Molecular Models of Toxoplasma gondii in Humans and Animals.

Toxoplasma gondii (T. gondii) is an obligate intracellular, zoonotic protozoan parasite of interest to physicians and veterinarians with its highly complex structure. It is known to infect about one-third of the world's population. Since it is a zoonotic disease, it is necessary to keep the animal population under control in order to prevent human exposure. Many studies have been conducted on the detection of T. gondii and it has been determined that there are three clonal groups consisting of types 1, 2, 3. Developments in molecular studies have led to changes in the taxonomy and new developments in parasitic diseases. It has helped in diagnosis, treatment, development of antiparasitic drugs and research on resistance. They also provided research on vaccine studies, genetic typing and phylogenetics of parasitic diseases. Conventional polymerase chain reaction (PCR), real-time PCR and genotyping studies conducted today increase our knowledge about T. gondii. Methods such as B1, SAG1, SAG2, GRA1, 529-bp repeat element, OWP genes and 18S rRNAs are mostly used in PCR, and methods such as MS, MLST, PCR-RFLP, RAPD-PCR and HRM are used in genotyping. Toxoplasmosis is a disease that is within the framework of the concept of one health and must attract attention, has not yet been eradicated in the world and needs joint studies for humans, animals and ecosystems to be eradicated. This can only be possible by establishing interdisciplinary groups, conducting surveys and training.

Molecular Detection of Canine Leishmaniasis in Northern Anatolia, Turkiye.

Background: Canin leishmaniasis (CanL), mostly caused by Leishmania infantum, is one of the most important vector-borne diseases in dogs in the Mediterranean region. In this study, we aimed to determine the disease profile in this region by firstly making microscopic and then molecular analyzes in the samples taken from the dogs. Methods: Overall, 112 whole blood samples taken from dogs for clinical applications by a veterinarian in Cankiri between December 2021 and November 2022 were used. After blood collection, both thin and thick drop blood smear preparations were prepared and evaluated for Giemsa staining. L. infantum was investigated by Real time-PCR (RT-PCR) method from all blood samples. Sequence analysis and phylogenetic tree study were performed on positive samples. Results: Both microscopic and RT-PCR analyzes were performed. In both studies, 3 of the 112 samples were positive. Because of the sequence analysis, they were L. infantum. Sequence analysis was performed from the samples found 3 positive. The phylogenetic tree was drawn by making NCBI (National Center for Biotechnology) data entries of the positive samples (Accession numbers: OQ184728, OQ184729, OQ184730). Conclusion: Dogs are important, as they are reservoir of this disease. In this study, 3 (2.7%) positive Leishmaniasis was detected in dogs in Cankiri. Ultimately, this should prompt discussion about new strategies going forward to combat infection caused by Leishmania.

Evaluation of Echinococcosis Pre-Diagnosis Patients Admitted to the National Parasitology Reference Laboratory of Turkey from 2014-2019.

Background: Echinococcosis is a common parasite with zoonotic character created by a small cestode, Echinococcus spp., and is an important public health problem in Turkey as well as all over the world. We aimed to investigate antibodies in serum samples of suspected Echinococcosis patients sent to the National Parasitology Reference Laboratories of the General Directorate of Public Health. Methods: Serum samples of 2390 patients sent to our laboratory between January 1, 2014 and May 01, 2019, evaluated by ELISA, Indirect Hemagglutination Test (IHA) and Western Blot (WB) methods are presented. Our laboratory is the national reference laboratory. All kinds of tests requested from suspected patients can be performed. Results: Overall, 1199 (50.2%) of 2390 serum samples were female and 1191 (49.8%) were male. It was observed that 178 (14.9%) of men and 210 (17.5%) of women were seropositive. There was no statistical difference between the sexes in terms of seropositivity. Of all samples, 1941 (81.2%) were negative, 388 (16.2%) were positive, and 61 (2.6%) were borderline. Results determined as borderline are considered suspicious and a recommendation is made to repeat the test after 15 days. A statistical difference was found in the distribution of seropositivity by years. While seropositivity was lowest in 2014, it was found to be highest in 2018 and 2019. Conclusion: Despite all the precautions taken, it is seen that echinococcosis still continues to exist in Turkey as a zoonotic disease. Hence, CE has been involved in Turkey Zoonotic Diseases Action Plan (2019-2023) and decided to carry out studies for the protection and prevention of the disease.

