Genetic diversity and haplotype analysis of Leishmania tropica identified in sand fly vectors of the genera Phlebotomus and Sergentomyia using next-generation sequencing technology.

Next-generation sequencing (NGS) was used to investigate the genetic diversity of Leishmania tropica in the sand fly vector, targeting the internal transcribed spacer 1 (ITS1) of the genus Leishmania. Bioinformatics analyses were conducted using Galaxy, MEGA version X, DnaSP ver. 6.12.03, and PopART 1.7 software for NGS analysis, phylogenetic tree, genetic diversity, and haplotype networking, respectively. A total of 307 engorged sand flies were trapped, with an overall Leishmania infection rate of 9.4 (29/307) and 6.8% by NGS and ITS1-PCR, respectively. Two Leishmania-infected sand fly genera were identified: Phlebotomus (10.2%, 26/254) and Sergentomyia (5.7% (3/53). The phylogenetic tree showed two clusters, cluster I included the four study sequences along with 25 GenBank-retrieved DNA sequences. Cluster II consisted of three sequences from Iran and Pakistan. The genetic diversity analysis for the 29 L. tropica sequences showed high haplotype (gene) diversity index (Hd) (0.62 ± 0.07) but low nucleotide diversity index (π) (0.04 ± 0.01). Tajima's D, a neutrality test, is more negative in cluster I (D = - 2.0) than in total population (D = - 1.83), but both are equally significant (P < 0.001), indicating that observed variation in cluster I and whole population is less frequent than expected. The median-joining haplotype network produced a total of 11 active haplotypes. In conclusion, L. tropica from sand flies in Palestine is monophyletic that assembled in one main phylogroup and one haplotype.

[Evaluation of Ex Vivo Cultivation Potentials of Trypanosoma cruzi, Leishmania tropica ve Toxoplasma gondii Parasites in J774, Vero and HeLa Cell Lines]. / Trypanosoma cruzi, Leishmania tropica ve Toxoplasma gondii Parazitlerinin J774, Vero ve HeLa Hücre Hatlarinda Ex Vivo Kültivasyon Potansiyellerinin Degerlendirilmesi.

Three obligate intracellular protozoan parasite species, namely Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii, causative agents of Chagas disease, Leishmaniasis and toxoplasmosis, respectively, which are responsible for significant morbidity and mortality and reside in macrophage cells, affect more than half of the world's population in connection with socio-economic and geographical factors and also causes neglected parasitic diseases of increasing importance. This study aimed to evaluate the ex vivo cultivation potential of T.cruzi, L.tropica and T.gondii parasites in J774, Vero and HeLa cells and to reproduce in a short time and in large amounts without losing their virulence properties. Ex vivo experimental models were created by infecting J774, Vero and HeLa cell lines confluently produced in cell culture flasks with T.cruzi, L.tropica and T.gondii parasites. In ex vivo cultivation, one passage was applied for seven days and three times in a row. Cells removed from the surface after each passage were plated on eight-well chamber slides. Giemsa stained slides were prepared and infection rates were evaluated by light microscopic examination. At the end of the study, it was observed that all three cell lines could be infected with T.cruzi, L.tropica and T.gondii parasites, and infection rates increased in all cell lines after consecutive passages. As a result of ex vivo cultivation, the best cell lines from which T.cruzi and L.tropica strains grew, were J774, Vero and HeLa, and HeLa, J774 and Vero cell lines for T.gondii strain, respectively (p<0.05). Trypanosoma cruzi, L.tropica and T.gondii parasites were successfully grown in J774, Vero and HeLa cell lines by ex vivo culture method in a short time and in large amounts without losing their virulence properties. Cell lines with the best ex vivo cultivation potential for T.cruzi and L.tropica parasites were J774, Vero and HeLa, respectively, while HeLa, J774 and Vero for T.gondii. It is thought that the data obtained in this regard will contribute to many studies on the development of vaccines, drugs and new diagnostic kits.

Poor adherence is a major barrier to the proper treatment of cutaneous leishmaniasis: A case-control field assessment in Iran.

