RESUMO
According to the World Health Organization (WHO), leishmaniasis is a zoonotic/anthroponotic parasitic disease endemic in 99 countries. It is estimated that approximately 12 million people are infected with Leishmania spp. and 350 million people live at risk. Every year, two million new cases are added to these figures. One and a half million cases of zoonotic/anthroponotic cutaneous leishmaniasis and 500 000 cases of visceral leishmaniasis are reported annually. One person is estimated to to be infected with cutaneous leishmaniasis in every 20 seconds and visceral leishmaniasis causes 60 000 deaths. In this report, two pediatric cases diagnosed with visceral leishmaniasis were presented. In the study, bone marrow aspirations were performed to determine the etiology of the disease in an eight-month-old male patient with fever and hepatosplenomegaly who had been followed up in Manisa Celal Bayar University, Department of Pediatrics, Division of Pediatric Hematology with the diagnosis of severe glucose-6-phosphate dehydrogenase (G-6PD) deficiency since the neonatal period and in a nine-month-old female patient who had had a high fever and bicytopenia for two weeks. Bone marrow aspirations were cultured in NNN medium and their smears were stained and examined with Giemsa. rk-39 and Leishmania IFAT tests were performed by using patients' sera. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis was also performed for Leishmania spp. Leishmania spp. amastigotes were observed in Giemsa-stained smear preparations, Leishmania spp. promastigotes were grown in NNN medium, rk39 rapid diagnostic kit was weakly positive, Leishmania IFAT was positive at a titer of 1/1024 and Leishmania tropica was identified as the causative agent by RT-qPCR analysis for both cases. These two cases suggested that fatal cases of visceral leishmaniasis may increase with the spread of visceralized isolates of L.tropica, the most common causative agent of cutaneous leishmaniasis in Türkiye, and this issue may create a significant public health problem.
Assuntos
Leishmania tropica , Leishmaniose Visceral , Humanos , Masculino , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Leishmania tropica/isolamento & purificação , Leishmania tropica/genética , Feminino , Lactente , Medula Óssea/parasitologia , Deficiência de Glucosefosfato Desidrogenase/complicações , AnimaisRESUMO
In recent years, isolation of resistant Leishmania species to drugs in use has made it necessary to search alternative molecules that may be drug candidates. In this study, it was aimed to investigate the cytotoxic and in vitro antileishmanial activity of hybrid silver nanoparticle (AgNP) complexes. In this study, three types of nanoparticles (NPs), oxidized amylose-silver (OA-Ag) NPs, oxidized amylose-curcumin (OA-Cur) NPs and oxidized amylose-curcumin-silver (OA-CurAgNP) nanoparticles were synthesized. The cytotoxic activity of the synthesized nanoparticles was determined against L929 mouse fibroblasts and the in vitro antileishmanial activity was determined against Leishmania tropica, Leishmania infantum and Leishmania donovani isolates by the broth microdilution method. It was observed that the hybrid OA-CurAgNP complex obtained by combining curcumin and silver nanoparticles showed cytotoxic effects against L929 mouse fibroblasts at concentrations of 1074 µg/mL and above. IC50 values expressing the antileishmanial activity of the hybrid OA-CurAgNP complex against L.tropica, L.infantum and L.donovani isolates, were found to vary between 95-121 µg/mL, 202-330 µg/mL and 210-254 µg/mL, respectively. Resistance development has emerged as a major challenge in the treatment of leishmaniasis in recent times. Metallic nanoparticles are considered excellent candidates for medical applications due to their chemical and physical properties, as well as their prolonged circulation in the body. The current drugs used for leishmaniasis treatment are highly toxic, while nanoparticles offer advantages such as low toxicity and easy cellular uptake due to their nanoscale dimensions. The identification of strong efficacy in these particles may contribute scientific evidence for their potential use in leishmaniasis treatment. Therefore, the therapeutical value of OA-CurAgNP complex alone in combination with existing drugs should be examined.
