of CL were identified and analyzed based on clinical presentation, pre-diagnosis, histopathological findings, and molecular diagnostics. DNA extraction and PCR were performed on paraffin-embedded tissue samples to identify the Leishmania species involved. Results: Out of the twelve CL cases reviewed, seven exhibited morphological findings strongly suggestive of CL (MFSS of CL), warranting further microbiological evaluation. All patients presented with non-healing skin lesions characterized by central ulceration, crater-like formations, or papulonodular lesions. Notably, CL was included in the clinical pre-diagnosis in only 58.3% of cases, while it was not considered in the remaining 41.7% of cases. Clinicians initially pre-diagnosed skin tumors in six cases (50%), four of which led to wide surgical excision. Histopathological examination in all cases revealed chronic or mixed (acute/chronic) inflammation, predominantly rich in histiocytes. To further investigate the role of Leishmania species in the pre-diagnosis, DNA extraction and PCR were performed on paraffin-embedded tissue samples, identifying L. infantum as the causative agent in 10 cases and L. major in two cases. Notably, L. infantum was the causative agent in all five cases initially misdiagnosed as skin tumors, which were also associated with a granulomatous type of chronic inflammation.

Genetic diversity and epidemiological insights into cutaneous leishmaniasis in Pakistan: a comprehensive study on clinical manifestations and molecular characterization of Leishmania species.

Cutaneous leishmaniasis (CL) stands out as a significant vector-borne endemic in Pakistan. Despite the rising incidence of CL, the genetic diversity of Leishmania species in the country's endemic regions remains insufficiently explored. This study aims to uncover the genetic diversity and molecular characteristics of Leishmania species in CL-endemic areas of Baluchistan, Khyber Pakhtunkhwa (KPK), and Punjab in Pakistan. Clinical samples from 300 CL patients were put to microscopic examination, real-time ITS-1 PCR, and sequencing. Predominantly affecting males between 16 to 30 years of age, with lesions primarily on hands and faces, the majority presented with nodular and plaque types. Microscopic analysis revealed a positivity rate of 67.8%, while real-time PCR identified 60.98% positive cases, mainly L. tropica, followed by L. infantum and L. major. Leishmania major (p = 0.009) showed substantially greater variation in nucleotide sequences than L. tropica (p = 0.07) and L. infantum (p = 0.03). Nucleotide diversity analysis indicated higher diversity in L. major and L. infantum compared to L. tropica. This study enhances our understanding of CL epidemiology in Pakistan, stressing the crucial role of molecular techniques in accurate species identification. The foundational data provided here emphasizes the necessity for future research to investigate deeper into genetic diversity and its implications for CL control at both individual and community levels.

First Molecular Evidence of Leishmania Infantum in Domestic Cats and Associated Risk Factors from the Black Sea Region of Türkiye.

PURPOSE: The objectives of the present study are to determine the molecular prevalence of Leishmania spp. in the owned domestic cats in the Black Sea Region of Türkiye and analyze the associated risk factors in FeL. METHODS: Conjunctival swabs (CS), blood, demographic, and clinical data were collected from 150 owned cats brought to the Veterinary Teaching Hospital during 2020-2022. Leishmania kinetoplast DNA (kDNA) from CS was screened by TaqMan Real-Time PCR (qPCR) with the genus-specific primers and a probe. RESULTS: All qPCR positive products were also amplified and sequenced to identify Leishmania species by ITS1 primers. Molecular prevalence of L. infantum found as 12.6% (19/150) in the observed cats in the Black Sea Region of Türkiye. There was a significant difference (p < 0.05) between neutered and intact cats with regarding to L. infantum positivity. Intact cats found to be 0.368 times more prone to be L. infantum-positive (L+). Dermatological lesions were found the most common (26.3%) problems in the L + cats. The median leucocyte count was the only parameter that was found statistically (p < 0.05) lower in the L + group (6.60) than the negative group (L-) (8.96), when comparing the WBC, NEU/LYM, MONO/LYM, EOS/LYM and PLT/LYM values. CONCLUSION: This study presented the molecular occurrence of FeL in the Black Sea Region of Türkiye for the first time indicating that the carrier status of the cats makes them alternative reservoirs for possible zoonotic transmission of L. infantum in this zone.

