Potential associations between alterations in gut microbiome and obesity-related traits after the bariatric surgery.

BACKGROUND: This study aimed to examine the effects of both obesity and bariatric surgery on gut microbiome, dietary intake, as well as metabolic and inflammatory parameters. METHODS: All participants (15 with morbid obesity who had bariatric surgery, 8 with morbid obesity and 11 non-obese) were followed up for a 6-month period with interviews at baseline (M0), at the end of 3 (M3) and 6 months (M6). Dietary assessment was done, and blood and faecal samples were collected. RESULTS: Dietary energy and nutrient intakes as well as serum glucose levels, total cholesterol, low-density lipoprotein (LDL)-cholesterol and high sensitivity C-reactive protein (hs-CRP) levels decreased after surgery (p < 0.05, for each). Participants with morbid obesity had higher levels of Firmicutes and lower levels of Bacteroidetes at M0 compared to non-obese participants. The abundances of Bacteroidetes increased (p = 0.02), whereas that of Firmicutes decreased (p > 0.05) after the surgery, leading to a significant decrease in Firmicutes/Bacteroidetes ratio (p = 0.01). At sub-phylum level, the abundances of Lactobacillus and Bifidobacterium decreased, whereas those of Akkermansia increased after the surgery (p < 0.01, for each). Although participants who were morbidly obese had a distinct profile according to ß-diversity indices at M0, it became similar with the profile of non-obese participants (p > 0.05) at M3 and M6. Similarly, α-diversity indices were lower in subjects with morbid obesity at M0, but became similar to levels in non-obese controls at M6. CONCLUSION: This study confirmed that bariatric surgery has substantial impacts on gut microbiome's composition and diversity, as well as anthropometrical measurements and biochemical parameters, which were associated with the alterations in dietary intake patterns.

Investigation of intestinal parasites by conventional and molecular methods in children with gastrointestinal system complaints.

Intestinal parasitic infections are a global health problem that causes morbidity and mortality, especially in children living in rural areas. In this study, stool samples of pediatric patients with gastrointestinal complaints were examined by conventional and molecular methods to determine the prevalence of intestinal parasites. A total of 100 pediatric patients with gastrointestinal complaints and 50 healthy children were included in the study. Stool samples were collected from each child and examined by direct microscopic examination (native-Lugol method), formol-ethyl acetate concentration technique, Kinyoun's acid-fast staining, and Wheatley trichrome staining methods. Real-time PCR was used for the detection of Blastocystis spp. and D. fragilis in the stool samples. Sanger sequencing was used to identify Blastocystis spp. subtypes. One or more intestinal parasites were found in 12% (n = 100) of the patient group and 1% (n = 50) of the control group using conventional techniques. By using real-time PCR, Blastocystis spp. was discovered in 14% (14/100) of the patient group and 8% (4/50) of the control group. There was no significant difference in the frequency of Blastocystis spp. between the two groups. The most prevalent Blastocystis subtype was ST1 and the most frequent allele was a2 among the samples successfully amplified and sequenced. D. fragilis was detected in 17% (17/100) of the patient group and 8% (4/50) of the control group by real-time PCR. The prevalence of D. fragilis was not significantly different between the patient and control groups, as well. Blastocystis spp. and D. fragilis were found in high prevalence in pediatric patients with gastrointestinal complaints in this study. Although the role of these protists as a pathogen in humans is still controversial, it is supposed to the presence of the parasites are associated with gastrointestinal disorders such as diarrhea, abdominal pain, nausea, and vomiting. More case-control studies are needed to understand the pathogenic or commensal role of these parasites on the intestinal microbiota, especially in both patients with gastrointestinal disorders and healthy individuals.

Prevalence of Blastocystis and Dientamoeba fragilis in diarrheal patients in Corum, Türkiye.