Assuntos
Antiprotozoários , Curcumina , Fibroblastos , Leishmania infantum , Leishmania tropica , Nanopartículas Metálicas , Prata , Animais , Camundongos , Prata/farmacologia , Prata/química , Nanopartículas Metálicas/química , Curcumina/farmacologia , Curcumina/química , Leishmania tropica/efeitos dos fármacos , Leishmania infantum/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Antiprotozoários/farmacologia , Antiprotozoários/química , Antiprotozoários/toxicidade , Leishmania donovani/efeitos dos fármacos , Concentração Inibidora 50 , Linhagem CelularRESUMO
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.
Assuntos
Doença de Chagas , Leishmania tropica , Parasitos , Toxoplasma , Trypanosoma cruzi , Animais , Humanos , Células HeLaRESUMO
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.
Assuntos
Antimaláricos , Canabidiol , Malária , Animais , Camundongos , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Canabidiol/farmacologia , Canabidiol/uso terapêutico , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Plasmodium berghei , Extratos Vegetais/farmacologia , Malária/tratamento farmacológico , Malária/parasitologia , Tetraciclina/farmacologia , Tetraciclina/uso terapêuticoRESUMO
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.
Assuntos
Tricomoníase , Vaginite por Trichomonas , Trichomonas vaginalis , Gravidez , Feminino , Humanos , Metronidazol/farmacologia , Metronidazol/uso terapêutico , Trichomonas vaginalis/genética , Vaginite por Trichomonas/diagnóstico , Vaginite por Trichomonas/tratamento farmacológico , Tricomoníase/diagnóstico , Nitrorredutases/uso terapêuticoRESUMO
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.
Assuntos
Leucemia , Malária Vivax , Malária , Pancitopenia , Plasmodium , Adolescente , Animais , Feminino , Humanos , Malária/diagnóstico , Malária/tratamento farmacológico , Malária/parasitologia , Malária Vivax/diagnóstico , ViagemRESUMO
Origanum sipyleum is used in folk medicine due to its anti-inflammatory, antimicrobial, and antioxidant properties. Ponatinib, an effective tyrosine kinase inhibitor in the treatment of chronic myeloid leukemia (CML), has severe side effects. Thus, we aimed to determine a novel herbal combination therapy that might not only increase the anti-leukemic efficacy but also reduce the dose of ponatinib in targeting CML cells. Origanum sipyleum was extracted with methanol (OSM), and secondary metabolites were determined by phytochemical screening tests. The cytotoxic effects of OSM on K562 cells were measured by WST-1 assay. Median-effect equation was used to analyze the combination of ponatinib and OSM (p-OSM). Apoptosis, proliferation, and cell-cycle were investigated by flow-cytometry. Cell-cycle-related gene expressions were evaluated by qRT-PCR. OSM that contains terpenoids, flavonoids, tannins, and anthracenes exhibited cytotoxic effects on K562 cells. The median-effect of p-OSM was found as synergistic; OSM reduced the ponatinib dose â¼5-fold. p-OSM elevated the apoptotic and anti-proliferative activity of ponatinib. Consistently, p-OSM blocked cell-cycle progression in G0/G1, S phases accompanied by regulations in TGFB2, ATR, PP2A, p18, CCND1, CCND2, and CCNA1 expressions. OSM enhanced the anti-leukemic activity of ponatinib synergistically via inducing apoptosis, suppressing proliferation, and cell-cycle. As a result, OSM might offer a potential strategy for treating patients with CML.
Assuntos
Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Origanum , Antineoplásicos/uso terapêutico , Apoptose , Resistencia a Medicamentos Antineoplásicos , Humanos , Imidazóis , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Metanol/farmacologia , Metanol/uso terapêutico , Inibidores de Proteínas Quinases/efeitos adversos , PiridazinasRESUMO
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.
Assuntos
Leishmania infantum , Leishmania tropica , Leishmaniose Cutânea , Leishmaniose Visceral , Parasitos , Animais , Humanos , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , ProteômicaRESUMO
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.
Assuntos
Antiprotozoários , Ferula , Leishmania tropica , Leishmaniose , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Clorofórmio/farmacologia , Clorofórmio/uso terapêutico , Etanol/farmacologia , Etanol/uso terapêutico , Humanos , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , TurquiaRESUMO
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.
Assuntos
Malária , Animais , Cloroquina , Seguimentos , Humanos , Malária/diagnóstico , Malária/tratamento farmacológico , Masculino , Camundongos , Microscopia , Plasmodium berghei/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
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.