Seroprevalence of Encephalitozoon cuniculi, Francisella tularensis and Toxoplasma gondii in Zoonotic Diseases in European Hares (Lepus europaesus)

Objective: European hares (Lepus europaeus) are among the most important animals that are connected with humans in many countries and natural life. Hares are important for public health, since they carry many zoonotic diseases, such as Encephalitozoon cuniculi, Francisella tularensis and Toxoplasma gondii. The study aimed to determine the seroprevalence of Encephalitozoon cuniculi, Francisella tularensis and T. gondii and the potential zoonotic risk posed by hares that live in provinces of Turkey. Methods: Blood samples were collected from hares during the official hunting season. Serum samples were examined serologically by enzyme-linked immunosorbent assay for E. cuniculi, Sabin-Feldman dye test was used to examine T. gondii, while micro-agglutination test was used to examine F. tularensis. Results: Of the total of 42 hares examined, one (2.4%) was found positive for E. cuniculi, two (4.8%) were found positive for T. gondii and one (2.4%) was found positive for F. tularensis. Conclusion: Anti-T. gondii and anti-E. cuniculi antibodies were serologically detected in hares for the first time in Turkey. Furthermore, this is the first study reporting the seropositivity of F. tularensis infection in hares.

Evaluation of Seropositivity of Toxoplasmosis Suspected Patients Admitted to the National Parasitology Reference Laboratory Between 2009 and 2019

Objective: Toxoplasmosis caused by Toxoplasma gondii (T. gondii), which is an obligatory intracellular parasite, is a worldwide zoonotic parasitic disease. In this study, results of T. gondii test conducted between January 2009 and May 2019 were analysed. This study aimed to evaluate the results of T. gondii test of patients who were admitted to the General Directorate of Public Health, National Parasitology Reference Laboratory between 2009 and 2019. Methods: The results of anti-T. gondii IgG, IgM, IgG avidity and Sabin-Feldman dye tests (SFDT), which are used to detect the presence of T. gondii, were examined. ELISA was used for anti-T. gondii IgG, IgM and IgG avidity tests. SFDT, which is the reference test in the diagnosis of T. gondii, is still the gold standard. In addition to laboratory analyses, information on gender, age, city of origin, year distribution of all cases and type of sample sent was also collected. Results: Of the 2.778 patients evaluated, 25.4% were males and 74.6% were females. Moreover, 47.1% and 10.2% of the patients were positive for anti-T. gondii IgG and anti-T. gondii IgM antibodies, respectively. In SFDT, 1.228 (52%) patients were found to be positive, including 319 (59.4%) of 537 men and 909 (49.8%) of 1.824 women. In this 10-year study, the most common seropositivity titre of SFDT was at the level of 1/64. In our study, IgG levels were found to be positive in all cases in which IgG avidity was studied when all the cases in which all three of the anti-T. gondii IgG, IgM and IgG avidity tests were studied together in one patient were evaluated. In addition, of the 293 patients with positive anti-T. gondii IgG, 62.8% had high avidity, 24.2% had a limit value and 13% had a low avidity. In cases involving both mother and baby, anti-T. gondii IgG and IgM seropositivity rates were 80% and 5% for both, respectively. These high rates support the transfer of antibodies from the mother to the baby. Regarding the distribution of provinces from which the samples originated, the highest number of cases came from Ankara (80.7%). Blood is the most predominant sample, followed by cerebrospinal fluid. Conclusion: T. gondii maintains its importance in public health, owing to its high positivity rates. This study, in which 10-year data were collected, showed that despite an increase in awareness, high seropositivity still continues. Therefore, systematic collection and evaluation of laboratory analysis results for toxoplasmosis diagnosis will contribute in taking control measures.

Gingival Myiasis on Oral Squamous Cell Carcinoma in Turkey: A Case Report

Myiasis is a disease caused by tissue invasion of diptera larvae and eggs. Oral myiasis is mostly related to old age, poor oral hygiene, suppurative lesions, anatomical disorders and cancer cases. Oral squamous cell carcinoma (OSCC) is an important risk factor for myiasis. This report presents the case of an 82-year-old woman who presented with gingival myiasis developing on the background of OSSC. The patient was diagnosed with OSSC in the hospital. Myiasis larvae were identified and sent to the National Parasitology Reference Laboratory for identification. Thus, development of myiasis on OSCC background was shown in Turkey for the first time. Myiasis larvae have been identified as the 3rd phase of the larvae Sarcophaga sp. development (Diptera: Sarcophagidae). As a result, myiasis cases are sporadic in Turkey, and it can be avoided by controlling fly population and by paying attention to hygiene. Controlling myiasis is an important public health problem and should be considered in a single health concept, as it causes health problems in both humans and animals. The findings of this case will draw attention to the importance of dealing with myiasis factors, which is a public health problem.

Whole-genome sequencing of a Toxoplasma gondii strain from a Turkish isolate using next-generation sequencing technology.

