Unpleasant Souvenir: Imported Plasmodium falciparum Malaria in Türkiye.

Objective: Each year, approximately 125 million people visit malaria-endemic countries. This study aimed to investigate the clinical characteristics of imported Plasmodium falciparum malaria infections in Türkiye. Methods: The study included patients diagnosed with P. falciparum malaria between 1996 and 2022. A retrospective evaluation was conducted on whole blood samples and/or blood smears, as well as detailed medical histories, clinical manifestations, and laboratory findings. A total of 131 imported cases of P. falciparum were included in the study. Results: Among the patients, 121 were male. Of these, 101 had traveled to Africa, while 30 had visited Asia. Among the patients, 109 were returned travelers, and 22 were refugees/migrants. Early trophozoites were observed in all patients, while gametocytes were detected in 30 patients. Cerebral malaria developed in 15 patients, resulting in the death of two individuals. Additionally, 10 patients received preventive chemoprophylaxis. Conclusion: Turkey is situated on migration routes that connect two continents to Europe, where more than 95% of the global malaria burden exists. The importation of malaria through returned travelers poses a risk of malaria reintroduction in our country, given the presence of suitable vectors, climate conditions, and environmental factors. Importantly, 30 patients (22.9%) exhibited gametocyte forms of P. falciparum, which have the potential to infect Anopheles species, thus establishing a basis for local malaria transmission.

In Vitro and In Silico Evaluations of the Antileishmanial Activities of New Benzimidazole-Triazole Derivatives.

Benzimidazole and triazole rings are important pharmacophores, known to exhibit various pharmacological activities in drug discovery. In this study, it was purposed to synthesize new benzimidazole-triazole derivatives and evaluate their antileishmanial activities. The targeted compounds (5a-5h) were obtained after five chemical reaction steps. The structures of the compounds were confirmed by spectral data. The possible in vitro antileishmanial activities of the synthesized compounds were evaluated against the Leishmania tropica strain. Further, molecular docking and dynamics were performed to identify the probable mechanism of activity of the test compounds. The findings revealed that compounds 5a, 5d, 5e, 5f, and 5h inhibited the growth of Leishmania tropica to various extents and had significant anti-leishmanial activities, even if some orders were higher than the reference drug Amphotericin B. On the other hand, compounds 5b, 5c, and 5g were found to be ineffective. Additionally, the results of in silico studies have presented the existence of some interactions between the compounds and the active site of sterol 14-alpha-demethylase, a biosynthetic enzyme that plays a critical role in the growth of the parasite. Therefore, it can be suggested that if the results obtained from this study are confirmed with in vivo findings, it may be possible to obtain some new anti-leishmanial drug candidates.

[Investigation of the Efficacy of Cinnamaldehyde, Cannabidiol and Eravacycline in a Malaria Model]. / Sitma Modelinde Cinnamaldehyde, Cannabidiol ve Eravacycline'in Etkinliginin Arastirilmasi.

In this study, it was aimed to investigate the antimalarial activity of cinnamaldehyde (CIN) and cannabidiol (CBD) which have shown various biological activities such as potent antimicrobial activity and eravacycline (ERA), a new generation tetracycline derivative, in an in vivo malaria model. The cytotoxic activities of the active substances were determined by the MTT method against L929 mouse fibroblasts and their antimalarial activity were determined by the four-day test in an in vivo mouse model. In this study, five groups were formed: the CIN group, the CBD group, the ERA group, the chloroquine group (CQ) and the untreated group (TAG). 2.5 x 107 parasites/mL of P.berghei-infected erythrocyte suspension was administered IP to all mice. The determined doses of active substances were given to the mice by oral gavage in accordance with the four-day test and the parasitemia status in the mice was controlled for 21 days with smear preparations made from the blood taken from the tail end of the mice. The IC50 values, which express the cytotoxic activity values of the active substances were determined as 27.55 µg/mL, 16.40 µM and 48.82 µg/mL for CIN, CBD and ERA, respectively. The mean parasitemia rate in untreated mice was 33% on day nine and all mice died on day 11. On the ninth day, when compared with the TAG group, no parasites were observed in the CIN group, while the average parasitemia was 0.08% in the CBD group and 17.8% in the ERA group. Compared to the mice in the TAG group, the life expectancy of the other groups was prolonged by eight days in the CIN group, 12 days in the CBD group and eight days in the ERA group. It has been determined that all three active subtances tested in this study suppressed the development of Plasmodium parasites in an in vivo mouse model and prolonged the life span of the mice. It is thought that the strong antimalarial activity of CIN and CBD shown in the study and the possible positive effect of ERA on the clinical course can be improved by combining them with the existing and potential antimalarial molecules.