Assuntos
Antiprotozoários/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Leishmania major/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Pirimidinas/farmacologia , Tiazóis/farmacologia , Animais , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Leishmania major/enzimologia , Macrófagos/efeitos dos fármacos , Camundongos , Modelos Moleculares , Estrutura Molecular , Oxirredutases/metabolismo , Testes de Sensibilidade Parasitária , Pirimidinas/síntese química , Pirimidinas/química , Células RAW 264.7 , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/químicaRESUMO
Malaria still remains to be a public health threat and one of the most important infectious diseases to get attention from World Health Organization. No domestic malaria cases have been reported on the island of Cyprus since 1948, as a result of successful elimination process. All of the malaria cases detected in recent years are imported cases. As known, hundreds of medicines are obtained from plants and traditional medicine are used in endemic places of malaria. The cause of malaria - Plasmodium parasites, are developing resistance to antimalarial drugs. Hence, research on plant extracts and essential oils have gained great interest in recent years to obtain new and safe agents/substances. In our study, it was aimed to investigate the in vivo antimalarial activities of essential oils obtained from Origanum dubium, Origanum majorana, Salvia fruticosa and Laurus nobilis plants which grows in Northern Cyprus against Plasmodium berghei - the rodent malaria agent. Plants were collected in appropriate seasons and were dried to obtain and analyze essential oils via Clevenger Apparatus system. L929 mouse fibroblast cell line and MTT [3-(4.5-dimethylthiazole-2-yl) -2.5-diphenyltetrazolium bromide] kit were used to determine the cytotoxic activities of the essential oils obtained. In our study, total of 36 mice (Balb/c) of 6 groups (6 mice in each group) were formed: chloroquine group (CG) (50 mg/kg) as malaria reference group, untreated control group (UTCG), O.dubium (OD) (20 mg/kg), O.majorana (OM) (20 mg/kg), S.fruticosa (SF) (20 mg/kg) and L.nobilis (LN) (20 mg/kg). The essential oils were given to mice infected with P.berghei strain orally on 0, 1, 2 and 3rd days (4 times in total). Blood was taken from the tail end of each mouse 24 hours after the last treatment and blood collection was continued every two days until the mice died. Withdrawn blood taken from the mice were prepared as a thin smear and stained with Giemsa. Then, parasitemia percentages in each smear were calculated. As a result of the cytotoxicity tests, cytotoxic activity was not found at 100 µg/ml (20 mg/kg) in all oils except OD essential oil. While the mice receiving chloroquine continued their lives with the disappearance of the parasite on the 6thday, the mice in the UTCG died on the 9th day. The parasitemia rate reached 35% in the OM group on the 23rd day, in the OD group on the 21st day and in the other groups (SF and LN) on the 14th day and the mice have died. In our study, the difference between the life span in all groups was found statistically significant (p≤ 0.001). As a result, the essential oils O.majorana (14 days increase according to UTCG) an endemic plant of Cyprus and O.dubium (12 days increase according to UTCG) which had an antimalarial effect, decreased parasitemia and increased the life span of mice more than two times, indicated that they could be a source for the acquisition of new antimalarial molecules.
Assuntos
Antimaláricos , Malária , Óleos Voláteis , Origanum , Extratos Vegetais , Animais , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Chipre , Células Alimentadoras , Malária/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Óleos Voláteis/farmacologia , Óleos Voláteis/uso terapêutico , Origanum/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Plasmodium berghei/efeitos dos fármacosRESUMO
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.
Assuntos
Variação Genética , Leishmania , Leishmaniose Cutânea , Leishmaniose Visceral , Animais , Modelos Animais de Doenças , Humanos , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Camundongos , TurquiaRESUMO
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.
Assuntos
Resistência a Medicamentos , Leishmania tropica , Leishmaniose Cutânea , Antimoniato de Meglumina , Resistência a Medicamentos/genética , Humanos , Leishmania tropica/efeitos dos fármacos , Leishmaniose Cutânea/tratamento farmacológico , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , TurquiaRESUMO
Leishmania virus (LRV) has previously been identified in different Leishmania species. Host-LRV interaction is associated with exacerbated clinical manifestations of cutaneous leishmaniasis (CL) and may cause poor therapeutic response. CL cases due to L. major with large skin lesions resistant to routine therapy were recently identified in Turkey. Here, we report the first autochthonous case of cutaneous leishmaniasis caused by LRV-positive Leishmania major, using conventional PCR targeting the viral capsid protein of LRV. The lesion of the case was 6 months old, relatively large (4 cm), and did not recover despite three consecutive intralesional applications of glucantime. Assessment of LRV's influence on prognosis and clinical outcomes of leishmaniasis, based on additional studies, is required.