Leishmaniasis is an overlooked, poverty-stricken, and complex disease with growing social and public health problems. In general, leishmaniasis is a curable disease; however, there is an expansion of unresponsive cases to treatment in cutaneous leishmaniasis (CL). One of the effective and ignored determinants in the treatment outcome of CL is poor treatment adherence (PTA). PTA is an overlooked and widespread phenomenon to proper Leishmania treatment. This study aimed to explore the effect of poor adherence in unresponsiveness to treatment in patients with anthroponotic CL (ACL) by comparing conventional statistical modalities and machine learning analyses in Iran. Overall, 190 cases consisting of 50 unresponsive patients (case group), and 140 responsive patients (control group) with ACL were randomly selected. The data collecting form that included 25 queries (Q) was recorded for each case and analyzed by R software and genetic algorithm (GA) approaches. Complex treatment regimens (Q11), cultural and lay views about the disease and therapy (Q8), life stress, hopelessness and negative feelings (Q22), adverse effects of treatment (Q13), and long duration of the lesion (Q12) were the most prevalent significant variables that inhibited effective treatment adherence by the two methods, in decreasing order of significance. In the inherent algorithm approach, similar to the statistical approach, the most significant feature was complex treatment regimens (Q11). Providing essential knowledge about ACL and treatment of patients with chronic diseases and patients with misconceptions about chemical drugs are important issues directly related to the disease's unresponsiveness. Furthermore, early detection of patients to prevent the long duration of the disease and the process of treatment, efforts to minimize side effects of treatment, induction of positive thinking, and giving hope to patients with stress and anxiety by medical staff, and family can help patients adhere to the treatment.

[The Effect of Oleuropein on The Mitochondrial Membrane Potential and Generation of Reactive Oxygen Species on Leishmania tropica Promastigotes]. / Oleuropein'in Leishmania tropica Promastigotlari Üzerinde Mitokondri Membran Potansiyeli ve Reaktif Oksijen Türlerinin Olusumuna Etkisi.

Leishmania parasites, which are reported to be endemic in 98 countries around the world, infect humans as well as wild and domestic carnivores and small mammals, and are transmitted by sand flies (Phlebotomus, dwarf sandflies). It is reported that 350 million people are at risk and two million new cases are seen in the world every year. It has been reported that different drugs (topical paromomycin, oral miltefosine, ketoconazole, rifampin, and zinc) have been tried in studies especially in endemic regions in the treatment of cutaneous leishmaniasis, and response to treatment has been obtained at different rates. Today, the search for alternative treatments continues and many studies have been carried out for this purpose. For centuries, olive leaf extracts have been used to maintain health. Oleuropein has numerous health benefits, including antioxidant, antimicrobial, anti-inflammatory, antiatherogenic, anticarcinogenic, antiviral activities, cardio- and neuroprotective, hepatoprotective effects. The aim of this study was to determine and understand the mode of action of oleuropein, the cell death mechanisms caused by oleuropein in L.tropica promastigotes. In this study, the phenolic and flavonoid content of oleuropein was determined by HPLC method. The antioxidant capacity and the amount of oleuropein were determined. Afterwards, morphological and physiological (mitochondrial membrane potential, formation of reactive oxygen species, Annexin V binding) changes triggered by oleuropein in L.tropica promastigotes were investigated using flow cytometry. Our studies revealed that apoptotic properties such as mitochondrial dysfunction, production of reactive oxygen species, flip-flop action of phosphatidylserine could induce cell death in L.tropica promastigotes. It has been observed that oleuropein induced typical apoptotic morphological features in L.tropica promastigotes. Total phenolic content and total flavonoid content values of oleuropein extract were determined as 33 mg/g and 229 mg/g. The radical removal method was used to investigate the antioxidant capacity of methanol extracts against free radicals. Total antioxidant content of oleuropein extract was determined as 87%. In addition, the amount of oleuropein in the oleuropein extract was determined as 21. 1% by HPLC. The oleuropein dose that killed 50% of L.tropica promastigotes, that is the IC50 value, was detected as 46.6 µg/mL after 24 hours. It was observed that the parasites in the control group preserved their typical morphological features with a single nucleus, flagella, kinetoplast and narrow cell body at both 24 and 48 hours. It was observed that as oleuropein concentrations increased, the and kinetoplasts of L.tropica promastigotes could not be distinguished from each other, they moved away from the narrow cell body structure, they lost their flagella and turned into a round form, and they moved away from the typical form of the parasite. The percentage of Annexin V+ apoptotic cells was found to be 2.9 ± 0.4% in the untreated control group, and 38.1 ± 6.9% in the oleuropein-treated group. Polarization in the mitochondrial membrane of healthy promastigotes caused an approximately 1.7-fold change in the direction of depolarization in oleuropein-treated promastigotes. According to these findings, oleuropein triggered mitochondria-related death in L.tropica promastigotes. Moreover, 1.4 ± 0.2 fold increase in reactive oxygen species production was detected in oleuropein-treated promastigotes compared to the untreated control group. Comparisons between groups were made using the independent sample t test method. In conclusion, phenolic compounds of olive leaf extract oleuropein induced apoptotic cell death in L.tropica promastigotes. Our results support that olive products such as oleuropein may have anti-parasitic effects.