To investigate the prevalence of Blastocystis and Dientamoeba fragilis in diarrhea patients and healthy individuals in Corum, Türkiye, fecal samples from 92 diarrhea patients and 50 healthy individuals were collected and evaluated using direct microscopy and molecular methods to screen for bacteria, protozoa, and viruses. The prevalence of Blastocystis was 24.6% in total and more frequent in the healthy group (30.0%). The commonly detected STs (subtypes) were ST3 (40.0%) and ST2 (34.2%). The distribution of Blastocystis STs in the healthy and diarrheal groups did not show any difference in sex and age, but ST3 was detected more frequently in patients aged from 40 to 59 years (p < 0.05). Alleles 4 (8/12) and 2 (4/12) were present in ST1; 9 (3/5) and 12 (2/5) in ST2; 34 (9/14), 36 (3/14), and 38 (2/14) in ST3; and only allele 42 (2/2) in ST4. D. fragilis was present in 8.4% of the population. However, there was no statistically significant difference between the healthy and diarrheic groups (12.0% and 6.5%, respectively), neither with respect to age nor sex. Co-infection was 58.3% and was more frequent in healthy individuals (33.3%) than in diarrhea patients (25.0%). Blastocystis ST3 was the most common subtype detected, with D. fragilis at 33.3%. Salmonella, Shigella, or helminth eggs were not observed in all groups, but Entamoeba histolytica, Giardia intestinalis, Cryptosporidium, Rotavirus, Adenovirus, and Clostridium difficile toxin were found only in diarrhea patients. These findings support the hypothesis that Blastocystis and D. fragilis may be part of the healthy human gut microbiome.

Detection of enteric parasites and molecular characterization of Giardia duodenalis and Blastocystis sp. in patients admitted to hospital in Ankara, Turkey.

This epidemiological study assesses the occurrence of enteric parasites in 4303 patients attended at two public hospitals in Ankara (Turkey) during 2018-2019. Microscopy was used as a screening test. Giardia duodenalis was also identified using a commercial ELISA for the detection of parasite-specific coproantigens. Giardia-positive samples by microscopy/ELISA were confirmed by real-time PCR and characterized using a multilocus genotyping scheme. Blastocystis sp. was genotyped in a sample subset. Blastocystis sp. (11.1%, 95% CI 11.4‒14.8%) and G. duodenalis (1.56%, 95% CI 1.22‒1.96) were the most prevalent pathogens found. Cryptosporidium spp., Entamoeba histolytica and intestinal helminths were only sporadically (<0.5%) found. For G. duodenalis, sequence (n = 30) analyses revealed the presence of sub-assemblages AII (23.3%), discordant AII/AIII (23.3%) and mixed AII + AIII (6.7%) within assemblage A, and BIII (10.0%), BIV (3.3%) and discordant BIII/BIV (23.3%) within assemblage B. Two additional sequences (6.7%) were assigned to the latter assemblage but sub-assemblage information was unknown. No associations between G. duodenalis assemblages/sub-assemblages and sociodemographic and clinical variables could be demonstrated. For Blastocystis sp., sequence (n = 6) analyses identified subtypes ST1, ST2 and ST3 at equal proportions. This is the first molecular characterization of G. duodenalis based on MLG conducted in Turkey to date.

[Investigation of Microsporidia prevalence with calcofluor white and uvitex 2B chemiluminescence staining methods and molecular analysis of species in diarrheal patients]. / Ishalli olgularda Microsporidia sikliginin Calcofluor beyazi ve uvitex 2B kemiluminesans boyama yöntemleriyle arastirilmasi ve türlerinin moleküler yöntemle tiplendirilmesi.

