Antiprotozoal activity of auranofin on Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii: in vitro and ex vivo study.

BACKGROUND: Three obligate intracellular protozoan parasite species, which are responsible for significant morbidity and mortality and settle in macrophage cells, affect more than one-half of the world's population, namely, Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii, which are causative agents of Chagas disease, leishmaniasis and toxoplasmosis, respectively. In the current study, it was aimed to investigate the in vitro and ex vivo antiprotozoal activity of auranofin on T. cruzi, L. tropica and T. gondii. METHODS: The in vitro drug efficacy (IC50) of auranofin was investigated by haemocytometry and the CellTiter-Glo assay methods and the ex vivo drug efficacy (IC50) by light microscopic examination of Giemsa-stained slides. Also, the cytotoxic activity (CC50) of auranofin was examined by the CellTiter-Glo assay. The selectivity index (SI) was calculated for auranofin. RESULTS: According to IC50, CC50 and SI data, auranofin did not exhibit cytotoxic activity on Vero cells, but exhibited antiprotozoal activity on epimastigotes and intracellular amastigotes of T. cruzi, promastigotes and intracellular amastigotes of L. tropica and intracellular tachyzoites of T. gondii (p<0.05). CONCLUSIONS: The detection antiprotozoal activity of auranofin on T. cruzi, L. tropica and T. gondii according to the IC50, CC50 and SI values is considered an important and promising development. This is significant because auranofin may be an effective alternative treatment for Chagas disease, leishmaniasis and toxoplasmosis in the future.

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

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

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.

Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

Is Toxoplasma gondii a potential risk for traffic accidents in Turkey?

In Turkey, traffic accidents are the major causes of mortality and morbidity. According to the statistics made by Turkish Security Headquarters in the year 2000, 7500 people are killed in the traffic accidents every year. Drivers are mostly blamed for the traffic accidents. Tired, weary, sleepy and absentminded driving are common reasons for traffic accidents due to many reasons. Among these reasons mental and reflex conditions of drivers are significant. Toxoplasmosis is one of the most common zoonoses throughout the world. In immunocomponent adults, toxoplasmosis has no clinical signs or symptoms and infection in such people can only be detected by serological examinations. This kind of latent or dormant toxoplasmosis can be located in neural or muscular tissues and cause prolonged reaction times of the muscles. Extended reaction times also lead to deceleration of the reflexes which could be a major cause of the traffic accidents. By the light of this situation, the incidence of Toxoplasma gondii is investigated among the population who were involved in a traffic accident while driving. A total of 185 people (100 men and 85 women), aged between 21 and 40 years, living in Izmir and Manisa cities, were enrolled in the study group (SG) during a period of 6 months time. Their informed consents were taken initially and the laboratory tests of all these individuals related to blood alcohol levels after the accidents were found negative. The control group (CG) were also consisted 185 people (95 men and 90 women), residents of the same region, in same age group. The people in CG had no clinical signs or symptoms concerning toxoplasmosis. All collected sera were examined using a commercial IgG and IgM ELISA kit. According to the results of serological tests of the SG, 45 (24.32%) sera were IgG positive, six (3.24%) were IgM positive and 10 (5.40%) were both IgG and IgM positive. On the other hand in CG, 12 (6.48%) people found IgG positive, one (0.54%) found IgM positive and three (1.62%) found both IgG and IgM positive. This data was considered as statistically significant by Yates corrected chi2-test. (p < 0.05). In conclusion, there is an increased risk for traffic accidents for drivers owing to these high seroprevalence of latent toxoplasmosis. Prospective studies are still necessary on this subject but latent toxoplasmosis of drivers should be taken into account while developing strategies to prevent traffic accidents in Turkey.

Prevalence of intestinal parasitosis in children with gastrointestinal symptoms associated with socio-economic conditions in Manisa region.

Intestinal parasitosis is still an important public health problem. The aim of this study was to determine the prevalence of intestinal parasitosis in children with gastrointestinal symptoms, and to evaluate its association with socio-economic and environmental factors. Stool samples of 3,216 children were examined by the saline-iodine method and trichrome staining. The cellophane tape method was also performed on 2,160 children. According to the educational levels and the economic status of families, the patients were classified as coming from underdeveloped, developing and developed areas. In 770 (23.9%) of 3,216 stool samples, various parasites were detected by the saline-iodine method and trichrome staining. The most common parasite was Giardia intestinalis (40.1%), followed by Entamoeba coli (10.2%). Enterobius vermicularis eggs were detected by the cellophane tape method in 221 (10.3%) out of 2,160 patients. The positive cases were evaluated according to the socio-economic and the environmental criteria; and most of them were found to have come from underdeveloped and developing areas. Health care and governmental officers should cooperate in order to improve the living conditions, and also people should be informed about the signs, symptoms and prevention methods of the parasitic diseases.