Assuntos
Leishmania major , Leishmaniose Cutânea , Proteínas do Capsídeo/genética , Vírus de DNA/genética , Vírus de DNA/isolamento & purificação , Humanos , Lactente , Leishmania major/virologia , Leishmaniose Cutânea/microbiologia , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/virologia , Reação em Cadeia da Polimerase , Falha de Tratamento , TurquiaRESUMO
Plasmodium falciparum malaria causes about 450.000 deaths every year, mostly in children around the world. The infection is seen in cases coming from abroad and may lead to deaths in Turkey. Many native P.falciparum malaria cases and deaths due to this infection were observed in Turkey during mid 1900's when malaria was epidemic. But only two native cases were reported in the last 50 years, both from Manisa. First case was a one-year old baby who has come to Manisa from Urfa with his family and has never been abroad. He has diagnosed with Plasmodium vivax malaria and treated with chloroquine and primaquine. A previously obtained thin blood film was examined and characteristic P.falciparum rings in red blood cells were observed and the case was published together with photographs as probable P.falciparum and P.vivax mixed infection. After this case, microscopists working in Malaria Control Unit of Manisa were informed about the differentiation of malaria species in thin blood samples. Soon afterward, another case who have never been abroad before were also diagnosed with P.falciparum and P.vivax mixed infection and this case was also published with photographs taken from thin blood samples. As molecular diagnostic methods were not improved and widespread in those years, it could not be applied in both cases. A Giemsa stained thin blood sample of the baby case was incidentally found 22 years afterwards and with the aim of molecular diagnosis, the blood sample on the slide previously processed for DNA isolation, then analysed with "FTD Malaria Differentiation (Fast Track Diagnostics, Luxembourg)" multiplex kit with real-time polymerase chain reaction by using probes special for P.falciparum, P.ovale, P.malariae, P.vivax species. DNA's belonging to P.falciparum and P.vivax were found to be positive, the case is molecularly proved to have P.falciparum and P.vivax mixed infection. This case indicated that Turkey is convenient for the expansion of P.falciparum malaria in terms of the climate and vectors and suggested that the potential danger may increase with the effects of global warming, wars and migrations and may jump to Europe over Turkey. The case which molecularly proved the existence of native P.falciparum malaria in the near future in Turkey, was presented to draw attention to the danger of this infection for Turkey and Europe.
Assuntos
Malária Falciparum , Malária Vivax , Plasmodium falciparum , Plasmodium vivax , Criança , Coinfecção/parasitologia , Europa (Continente) , Humanos , Lactente , Malária Falciparum/diagnóstico , Malária Falciparum/parasitologia , Malária Vivax/diagnóstico , Malária Vivax/parasitologia , Masculino , Patologia Molecular , Plasmodium falciparum/genética , Plasmodium vivax/genética , TurquiaRESUMO
Cutaneous leishmaniasis (CL) is a parasitic disease transmitted by vector sand flies Phlebotomus and Lutzomyia. This disease is characterized by long time non-healing skin lesions, and caused by Leishmania species. CL is the most common infection in Eastern and Southeastern Anatolia in Turkey and L.tropica is known as the main agent of the disease. Number of cases is increasing in our country in time because of malnutrition, migration, travel, low socioeconomic level and ecological changes. For the treatment, the pentavalent antimonials are often used as intralesionally for many years, and it was reported that resistant cases have increased in recent years. New treatment methods and anti-Leishmanial activity of new agents have been investigated because of side effects, resistance development and toxic reactions of the present drugs. These studies are first carried out in vitro and afterwards with in vivo experimental animal models. Reporter gene technology has been used to investigate a variety of purposes like biological events in microorganisms and the efficacy and resistance of drugs in recent years. The major areas that green fluorescent protein (gfp) used are that they can be incorporated into different genes to determine the amount of expression of these genes in different organisms and can be used as markers in living cells. Especially gfp gene, which encodes the green fluorescent protein, is widely used nowadays. Gene-based assays have several advantages like being easy to follow-up, inexpensive and have improved biosecurity. The aim of the present study was to perform the transfection of L.tropica with "enhanced gfp (egfp)" and in vitro usefulness of gfp-transfectants as a drug screening model in comparison to the conventional methods. Promastigotes of L.tropica were transfected with p6.5/egfp by electroporation and selected for tunicamycin-resistance as previously described. L.tropica promastigotes transfected with gfp and in vitro effect of meglumine animoniate was assessed using different methods such as fluorescence microscopy, fluorometer and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide) assay. The use of gfp-transfected Leishmania strains was found more rapid and more sensitive by fluorescent microscopy and fluorometry than conventional assays for the evaluation of potential anti-leishmanial agents. Consequently, stable gfp-transfected Leishmania species will be used in vitro and in vivo for screening of anti-leishmanial drugs and vaccine development as well as for understanding the biology of the host-parasite interactions at the cellular level. As a result ot this study, gfp transfected model using a Turkish L.tropica isolate was established to be used in further studies.