[An In Vitro Study on Sonodynamic Therapy of Leishmania tropica Using Curcumin]. / Kurkumin Kullanilarak Leishmania tropica'nin Sonodinamik Tedavisi Üzerine Bir In Vitro Çalisma.

Leishmaniasis is an infectious disease that is transmitted by Phlebotomus, 400 thousand new cases appearing every year, and approximately 350 million people are at risk, and accepted by the World Health Organization as one of the six important tropical diseases. Cutaneous leishmaniasis is a disease that occurs on exposed areas of the body and is characterized by long-term non-healing skin lesions. Although the treatment methods applied today vary according to the clinical picture of the patient, the immune system of the person and the causative agent Leishmania species, there is still no standard treatment scheme that has few side effects and can be used in the treatment of leishmaniasis. Therefore, alternative treatment methods with less side effects are being tried. Sonodynamic therapy (SDT) has also emerged as an active antimicrobial research area in recent years. SDT, a new modality for antibacterial therapy, aims to increase antibacterial effects with the simultaneous combination of low-intensity ultrasound and sonosensitizer. There is no information in the literature about the effect of SDT on parasites. In this study, it was aimed to demontrate the anti-leishmanial effect and possible mechanisms of curcumin mediated SDT on L.tropica promastigotes in vitro. Parasites were incubated with 0.25, 1.0, 4.0 and 15.6 micromolar (µM) of curcumin for one hour and subjected to 1 MHz frequency, 50% duty cycle and 3 W/cm2 intensity ultrasound irradiation. XTT assay was used to evaluate the viability of the cells and morphological changes were analyzed by Giemsa staining. Flow cytometry was used to quantify the fluorescence emitted by intracellular reactive oxygen species (ROS) signal, JC-1, cell cycle, Annexin V/PI staining reagents. With the combination of curcumin (15.6 µM) and ultrasound (3 W/cm2 intensity, seven minutes), L.tropica promastigote viability was found to be significantly decreased compared to the control group. Giemsa staining results showed that 15.6 µM curcumin mediated SDT induced several morphological alterations in L.tropica promastigotes typical for apoptosis. Late apoptosis was observed in 15.6 µM curcumin combined SDT treated parasites according to Annexin/PI staining. Besides, curcumin mediated SDT caused mitochondrial membrane potential (∆á´ªm) loss. Cell cycle analysis data indicated that curcumin based SDT caused an subG1 arrest in the cell cycle of L.tropica promastigotes. The generation of intracellular ROS detected by flow cytometry was increased in L.tropica promastigotes treated with curcumin mediated SDT. This study provided new data elucidating the molecular mechanism underlying the anti-leishmanial effect of curcumin mediated SDT. Curcumin mediated SDT has the potential to inactivate L.tropica promastigotes. However, further testing with amastigote or animal models is needed.

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR.

BACKGROUND: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. METHODS: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. RESULTS: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. CONCLUSIONS: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

The efficacy of AuNP-probe conjugate nanobiosensor in non-amplification and amplification forms for the diagnosis of leishmaniasis.