[Comparison of Conventional Methods with Molecular Methods in the Diagnosis of Trichomonas vaginalis and Investigation of Metronidazole Resistance]. / Trichomonas vaginalis'in Tanisinda Konvansiyonel Yöntemlerin Moleküler Yöntemlerle Karsilastirilmasi ve Metronidazol Direncinin Arastirilmasi.

Trichomoniasis is a sexually transmitted parasitic infection caused by Trichomonas vaginalis. In the diagnosis of trichomoniasis, direct microscopy (DM) is preferred, which is a cheap and fast method, although it has low sensitivity. Culture methods, which are accepted as the gold standard, can only be applied in certain centers due to the need for experienced personnel and the ability to get results within 2-7 days, despite their high sensitivity. In this study, it was aimed to compare conventional microscopic and culture methods used in the routine diagnosis of T.vaginalis with polymerase chain reaction (PCR) method and to investigate ntr4 and/or ntr6 gene polymorphism in the nitroreductase gene region, which are thought to be associated with metronidazole resistance in T.vaginalis strains isolated from clinical specimens. Vaginal swab specimens were collected from the posterior fornix of the vagina with two sterile ecuvion sticks during the gynecological examinations of 200 patients who applied to the Balikesir University Health Practice and Research Hospital, Obstetrics and Gynecology Polyclinic between March 2019 and August 2021. The first swab sample was used for direct microscopic examination, Giemsa staining and conventional PCR analysis, while the second swab specimen was taken into trypticase-yeast-extract-maltose (TYM) medium for T.vaginalis culture and followed for eight days at 37 °C. All specimens were screened for the presence of T.vaginalis using primers specific to the ß-tubulin (btub1) gene region and clinical isolates grown in TYM medium were examined for metronidazole resistance using primers specific for the nitroreductase gene region by using conventional PCR. Drug resistance test was also performed for the isolates in which polymorphism associated with metronidazole resistance was detected. Eight (4%) of 200 patient specimens were found positive by both culture/staining and PCR methods. The mean age of the patients included in the study was 39.9, while the mean age of the patients with positive T.vaginalis was 41.8. The most common clinical findings in the patients were foul-smelling vaginal discharge (36%), groin pain (21%), vaginal itching (19%), and burning sensation during urination (18%). In three out of eight T.vaginalis strains isolated from clinical samples, the presence of polymorphism in the ntr6 gene, which is thought to be associated with metronidazole resistance, was demonstrated by PCR. It was observed that three isolates with ntr6 gene polymorphism were phenotypically resistant to metronidazole (MLK= 390 µM). In this study, the fact that three of eight clinical isolates that were resistant to metronidazole by the broth microdilution method and as well as showing ntr6 gene polymorphism supported the thesis that there might be a close relationship between metronidazole resistance and ntr6 gene polymorphism. As a result, the use of culture and molecular methods in the diagnosis of T.vaginalis, in addition to the microscopy method, may contribute to a more accurate laboratory diagnosis of the agent, to detect metronidazole resistance molecularly and phenotypically, to determine metronidazole resistance rates in our country and to update treatment protocols within the framework of these data.

[Autochthonous Case of Malaria Prediagnosed as Leukemia]. / Lösemi Ön Tanili Yerli Sitma Olgusu.