Microsporidia, obligate intracellular parasites, were first defined by Nageli in 1857. Microsporidia phylum consists of 200 genus and 1500 species. They have a wide host spectrum including insects, fish, and mammals. It has been shown that they may also infect humans and may be existed both in symptomatic and asymptomatic forms. There are eight species infecting humans, which include Anncaliia (Brachiola, Nosema), Encephalitozoon, Entrocytozoon, Microsporidium, Nosema, Pleistophora, Trachipleistophor, and Vittaforma. The species most commonly infect humans are Encephalitozoon intestinalis and Enterocytozoon bieneusi. The aim of this study was to determine the prevalence of Microsporidia by using two different chemiluminescence stains, namely uvitex 2B and calcoflour and detect species by molecular analysis in diarrheal patients. For this purpose, we studied stool samples of 200 patients with diarrhea sent to Gazi University Health Practice and Research Hospital, Microbiology Laboratory and Ankara Numune Training and Research Hospital Microbiology Laboratory between 2012-2013. The stool samples were stained with chemiluminescent stains uvitex 2B and calcoflour methods; the Microsporidia prevalence was found to be 38% (77/200) by fluorescent microscopic examination. Statistically an excellent consistency was found between the chemiluminescent stains uvitex 2B and calcoflour (Cohen's kappa= 0.881). A statistical analysis for the consistency of uvitex 2B and calcoflour in terms of numerical density (low, high) and luminescence of spores (dim, bright) showed a moderate consistency between the two stains with respect to determining numerical density of spores (Cohen's kappa= 0.354), while there was no consistency in terms of luminescence of spores (Cohen's Kappa= 0.001). No significant difference was found between the Microsporidia prevalence with respect to age group or clinics (p > 0.05). A sex-based analysis showed that Microsporidia prevalence was more common in women (50%) than men (30.8%) (p< 0.05). In 77 samples that were detected positive for Microsporidia with uvitex 2B and calcoflour stains determination of genus and species level were done by using multiplex nested polymerase chain reaction (PCR) method. With this technique, seven (9.1%) of 77 isolates were detected as E.bieneusi, and 70 (90.9%) as Encephalitozoon spp. When the Microsporidia genus was considered, the Microsporidia prevalence did not show differences with respect to age, sex, and referring clinics (p> 0.05). In our study 44 (62.9%) of 70 Encephalitozoon spp. were E.intestinalis, 22 (31.4%) were E.cuniculi, and 4 (5.7%) were E. hellem. No statistical difference was found in the distribution of Encephalitozoon spp. with age, sex, and referring clinic (p> 0.05). We concluded that examination of stool samples with the chemiluminescent stain uvitex 2B and/or calcoflour would be useful for the initial stage of Microsporidia diagnosis; furthermore, the multiplex nested PCR method was considered useful for determination of genus and species. In our country, there is a small number of molecular reports about Microsporidia prevalence in stool samples. Molecular methods should be used more commonly for the evaluation of treatment options in diarrheal patients and detection of Microsporidia epidemiology.

Demodex spp. as a possible aetiopathogenic factor of acne and relation with acne severity and type.

INTRODUCTION: Acne is a very common skin disease in adolescents and young adults, but it also affects adults. However, its aetiology is not yet fully understood. Demodex appears to be associated with multiple skin disorders, but controversy persists. Some reports indicate a connection between acne vulgaris and demodicosis. AIM: To confirm the association between Demodex infestation and acne vulgaris. MATERIAL AND METHODS: A total of 108 patients were enrolled in the acne group. Acne severity was calculated as GASS and acne type (adolescent and post adolescent) was recorded. An age-sex matched healthy control group comprising 65 individuals were included in the study. Dermatological examinations were performed and an SSSB was used to determine the presence of Demodex. RESULTS: In our study, Demodex positivity was seen in 46 (42.6%) patients in the acne group and 8 (12.3%) in the control group; this difference was statistically significant (p < 0.001). A multivariate Backward Step-By-Step Logistic Regression analysis identified the most effective factors for acne development such as Demodex positivity (OR = 5.565, 95% CI: 2.384-12.99 and p < 0.001) and age under 25 years (OR = 2.3 and 95% CI: 1.183-4.473 and p = 0.014). Alcohol consumption was related to Demodex positivity (p = 0.019) in post adolescent acne. CONCLUSIONS: Our study is the first one to evaluate acne severity, acne type and the relationship to Demodex prevalence. We suggest that Demodex infestation should be considered when the classical therapies are ineffective especially in cases of post adolescent acne.

A novel ELISA test for laboratory diagnosis of Blastocystis spp. in human stool specimens.