Nanobiosensor platforms have emerged as convenient and promising approaches with remarkable efficacy for the diagnosis of infectious diseases. Gold nanoparticles (AuNPs) have been widely used due to numerous advantageous properties such as optical, electrical, physicochemical and great biomolecules binding capabilities. This study aimed to apply AuNP-Probe Conjugate for the detection of Leishmania spp., using colorimetric and amplification methods targeting parasitic ITS2 fragment. The first method was carried out by hybridization of 10µL of DNA with 4 µL of probe and addition of 5 µL of 0.2 N HCl (non-amplification method). Second method was followed by polymerase chain reaction (PCR) amplification using thiolated primer, 5 µL of AuNP and 5 µL of 0.2 N HCl. The appearance of red and purple colors indicated positive and negative results, respectively. The minimum of detection for non-amplification and amplification methods for three strains of Leishmania namely L. major, L. tropica and L. infantum were determined to be 32 fg/µL and 16 fg/µL, respectively. Sensitivity for detection of visceral leishmaniasis (VL) for non-amplification and amplification methods included 96% and 100%, respectively and for cutaneous leishmaniasis (CL) included 98% and 100%, respectively. The results of this investigation revealed that sensitivity of amplification method was the same as RT-qPCR, while that of non-amplification method was lower. However, this method was promising because of no need for any equipment, high specificity, enough sensitivity, low cost and rapidity (less than 30 min) to complete after genomic DNA extraction.

Failure to Detect Leishmania in the Blood of Patients with Old-World Cutaneous Leishmaniasis: Implications for Blood Donation.

Cutaneous leishmaniasis (CL) is endemic in Israel, caused mainly by Leishmania major (L. major) and L. tropica. In addition, returning travelers import another leishmanial species such as L. braziliensis. Although we are dealing with a skin disease, the blood bank in Israel does not accept blood donations from people infected with CL in cases of multiple lesions due to the possibility of transfusion. Our purpose was to investigate the prevalence of Leishmania in the blood of patients with active or previous CL. This pilot study screened patients with active or previous CL for parasites in their blood. All patients were infected in Israel or were returning travelers with leishmaniasis acquired in Latin America. Patients were seen at the Sheba Medical Center. In addition, patients were seen at their homes in L. tropica and L. major endemic regions in Israel. Blood samples were taken from each patient for culture and polymerase chain reaction (PCR). Altogether 62 blood samples were examined (L. tropica = 26, L. major = 33, and L. braziliensis = 3). Twenty-seven patients had an active disease and 35 were recovered. All blood cultures and PCR were negative for parasites except one blood sample that was PCR positive for L. braziliensis. The findings of our study, although a small sample, suggest that people with active or recent CL caused by L. major and L. tropica, do not harbor parasites in their blood. Thus, their exclusion from blood donation should be revisited. Further studies are needed with larger sample size and highly sensitive tests.

In vitro Evaluation of the Effect of Sonodynamic Therapy Using Rose Bengal on Leishmania tropica Promastigotes'

Objective: There is a need for new treatment options for treating Leishmaniasis, since there is no standard treatment scheme with few side effects. Sonodynamic therapy (SDT) is also a candidate to be one of these options. SDT is a treatment method based on the simultaneous combination of low-intensity ultrasound and a sonosensitizer, and the generation of reactive oxygen species in cells in the presence of molecular oxygen. Sonosensitizer, ultrasound, and molecular oxygen individually, these components are not toxic, but when combined form cytotoxic reactive oxygen species In this study, we evaluated the effect of rose bengal (RB)-mediated SDT on Leishmania tropica (L. tropica) promastigotes. Methods: SDT was performed using different concentrations of RB (20, 40, and 80 µM) and ultrasound at a frequency of 1 MHz with an intensity of 1, 1.5, and 2 W/cm2 for 10, 20, and 30 min. Results: Incubation with different RB concentrations applied alone had no effect on L. tropica promastigotes. Ultrasound application time for L. tropica promastigotes alone was determined as 10 min. Ultrasound application intensity showed more significant results at 2 W/cm2. It was determined that the number of promastigotes was lower than that of the control group after 10 min of exposure to ultrasound at 2 W/cm2 at 1 MHz frequency for 10 min with RB (80 µM). Morphologically, round, swollen, atypical forms of the parasite with indistinguishable nuclei are observed, but typical narrow cell body forms have also been detected. Conclusion: These results showed that RB-mediated SDT on L. tropica could be a candidate treatment approach. This approach can be used for both superficial and deeply located lesions. This study emphasized the biophysical mechanisms, ultrasound exposure strategies, reliability and difficulties in the clinical practice of RB-mediated SDT on L. tropica promastigotes.

Could RPMI-1640 Medium be Used in the Diagnosis and Isolation of Cutaneous Leishmaniasis Lacking NNN Medium?

Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.

Domestic dogs carriers of Leishmania infantum, Leishmania tropica and Crithidia fasciculata as potential reservoirs for human visceral leishmaniasis in northeastern Iran.