Assuntos
Proteínas de Fluorescência Verde , Leishmania tropica , Transfecção , Animais , Antiparasitários/farmacologia , Proteínas de Fluorescência Verde/genética , Leishmania tropica/efeitos dos fármacos , TurquiaRESUMO
Leishmaniasis is a zoonotic/anthroponotic vector borne parasitic infection which is caused by Leishmania species and transmitted by sand flies (Phlebotomus spp.) The reservoirs of Leishmania species in nature are various wild and domestic carnivores, rodents and human. The aim of this study was to investigate whether the rodents in genera Meriones, Mesocricetus, Rattus and Mus which inhabit in the natural habitat of our country could be natural reservoirs of Leishmania tropica, Leishmania infantum, Leishmania major and Leishmania donovani for cutaneous Leishmaniasis (CL)., The rodents Mus musculus (Balb/C mouse), Mesocricetus auratus (hamster), Meriones unguiculatus (gerbil) and Rattus norvegicus (rat) which are part of the natural habitat in Turkey were used in the study. L.tropica, L.infantum, L.major and L.donovani promastigote isolates obtained from CL patients and cultured in enriched media were injected in the footpads of the animals intradermally using the density of 108 promastigote/ml. The scale of the lesions on the footpads of the animals were measured for 12 weeks. At the end of the experiment, the animals were sacrificed and "touch preparations" were prepared using footpad, liver, spleen and testicles of the sacrified animals and were examined using Giemsa stained slides following culturing in enriched NNN medium. Leishmania amastigotes were seen in the slides prepared from the footpads of the all experimental animals and all cultures were positive for promastigotes prepared from the same clinical material. But not all the experiment groups were positive for the liver, spleen and testicle preparations. According to these results it was concluded that while all rodents in the experiment groups were positive for CL, only a part of the experiment groups were positive for internal organ involvement. Accordingly, (a) All Leishmania strains caused both CL and internal organ involvement in M.unguiculatus and M.musculus, (b) only L.tropica caused CL and internal organ involvement in R.norvegicus, while other Leishmania strains only caused CL in this group, (c) in M.auratus only L.donovani caused CL while other strains caused both CL and internal organ involvement. In our study, it was determined that the rodents Meriones, Mesocricetus, Rattus and Mus genera which are part of our country's natural habitat could serve as natural reservoirs of L.tropica, L.infantum, L.major and L.donovani, thus having the potential for the spreading of Leishmaniasis in our country and important information were gathered concerning the clinical aspects of the infection caused by Leishmania species in their potential reservoir hosts.