Malaria is a parasitic disease transmitted by infected female Anopheles mosquitoes. There are five species of Plasmodium species that can infect humans. Of these species, especially P.falciparum and P.vivax pose the greatest threat to human health. In the 2014 report of the World Health Organization, it was reported that there were no locally acquired cases of malaria in 16 countries including Türkiye. Malaria cases originating from outside the country and imported due to migration, travel and working abroad are reported as import cases. In this report, a case of non-imported malaria followed with a preliminary diagnosis of leukemia was presented. A 14-year-old female patient who was admitted to a health institution with complaints of high fever, headache, chills, nausea-vomiting, and diarrhea that had been going on for two weeks, was pre-diagnosed as leukemia and was referred to Manisa Celal Bayar University Faculty of Medicine, Hafsa Sultan Hospital, Department of Pediatric Hematology and after pancytopenia was detected in the complete blood count. The anamnesis of the patient revealed that she had no history of international travel and that she had been prescribed medications such as paracetamol, amoxicillin, and metoclopramide for flu-like complaints while working in the Southeastern Anatolia, Aegean, and Mediterranean Regions of Türkiye. Bone marrow aspiration was performed for the etiological examination of pancytopenia. Giemsa-stained blood smears, rapid diagnostics, and real-time quantative polymerase chain reaction (qRt-PCR) analyses were performed in the medical parasitology laboratory and malaria was suspected in both bone marrow and peripheral blood smears. P.vivax erythrocytic forms and gametocytes were present in abundance in smear preparations stained with Giemsa, and rapid diagnosis kit was positive for P.vivax. The strain was genotyped as P.vivax by qRt-PCR analysis. For the treatment of the patient, airalam (artemether + lumefantrine) tablets were provided with 2 x 4 daily posology for three days after the diagnosis, and primaquine was provided after one week of the diagnosis as 1 x 2 tablets (1 x 15 mg) for 14 days, and the patient was discharged without complications following the treatment regimen. The fight against malaria continues uninterruptedly since the establishment of the Republic of Türkiye. Tropical diseases, especially malaria, is of great importance for Türkiye due to numerous reasons such as its location in the subtropical region where Anopheles mosquitoes are capable of malaria transmission, it is situated at the crossroads on the migration routes between continents where human traffic is busy, there are many people who go abroad for work and most importantly rising temperatures due to climate change. For this reason, this case report is important to emphasize the importance of malaria for the country and to increase the awareness of clinicians and laboratories about malaria and the possibility of autochthonous malaria transmission in Türkiye.

Visceral Leishmaniasis Caused by Leishmania Tropica.

PURPOSE: In Turkey, the main causative agent of visceral leishmaniasis (VL) is Leishmania. infantum and the main causative agent of cutaneous leishmaniasis (CL) is Leishmania tropica. In this study, we aimed to discuss the possible mechanisms, clinical aspects, and threat of visceralizing L. tropica. METHODS: This study includes seven cases of VL caused by L. tropica.Five patients were male (71%) and four were adults (57%). RESULTS: All the VL patients complained of fever and splenomegaly. Fatigue, pancytopenia, and hepatomegaly were present in six patients each (86%), while weight loss and gastrointestinal system (GIS) symptoms were present in 5 patients (71%). CONCLUSIONS: In this study, we have evaluated seven cases of visceralized L. tropica (VLT) in the context of the changing leishmaniasis epidemiology in Turkey. We have evaluated the possible mechanisms of visceralization; inter- and intraspecies genetic exchange with all the old world leishmaniasis agents present in the region, stress induced by inappropriate use of drugs, and possible ongoing adaptation mechanisms of Leishmania spp. The threat posed by VLT is significant as L. tropica is the most widespread and most common cause of leishmaniasis in Turkey. We do not know the vectorial capacity of the sand flies for the transmission of VLT strains or if these strains are in circulation in Turkey. Future studies should be carried out to investigate these issues as the transition of L. tropica from a mild disease-causing agent to a mortal one poses a significant public health concern for Turkey and Europe.

Design, synthesis, in vitro - In vivo biological evaluation of novel thiazolopyrimidine compounds as antileishmanial agent with PTR1 inhibition.