PURPOSE: Toxoplasma gondii is an intracellular parasite that can affect all vertebrae and is the causative agent of toxoplasmosis. At present, the United States CDC (Centers for Disease Control and Prevention) recognizes this infection as a neglected disease. Toxoplasma gondii infection profiles exhibit differences because of the different regional and climatic responses to these parasites in Turkey, and these protozoan infections are notably common in this country. In this study, we attempted to obtain the whole-genome sequence of T. gondii using next-generation sequencing technology. METHODS: Toxoplasma gondii isolates were isolated from an infant with congenital toxoplasmosis by Ekmen et al. (1974) in Ankara, Turkey. Whole-genome sequencing (WGS) was performed using the Illumina HiSeq 2500 and HiSeq SBS Kit v2. A T. gondii library was created on this device in the initial stage. After the completion of the library phase, sequence analysis was begun with a next-generation sequencing device. The resulting fragments were combined using paired-end (PE) reading and converted into a single DNA fragment. Bioinformatic analysis was performed using the Geneious 2.1. RESULTS: In our study, WGS was successfully performed on T. gondii. The T. gondii whole-genome sequence has a coverage value of 50x, a size of 61,5763 Mb and a GC ratio of 52.6%. Data from this sequence were submitted to the National Center for Biotechnology Information (NCBI) GenBank (www.ncbi.nlm.nih.gov) database under the name Toxoplasma gondii TR01 (TG_TR01). The accession number of the genome obtained in this study is WOEV00000000.1. The biological sample access number is SAMN13338796. The genome of the T. gondii strain obtained in this study was compared with the reference genome, and 8312 CDSs (coding sequences), 183 tRNAs, 294 rRNAs and 8789 genes were identified. Among the 8312 CDSs, 4284 encoded hypothetical proteins (hypothetical protein CDSs/proteins of unknown function). The entire genome sequence of T gondii TR01 was compared with that of Toxoplasma gondii ME49. The results of this comparison demonstrate that the analyzed genome was 99,98% similar to the reference genome. The accession numbers of 14 chromosomes belonging to the genome sequences of T. gondii TR01 (TG_TR01) are CM019722.1, CM019723.1, CM019724.1, CM019725.1, CM019726.1, CM019727.1, CM019728.1, CM019729.1, CM019730.1, CM019731.1, CM019732.1, CM019733.1, CM019734.1, and CM019735.1. CONCLUSION: In this study, a whole-genome sequences of T. gondii was conducted for the first time in Turkey. The analyzed strain was named T. gondii TR01. The data obtained from this study may contribute to a better understanding of T. gondii. T. gondii is an important pathogen with an unusual population structure. Although T. gondii is highly zoonotic and has a complicated life cycle, some strains of this parasite have exhibited high genetic sequence similarity, and our study supports this knowlegde. The characterization of this strain may be very useful for the scientific community of our country and may help to establish a foundation for further research investigating the genome of T. gondii.

Investigation of Anti-Toxoplasma gondii Antibodies in Cats Using Sabin-Feldman Dye Test in Ankara in 2016

Objective: Toxoplasmosis, in which obligate intracellular protozoa Toxoplasma gondii (T.gondii) is the causative organism, is a multisystemic disease that can be seen all over the world and can impair all vertebrates. The only hosts known for T.gondii are members of Felidae family. Our study aimed to determine anti-Toxoplasma gondii antibodies with Sabin-Feldman Dye Test (SFDT) in cats in Ankara. It's aimed to evaluate the current situation in terms of Toxoplasmosis spread by comparing our findings with previous studies in the same region. Methods: Rh strain of Toxoplasma used in our study is maintained in our laboratory. SFDT is still accepted as the gold standard. Material of the study was obtained by taking blood samples from cats who were admitted to the clinics between March 2016 and October 2016 in Ankara. Blood samples were inactivated and measurements were done with SFDT 1/4, 1/16, 1/64, 1/256, 1/1024 titers. Results: SFDT resulted positive in 56 (43.4%) cats at a dilution of 1/16, in 7 (5.4%) cats at a dilution of 1/64, in 23 (17.8%) cats at a dilution of 1/256 and negative in 43 (33.3%) cats. Comparison of demographic data with SFDT results showed that positive test results did not differ according to gender and age (P=0.803 and P=0.991, respectively). Seropositivity was higher in stray cats than house cats (P<0.001). Test results were negative in the cats that fed only by commercial dry food (P<0.001). Positivity in hunter cats was more than in non-hunters (P<0.001). Conclusion: Seropositivity was detected in 66.6% of the cats, which was quite a high rate. As a result, taking precautions in terms of Toxoplasma for stray cats that are hunting and feeding naturally is a necessity for public health.

Laboratory Safety in Parasitology Laboratory.

Working in a laboratory is very difficult and needs special attention. Laboratory workers can be exposed to numerous potential hazards including chemical, biological, physical, and radioactive. That is why it is really important to follow the working principles in laboratories for the sake of the lab analyzers and others who work with them in the lab. Laboratory safety includes the use of certain laboratory rules, methods, infrastructures, and devices during work to protect the working person and the working material. All studies show that > 70% of medical decisions are based on laboratory results. In such important laboratories, it is must to get safe and reliable results. This requires a well-established working system and strict observance of laboratory safety. Biosafety is very important in parasitology laboratories as well as in all microbiology laboratories. Usually, it takes a long time for people to detect parasitic diseases through laboratory accidents, who are working in laboratories. That is why, especially in parasitology laboratories, the issue of laboratory safety should be emphasized more sensitively. We will be reviewing the hazards, parasites, exposure routes, and protective measures imposed in parasitology laboratories.