Detection of Blastocystis is routinely performed by microscopy, culture, and formyl-ether (ethyl acetate) concentration technique (FECT). Yet, these methods require special skilled personnel, are time consuming, and often involve processing that may cause misdiagnosis. The aim of this work is to demonstrate the usefulness of a newly introduced ELISA test for the detection of Blastocystis antigens in stool samples (CoproELISA(TM) Blastocystis, Savyon Diagnostics) as a proper alternative to currently used methods, especially microscopy. A cohort of 179 fresh/frozen clinical stool samples was tested by the ELISA test, and results were compared to consensus methods comprised of microscopic examination of Lugol's iodine staining, culture, and immunofluorescence assay (IFA). The new ELISA test was able to detect fewer than 10(3) cells, recognized subtypes 1, 2, 3, and 5 (comprising >95 % of human Blastocystis infections), and exhibited similar reactivity when comparing formalin-preserved samples to fresh/frozen samples. The test demonstrated 92 % sensitivity, 87 % specificity, and 89 % accuracy when culture, and IFA or microscopy consensus results were taken as reference. When the consensus was comprised of culture and IFA, the test demonstrated sensitivity, specificity, and accuracy of 82, 86, and 84 %, respectively. In contrast, the sensitivity of Lugol staining microscopy was only 18 %. This work presents a unique ELISA test that provides an alternative to the use of microscopy, currently most widely used method. The test enables high-throughput screening and diagnosis of Blastocystis, adaptation to automatic procedures.

[Investigation of the presence of Blastocystis spp. in stool samples with microscopic, culture and molecular methods]. / Diski örneklerinde Blastocystis spp. varliginin mikroskobik, kültür ve moleküler yöntemlerle arastirilmasi

Blastocystis species are enteric protozoa frequently detected in human and animals. Seventeen subtypes (STs) have now been identified, nine of them isolating from humans. The pleomorphic structure and genetic diversity of Blastocystis spp. and the absence of standardized diagnostic methods complicate the evaluation of current data. Microscopic methods such as native-lugol and trichrome staining are most frequently used methods in routine diagnosis, while culture and molecular methods are preferred for research purposes. The aims of this study were to investigate the presence of Blastocystis spp. in the stool samples of patients with gastrointestinal complaints by microscopic and culture methods, and to detect the subtypes of isolates by polymerase chain reaction (PCR). A total of 350 stool samples collected from patients with diarrhea (n= 157) and without diarrhea (n= 193) were included in the study. Presence of Blastocystis spp. in the samples were investigated by native-lugol examination, trichrome staining and direct fluorescent antibody (DFA) methods. Ringer's solution containing 10% horse serum and 0.05% asparagine was used for cultivation. The cultures were evaluated after 3-4 days of incubation at 37°C by microscopic examination. The subtypes of Blastocystis spp. strains isolated from the cultures have been identified by PCR using sequence-tagged site primers. A total of 66 (19%) stool samples, of them 26 (16.6%) were from diarrheal and 40 (21%) from non-diarrheal cases, yielded Blastocystis sp. growth in culture. Among the evaluated samples, 12% (42/350) were found positive with native-lugol examination, 17% (58/350) with trichrome staining, and 19% (66/350) with DFA method. The agreement of culture and native-lugol method was estimated as strong (κ= 0.752), while it was very strong between culture with trichrome staining and DFA methods (κ= 0.922 and κ= 1.00, respectively). When the culture was accepted as reference method, the sensitivity and specificity rates of native-lugol method were 65% and 100%, trichrome staining method were 88% and 100%, and DFA method were 100% and 100%, respectively. Forty-three (65%) of Blastocystis spp. positive samples were subtyped by PCR, while 23 isolates could not be subtyped. The most frequent detected subtype was ST3 (12/43; 28%), followed by ST1 (6/43; 13.9%), ST4 (5/43; 11.6%) and ST7 (5/43; 11.6%), ST2 (3/43; 7%) and ST6 (1/43; 2.3%). ST5 was not detected in this study and 11 (25.6%) samples have been identified to have mixed subtypes. The differences of Blastocystis spp. positivity rates and the distribution of the subtypes between the patients with or without diarrhea were not found statistically significant (p> 0.05). In our study, ST3 was the most frequently identified Blastocystis spp. subtype, similar to the previous national studies, however ST6 and ST7 have been identified for the first time. In conclusion, as the sensitivity of native-lugol examination is low, culture is time-consuming and laborious and PCR methods are costly and non-standardized, rapid, practical and high sensitive DFA is considered as the favourable method in the diagnosis of Blastocystis spp. in routine laboratories.