The leishmaniasis are a group of vector-borne diseases caused by a protozoan parasite from the genus Leishmania. In this study, a series of thiazolopyrimidine derivatives were designed and synthesized as novel antileishmanial agents with LmPTR1 inhibitory activity. The final compounds were evaluated for their in vitro antipromastigote activity, LmPTR1 and hDHFR enzyme inhibitory activities, and cytotoxicity on RAW264.7 and L929 cell lines. Based on the bioactivity results, three compounds, namely L24f, L24h and L25c, were selected for evaluation of their in vivo efficacy on CL and VL models in BALB/c mice. Among them, two promising compounds, L24h and L25c, showed in vitro antipromastigote activity against L. tropica with the IC50 values of 0.04 µg/ml and 6.68 µg/ml; against L. infantum with the IC50 values of 0.042 µg/ml and 6.77 µg/ml, respectively. Moreover, the title compounds were found to have low in vitro cytotoxicity on L929 and RAW264.7 cell lines with the IC50 14.08 µg/ml and 21.03 µg/ml, and IC50 15.02 µg/ml and 8.75 µg/ml, respectively. LmPTR1 enzyme inhibitory activity of these compounds was determined as 257.40 µg/ml and 59.12 µg/ml and their selectivity index (SI) over hDHFR was reported as 42.62 and 7.02, respectively. In vivo studies presented that L24h and L25c have a significant antileishmanial activity against footpad lesion development of CL and at weight measurement of VL group in comparison to the reference compound, Glucantime®. Also, docking studies were carried out with selected compounds and other potential Leishmania targets to detect the putative targets of the title compounds. Taken together, all these findings provide an important novel lead structure for the antileishmanial drug development.

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.

Investigation of in vitro Efficacy of Miltefosine on Chronic Cutaneous Leishmaniasis

Objective: Leishmaniasis is the second deadliest parasitic disease in the World Health Organisation's list of neglected diseases, following malaria. Cutaneous leishmaniasis (CL) is the most common form of the disease and it is one of the few communicable diseases with increasing incidence rates owing to factors like armed conflicts and climate change. CL can be divided into two major groups: Acute CL (ACL) and chronic CL (CCL). The aim of this study was to compare the in vitro efficacy of miltefosine and pentavalent antimony compounds in the CCL patient samples. Methods: Five isolates previously isolated from 5 CCL patients were included in this study. Genotyping is performed using internal transcribed spacer 1 (ITS 1) gene region real-time PCR. In vitro drug efficacy tests were applied to determine their activity against meglumine antimoniate (MA) and miltefosine. Serial dilutions (512, 256, 128, 64, 32, 16, 8 and 4 µg/mL) prepared from MA and miltefosine were prepared in 96-well flat-bottom cell culture plates and incubated at 24 °C for 48 hours. The efficacy of the drug on Leishmania spp. promastigotes after 24 and 48 hours was evaluated by hemocytometer slide and XTT cell viability test. Results: All of the samples were genotyped as L. tropica. Evaluation of 24 and 48 hours showed, 128 µg/mL and 256 µg/mL and 32 µg/mL and 64 µg/mL concentrations of miltefosine and MA were enough to kill all the promastigotes respectively. The results of the hemocytometer slide and XTT were consistent. Conclusion: There are no studies investigating the in vitro efficacy of miltefosine with the CCL patient group. To overcome the treatment challenges experienced in this special patient group, more studies are needed. According to our results, it is concluded that miltefosine is efficient for the treatment of CCL and further clinical studies with miltefosine will reveal valuable data.

[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.

Autochthonous transmission of Leishmania donovani and Leishmania major with all the components of infection cycle at Europe's doorstep.

OBJECTIVES: Leishmaniasis is a vector-borne disease and dogs may act as urban reservoirs. Turkey and most of the Mediterranean basin countries are endemic for leishmaniasis. In this study, it is aimed to report the autochthonous leishmaniasis cases, with all the components of the infection cycle (reservoir, vector, and the host) in a region close to Europe. METHODS: Nine human and four canine autochthonous leishmaniasis cases were included in the study. Direct microscopy, culture methods, serological, and molecular tests were applied to the samples obtained from the cases. RESULTS: VL and CL patients consisted of 2 L.infantum, 1 L. donovani, 2 L. tropica, and 2 L. tropica,1 L. major,1 L. infantum infected patients respectively. CanL cases were infected with L. infantum, L. donovani, L. tropica, and L. major. CONCLUSIONS: All the cases were autochthonous cases located in Manisa province. As Greece and all the Mediterranean basin countries in Europe share competent vectors, it is concluded that the detection of all 4 species of Leishmania parasites in such proximity to Europe poses an important public health threat for Europe. This study reports all four species of Leishmania spp., including L. major and L.donovani in close proximity to continental Europe.

Comparative proteomic analysis of Leishmania parasites isolated from visceral and cutaneous leishmaniasis patients.