Comparative analysis of the severity and progression of cutaneous leishmaniasis caused by Leishmania tropica in untreated and glucantime-treated patients.

Millions of people worldwide are affected by cutaneous leishmaniasis (CL), a disease that has a significant impact on morbidity and mortality. Understanding the immune responses responsible for tissue damage or the process of lesion healing plays a pivotal role in shaping optimal treatment strategies. In this study, we investigated immunological phenotypes for three groups: glucantime treated (n = 30) and untreated (n = 30) CL patients infected with Leishmania tropica (L. tropica), and healthy controls (n = 20). T-lymphocytes (CD4+ and CD8+), and B lymphocytes (CD14+ and CD19+) were isolated using antibody-conjugated microbeads and magnetic field isolation to achieve high purity. A higher significant difference was observed between T-lymphocytes (CD4+ and CD8+), and B-lymphocytes (CD14+ and CD19+) cells in CL-infected groups before and after treatment (p < 0.0001). When compared, there was also a significant difference among T-lymphocytes (CD4+ and CD8+), B lymphocytes (CD14+ and CD19+) p < 0.0001, p < 0.0005, and p < 0.0007, respectively between CL-infected individuals (before and after treatment) to controls. Our findings suggest that an increased proportion of these cells seen in treated patients may mediate healing, while it is also possible that they may contribute to tissue injury. Understanding the immune system and lesion size of CL can help develop immunotherapies and comprehend the evolution of this parasitic disease.

The estimated distribution of autochthonous leishmaniasis by Leishmania infantum in Europe in 2005-2020.

BACKGROUND: This study describes the spatial and temporal distribution between 2005 and 2020 of human and animal leishmaniasis by Leishmania infantum in European countries reporting autochthonous cases, and highlights potential activities to improve disease control. METHODOLOGY/PRINCIPAL FINDINGS: It was based on a review of the scientific literature and data reported by the World Health Organization (WHO), the World Organization for Animal Health (WOAH) and the Ministries of Health, including hospital discharges in some countries. Autochthonous infections were reported in the scientific literature from 22 countries, including 13 and 21 countries reporting human and animal infections, respectively. In contrast, only 17 countries reported autochthonous human leishmaniasis cases to the WHO and 8 countries animal infections to the WOAH. The number of WOAH reported cases were 4,203, comprising 4,183 canine cases and 20 cases in wildlife. Of 8,367 WHO reported human cases, 69% were visceral leishmaniasis cases-of which 94% were autochthonous-and 31% cutaneous leishmaniasis cases-of which 53% were imported and mostly in France. The resulting cumulative incidence per 100,000 population of visceral leishmaniasis between 2005-2020, was highest in Albania (2.15 cases), followed by Montenegro, Malta, Greece, Spain and North Macedonia (0.53-0.42), Italy (0.16), Portugal (0.09) and lower in other endemic countries (0.07-0.002). However, according to hospital discharges, the estimated human leishmaniasis incidence was 0.70 in Italy and visceral leishmaniasis incidences were 0.67 in Spain and 0.41 in Portugal. CONCLUSIONS/SIGNIFICANCE: Overall, there was no evidence of widespread increased incidence of autochthonous human leishmaniasis by L. infantum in European countries. Visceral leishmaniasis incidence followed a decreasing trend in Albania, Italy and Portugal, and peaked in Greece in 2013, 2014 and 2017, and in Spain in 2006-2007 and 2011-2013. Animal and human cutaneous leishmaniasis remain highly underreported. In humans, hospital discharge databases provide the most accurate information on visceral leishmaniasis and may be a valuable indirect source of information to identify hotspots of animal leishmaniasis. Integrated leishmaniasis surveillance and reporting following the One Health approach, needs to be enhanced in order to improve disease control.