BACKGROUND: In recent years, cases of human visceral leishmaniasis (HVL) have been reported in some districts of Golestan Province, northeastern Iran, particularly in rural areas. Recent epidemiological evidence in Leishmania infantum endemic regions of in Iran indicates approximately 50%-80% of seropositive dogs are asymptomatic for Leishmania infection. OBJECTIVES: The goal in this study was to determine Leishmania species infecting domestic dogs in Golestan Province, Iran. METHODS: Between 2015 and 2016, blood samples were obtained from 100 domestic dogs in rural regions of Golestan Province, northeastern Iran. All samples were tested for anti-Leishmania antibodies using a direct agglutination test (DAT), and for Leishmania spp. kinetoplast DNA (kDNA) using PCR. RESULTS: Seven (7%) dogs were antibody positive and 25 dogs (25%) were Leishmania spp. DNA positives by PCR positive for leishmaniasis. Four of the seven (71%) antibody-positive dogs and 19 of the 25 (76%) PCR-positive dogs were asymptomatic. The rate of infection detected by PCR was significantly higher in male dogs (21/75, 28%) than that in female dogs (4/25, 16%). The ITS1 PCR-RFLP assay identified the presence of L. infantum, L. tropica or Crithidia spp. in the 25 PCR-positive samples. CONCLUSIONS: The high proportion of asymptomatic dogs in the study areas represent they act as potential reservoirs in the transmission cycle of Leishmania spp. and also Crithidia fasciculata as an emerging agent for the first time. Moreover, our data showed that PCR is a more reliable assay than DAT for detecting Leishmania spp. infection among asymptomatic dogs.

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.

In vivo comparative study of the efficacy of ß-sitosterol, ketoconazole 2% and mupirocin for the treatment cutaneous leishmaniosis.

Cutaneous leishmaniosis (CL) is an important parasitic disease characterized by specific skin lesion, includes the vector that cause the CL and treatment in general. The study aimed to identify the effect of three different drugs which are ß-sitosterol, ketoconazole 2% and mupirocin in the treatment of cutaneous leishmaniosis. The study was conducted at Dermatological Unit of Al Hussein Teaching Hospital/Thi-Qar/Iraq from October to November 2021. The patients presented with a lesion will be involved in this study and its involved isolation of parasites from the lesion of patients and the parasite was replicated in (NNN) media then the inoculum was concentered. A total of 40 male of mice (Mus musculus) of BALB/c strain injected by parasite suspension, after the appearance of lesion, ß-sitosterol, ketoconazole 2% and mupirocin was applied on a lesions daily for 2 weeks, the statistical analysis was done by SPSS program. In the current study, the ß-sitosterol was most effective in the treatment of skin leishmanial lesion than the other drugs with mean is (11.9±1.449 mm) in compared with the other drugs under P-value < 0.046 with the complete recovery. ß-sitosterol was highly effect on the L. tropica infections with complete recovery and no scar appearance than ketoconazole and mupirocin and can be used for treatment of the disease lesion.

Treatment outcome of imported cutaneous leishmaniasis among travelers and migrants infected with Leishmania major and Leishmania tropica: a retrospective study in European centers 2013 to 2019.

OBJECTIVES: Cutaneous leishmaniasis (CL) in Asia, Northern, and Sub-Saharan Africa is mainly caused by Leishmania major and Leishmania tropica. We describe and evaluate the treatment outcome of CL among travelers and migrants in Europe. METHODS: We conducted a retrospective study of parasitological confirmed CL cases caused by L. major and L. tropica during 2013-2019 in Europe. Data were collected from medical records and databases within the LeishMan network. RESULTS: Of 206 included cases of CL, 75 were identified as L. major and 131 as L. tropica. Of patients with L. tropica infection, 80% were migrants, whereas 53% of patients with L. major infection had been visiting friends and relatives. Among patients with L. tropica, 48% were younger than 15 years. Pentavalent antimony cured 73% (L. major) and 78% (L. tropica) of patients. The cure rate for intralesional administration was 86% and 67% for systemic, on L. tropica. Liposomal amphotericin B had a cure rate of 44-63%. CONCLUSION: L. major infections were mostly found in individuals visiting friends and relatives, whereas L. tropica were mainly identified in migrants. No patients with L. major relapsed. Pentavalent antimony, liposomal amphotericin B, and cryotherapy had cure rates in accordance with previous studies.