Incidence of giardiasis among siblings in Turkey.

BACKGROUND: Giardiasis, a common infection among children, is caused by a flagellated protozoan called Giardia lamblia. It is well known to be contagious in common living places. This is an epidemiologic study investigating the incidence of giardiasis among the siblings of patients with giardiasis living in the same household. METHODS: Stool samples of 50 patients with a G. lamblia positive sibling in the same household, and 50 patients with a G. lamblia negative sibling in the same household, were examined for giardiasis by saline-Lugol, formalin-ethyl acetate concentration and trichrome staining methods. Other causes of diarrhea were excluded by microbiologic laboratory tests. RESULTS: Thirty-eight per cent of siblings of G. lamblia positive patients and 8% of siblings of G. lamblia negative children were found to be positive for G. lamblia cysts and/or trophozoites. The differences between the groups were statistically significant (P < 0.001). CONCLUSION: The results suggested that the examination of G. lamblia among the siblings of patients with giardiasis both in the same age group and living in the same household should be considered.

Serological levels of zinc, copper and iron elements among Giardia lamblia infected children in Turkey.

BACKGROUND: Giardiasis, an intestinal protozoan infection caused by Giardia lamblia, is common in Turkey, especially among children aged between 2- and 14-years-old. Effects of giardiasis on serological levels of zinc, copper and iron elements were assessed in this study. METHODS: A total of 45 children, aged between 2- and 14-years-old, who were admitted to the Pediatrics Department of Celal Bayar University Medical School with gastrointestinal complaints and diagnosed as having giardiasis by stool examinations in the Parasitology Department, were enrolled as the study group (SG). The control group (CG) consisted of 45 age-matched healthy children. Serological levels of zinc, copper and iron were measured by atomic absorption spectrophotometer in all samples. RESULTS: As a result of the study, serum zinc levels were 67.43 +/- 17.72 microg/dL and 145.20 +/- 9.13 microg/dL, copper levels were 198.45 +/- 39.14 microg/dL and 150 +/- 21.14 microg/dL and iron levels were 87.98 +/- 18.31 microg/dL and 160.45 +/- 45.40 microg/dL, in SG and CG, respectively. When compared separately as SG and CG, there was a statistically significant difference between the serological levels of all these elements. CONCLUSION: These results revealed that giardiasis increased the serological levels of copper, like other infectious agents. However, zinc and iron levels decreased during giardiasis due to malabsorption.

Giardiasis treatment in Turkish children with a single dose of ornidazole.

This study was designed to compare the treatment efficacy of single dose of ornidazole with 5 d treatments of ornidazole and metronidazole in children with giardiasis. 175 children, between 2 and 15 y old, whose stool samples were found to be positive for Giardia lamblia cysts and/or trophozoites by either saline-Lugol, formalin-ethyl acetate or trichrome staining, were enrolled in the study. Of these children, 105 were treated with a single dose of ornidazole: 35 with 30 mg/kg, 35 with 25 mg/kg and 35 with 20 mg/kg; 35 were treated with 25 mg/kg per day of ornidazole for 5 d in 2 doses and 35 children were treated with 20 mg/kg per day metronidazole for 7 d in 3 doses. All cases were examined on the 7th, 10th and 14th days after treatment by the same methods; clinical symptoms were also evaluated. Giardia lamblia was eradicated in 34 of 35 (97%), 34 of 35 (97%) and 33 of 35 (94%) patients treated with 30, 25 and 20 mg/kg single doses of ornidazole, respectively. Eradication was achieved in all 35 patients treated with 25 mg/kg per day ornidazole for 5 d and in 31 of 35 (89%) patients treated with metronidazole. There was no statistically significant difference among doses of ornidazole (p > 0.05); however, all ornidazole treatment regimens were significantly more effective than metronidazole treatment (p < 0.05). No important side-effects were detected in any patients and clinical symptoms disappeared in all. Single-dose ornidazole treatment could be considered as a proper and effective alternative method for the treatment of giardiasis in children.