Arthropod vectors of disease agents: Their role in public and veterinary health in Turkiye and their control measures.

Mosquitoes, sandflies, and ticks are hematophagous arthropods that pose a huge threat to public and veterinary health. They are capable of serving as vectors of disease agents that can and have caused explosive epidemics affecting millions of people and animals. Several factors like climate change, urbanization, and international travel contribute substantially to the persistence and dispersal of these vectors from their established areas to newly invaded areas. Once established in their new home, they can serve as vectors for disease transmission or increase the risk of disease emergence. Turkiye (formerly Turkey) is vulnerable to climate change and has experienced upward trends in annual temperatures and rising sea levels, and greater fluctuations in precipitation rates. It is a potential hotspot for important vector species because the climate in various regions is conducive for several insect and acari species and serves as a conduit for refugees and immigrants fleeing areas troubled with armed conflicts and natural disasters, which have increased substantially in recent years. These people may serve as carriers of the vectors or be infected by disease agents that require arthropod vectors for transmission. Although it cannot be supposed that every arthropod species is a competent vector, this review aims to (1) illustrate the factors that contribute to the persistence and dispersal of arthropod vectors, (2) determine the status of the established arthropod vector species in Turkiye and their capability of serving as vectors of disease agents, and (3) assess the role of newly-introduced arthropod vectors into Turkiye and how they were introduced into the country. We also provide information on important disease incidence (if there's any) and control measures applied by public health officials from different provinces.

Covid-19 vaccination acceptance and hesitancy among the Turkish adult population.

Objective: The aim of this study was to determine the acceptance of Covid-19 vaccine among the Turkish adult population. Methods: A total of 2023 persons participated in this cross-sectional study between October 2020 and January 2021. The questionnaire, which was delivered via social media, was filled out by the participants over "Google Forms". Results: Questionnaire results showed that 68.7% of the participants might agree to vaccinated against COVID-19. According to univariate analysis, the age group of 50-59, urban residents, healthcare workers, non-smokers, and those with chronic diseases, those who were vaccinated against influenza, pneumonia, and tetanus were all willing to be vaccinated against COVID-19. Conclusions: It is very important to determine a community's willingness to be vaccinated against COVID-19 so that interventions can be made to solve related problems. Risk of exposure and importance of Prevention play a critical role in vaccination acceptance.

Immunoinformatics Approach to Design a Multi-Epitope Vaccine against Cutaneous Leishmaniasis.

Cutaneous Leishmaniasis (CL), a neglected vector-borne disease caused by protozoan parasite Leishmania major (L. major), is a major public health concern, and the development of new strategies to reduce the disease incidence has become a top priority. Advances in immunoinformatics and in-silico epitope prediction could be a promising approach to designing a finest vaccine candidate. In this study, we aimed to design a peptide-based vaccine against CL using computational tools and identified ten B-cell-derived T-cell epitopes from the glycoprotein gp63 of L. major. All of the potential immunodominant epitopes were used to design a vaccine construct along with a linker and an adjuvant at the N-terminal for enhancing its immunogenicity. Additionally, many characteristics of the proposed vaccine were examined, and it was confirmed to be non-allergenic, non-toxic, and thermally stable. To assess the vaccine interaction with the innate immune toll-like receptor-4 (TLR-4), a 3D structure of the vaccine construct was developed. Molecular docking and molecular dynamic simulation were used to confirm the binding and to assess the stability of the vaccine-TLR4 complex and interactions, respectively. In conclusion, our multi-epitope vaccine will provide a gateway to analyze the protein function of a potential vaccine candidate against CL.

Entomological surveillance and spatiotemporal risk assessment of sand fly-borne diseases in Cyprus.