Genotypic and phylogenic analyses of cutaneous leishmaniasis in Al Ahsa, Eastern Saudi Arabia during the coronavirus disease 2019 pandemic: First cases of Leishmania tropica with the predominance of Leishmania major.

During the coronavirus disease 2019 lockdown period, a surge in sandflies and cutaneous leishmaniasis (CL) cases was observed in Al-Ahsa, Saudi Arabia. Skin punch biopsies were obtained from 100 patients clinically diagnosed with CL in Al-Ahsa who had no travel history in the last 6 months. Impression smears were used following a three-step polymerase chain reaction (PCR) protocol using genus-specific primers targeting kDNA and ITS1. Leishmania speciation was determined by ITS1 PCR/nested PCR-restriction fragment length polymorphism and sequencing. A phylogenetic tree was constructed. The associated patient characteristics were analyzed. Using internal transcribed spacer one (ITS1)-PCR/nested PCR, 98 cases were considered true-positive CL. Leishmania major was the predominant species, and Leishmania tropica was identified in three cases. Microscopy had poor sensitivity and perfect specificity. Direct ITS1-PCR missed nine cases. Sex, residence, and treatment outcome were significantly associated with the occurrence of Leishmania; distribution of skin lesion(s) and treatment outcome were significantly associated with Leishmania genotype. This is the first time that L. tropica was identified as a cause of CL in human in Al-Ahsa, in addition to the predominant zoonotic species, L. major. We recommend using ITS1-nested PCR for negative cases by ITS1-PCR. Further exploration of Leishmania transmission dynamics in vectors and reservoir animals is essential for designing effective preventive measures.

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 in vitro anti-Leishmania Effect of Zingiber officinale Extract on Promastigotes and Amastigotes of Leishmania major and Leishmania tropica

Objective: Recently, the use of pentavalent antimony compounds for Leishmaniasis treatment has been associated with disease recurrence, drug resistance, and severe side effects. Therefore, there is a need to develop alternative treatment strategies. This study investigates the in vitro effects of Zingiber officinale on promastigotes and amastigotes of Leishmania major and Leishmania tropica. Methods: Promastigotes and amastigotes of Leishmania major and Leishmania tropica were cultured and mass-produced in an RPMI1640 medium enriched with other necessary compounds. The MTT colorimetric method and calculating the IC50 value were used to evaluate the anti-leishmania activity of hydroalcoholic extract of Zingiber officinale. Results: The hydroalcoholic extract of Zingiber officinale inhibited the growth of Leishmania major and Leishmania tropica promastigotes in 24, 48, and 72 hours after in vitro incubation. The IC50 of hydroalcoholic extract of Zingiber officinale was 56 µg/mL for Leishmania major and 275 µg/mL for Leishmania tropica promastigotes after 72 hours. The IC50 of hydroalcoholic extract of Zingiber officinale was 75 µg/mL for Leishmania major and 325 µg/mL for Leishmania tropica amastigotes after 72 hours. Conclusion: The results showed that hydroalcoholic extract of Zingiber officinale has cytotoxicity properties, and Leishmania tropica has a higher resistance to hydroalcoholic extract of Zingiber officinale than Leishmania major. Further research is recommended.

Leishmania tropica and Leishmania infantum infection in dogs and cats in central Israel.

BACKGROUND: Three species of Leishmania cause disease in humans in Israel and are endemic in the Middle East: Leishmania infantum, Leishmania tropica and Leishmania major. These species infect dogs and cats, but little is known about their prevalence in pet populations and their clinical manifestations. A study on dog and cat Leishmania infection was conducted in a focus of human L. tropica infection in central Israel with the aim of getting insight on leishmaniosis in pets in an area where human infection is highly prevalent. METHODS: Blood, demographic and clinical data were collected from dogs and cats brought for veterinary care in a focus of human L. tropica infection during 2018-2020. kDNA PCR and internal transcribed spacer1 high-resolution melt analysis PCR (ITS1 HRM PCR) with DNA sequencing were performed for the detection of Leishmania and species determination. RESULTS: Forty-three of 189 dogs (22.8%) and 44 of 152 cats (28.9%) were positive for Leishmania spp. infection by kDNA PCR. The ITS1 HRM PCR detected six dogs (3.3%) infected with L. infantum and one (0.5%) with L. tropica, whereas six cats (3.9%) were found infected by L. infantum and five (3.3%) by L. tropica. Four of the five L. tropica-positive cats suffered from weight loss, four had azotemia, two with mild and two with severe azotemia and progressive renal disease. Three cats had gingivostomatitis; three had skin lesions with abscess and ulcers in two and scales and hair loss in another cat, which was also FIV +. This is the first report of feline L. tropica infection in Israel. Clinical information on cats with this infection from previous studies elsewhere is scarce. CONCLUSIONS: A high rate of Leishmania spp. infection, mostly estimated as sub-clinical, was found in dogs and cats admitted for veterinary care in an L. tropica focus. Among the animals in which infection could be characterized to the species level, more dogs were infected with L. infantum than with L. tropica while 5 of 11 cats were infected with L. tropica and had signs of systemic and skin disease not described before in feline L. tropica infection.