Detection of Blastocystis sp. and Dientamoeba fragilis using conventional and molecular methods in patients with celiac disease.

Blastocystis sp. and Dientamoeba fragilis are intestinal protists, which are common worldwide, but the pathogenic role of these organisms in gastrointestinal diseases is still controversial. This study aimed to investigate the frequency of Blastocystis sp. and D. fragilis in stool samples from adult patients with celiac disease (CD) by using conventional and molecular methods. A total of 75 patients with CD and 75 healthy individuals were included in this study. Fresh stool specimens collected from each individual were analyzed by conventional and molecular methods. The overall prevalence of Blastocystis sp. and D. fragilis was 41.3% (31/75) and 24% (18/75) in patients with CD, and 46.7% (35/75) and 13.3% (10/75) in healthy controls, respectively. There was no statistically significant difference in the prevalence of Blastocystis sp. and D. fragilis between CD patients and healthy individuals. Blastocystis sp. subtypes were identified in 20 CD and 16 control patients and the overall subtype distribution was observed as ST1 13.9%, ST2 30.6%, and ST3 55.6%. The prevalence of Blastocystis sp. and D. fragilis in adults with CD is similar to the prevalence of protozoa in healthy adults. In this study, the most prevalent Blastocystis subtype was ST3 and the most frequent allele was a34 in both CD patients and healthy individuals. No significant difference was found between the two groups in terms of the detection rates of Blastocystis sp. and D. fragilis, and it is thought that both protists may be colonisers of the intestinal microbiome.

Blastocystis: A Mysterious Member of the Gut Microbiome.

Blastocystis is the most common gastrointestinal protist found in humans and animals. Although the clinical significance of Blastocystis remains unclear, the organism is increasingly being viewed as a commensal member of the gut microbiome. However, its impact on the microbiome is still being debated. It is unclear whether Blastocystis promotes a healthy gut and microbiome directly or whether it is more likely to colonize and persist in a healthy gut environment. In healthy people, Blastocystis is frequently associated with increased bacterial diversity and significant differences in the gut microbiome. Based on current knowledge, it is not possible to determine whether differences in the gut microbiome are the cause or result of Blastocystis colonization. Although it is possible that some aspects of this eukaryote's role in the intestinal microbiome remain unknown and that its effects vary, possibly due to subtype and intra-subtype variations and immune modulation, more research is needed to characterize these mechanisms in greater detail. This review covers recent findings on the effects of Blastocystis in the gut microbiome and immune modulation, its impact on the microbiome in autoimmune diseases, whether Blastocystis has a role like bacteria in the gut-brain axis, and its relationship with probiotics.

Oxidative stress and tryptophan degradation pattern of acute Toxoplasma gondii infection in mice.

Toxoplasma gondii is a very common obligate single-cell protozoan parasite which induces overproduction of interferon (IFN)-gamma and of other proinflammatory cytokines. Although immunomodulatory role of IFN-gamma favors tryptophan (Trp) degradation via indoleamine-2,3-dioxygenase (IDO) activity and is related with nitric oxide (NO) synthesis, the mechanism of antitoxoplasma activity is complex. In order to characterize the Trp degradation pattern during the acute T. gondii infection, serum Trp, kynurenine (Kyn), and urinary biopterin levels of mice were measured. The possible oxidative status was evaluated by the liver, spleen, brain, and serum malondialdehyde (MDA) and NO levels. Increased free radical toxicity may cause elevation in tissue MDA in T. gondii-infected mice, while unchanged serum MDA might indicate the increased oxidative stress due to T. gondii infection restricted to intracellular area. Elevated serum NO most probably might be due to the formation of reactive nitrogen radicals. The Kyn/Trp ratio was higher in T. gondii-infected mice compared to healthy animals (p < 0.05); however, it was not correlated with urinary biopterin. These results suggested that Trp degradation might be promoted by a pathway other than IDO during T. gondii infection and the reduction of Trp concentration favors the local immunosuppression and systemic tolerance.