Visceral and cutaneous leishmaniases are important public health concerns in Cyprus. Although the diseases, historically prevalent on the island, were nearly eradicated by 1996, an increase in frequency and geographical spread has recently been recorded. Upward trends in leishmaniasis prevalence have largely been attributed to environmental changes that amplify the abundance and activity of its vector, the phlebotomine sand flies. Here, we performed an extensive field study across the island to map the sand fly fauna and compared the presence and distribution of the species found with historical records. We mapped the habitat preferences of Phlebotomus papatasi and P. tobbi, two medically important species, and predicted the seasonal abundance of P. papatasi at unprecedented spatiotemporal resolution using a climate-sensitive population dynamics model driven by high-resolution meteorological forecasting. Our compendium holds a record of 18 species and the locations of a subset, including those of potential public and veterinary health concern. We confirmed that P. papatasi is widespread, especially in densely urbanized areas, and predicted that its abundance uniformly peaks across the island at the end of summer. We identified potential hotspots of P. papatasi activity even after this peak. Our results form a foundation to inform public health planning and contribute to the development of effective, efficient, and environmentally sensitive strategies to control sand fly populations and prevent sand fly-borne diseases.

Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia.

Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.

The Effect of BTK Inhibitor Ibrutinib on Leishmania infantum Infection In Vitro.

PURPOSE: Leishmaniasis is a neglected infectious disease affecting millions of people worldwide. Visceral leishmaniasis (VL), caused by Leishmania infantum and Leishmania donovani, is one of the main clinical forms of the disease and fatal if not treated promptly and properly. Despite being available for the last 70 years, current drugs used in the treatment of leishmaniasis have serious problems as they have high toxicity, require long-term administration and cause serious side-effects, leading to the emergence of resistant and relapse cases. Therefore, there is an urgent need for the discovery of novel antileishmanial molecules and the development of new treatment regimens. The drug used for chemotherapy of B-cell malignancies, Ibrutinib, an inhibitor of Bruton's Tyrosine Kinase (BTK), can offer a new therapeutic perspective due to the functions of BTK on intracellular signaling mechanism of macrophages, which are the primary resident cell for Leishmania. Hence, the study aimed to evaluate ibrutinib as a potential anti-Leishmanial drug. METHOD: In this study, we evaluated the antileishmanial effect of Ibrutinib by in vitro L. infantum infection model using macrophages, with cell viability assay, parasite rescue assay, real-time qPCR. RESULTS: We showed that Ibrutinib was significantly more effective than the Glucantime against L. infantum. In addition, our data revealed that Ibrutinib inhibited parasite growth and load without impairing macrophage viability. CONCLUSIONS: Consequently, due to its efficacy and safety, Ibrutinib may be a promising candidate for the treatment of VL caused by L. infantum as a host-targeted drug.

Multiparametric approach to assess the disease severity and progression of cutaneous leishmaniasis infection.

The pathophysiology of Cutaneous Leishmaniasis (CL), an infection caused by Leishmania tropica (L. tropica) and Leishmania major (L. major) is primarily determined by inflammation-mediated immune cells. The immune response mainly depends on cells and molecules related to T-cells that influence susceptibility and disease development. Understanding the immunological mechanisms that cause tissue injury or lesion healing is critical for developing appropriate treatment strategies. In the present study, T-cells profile and cell-free mitochondrial DNA (CF mt-DNA) were investigated in CL patients infected with L. tropica (n = 34) and L. major (n = 2) and compared with non-infected healthy controls (n = 20). There was a significant (p<0.0001) difference between CD4+ T-cells among L. tropica and L. major CL-infected groups as compared to control while no significant difference (p = 0.8597) was found in the percentages of CD8+ T-cells. When L. tropica and L. major CL-infected individuals were compared to controls, the levels of IL-4 and expression of CF mt-DNA were significantly higher (p<0.0001). Higher levels of CF mt-DNA were detected in CL patients, irrespective of the infective Leishmania species. We proposed that the levels of CF mt-DNA and IL-4 in CL-infected individuals can be used to determine the disease progression. A better understanding of these biomarkers and evaluation of the immune responses in CL patients might benefit the development of vaccines and immunotherapies.