Leishmaniasis is an infectious disease in which different clinical manifestations are classified into three primary forms: visceral, cutaneous and mucocutaneous. These disease forms are associated with parasite species of the protozoan genus Leishmania. For instance, Leishmania infantum and Leishmania tropica are typically linked with visceral (VL) and cutaneous (CL) leishmaniasis, respectively; however, these two species can also cause other form to a lesser extent. What is more alarming is this characteristic, which threatens current medical diagnosis and treatment, is started to be acquired by other species. Our purpose was to address this issue; therefore, gel-based and gel-free proteomic analyses were carried out on the species L. infantum to determine the proteins differentiating between the parasites caused VL and CL. In addition, L. tropica parasites representing the typical cases for CL were included. According to our results, electrophoresis gels of parasites caused to VL were distinguishable regarding the repetitive down-regulation on some specific locations. In addition, a distinct spot of an antioxidant enzyme, superoxide dismutase, was shown up only on the gels of CL samples regardless of the species. In the gel-free approach, 37 proteins that were verified with a second database search using a different search engine, were recognized from the comparison between VL and CL samples. Among them, 31 proteins for the CL group and six proteins for the VL group were determined differentially abundant. Two proteins from the gel-based analysis, pyruvate kinase and succinyl-coA:3-ketoacid-coenzyme A transferase analysis were encountered in the protein list of the CL group.

[The Follow-Up of Treatment Process of Malaria By Real-Time Polymerase Chain Reaction: In Vivo Model]. / Sitmada Tedavi Sürecinin Gerçek Zamanli Polimeraz Zincir Reaksiyonu ile Takibi: In Vivo Model.

Microscopic methods are accepted as the gold standard in the diagnosis of malaria and in the followup of treatment. However, as the microscopical methods require experienced personnel, it is important to confirm the diagnosis with a different method for accurate diagnosis and treatment follow-up. In our study, we aimed to investigate the utility of the use of real time reverse transcriptase polymerase chain reaction (rRT-PCR), as well as microscopic methods for malaria treatment follow-up. In our study, we formed five groups each consisting of five male Balb/c mice. Each mouse was injected intraperitoneally with 107/ml Plasmodium berghei parasites. After 48 hours following the injection, the mice in the first, second and third groups received 50 mg/kg/day of chloroquine treatment for one, two and three days, respectively. The fourth group was not treated and the fifth group of mice received saline for three days. The parasitemia was monitored for 21 days by blood smears prepared from the end of tail of the mice and searching the presence of the target gene region of the parasite by rRT-PCR. Both the blood smears and rRT-PCR results were positive for groups I, II, IV and V. Both blood smears and rRT-PCR results of mice in groups other than the third group were found to be positive. Blood smears of the mice in third group were found to be positive on the 5th and 7th days of the infection, and the subsequent preparations were evaluated as negative. rRT-PCR results showed positivity on day seven, but no presence of the target gene region of the parasite was detected on the other days. The comparison of microscopy and rRT-PCR methods, had shown parallel results. Apart from the microscopic examination method, it was concluded that the rRT-PCR method is important in the diagnosis of malaria and in the follow-up of the patient during the treatment process, and that different methods that support each other should be used.

In vitro Anti-Leishmanial Activity of Essential Oils Extracted from Plants Growing in Northern Cyprus Against Leishmania tropica

