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

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

Retrospective Analysis of Cystic Echinococcosis Results in Aydin Adnan Menderes University Training and Research Hospital Parasitology Laboratory Between 2005 and 2017

Objective: Echinococcus granulosus is the causative helminth of cystic echinococcosis (CE). The parasite is known to form fluid-filled cysts that grow slowly in the internal organs, particularly the liver and/or lungs. This disease is still important in terms of public health and economically in Turkey and other countries where animal husbandry is widespread. The aim of our study was to retrospectively evaluate the cases that were admitted to the Adnan Menderes University, Training and Research Hospital Parasitology laboratory on suspicion of CE between January 2005 and January 2017. Methods: Totally, 3446 sera (from 2019 female and 1427 male) were tested with an in-house ELISA for the presence of E. granulosus specific IgG antibodies at the timeswhen they were sent. Socio-demographic characteristics (age, gender, residence, and dog ownership), positivity titers, and cyst locations of pathologically confirmed CE patients were analyzed retrospectively. Results: The ages of patients varied between 4-87 years. It was found that 1104 (32%) of the 3446 sera were positive, and of them, 642 (58.1%) were female and 462 (41.9%) were male. Patients who had pathologically confirmed CE diagnosis constituted 247 (22.3%) of the total seropositive sera. Liver was the most commonly affected organ (81.8%), followed by lungs (6.1%). Conclusion: CE remains an important public health problem in our city; therefore, it is once again emphasized that preventive studies should be planned.

[Transfection of Leishmania tropica with green fluorescent protein (gfp) gene and investigation of the in vitro drug effect]. / Leishmania tropica'nin yesil floresan protein (gfp) geni ile transfeksiyonu ve in vitro ilaç etkisinin arastirilmasi.

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.

Investigation of the Applicability of a Rapid Diagnosis Test in the Diagnosis of Cystic Echinococcosis

OBJECTIVE: Echinococcus granulosus, the etiological agent of cystic echinococcosis (CE) in humans and livestock, is a widely distributed zoonotic pathogen tapeworm. The infection is transmitted to humans by the ingestion of E. granulosus eggs released in the feces of definitive hosts such as dogs. The larval stage of the parasite develops a slowly enlarging cyst in the visceral organs, particularly in the liver and/or lung. The aim of the present study was to evaluate the diagnostic value of an immunochromatographic test (ICT) for CE. METHODS: A total of 50 sera from surgically and/or pathologically confirmed patients with CE were included in the study as the study group; the control group comprised patients who tested negative for enzyme-linked immunosorbent assay (ELISA). Sera were selected from the collection at Adnan Menderes University, Faculty of Medicine, Parasitology Laboratory, by simple random sampling. The collection included sera obtained between 2010 and 2014; antibody titers of each serum sample were determined using in-house ELISA, before storage at -20°C. The presence of E. granulosus antibody in the sera was determined using a commercially available ICT (VIRAPID® HYDATIDOSIS) kit method. RESULTS: In the study group (E. granulosus-confirmed cases), two (4%) of the 50 sera were negative and 48 (96%) were positive with ICT. In the control group (ELISA-negative), all were negative with ICT. CONCLUSION: The rapid diagnostic test has been evaluated as a practical, easy-to-use method for detecting CE, and it can be used as a screening test in routine diagnosis and research.

Use of Internal Transcribed Spacer Sequence Polymorphisms as a Method for Trichomonas vaginalis Genotyping.

OBJECTIVE: Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen with a worldwide distribution. The aim of the present study was to design a new genotyping tool for T. vaginalis isolates using internal transcribed spacer (ITS) sequences. METHODS: First, a total of 20 cryopreserved T. vaginalis isolates were thawed and genomic DNA was isolated from fresh cultures. A polymerase chain reaction (PCR) was performed to amplify the ITS regions and the amplicons were sequenced. These sequences were aligned with others from Genbank and polymorphisms were detected. At last, each ITS sequence was given a different sequence type. RESULTS: More than 99% homology was observed among sequences. Of 20 isolates, five had identical ITS sequence to reference (L29561) defined as ITST1. Moreover, 13 had A58 deletion (ITST10), one had C203T mutation (ITST2), and one had both A58 deletion and C203T mutation (ITST11). ITS typing of T. vaginalis sequences on Genbank revealed a total of 11 ITS types with the predominance of ITST1 (44.4%) globally. CONCLUSIONS: ITS typing seems to be an applicable and useful tool for a better understanding of molecular epidemiology as well as for the dissemination of T. vaginalis clones.

[Determination of Leishmania species by PCR-RFLP in the smear samples taken from the lesions of cutaneous leishmaniasis cases]. / Kütanöz leysmanyazli olgularda lezyondan alinan yayma örneginden PCR-RFLP ile Leishmania türünün arastirilmasi.

The forms of the disease caused by Leishmania species in Turkey as well as in Aegean region are cutaneous and visceral leishmaniasis (CL and VL, respectively), and the agent of CL is commonly L.tropica. However, L.infantum was also reported as being CL agent recently. Direct microscopic examination, serological tests and culture are the conventional methods used for the diagnosis of CL. Since the specificities of these methods are high their sensitivities are variable and identification at species level is not possible. Recently, the use of polymerase chain reaction (PCR)-based molecular methods enabled the rapid and reliable diagnosis and species identification. The aim of this study was to investigate the performance of PCR-restriction fragment length polymorphism (RFLP) method both for the detection and identification of Leishmania species simultaneously in CL patients. A total of 30 smear samples that were positive for Leishmania amastigotes with microscopic examination, obtained from CL-suspected cases admitted to Adnan Menderes University Medical School Hospital, Parasitology Laboratory (located at Aydin, in the Aegean region of Turkey) between 2012-2014 period were included in the study. Ten samples taken from the skin lesions caused by Staphylococcus aureus (n= 5) and Candida albicans (n= 5) were also included as negative controls. DNA extractions from the smears were performed by the use of a commercial kit (Macherey-Nagel NucleoSpin Tissue® Kit, Germany). DNA isolation was also performed from L.major, L.infantum and L.tropica promastigotes that were grown in culture as positive controls. In PCR method LITSR and L5.8S primers targeting to ITS (internal transcribed spacer)-1 region were used. In RFLP method, the amplified PCR products were cleaved by BsuRI (HaeIII) restriction enzyme for the species identification. As a result, restriction profiles of all samples (n= 30) were in accordance with L.tropica restriction profile. No band was observed in the control samples (n= 10). The data of this study showed that the most common CL agent in Aydin is L.tropica. In conclusion, ITS-1 PCR-RFLP method may be used directly as a single routine procedure for both the detection and identification of Leishmania species in the clinical samples of CL patients, in laboratories with adequate facilities.