Assuntos
Reservatórios de Doenças , Leishmaniose Cutânea , Roedores , Animais , Cricetinae , Humanos , Leishmaniose Cutânea/transmissão , Camundongos , Ratos , Roedores/parasitologia , TurquiaRESUMO
Leishmaniasis, seen in tropical and subtropical regions, is an infectious disease caused by the protozoan parasite Leishmania species. There are three main forms of leishmaniasis: cutaneous, mucocutaneous and visceral leishmaniasis. Cutaneous leishmaniasis (CL) has become an increasing problem as the number of travels around the world increases and people go to work in endemic areas. Turkey has received great numbers of immigrants in recent years, from its neighboring countries like Iraq, Islamic Republic of Iran, Afghanistan, Turkmenistan and the Syrian Arab Republic because of the political instabilities in these countries as well as the job opportunities caused by large-scale development projects undertaken by Turkey. In this report, imported CL cases detected in five truck drivers transporting from Hatay to Turkmenistan, Syria, Saudi Arabia, Iran and Georgia, Uzbekistan and Azarbaijan countries were presented. The patients admitted to Mustafa Kemal University, Faculty of Medicine Dermatology Policlinic, with wound complaints on their bodies were directed to the Department of Parasitology to obtain smear samples from their wounds. The age range of the patients were 38 to 43 years. Patients with wound trail for a period ranging from one month to one year had a number of lesions varying between 2-7 and in all cases, a smear preparation was prepared from the lesions for diagnostic purposes. Clinical material obtained from five patients with pre-diagnosis of CL was firstly examined with Giemsa stain. Samples taken from the patients were inoculated into modified NNN (Novy-MacNeal-Nicolle) medium for the evaluation of the presence of the promastigotes. Promastigotes obtained from the inoculated medium were also genotyped using the ITS1 region. In all of the slides prepared from the clinical material taken from the patients amastigotes were determined. The growth of promastigotes were observed in only three of the clinical specimen inoculated media. The genotyped three species were Leishmania tropica, Leishmania infantum/donovani and Leishmania major. In this study, the importance of support for the diagnosis of different microbiological methods used in the diagnosis of leishmaniasis infection which occurred during the outbreaks of the disease has been put forward. In addition, it was aimed to draw attention to the importance of imported CL cases in our country diagnosed in five truck drivers making transportation from Hatay to Turkmenistan, Syria, Saudi Arabia, Iran, Georgia, Uzbekistan and Azerbaijan.
Assuntos
Leishmania , Leishmaniose Cutânea , Adulto , Genótipo , Humanos , Leishmania/genética , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Veículos Automotores , Síria , TurquiaRESUMO
Leishmaniasis is a vector-borne zoonotic disease that shows different clinical features like cutaneous, mucocutaneous, visceral and viscerotropic forms. The protocols used in the treatment of leishmaniasis are toxic and have many limitations during administration. One of the limitations of treatment is the resistance against the protocols in practice. There is also a need to define new treatment options especially for resistant patients. Ex-vivo models using primary cell cultures may be a good source for evaluating new drug options in patients with antimony resistance, in addition to in-vitro and in-vivo studies. In this study, it was aimed to define a new ex-vivo culture model to evaluate treatment options in patients with cutaneous leishmaniasis who did not respond to treatment. In our experimental model of ex-vivo infection, Leishmania tropica promastigotes isolated from a case previously diagnosed with cutaneous leishmaniasis were used. The primary astroglial cell culture used for the ex-vivo model was prepared from 2-3 days old neonatal Sprague Dawley rat brains under sterile conditions by the modification McCarthy's method. The astroglia cells, which reached sufficient density, were infected with antimony resistant L.tropica promastigotes. After 24 hours of incubation, the supernatant on the cells were collected, the cell culture plate was dried at room temperature, then fixed with methyl alcohol and stained with Giemsa to search for L.tropica amastigotes. Amastigotes were intensely observed in glia cells in primary cell cultures infected with L.tropica promastigotes. No promastigotes were seen on Giemsa stained preparations of the precipitates prepared from the bottom sediment after the centrifugation of the liquid medium removed from the infected plates. In this study, promastigotes from a cutaneous leishmaniasis patient unable to respond to pentavalent antimony therapy were shown to infect rat glia cells and converted to amastigote form. This amastigote glial cell model, as far as we know, is the first model in the literature produced by L.tropica. The occurrence of L.tropica amastigote forms in glia cells may be indicative of the ability of Leishmania species to infect the central nervous system. The central nervous system may be an area for the Leishmania amastigotes to escape from the immune system in cases of leishmaniasis without a treatment response. Our study is important because it is the first study to show the infection of glia cells with L.tropica amastigotes.