Imported case ofLeishmania tropica cutaneous leishmaniasis in a 10-year-old child in Malaysia.

The present paper reported a first imported case of cutaneous leishmaniasis in a 10-year- old child who returned from Saudi Arabia to Malaysia. Six weeks after his travel to Malaysia, two erythematous dermal nodules were developed over his right cheek and chin. Occurrence of intracellular amastigote of Leishmania was observed through examination of skin biopsy with hematoxylin and eosin stain. Furthermore, molecular analysis of ribosomal internal transcribed spacer 1 (ITS1) of Leishmania spp. confirmed the child was infected with Leishmania tropica. The child was given oral fluconazole and he had a 80% recovery before he went back to Saudi Arabia.

[Investigation of the Anti-Leishmanial Effects of Prangos ferulacea and Ferula orientalis Extracts Collected from Sirnak Province Against Leishmania tropica Isolated in Turkey]. / Sirnak Ili ve Çevresinden Toplanan Prangos ferulacea ve Ferula orientalis Ekstrelerinin Türkiye'den Izole Edilmis Leishmania tropica'ya Karsi Anti-Leishmanial Etkilerinin Arastirilmasi.

Leishmaniasis is a vector-borne disease that is caused by the protozoa of Leishmania genus. Leishmaniasis is endemic in tropical, subtropical, and large areas of the Mediterranean basin, and covers a total of 98 countries worldwide. It is estimated, according to the World Health Organization (WHO) data, that approximately 350 million people are at risk in these areas, and approximately 12 million people are infected. Increased drug resistance has been documented lately, in the treatment of leishmaniasis which causes almost 1.2 million new cases annually. Thus, interest in plant-derived active substances has increased in recent years, and new anti-leishmanial agents are investigated with in vitro studies. The aim of the present study was to investigate the anti-leishmanial effects of Prangos ferulacea and Ferula orientalis plant extracts collected from the rural areas of Sirnak province against Leishmania tropica. The water, chloroform, and ethanol extracts of the roots, stems, and fruits of P.ferulaceae and F.orientalis plants were obtained, and the cytotoxic activity tests of the extracts were performed. L.tropica isolate obtained from the Parasite Bank in Manisa Celal Bayar University in Turkey (MHOM/TR/2012/CBCL-LT) was grown on NNN and RPMI 1640 broth medium. The cytotoxicity of each extract on the L.tropica isolate was evaluated with the XTT test. Amphotericin B (AmpB) was used as the positive control, and the IC50 values were determined. The lowest IC50 values of the plant extracts were found to be as follows: P.ferulaceae root chloroform extract 36 µg/ml and fruit chloroform extract 20 µg/ml, F.orientalis root ethanol extract 2.5 µg/ml, and fruit ethanol extract 48 µg/ml, stem chloroform extract 24 µg/ml, and fruit chloroform extract 3.1 µg/ml. It was also determined in our study that only P.ferulaceae root ethanol extract showed cytotoxic activity on the WI-38 fetal lung fibroblast cell line at 65.19 µg/ml at 72 hours. This is the first study that assessed the anti-leishmanial activities of P.ferulaceae and F.orientalis plants that grow in high altitude areas of our country. It was determined that P.ferulaceae root ethanol extract and fruit chloroform extract had the lowest IC50 values among the 18 plant extracts that we examined for their anti-leishmanial activities. The outcomes of this study will be useful in further studies for the determination of active compounds in P.ferulaceae and F.orientalis plant extracts.