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Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba , B . mandrillaris , and N . fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N . fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x10 5 -1.4x10 2 plasmid copies/l and for N . fowleri in the range of 8x10 3 -11x10 2 plasmid copies/l. The highest concentration of Acanthamoeba and N . fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N . fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N . fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N . fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N . fowleri genotypes in various water sources in Turkey.

Dirofilariasis is a vector-borne disease that is present worldwide. This report describes a giant subconjunctival granuloma which mimics scleritis, caused by D. immitis. A 60-year-old man was referred with the complaints of irritation, redness, and swelling at the medial part of the right eye. He was living in Izmir province located in western Turkey. Slit-lamp examination showed a firm, immobile mass measuring 13.0 × 5.0 × 5.0 mm with yellowish creamy color. The mass was completely removed surgically under local anesthesia mainly for diagnosis. Histopathology revealed typical morphological features of a filarioid nematode in favor of Dirofilaria as characterized by the external smooth cuticular surface, cuticular layer, muscle layer, and intestinal tubule. Molecular study was performed using DNA isolated from paraffin-embedded tissue sections of the worm. PCR amplification and then DNA sequence analysis of cytochrome c oxidase subunit 1 (cox1) gene fragment confirmed that the worm was D. immitis. It is suggested that this may represent the first human case of D. immitis occurring in subconjunctival granuloma in Turkey. Although rare, D. immitis caused by ocular dirofilariasis in humans should be considered.

The aims of this study were to identify Blastocystis subtypes (STs) in a cohort of Turkish patients with various gastrointestinal symptoms using a novel Real Time PCR method developed recently for Blastocystis detection and assess the relationship between Blastocystis STs and patient symptoms. Totally, 617 stool samples of patients with gastrointestinal symptoms were examined with microscopy and inoculated in Jones medium. Blastocystis-positive samples were further assessed to identify coinfections with other possible pathogens, including bacteria and viruses. Diagnostic efficacies of microscopy, culture and Real-Time PCR were compared. PCR products were sequenced to identify the subtypes of Blastocystis isolates. Totally 94 (15.24%) samples were positive for Blastocystis after all methods. Among these, 83 of 94 (88.3%) samples were identified with all methods, while 11 were positive only with Real Time PCR. Diarrhea and abdominal pain were the leading symptoms in the patients. The only pathogenic agent identified in 76 of 94 (80.9%) patients was Blastocystis. Subtype 3 was the leading Blastocystis subtype (44.6%), while subtypes 6 and 7 were firstly isolated from symptomatic patients in our region. Comparison of three diagnostic methods indicated Real Time PCR as the most sensitive and specific method. Blastocystis was the only pathogenic agent among symptomatic patients, with subtype 3 being predominant. Patients with subtypes 6 and 7 need further assessments concerning the zoonotic potential of Blastocystis.

Over the past decade, the number of international travels has increased. Hence, the risk of transmission of parasitic diseases has also increased. One of the risk infections is malaria; Plasmodium vivax and P. falciparum species can be transmitted. The distribution of leishmaniasis cases has been reported from the south of USA to the north of Argentina. Approximately 57,000 cases of cutaneous and mucocutaneous leishmaniasis occur annually, and approximately 4000 visceral leishmaniasis cases are observed. It is reported that Chagas disease is endemic in 21 countries, and approximately 6 million people are affected every year. In this continent, 25 million people are at a risk of schistosomiasis, and most (90%) are living in Brazil. According to the World Health Organization, individuals travelling to Ecuador, Colombia, Brazil, Guatemala, Mexico, and Venezuela are at a risk of onchocerciasis as well as infecting approximately 12.6 million individuals with lymphatic filariasis (80% in Haiti). Significant mortality and morbidity can be observed in cases where necessary precautions are not taken in individuals travelling to these regions and where appropriate prophylactic drugs are not administered.

Vermamoeba vermiformis ( V. vermiformis ) is one of the most common free-living amoeba (FLA) and is frequently found in environments such as natural freshwater areas, surface waters, soil, and biofilms. V. vermiformis has been reported as a pathogen with pathogenic potential for humans and animals . The aim is to report a case of non- Acanthamoeba keratitis in which V. vermiformis was the etiological agent, identified by culture and molecular techniques. Our case was a 48-year-old male patient with a history of trauma to his eye 10 days ago. The patient complained of eye redness and purulent discharge. A slit-lamp examination of the eye revealed a central corneal ulcer with peripheral infiltration extending into the deep stroma. The corneal scraping sample taken from the patient was cultured on a non-nutritious agar plate (NNA). Amoebae were evaluated according to morphological evaluation criteria. It was investigated by PCR method and confirmed by DNA sequence analysis. Although no bacterial or fungal growth was detected in the routine microbiological evaluation of the corneal scraping sample that was cultured, amoeba growth was detected positively in the NNA culture. Meanwhile, Acanthamoeba was detected negative by real-time PCR. However, V. vermiformis was detected positive with the specific PCR assay. It was confirmed by DNA sequence analysis to be considered an etiological pathogenic agent. Thus, topical administration of chlorhexidine gluconate %0.02 (8 × 1) was initiated. Clinical regression was observed 72 h after chlorhexidine initiation, and complete resolution of keratitis with residual scarring was noticed in 5 weeks. In conclusion, corneal infections due to free-living amoebae can occur, especially in poor hygiene. Although Acanthamoeba is the most common keratitis due to amoeba, V. vermiformis is also assumed to associate keratitis in humans. Clinicians should also be aware of other amoebic agents, such as V. vermiformis , in keratitis patients.