The Designing of a Gel Formulation with Chitosan Polymer Using Liposomes as Nanocarriers of Amphotericin B for a Non-invasive Treatment Model of Cutaneous Leishmaniasis.

PURPOSE: Leishmaniasis is a disease caused by different Leishmania spp., which are transmitted to humans by a bite of infected female sand flies. Cutaneous leishmaniasis (CL, oriental sore), visceral leishmaniasis (VL), and mucocutaneous leishmaniasis (MCL) are three main clinical forms, however, only CL and VL are seen in Turkey. Cutaneous leishmaniasis is characterized by skin lesion(s) and is one of the most important vector-borne diseases in Turkey with over 2000 cases reported annually in 40 out of 81 provinces. The treatment is usually made invasively and painfully by intralesional injection of pentavalent antimony compounds. Non-invasive and innovative treatment methods are needed as aimed in this study. METHODS: In the present study, one of the classical antileishmanial drugs, amphotericin B (AmB), encapsulated in liposomes was evaluated using non-invasive design based on chitosan, which is a nontoxic, biocompatible and biodegradable polymer. To avoid the invasive effect of conventional intralesional needle application, the drug was encapsulated in liposomes and incorporated into a chitosan gel for applying topically on the skin lesion. The efficacy of encapsulation of amphotericin B into liposomes and the drug release from liposomes were studied. The chitosan gel was evaluated for viscosity, flowability, appearance and pH. The efficacy of the drug embedded into chitosan gel, liposomal AmB alone and chitosan gel alone in four different concentrations was also tested using Leishmania spp. promastigotes in vitro. RESULTS: The findings have shown that AmB was encapsulated into the liposomes with high efficiency (86.6%) and long-term physical and chemical stability. Therefore, designed liposomal formulation was suitable for sustained release. The appearance of the drug-embedded chitosan gel was transparent and appropriate. Chitosan gels showed non- Newtonian behavior and plastic flow. The liposomal AmB also showed higher efficacy with no parasites in all concentrations while drug embedded into chitosan gel and chitosan gel alone were effective in two higher concentrations. The lower efficacy of the drug-embedded chitosan gel in 24 h in in-vitro study was probably due to slow release of the drug. CONCLUSION: The gel design created in this study will provide ease of use for the lesions of CL patients that do not have a specific number, size, and shape. Follow-up studies by the ex-vivo macrophage infection model with Leishmania intracellular amastigote forms and Leishmania-infected animal models are needed to understand the present design's efficacy better.

Increasing the Sensitivity of Leishmania RNA Virus 2 (LRV2) Detection with a Modification in cDNA Synthesis

Objective: Leishmania RNA virus was detected the first time in the New World Leishmania species. Recent studies were also showed the presence of Leishmania RNA virus 2 (LRV2) in Old Word Leishmania species including Turkish L. major and L. tropica isolates. This study aimed to increase the sensitivity of qPCR with a modification in the denaturation step of cDNA preparation protocol. Methods: In this study, LRV2+ three L. major, two L. tropica strains and L. major control strain (MHOM/SU/73/5-ASKH) were included. Total RNA isolation was done using different numbers of Leishmania promastigotes (108, 105 and 103). Before cDNA synthesis, samples were denatured at 95 °C for 2 min, as a modification of the kit procedure. qPCR was undertaken using 0.5 mM primers (LRV F-HR/LRV R-HR) diluted in SYBR Green Master mix. Results: We observed lower Ct values in amplicons with the modified version than with the classical kit protocol for cDNA synthesis, in all of the strains used in the study. The addition of pre-denaturation step at 95 °C showed lower Ct values meaning the sensitivity increased. Different parasite dilutions showed similar results. Conclusion: It is important to increase the sensitivity especially with the aim for detecting LRV in clinical samples obtained from patients probably have less number of parasites. The presence and burden of the virus can help to understand the relationship between the clinical findings and the pathogenicity of the parasite which may lead to changes in the course of treatment.