[Investigation of Microsporidia prevalence by different staining methods in cases of diarrhea]. / Ishalli Olgularda Microsporidia Sikliginin Farkli Boyama Yöntemleriyle Arastirilmasi

Microsporidia, depending on their different species, generally lead to self-limited, sporadic and mild infections such as diarrhea, corneal ulcer and myositis. They are considered as opportunistic pathogens in HIV-positive patients however in recent years Microsporidia have been detected also in immunocompetent individuals as a cause of diarrhea. Diagnosis of Microsporidia depends on the detection of spores or different developmental stages of protozoon in stool, urine, sinus aspirates, nasal discharge, bronchoalveolar lavage or tissue biopsies. Diagnosis of Microsporidia infections is usually achieved by the use of different staining methods, serological tests, polymerase chain reaction, and electron microscopic methods. The aims of this study were to detect the incidence of microsporidia in patients with diarrhea by using three different staining methods and to compare the performance of these methods. A total of 225 stool samples from diarrheal patients (84 were children, 141 were adults; 103 were female, 122 were male) admitted to Gazi University Medical Faculty Hospital between March-June 2009, have been evaluated in the laboratory of Medical Microbiology Department. Stool samples were examined in terms of the presence of Microsporidia spores by Weber's modified trichrom staining (MTS), calcofluor (CF) and acridine orange (AO) staining methods. Microsporidia positivity rate was 9.8% (22/225) in the diarrheal patients, the rate being 9.5% (8/84) in children and 9.9% (14/141) in adults. There was no statistically significant difference between age and gender groups (p> 0.05) regarding Microsporidia detection. When MTS was considered as the reference method, sensitivity, specifity and consistency of AO staining were estimated as 100%, 91.6% and 92%, respectively, while those rates for CF staining were 95.4%, 99.5% and 99%, respectively. There was very strong and significant correlation (r= 0.950, p< 0.001) between CF staining and MTS, while there was strong and significant (r= 0.719, p< 0.001) correlation between AO staining and MTS. Although AO staining is rapid and convenient, the positive predictive value was measured very low (56.4%) and the interpretation of stained slides was very difficult since background of the slides was stained orange and there were a lot of dye artefacts. In conclusion, screening Microsporidia in all diarrheal stool samples is of diagnostic value. To increase sensitivity and reliability in the detection of Microsporidia spores in diarrheal samples, initial application of calcofluor staining should be followed by the confirmatory MTS method.

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples.

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes.

Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods.

INTRODUCTION: The clinical significance of Blastocystis sp. and Dientamoeba fragilis in 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 of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. 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 for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. RESULTS: The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. 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 of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. CONCLUSION AND RECOMMENDATION: Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

Case Report: A Human Case of Onchocerca lupi Mimicking Nodular Scleritis.

We report a case of human ocular onchocerciasis by zoonotic Onchocerca lupi presenting as nodular scleritis. Molecular analyses were used to confirm diagnosis at species level. In addition to few existing reports of human infection by O. lupi in Turkey, this case further suggests that the pathogen might be more common than previously reported, thus requiring further attention and investigations.

Metabolic Fluctuations in the Human Stool Obtained from Blastocystis Carriers and Non-Carriers.

Blastocystis is an obligate anaerobic microbial eukaryote that frequently inhabits the gastrointestinal tract. Despite this prevalence, very little is known about the extent of its genetic diversity, pathogenicity, and interaction with the rest of the microbiome and its host. Although the organism is morphologically static, it has no less than 28 genetically distinct subtypes (STs). Reports on the pathogenicity of Blastocystis are conflicting. The association between Blastocystis and intestinal bacterial communities is being increasingly explored. Nonetheless, similar investigations extending to the metabolome are non-existent.Using established NMR metabolomics protocols in 149 faecal samples from individuals from South Korea (n = 38), Thailand (n = 44) and Turkey (n = 69), we have provided a snapshot of the core metabolic compounds present in human stools with (B+) and without (B-) Blastocystis. Samples included hosts with gastrointestinal symptoms and asymptomatics. A total of nine, 62 and 98 significant metabolites were associated with Blastocystis carriage in the South Korean, Thai and Turkish sample sets respectively, with a number of metabolites increased in colonised groups. The metabolic profiles of B+ and B- samples from all countries were distinct and grouped separately in the partial least squares-discriminant analysis (PLS-DA). Typical inflammation-related metabolites negatively associated with Blastocystis positive samples. This data will assist in directing future studies underlying the involvement of Blastocystis in physiological processes of both the gut microbiome and the host. Future studies using metabolome and microbiome data along with host physiology and immune responses information will contribute significantly towards elucidating the role of Blastocystis in health and disease.

