Introduction: The clinical significance ofBlastocystissp. andDientamoeba fragilisin patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes ofBlastocystissp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence ofBlastocystissp. andD.fragilisin the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identifyBlastocystissp. subtypes using next generation sequencing.Material and methods: Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened forBlastocystissp. andD.fragilisby conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of theSSUrRNA gene. Next generation sequencing was used to identifyBlastocystissp. subtypes.Results: The prevalence ofBlastocystissp. andD.fragiliswas 16.7% and 11.9%, respectively as measured by qPCR. The prevalence ofBlastocystissp. andD.fragiliswas lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). FiveBlastocystissp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution ofBlastocystissp. subtypes between immunocompetent and immunodeficient patients.Conclusion and recommendation: Our findings demonstrated thatBlastocystissp. andD.fragilisare commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence ofBlastocystissp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

Purpose: To evaluate the antiamoebic properties of photo-activated chromophore for keratitis (PACK)–corneal cross-linking (CXL) (PACK-CXL), in combination with riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%), for treatment of Acanthamoeba trophozoites and cysts. Materials and Methods: Cultures of Acanthamoeba castellanii were grown in a fluid medium at a concentration of 2.7 × 10⁵ cell/ml. PACK-CXL was used on A. castellani cells in combination with either riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%). Riboflavin-containing wells were irradiated with ultraviolet-A (UVA) light (365-nm wavelength). Rose bengal-containing wells were irradiated with green light (523-nm wavelength). A power density of 9 mW/cm² for 10 min and total irradiation dose of 5.4 J/cm² was used for both riboflavin and rose bengal. After UVA and green light irradiation, cell viabilities were evaluated, and percentage of dead cells calculated. Results: No significant amoebicidal activity was observed following PACK-CXL/riboflavin at either concentration. PACK-CXL/rose bengal, however, was observed to be highly effective in eradicating Acanthamoeba cells at either concentration, with no significant difference observed between the two concentrations. The percentage of dead cells was 63% following treatment at either rose bengal concentration. Conclusion: PACK-CXL with rose bengal demonstrated pronounced antiamoebic activity against A.castellanii. Further in vitro and in vivo studies are needed to confirm this finding.

Purpose: To evaluate the in vivo efficacy of rose bengal (RB)-mediated photodynamic antimicrobial therapy (PDAT) for treatment of Acanthamoeba castellanii keratitis (AK). Materials and Methods: An animal (rabbit) AK model was successfully achieved via intrastromal inoculation of a suspension of A. castellanii cells and trophozoites. Prior to RB-PDAT (pre-treatment, day-5), the severity of the induced corneal infection was graded numerically for epithelial defects, stromal edema, neovascularity, and stromal opacity/infiltration. The right eyes of rabbits (n=18) were divided equally into three groups (n=6/group): control (no treatment); 0.1% RB+518 nm irradiation (5.4 J/cm²); and 0.2% RB+518 nm irradiation (5.4 J/cm²). On post-treatment day-5, animals were euthanized, after which corneal buttons were excised and submitted for real-time polymerase chain reaction (RT-PCR) analysis. Results: Post-treatment clinical scores of the 0.1 and 0.2% RB groups indicated significant improvement compared to control group scores (pre-treatment clinical scores; 5.17±0.98, 7.50±0.62, and 6.17±0.70 and post-treatment clinical scores; 4.50±0.56, (p=0.043), 3.50±0.99 (p=0.039), 6.83±1.66 (p=0.34), respectively). RT-PCR analysis revealed that the mean cycle threshold (Ct) values were significantly higher in treated-group corneas compared to control-group corneas, with no significant differences between treated-groups (Mean Ct values; 34.33, 34.5, and 29.67 for 0.1 and 0.2% RB, and control groups). There was a statistically significant negative correlation between post-treatment clinical scores and Ct values (r=-0.474, p-value 0.047). Conclusions: Our results demonstrate that RB-PDAT is effective in decreasing the parasitic load and clinical severity of AK.

Purpose: Cystic echinococcosis (CE) is caused by the larval form of Echinococcus granulosus. Clinical, radiologic, pathologic, and serologic findings should be evaluated together for the diagnosis of CE. The sensitivity and specificity oalf serologic tests may vary depending on the method used. In this study, we aimed to detect IgG antibodies specific to E. granulosus using indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodies (IFA) and western blot (WB) tests. Methods: In our study, the serum samples of 74 patients sent to our laboratory with suspicion of CE were studied using two different commercial IHA tests, ELISA, IFA and WB test. The test results were evaluated along with radiological findings and histopathological examinations, the latter being the gold standard. Results: Of all the patients, 51 (69%) were female and 23 (31%) were male. There was a statistically significant difference between males and females (χ² = 9.7, p = 0.002). Out of 74 patients, positivity rates for Siemens IHA, Fumouze IHA, ELISA, IFA and WB test were positive as 33 (44.6%), 35 (47.3%), 43 (58.1%), 42 (56.7%) and 38 (51.3%), respectively. The sensitivity and specificity of the tests were as follows: 66.67 and 2.31% for Siemens IHA; 70.83% and 96.15% for Fumouze IHA; 85.42%, and 88.46% for ELISA; 83.33% and 88.46% for IFA; 72.92% and 88.46% for WB test. Conclusion: There were statistically significant differences in between all five methods (p < 0,001). While the tests with the highest specificity was Fumouze IHA, the test with the highest sensitivity was the ELISA test. It was concluded that IHA and ELISA tests were more practical in practice because of their greater applicability.

Background & Aims: Hepatic encephalopathy (HE) is a serious neuropsychiatric sequela emerging in the advanced stages of cirrhosis. The gut microbiota plays an important role in the development of HE. The aim of the study was to analyze the dynamic interplay between microbiota and Blastocystis in cirrhotic patients with or without encephalopathy. Methods: The study was designed as cross-sectional study. A total of 37 patients from the Ankara city, admitted to the University Hospital within a 6-month period prior to enrolment into the study were included in the study. After the regular health checks, clinical histories, clinical examinations, and Psychometric HE Score (PHES) points, patients’ MELD and CTP scores were recorded. The fecal microbiota configurations were characterized by targeting hypervariable regions V3 and V4 of the 16S rRNA gene using Illumina MiSeq System. Results: Blastocystis spp. were detected in 21.6% (n = 8) of all cirrhotic patients. When those were analyzed by subgroups, four of them were subtype 2, three were subtype 3 and one was subtype 1. Blastocystis spp. were not found in any of the patients with HE; however, they were detected in 38.1% of the patients without HE. Also the increase in the bacterial diversity was observed along with the absence of Blastocystis. It was suggested that there was an inverse relationship between Blastocystis spp. and advanced stages of HE and the structure and composition of gut microbiota. Conclusion: The absence of Blastocystis spp. is associated with the HE severity and dysbiosis in the gut microbiota. Abbreviations: Bl: Blastocystis; CTP: Child-Turcotte-Pugh; DNA: deoxyribonucleic acid; HE: hepatic encephalopathy; IBD: inflammatory bowel disease; IBS: irritable bowel syndrome; MELD: Model for End Stage Liver Disease; MHE: Minimal hepatic encephalopathy; NASH: Non-alcoholic steatohepatitis; PBC: Primary Biliary Cirrhosis; PCR: Polymerase Chain Reaction; PHES: Psychometric Hepatic Encephalopathy Score; rRNA: ribosomal ribonucleic acid.