Objective: Natural plant products are considered as a source of novel and effective compounds for the treatment of leishmaniasis. In this study, the in vitro activities of essential oils obtained from Origanum dubium (OD), Origanum majorana (OM), Salvia fruticosa (SF) and Laurus nobilis (LN) plants in Northern Cyprus were investigated against Leishmania tropica. Methods: Leishmania tropica strain (MHOM/TR/2012/CBCL-LT) was obtained. RPMI-1640 was added to 96-well plates in 100 µL aliquots, 100 µg/mL essential oil was added to the first well of each row and serial 2-fold dilutions were performed. A promastigote suspension was pipetted into all wells, and the plates were incubated. The promastigotes were enumerated using a haemocytometer. Results: OD essential oil was effective at killing all promastigotes at a minimum inhibitor height (MIC)=0.2 µg/mL and had high activity at the lowest concentrations. Both SF and LN oils had MIC=1.56 µg/mL and LD50=0.78 µg/mL. SF was observed to impair promastigote morphology at the lowest concentrations, while LN did not exert any effect at concentrations <0.2 µg/mL. OM essential oil was found to have a MIC=3.13 µg/mL and a LD50=1.56 µg/mL. Conclusion: All tested essential oils inhibited promastigotes of Leishmania tropica. OD essential oil demonstrated the highest anti-leishmanial activity. Amaç: Bitkilerden elde edilen dogal ürünlerin leishmaniasis tedavisi için yeni ve etkili bilesiklerin üretilmesine öncülük edecegi düsünülmektedir. Çalismamizda, Kuzey Kibris'ta yetisen Origanum dubium (OD), Origanum majorana (OM), Salvia fruticosa (SF) ve Laurus nobilis (LN) bitkilerinden elde edilen uçucu yaglarin Leishmania tropica'ya karsi in vitro etkinlikleri arastirilmistir. Yöntemler: Çalismamizda, Leishmania tropica susu (MHOM/TR/2012/CBCL-LT) kullanildi. Düz tabanli 96'lik plaklarda, tüm kuyucuklara 100 µL RPMI-1640 ve ilk kuyucuklara 100 µg/mL uçucu yaglar eklenerek, seri dilüsyonlari yapildi. Ardindan tüm kuyucuklara Leishmania tropica promastigot süspansiyonundan pipetlendi ve inkübe edildi. Hemositometre yöntemiyle promastigotlarin sayisi incelendi. Bulgular: OD yaginin minimum inhibitör konsantrasyonu (MIK)=0,2 µg/mL'de tüm promastigotlari öldürürken, en düsük konsantrasyonlarda bile etkili oldugu görülmüstür. SF ve LN uçucu yaglarinin ikisinde de MIK=1,56 µg/mL, LD50=0,78 µg/mL olarak saptanmistir. SF'nin en düsük konsantrasyonlarinin bile promastigot morfolojisini bozdugu görülürken, Laurus nobilis'in ise 0,2 µg/mL'den sonraki konsantrasyonlarda etkisini kaybettigi belirlenmistir. OM uçucu yaginin MIK=3,13 µg/mL, LD50=1,56 µg/mL oldugu görülmüstür. Sonuç: Kullanilan tüm uçucu yaglarin Leishmania tropica promastigotlarini inhibe ettigi görülürken, en yüksek anti-leishmanial etkinlik Origanum dubium uçucu yaginda bulunmustur.

Overcoming the Challenge; In Vivo Efficacy of Miltefosine for Chronic Cutaneous Leishmaniasis.

BACKGROUND: Cutaneous Leishmaniasis (CL) is the most common form of leishmaniasis. CL can be divided into two major groups: acute CL (ACL) and chronic CL (CCL). The aim of this study is to compare the efficacy of miltefosin and pentavalent antimony compounds in vivo with the CCL patient samples. MATERIALS: Three study groups were formed, each consisting of five male Mus musculus (Balb/C) mice. In this model, promastigotes from the culture of a CCL patient were utilized. 100 µL L. tropica promastigote suspension with a density of 108 promastigotes/ml were injected into the hint-right footpad of each experimental animal intradermally. Footpads of the mice were measured every two weeks until 24th week. From the 13th week, miltefosin 50 mg/kg/day was administered orally using gavage for 21 days, Meglumin antimoniate (MA) was administered by intramuscular (IM) injection daily for 21 days at 50 mg/kg/day and saline was administered IM for 21 days for the miltefosine, MA and control group, respectively. RESULTS: The footpad measurements of the miltefosine group were lower than the control group statistically. Between the MA group and the miltefosine group and MA group and the control group, there was no statistically significant difference. Giemsa stained slides revealed amastigotes in one, two and all of the slides for the miltefosine, MA and control group, respectively. Molecular tests were performed with the Rotor-Gene device and L. tropica consistent peaks were obtained in one of the miltefosine group, four in the MA group and all mice in the control group. CONCLUSIONS: Demonstration of both clinical and laboratory improvement in four of the five experimental animals provides strong evidence that miltefosine is an effective drug in the treatment of CCL. In the literature, no clinical or laboratory studies using miltefosine have been performed with CCL patients only.

In vitro Cultivation of Plasmodium falciparum.