Rose Bengal-Mediated Photodynamic Antimicrobial Treatment of Acanthamoeba Keratitis.

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 = .043), 3.50 ± 0.99 (p = .039), 6.83 ± 1.66 (p = .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.

Hepatitis B virus and hepatitis C virus seroprevalence in the elderly living in nursing homes.

BACKGROUND: : Communal living situations such as nursing homes create a risk for the spread of hepatitis B virus and hepatitis C virus (HCV). The aim of this study was to determine the seroprevalence of hepatitis B virus and HCV in the elderly living in 2 nursing homes in Ankara, Turkey. METHODS: : A total of 227 persons (mean age, 76.11 +/- 8.55 years) participated in this cross-sectional study. All individuals were investigated seroprevalence for hepatitis B surface antigen (HBsAg), anti-HBs immunoglobulin G (IgG), anti-hepatitis B core IgG, and anti-HCV IgG. RESULTS: : Positive seroprevalence was 11.9% for HBsAg, 48.0% for anti-HBs IgG, 25.1% for anti-hepatitis B core IgG, and 2.5% for anti-HCV IgG. Hepatitis B surface antigen positivity was 12.4% in males and 11.5% in females (P > 0.05); and the seroprevalence was 10.4% for those living in nursing homes for 1 year or less and 13.0% for those living in nursing homes for more than 1 year (P > 0.05). CONCLUSIONS: : The fact that nearly half of those living in nursing homes had not encountered hepatitis B infection or had not received hepatitis B vaccination indicates the need for administering hepatitis B vaccines in this group.

Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey.

Blastocystis infection has been reported to be associated with irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and chronic diarrhoea. The availability of data on the subtypes of Blastocystis found in these patient groups would be of interest in understanding the significance of Blastocystis infection in chronic illness. In this study, we identify Blastocystis subtypes found in patients presenting with IBS, IBD, chronic diarrhoea and asymptomatic patients in Ankara, Turkey. Blastocystis was detected in 11 symptomatic patients by microscopy and 19 by stool culture. Stool culture was more sensitive than microscopy in identifying Blastocystis. Using standard nomenclature adopted in 2007, Blastocystis sp. subtype 3 was the most common in all groups, followed by Blastocystis sp. subtype 2. Identical subtypes of Blastocystis are found in patients with IBS, IBD and chronic diarrhoea. These particular subtypes show low host specificity and are carried by humans and some farm animals. The subtypes of Blastocystis that are commonly found in rodents and certain wild birds were not found in these patients. We suggest a model in which the severity of enteric protozoan infection may be mediated by host factors.

PCR-based subtyping of Blastocystis isolates from symptomatic and asymptomatic individuals in a major hospital in Ankara, Turkey.

The stool samples obtained from 94 patients with gastrointestinal symptoms and 109 asymptomatic individuals, who checked in due to other reasons, admitted at a major hospital in Ankara, Turkey were examined with native Lugol's iodine, trichrome, and Kinyoun's acid-fast stainings for parasitology examinations and with in vitro culture method for detection of Blastocystis. In a total of 203 stool samples tested, native Lugol's iodine and trichrome stainings could detect 12 (5.9%) and 20 (9.9%) positive samples for Blastocystis, respectively. Conversely, culture method could detect 66 (32.5%) positive samples, and this method was more sensitive compared to the both microscopic examinations (p < 0.001). Among 66 positive samples for Blastocystis, 27 were from symptomatic patients and 39 were from asymptomatic group. Subtypes (STs) were determined by PCR using seven different sequence-tagged site primers. ST3 was the most dominant in both symptomatic and asymptomatic groups and followed by ST1 or ST2. There were mixed infections with STs 1 and 2 or STs 1 and 3 in nine isolates. There was no statistical significance of the distribution of Blastocystis sp. subtypes between symptomatic and asymptomatic individuals (p > 0.05).