The current epidemiology of leishmaniasis in Turkey, Azerbaijan and Georgia and implications for disease emergence in European countries.

Leishmania spp. are sand fly-borne protozoan parasites causing leishmaniasis in humans and animals. The aim of the study was to analyse the epidemiology of leishmaniasis in Turkey, Azerbaijan and Georgia from 2005 to 2020 and evaluate the associated risk for disease emergence in European countries. It is based on an analysis of WHO and OIE reported cases between 2005 and 2020, a review of scientific articles published in SCOPUS between 2009 and 2020 and a questionnaire survey to public health and veterinary authorities in these countries. Endemic Leishmania spp. include L. infantum in the three countries, L. major in Azerbaijan and Turkey and L. tropica and L. donovani in Turkey. Leishmaniasis is reported in humans, animals and sand flies and incidence is spatially and temporarily variable. In the southern Caucasus and particularly in Georgia, reported incidence of human visceral leishmaniasis by L. infantum remains high. However, whilst Georgia experienced a gradual decrease from >4.0 cases per 100,000 population in 2005-09 to 1.13 cases per 100,000 population in 2020, the period with highest incidence in Azerbaijan, which ranged between 0.40 and 0.61 cases per 100,000 population, was 2016-2019, and no cases have so far been reported for 2020. Visceral leishmaniasis in the Southern Caucasus affects mostly young children from deprived urban areas and its closely associated to canine leishmaniasis. Turkey reported cases of visceral leishmaniasis between 2005 and 2012 and in 2016 only, and incidence ranged between 0.02 and 0.05 per 100,000 population. In contrast, the reported annual incidence of cutaneous leishmaniasis in Turkey was much greater and peaked at 7.02 cases per 100,000 population in 2013, associated to imported cases from cutaneous leishmaniasis endemic Syria. Leishmaniasis by L. infantum in Azerbaijan and Georgia represents a regional public and animal health challenge that requires support to improve diagnosis, treatment and control. The unprecedented rise of cutaneous leishmaniasis and the spread of L. tropica and L. donovani in Turkey is an important risk factor for their emergence in Europe, especially in Mediterranean countries where competent vectors are widespread.

Blood Meal Analysis and Molecular Detection of Leishmania DNA in Wild-Caught Sand Flies in Leishmaniasis Endemic Areas of Turkey and Northern Cyprus.

INTRODUCTION: Phlebotomine sand flies (Diptera: Psychodidae) are known as the vector of diseases such as leishmaniasis, bartonellosis and viral diseases. The aim of this study is to detect the host feeding pattern of sand flies in the endemic areas for leishmaniasis in Turkey (Antalya, Kayseri) and Northern Cyprus (TRNC) as well as the presence of Leishmania DNA in the specimens. METHODS: One-hundred seventy-six blood-fed sand fly specimens were examined for blood meal analysis. A SYBR Green-PCR assay was performed with specific forward primers for each host and a universal reverse primer. Primers of human and goat were used together in multiplex PCR while goat and cow were studied separately. ITS-1 qPCR assay was also performed on both blood-fed and non-blood-fed females to detect Leishmania parasites. RESULTS: Blood sources could be detected in 69 out of 176 blood-fed sand fly specimens. The results of blood meal analysis showed that specimens were fed mostly on cows (22.2%) followed by humans (5.7%), goats (2.8%) and dogs (0.6%). Multiple feeding patterns were also detected as human + cow (3.4%), cow + goat (2.8%) and human + goat (1.7%). Five of the blood-fed specimens were Leishmania spp. positive: P. major s.l. (n = 1), P. tobbi (n = 2) were L. tropica positive from Antalya, P. simici was positive for L. infantum from Kayseri and P. papatasi (n = 1) was positive for L. major from Cyprus. Leishmania infection rates were determined as 3.79%, 1.69% and 2.63% among the blood-fed sand fly specimens in Antalya, Kayseri and TRNC, respectively. CONCLUSION: The SYBR-Green-based multiplex PCR assay is a cost-effective and promising tool for blood meal identification of wild-caught sand flies as well as other blood-sucking arthropods. Feeding patterns of important vector species detected in the present study show the high risk in these endemic areas. As a next step, to identify the blood source in a shorter time and to make the test more sensitive, development of this assay to probe-based and multiplex PCR will be also planned.