OBJECTIVE: Plasmodium falciparum is a protozoan parasite that causes many deaths worldwide. It's cultivation in an in vitro culture setting contributes significantly to scientific studies. However, there are no laboratories in Turkey that cultivate P. falciparum in vitro. Hence, the purpose of this study was to cultivate P. falciparum in vitro. METHODS: Five P. falciparum strains were used in our study and were kept frozen in liquid nitrogen tanks. These parasite strains were then thawed in a 37 °C water bath and transferred to the Albumax-complete medium that was previously prepared. After that, the petri dishes were placed in the chamber. For 30 seconds, a special gas mixture containing 5% CO2, 5% O2 and 90% N2 was added into the chamber which was placed in a 37 °C oven and left for incubation for 2 days. At the end of the incubation period, thin smear preparations were prepared from the medium, stained with Giemsa and examined using an immersion lens. RESULTS: Examination of the smears revealed that trophozoite and schizont forms of all P. falciparum isolates were present at a rate of 2% in in vitro culture medium. CONCLUSION: As a result of our study, the in vitro culture of P. falciparum was successfully developed. With this, several projects such as biological and chemical characteristics, pathogenicity, phenotypic and molecular-level drug sensitivities and parasite vaccination studies can be carried out more easily in our country.

Evaluation of Imported Malaria Cases in Northern Cyprus between 2016 and 2019: First Data Series

Objective: In present times, malaria remains an infectious disease with a high mortality rate in some regions of the world. It is predicted to preserve its importance as a disease in the future because of the traveling human populations from malaria-endemic African countries into the regions where malaria has been eradicated. The objective of this study is to evaluate the increasing imported malaria cases in the Turkish Republic of Northern Cyprus. Methods: In this study, we investigated 13 patients who were diagnosed with malaria between 2016 and 2019. We clinically evaluated all the cases. More importantly, we made the diagnosis of these patients by Giemsa-stained thin and thick blood smears, rapid malaria antigen tests, and genotyping (only for five patients) by real-time polymerase chain reaction. Additionally, we evaluated patients with malaria in terms of age, gender, and seasons. Results: In the diagnosed malaria cases, 11 (84.4%) of them were male and 2 (15.6%) were female. There was no significance between malaria infection and gender (p=0.358). Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale infection were detected in ten patients (76.9%), two (15.4%) patients, and one (7.7%) patient, respectively. There was a significant increase (p=0.003) in the malaria cases in 2019 (n=9). The seasonal comparison revealed that malaria infections are most common in autumn (8/13, 61.5%). Conclusion: Despite the eradication of malaria in Turkish Republic of Northern Cyprus, the rising number of recently imported cases increases the risk of emerging local cases. Malaria infection should be immediately suspected, particularly, in foreign patients who travel from the malaria-endemic region and present with symptoms such as fever and shivering if the laboratory findings especially detect thrombocytopenia.

[Diversity of Leishmania Strains Isolated from Cutaneous Leishmaniasis Patients in Turkey and its Reflection to Clinics in Mice Model]. / Türkiye'de Kutanöz Leysmanyazis Hastalarindan Elde Edilen Leishmania Izolatlarindaki Farkliliklar ve Bunlarin Fare Modeline Klinik Yansimasi.

Although asexual reproduction has been attributed to Leishmania species, genetic exchange has recently been demonstrated, which helped emerging of hybrid isolates. Situated on the crossroads between three continents, Leishmania hybrids may be present in Turkey. In Turkey, visceral leishmaniasis caused by Leishmania infantum is less common, while cutaneous leishmaniasis (CL) caused by Leishmania tropica and L.infantum could reach 2500 reported cases a year. Our aim was to investigate genetic variability of local Leishmania species and presence of hybrid Leishmania strains in Turkey. Twenty CL patients from Sanliurfa and Hatay, where only L.tropica and both L.tropica and L.infantum cause CL, respectively, were registered equally. All isolates were assessed with real-time polymerase chain reaction (Rt-PCR), isoenzyme analysis, gene sequencing, two-dimensional gel electrophoresis (2D-PAGE) and MALDI-TOF/TOFMS followed by in vivo analyses on mouse model. Identification of differentially expressed proteins was performed. These proteins were confirmed by sequence analysis. All isolates from Sanliurfa were found to be L.tropica which caused cutaneous infection in mice. However, one of 10 isolates from Hatay was found as Leishmania major which caused cutaneous infection. Five isolates were found as L.tropica with Rt-PCR and gene sequencing, one of which had one different protein from the reference L.tropica strain and caused cutaneous infection. Four of the five isolates had five different proteins compared to reference strain and caused both cutaneous and visceral infections. Remaining four isolates showed double melting curves in Rt-PCR, which were concordant with L.tropica and L.infantum. Their sequencing and isoenzyme analyses indicated them as L.infantum. They had six different proteins compared to reference L.infantum strain and caused cutaneous and visceral infections. It is concluded that the isolates with different proteins were hybrid Leishmania species. In the present study, outcomes of the proteomics, genomics, clinical manifestations and tissue tropism on animal models were evaluated together for the first time. In addition to L.tropica and L.infantum, L.major was identified as a causative agent for CL and hybrids of L.infantum/tropica were also shown to be present.