Purpose Blastocystis sp. is one of the most prevalent intestinal protozoa found in humans and many other animals. The present study aimed to examine the distribution and genetic diversity of Blastocystis sp. in stool samples from patients with gastrointestinal complaints in İzmir, Turkey. Methods All stool samples of 439 patients with gastrointestinal complaints were examined by native-Lugol and trichrome staining. To investigate the presence of Blastocystis sp. in stool samples, DNA was isolated, and PCR was performed with the barcode region in the SSU rRNA gene. PCR positive samples were sequenced to identify subtypes and alleles of Blastocystis sp. Results The prevalence of Blastocystis sp. was found to be 16.6% (73/439) in patients with gastrointestinal complaints in İzmir, Turkey. Three different Blastocystis sp. subtypes were identified. ST3 (28/55; 51.0%) was the most common subtype followed by ST2 (19/55; 34.5%) and ST1 (8/55; 14.5%). Itching and diarrhea were the most prominent clinical symptoms in Blastocystis sp. positive patients. When clinical symptoms and subtypes were compared, diarrhea was found in 62.5%, 47.4%, and 46.4% of patients with ST1, ST2, and ST3 subtypes, respectively. In addition, itching was found in 37.5%, 32.1%, and 21.1% of patients with ST1, ST3, and ST2, respectively. Six distinct alleles were identified by allele analysis of Blastocystis 18S rRNA gene: allele 4 for ST1, alleles 9, 11, and 12 for ST2, and alleles 34 and 36 for ST3. In this study, Blastocystis sp. was detected in 16 of 21 districts, including the central and rural districts of İzmir. Although ST1 was detected in central districts, it was not found in rural districts. Conclusion This study provides comprehensive data on the prevalence and molecular epidemiology of the genetic diversity at the level of subtypes and alleles of Blastocystis sp. in different districts of İzmir province in Turkey. To the best of our knowledge, this is the first study which evaluates the distribution of subtypes and alleles of Blastocystis sp. according to PCR and SSU rRNA gene sequencing in patients with gastrointestinal complaints in different districts of İzmir province in Turkey.

Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples.OBJECTIVEAcanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples.Acanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.METHODSAcanthamoeba strain grown in culture was diluted in 200 μL as 1x106 trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 °C in a total volume of 25 μL.To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.RESULTSTo determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%.It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.CONCLUSIONIt has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples.

Blastocystis hominis is one of the most common eukaryotic organisms in the intestinal tract of humans, while its pathogenic potential is still controversial. A total of 286 stool samples obtained from adult and pediatric patients with or without gastrointestinal symptoms in two hospitals in Manisa, Turkey, were cultured to detect B. hominis infection. Forty-one and 51 isolates were obtained from the adults and children, respectively, and these isolates were subjected to subtyping by polymerase chain reaction (PCR) with the known sequence-tagged site primers. The correlation between the genotype and the symptoms was evaluated. PCR subtyping indicated that subtype 3 was the most common genotype in both symptomatic and asymptomatic groups, and the second common genotype was subtypes 1 and 2 in symptomatic and asymptomatic groups, respectively. A significant correlation between subtype 2 and the asymptomatic groups was found among both in pediatric and adult patients (χ2cal = 4.38, df = 1, p = 0.044). However, there were no significant differences between the other genotypes and the symptomatic or asymptomatic groups, as well as both the age and sex of the patients. The present study suggests that subtype 2 is a non-pathogenic genotype of B. hominis.

Amaç: Serbest yasayan amiplerden biri olan Acanthamoeba, basta su, toprak ve hava olmak üzere birçok çevresel örnekte tespit edilmistir. Acanthamoeba'nin neden oldugu en önemli klinik tablolar içinde Acanthamoeba keratiti ve granülomatöz amibik ensefalit bulunmaktadir. Bu çalismada klinik ve çevresel örneklerde Acanthamoeba'nin varliginin daha hizli bir sekilde saptanabilmesi için Acanthamoeba 18S rRNA gen bölgesine özgü primerler ile gelistirilen hizli döngü aracili izotermal amplifikasyon (LAMP) testinin hassasiyetinin belirlenmesi amaçlanmistir.