Fauna, Seasonal Activity, and Altitudinal Distribution of Phlebotomine Sand Flies (Diptera: Psychodidae) in Leishmaniasis Endemic Area, Aydin Mountains, and Surroundings in Western Turkey

Objective: Human and canine leishmaniasis (CanL) are endemic in Turkey, and cutaneous leishmaniasis (CL) is a primary public health problem among vector-borne diseases, with 1500-2000 autochthonous cases per year. In this study, we aimed to perform a comprehensive entomological survey, including the detection of sand fly fauna, seasonal activity, and altitudinal distribution, together with the ecological features of Aydin Mountains and their surroundings that are endemic areas for CL caused by Leishmania tropica and CanL caused by L. infantum MON-1. Methods: The survey was carried out from June 2009 to July 2010. Ten localities with different altitudes were selected. Sand fly collection was done every 15 days in July and August when sand flies are most active and every 30 days between September and June (14 months, 16 samplings) using CDC light traps and sticky traps. During the collection, the temperature, humidity, and ecological and environmental features were recorded. Sand fly specimens were dissected and identified at species level using written keys. Results: A total of 6712 sand fly specimens (3268 females and 3444 males) were collected from 10 localities throughout all study periods. Species identification revealed that nine and three species belonging to Phlebotomus (P. major s.l. 30.38%, P. tobbi 22.93%, P. papatasi 5.88%, P. sergenti s.l. 4.51%, P. alexandri 4.26%, P. simici 3.50%, P. burneyi 0.63%, P. brevis 0.45%, and Transphlebotomus spp. 0.28%) and Sergentomyia (S. dentata 23.17%, S. minuta 2.43%, and S. antennata 1.58%) were found, respectively. The seasonal activities of these 12 sand fly species were determined, and P. major s.l. and P. tobbi, which were probable vectors of VL and CanL, were present in the study area. P. sergenti s.l., a probable vector of CL, was also found in the region and was active for seven months between April and October. P. sergenti s.l. was found in the 400-600 m altitude in July, while P. major and P. tobbi were found in high densities in the 200-400 m altitude in September. Conclusion: Our results showed that leishmaniasis vectors are present in different altitudes in the Aydin mountains and surroundings. The findings revealed the time intervals of parasite transmission in the area and the period of applying protective measures, such as insecticide application.

Host-Parasite Interactions: Regulation of Leishmania Infection in Sand Fly.

PURPOSE: Sand flies are the only proven vectors of leishmaniases, a tropical neglected disease endemic in at least 92 countries. Vector-parasite interactions play a significant role in vector-borne disease transmission. There are various bottlenecks to Leishmania colonization of the sand fly midgut. Such bottlenecks include the production of innate immune-related molecules, digestive proteases, parasite impermeable peritrophic membrane, and resident gut microbiota. These barriers determine the parasite load transmitted and, consequently, the disease outcome in mammalian host. Therefore, it is important to understand the molecular responses of both sand fly and Leishmania during infection. METHOD: Here, we reviewed the published literature on sand fly-Leishmania interactions bringing together earlier and current findings to highlight new developments and research gaps in the field. CONCLUSION: Recent research studies on sand fly-Leishmania interaction have revealed contrasting observations to past studies. However, how Leishmania parasites evade the sand fly immune response still needs further research. Sand fly response to Leishmania infection can be best understood by analyzing its tissue transcriptome. Better characterization of the role of midgut components could be a game changer in development of transmission-blocking strategies for leishmaniasis.