[Determination of Antimony Resistance Mechanism of Leishmania tropica Causing Cutaneous Leishmaniasis in Turkey]. / Türkiye'de Kutanöz Leysmanyazis Etkeni Leishmania tropica'da Antimon Direnç Mekanizmasinin Belirlenmesi.

World Health Organization reported that approximately one billion people are at risk in endemic areas, one million cases of cutaneous leishmaniasis (CL) and approximately 300,000 cases of visceral leishmaniasis (VL) were reported per year in the last five years. The number of deaths due to VL is reported to be approximately 20,000 per year. Approximately 2500 cases/year have been reported as CL, caused by Leishmania tropica and Leishmania infantum, in Turkey. The significant increase observed in many cities mainly in the provinces of Mediterranean and Aegean regions in cases and foci in recent years, suggests that there may be an increase in this infections in the following years as well. In Turkey, the causative agent of CL is L.tropica and meglumine antimoniate is used in the treatment of CL. We aimed to determine antimony resistance genes specific for L.tropica by comparing the gene and protein expressions of antimony-resistant and non-resistant L.tropica strains. L.tropica isolates obtained from 3 CL patients without antimonate resistance from Aegean, Mediterranean and Southeastern regions of Turkey were provided to transform into 3 resistant isolates against meglumine antimony in the laboratory conditions. Gene expression alterations by microarray method; protein profiles by two-dimensional gel electrophoresis (2D-PAGE) and relevant proteins by MALDI-TOF/TOF MS of these isolates were accomplished and compared. L.tropica isolates from 10 CL patients who did not respond to antimony therapy were analyzed for resistance to antimonial compounds and quantitative real-time polymerase chain reaction was performed to detect the expression of genes responsible for resistance development. Moreover, differences in protein expression levels in isolates with and without antimony resistance were determined by comparing protein profiles and identification of proteins with different expression levels was carried out. Enolase, elongation factor-2, heat shock protein 70, tripanthione reductase, protein kinase C and metallo-peptidase proteins have been shown to play roles in L.tropica isolates developing resistance to antimonial compounds and similar expression changes have also been demonstrated in naturally resistant isolates from patients. In conclusion, it was revealed that L.tropica strains in our country may gain resistance to meglumine antimoniate in a short time. It is foreseen that if the patients living in our country or entering the country are treated inadequately and incompletely, there may be new, resistant leishmaniasis foci that may increase the number of resistant strains and cases rapidly.

Design, synthesis, and in vitro biological evaluation of novel thiazolopyrimidine derivatives as antileishmanial compounds.

A series of thiazolopyrimidine derivatives was designed and synthesized as a Leishmania major pteridine reductase 1 (LmPTR1) enzyme inhibitor. Their LmPTR1 inhibitor activities were evaluated using the enzyme produced by Escherichia coli in a recombinant way. The antileishmanial activity of the selected compounds was tested in vitro against Leishmania sp. Additionally, the compounds were evaluated for cytotoxic activity against the murine macrophage cell line RAW 264.7. According to the results, four compounds displayed not only a potent in vitro antileishmanial activity against promastigote forms but also low cytotoxicity. Among them, compound L16 exhibited an antileishmanial activity for both the promastigote and amastigote forms of L. tropica, with IC50 values of 7.5 and 2.69 µM, respectively. In addition, molecular docking studies and molecular dynamics simulations were also carried out in this study. In light of these findings, the compounds provide a new potential scaffold for antileishmanial drug discovery.