Amaç: Serbest yasayan amiplerden biri olan Acanthamoeba, basta su, toprak ve hava olmak üzere birçok çevresel örnekte tespit edilmistir. Acanthamoeba'nin neden oldugu en önemli klinik tablolar içinde Acanthamoeba keratiti ve granülomatöz amibik ensefalit bulunmaktadir. Bu çalismada klinik ve çevresel örneklerde Acanthamoeba'nin varliginin daha hizli bir sekilde saptanabilmesi için Acanthamoeba 18S rRNA gen bölgesine özgü primerler ile gelistirilen hizli döngü aracili izotermal amplifikasyon (LAMP) testinin hassasiyetinin belirlenmesi amaçlanmistir. Yöntemler: Kültürde çogaltilan Acanthamoeba spp. susu 200 [micro]L'de 1x10 (6) trofozoit olacak sekilde sulandirilip DNA izolasyonu yapilarak DNA miktari Nano-Drop Spektrofotometre ile belirlenmistir. Saflastirilan DNA'lar 1000 pg'den 0,001 pg'ye kadar seyreltilip, kolorimetrik ve floresan tabanli iki farkli LAMP reaksiyonunda kullanilmistir. LAMP reaksiyon karisimi toplam 25 [micro]L hacimde 63 [degrees]C'de 60 dk olacak sekilde inkübe edilmistir. Bulgular: Gelistirilen testlerin hassasiyetinin saptanmasinda elde edilen Acanthamoeba spp. genomik DNA'sini hem kolorimetrik hem de floresan tabanli LAMP testlerinde 1, 10, 100 ve 1000 pg/reaksiyon pozitiflik gözlenmistir. Hem kolorimetrik hem de floresan LAMP testinin en düsük analitik hassasiyeti 1 pg/reaksiyon olarak tespit edildi. Ayrica, testin özgüllügünü degerlendirmek için diger parazit DNA'lari ile uygulanan LAMP reaksiyonu sonucunda kolorimetrik ve %1'lik agaroz jel elektroforezinde pozitif kontrol disinda LAMP ürünü tespit edilmemis olup, gelistirilen testin özgüllügü %100 olarak tespit edilmistir. Sonuç: Acanthamoeba'nin 18S rRNA gen bölgesi hedef alinarak gelistirilen LAMP testinin 1 pg genomik DNA saptama limitine sahip oldugu ortaya konulmustur. LAMP testinin kültür yöntemine göre daha hassas ve hizli olmasinin yani sira basit, ucuz ve yüksek hassasiyete sahip olmasi umut vericidir. Bu nedenle gelistirilen testin çevresel ve klinik örneklerde Acanthamoeba spp. tanisi için uygulanabilecegi düsünülmektedir. Anahtar Kelimeler: Acanthamoeba spp., 18S rRNA geni, hassasiyet, kolorimetrik LAMP, floresan LAMP Objective: Acanthamoeba, one of the free-living amoeba, has been detected in many environmental samples, mainly in water, soil and air. Acanthamoeba keratitis and granulomatous amoebic encephalitis are among the most important clinical manifestations caused by Acanthamoeba. In this study, it was aimed to determine the sensitivity of the rapid loop mediated isothermal amplification (LAMP) test designed with primers specific to Acanthamoeba 18S rRNA gene to detect more rapidly the presence of Acanthamoeba in clinical and environmental samples. Methods: Acanthamoeba strain grown in culture was diluted in 200 [micro]L as 1x10 (6) trophozoites and DNA was isolated, and the amount of DNA was determined by Nano-Drop Spectrophotometer. The purified DNAs were diluted from 1000 pg to 0.001 pg and used in colorimetric and fluorescence-based LAMP reactions. The LAMP reaction mixture was incubated for 60 minutes at 63 [degrees]C in a total volume of 25 [micro]L. Results: To determine the sensitivity of the test, positivity of Acanthamoeba genomic DNA was observed at 1, 10, 100 and 1000 pg/reaction in both colorimetric and fluorescence-based LAMP tests. The lowest analytical sensitivity of both calorimetric and fluorescent LAMP assay was determined as 1 pg/reaction. In addition, as a result of LAMP reaction applied with other parasite DNAs to evaluate the specificity of the test, no LAMP product was detected in calorimetric and 1% agarose gel electrophoresis, except for positive control, and the specificity of the test was determined as 100%. Conclusion: It has been demonstrated that the LAMP assay designed by targeting 18S rRNA gene of Acanthamoeba has a detection limit of 1 pg of genomic DNA. It is promising that LAMP test is more sensitive and faster than culture method, as well as simple, inexpensive and highly sensitive. For this reason, it is thought that developed test can be applied in the diagnosis of Acanthamoeba in environmental and clinical samples. Keywords: Acanthamoeba spp., 18S rRNA gene, sensitivity, colorimetric LAMP, fluorescence LAMP