Determination of sand fly fauna and molecular detection of Leishmania in sand flies in Antalya Province, Southern Turkey.

Visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) are diseases transmitted by infected female sand flies. Since the eradication of malaria in Turkey, CL is the main vector-borne disease in the country, with more than 2000 cases per year, making it a significant public health problem. The aims of this study were to carry out an entomological survey in Antalya Province, an endemic area for CL in the Mediterranean Region of Turkey, to identify sand fly fauna and to screen female specimens for the presence of Leishmania parasites (Leishmania infantum, L. tropica, L. major, and L. donovani) using molecular analysis. Sand flies were collected in 42 localities of seven districts in Antalya Province using CDC miniature light traps in two different periods, June 2012 and September 2013. The specimens were kept in 96% ethanol until the dissection was done. The head and genitalia of the specimens were cut for preparing individual slides for species identification. The rest of the body of female specimens was kept separately. The specimens were identified at the species level, and 27 pools were generated according to the locations and species for screening the presence of Leishmania. A commercial kit was used for DNA extractions. Real-time and conventional polymerase chain reaction (PCR) targeting the internal transcribed spacer region (ITS1) were then performed. In total, 1306 specimens comprising nine species belonging to the Phlebotomus genus were collected in the study region, with Phlebotomus neglectus/syriacus (38.82%) the most abundant, followed by P. alexandri (21.67%) and P. tobbi (20.44%). In the 27 pools, Leishmania infantum DNA was detected in four pools containing P. neglectus/syriacus and one pool containing P. tobbi. In conclusion, the sand fly fauna in the Antalya Province is diverse. The probable vector sand fly species are P. neglectus/syriacus and P. tobbi with high dominance (59.26%), which indicates a high risk of CL transmission. The data presented here may help to shed more light on the transmission cycles of the Leishmania parasite in this CL endemic area.

PpSP32-like protein as a marker of human exposure to Phlebotomus argentipes in Leishmania donovani foci in Bangladesh.

Phlebotomus argentipes is a predominant vector of Leishmania donovani, the protozoan parasite causing visceral leishmaniasis in the Indian subcontinent. In hosts bitten by P. argentipes, sand fly saliva elicits the production of specific anti-salivary protein antibodies. Here, we have utilised these antibodies as markers of human exposure to P. argentipes in a visceral leishmaniasis endemic area in Pabna district, Bangladesh. The use of whole salivary gland homogenate as an antigen to detect these antibodies has several limitations, therefore it is being superseded by the use of specific recombinant salivary proteins. We have identified three major P. argentipes salivary antigenic proteins recognised by sera of bitten humans, expressed them in a recombinant form (rPagSP04, rPagSP05 and rPagSP06) and tested their applicability in ELISA and immunoblot. One of them, PpSP32-like protein rPagSP06, was identified as the most promising antigen, showing highest resemblance and correlation with the IgG response to P. argentipes salivary gland homogenate. Furthermore, we have validated the applicability of rPagSP06 in a large cohort of 585 individuals and obtained a high correlation coefficient for anti-rPagSP06 and anti-P. argentipes saliva IgG responses. The anti-rPagSP06 and anti-P. argentipes salivary gland homogenate IgG responses followed a similar right-skewed distribution. This is the first report of screening human sera for anti-P. argentipes saliva antibodies using recombinant salivary protein. The rPagSP06 was proven to be a valid antigen for screening human sera for exposure to P. argentipes bites in a visceral leishmaniasis endemic area.