Development and Sensitivity Determination of 18S rRNA Gene-specific Fast Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Acanthamoeba.

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 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. 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.

Gut microbiota-derived metabolite trimethylamine N-oxide and biomarkers of inflammation are linked to endothelial and coronary microvascular function in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD), which is an umbrella term used for ulcerative colitis (UC) and Crohn's disease (CD), is associated with an increased risk for atherosclerotic cardiovascular disease (CVD). We aimed to investigate the association of local and systemic biomarkers of inflammation and gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) with endothelial and coronary microvascular dysfunction in IBD. METHODS: A total of 56 patients with IBD (20 with UC and 36 with CD) and 34 age and gender matched controls were included. For all participants, samples were collected to analyze faecal calprotectin, and TMAO concentrations. Ultrasound-based examinations were done to measure flow-mediated vasodilatation (FMD) and coronary flow velocity reserve (CFVR). RESULTS: Patients with IBD had lower CFVR (2.07 (1.82-2.40)) and FMD (8.7 ± 3.7) as compared to controls (2.30 (2.07-2.74), p = 0.005 and 11.9 ± 6.8, p = 0.03). In patients with IBD, TMAO concentration (r = -0.30, p = 0.03), C-reactive protein (r = -0.29, p = 0.03) and WBC count (r = -0.37, p = 0.006) had a significant negative correlation with CFVR, and TMAO (ß = -0.27, 95 % CI: -0.23 to -0.02) and WBC count (ß = -0.31, 95 % CI: -0.56 to -0.06) were significant predictors of CFVR after multivariate adjustment. None of the biomarkers of inflammation or TMAO showed significant correlations with FMD. In patients with UC, TMAO showed a significant correlation with both CFVR (r = -0.55, p = 0.01) and FMD (r = -0.60, p = 0.005) while only WBC count had a statistically significant correlation with CFVR (r = -0.49, p = 0.004) in patients with CD. CONCLUSIONS: Gut microbiota-derived metabolite TMAO and biomarkers of systemic inflammation are associated with measures of endothelial/coronary microvascular dysfunction in patients with IBD.

Construction of a multiepitope vaccine candidate against Fasciola hepatica: an in silico design using various immunogenic excretory/secretory antigens.

BACKGROUND: Fasciola hepatica is an important pathogen that causes liver fluke disease in definitive hosts such as livestock animals and humans. Various excretory/secretory products have been used in serological diagnosis and vaccination studies targeting fasciolosis. There are no commercial vaccines against fasciolosis yet. Bioinformatic analysis based on computational methods have lower cost and provide faster output compared to conventional vaccine antigen discovery techniques. The aim of this study was to predict B- and T-cell specific epitopes of four excretory/secretory antigens (Kunitz-type serine protease inhibitor, cathepsin L1, helminth defense molecule, and glutathione S-transferase) of Fasciola hepatica and to construct a multiepitope vaccine candidate against fasciolosis. METHODS AND RESULTS: Initially, nonallergic and the highest antigenic B- and T- cell epitopes were selected and then, physico-chemical parameters, secondary and tertiary structures of designed multiepitope vaccine candidate were predicted. Tertiary structure was refined and validated using online bioinformatic tools. Linear and discontinuous B-cell epitopes and disulfide bonds were determined. Finally, molecular docking analysis for MHC-I and MHC-II receptors was performed. CONCLUSION: This multi-epitope vaccine candidate antigen, with high immunological properties, can be considered as a promising vaccine candidate for animal experiments and wet lab studies.

Comparison of the New Multiepitope Recombinant Peptide Antigen with Cyst Fluid Antigen in the Follow-up of Patients with Cystic Echinococcosis

Objective: The follow-up of patients with cystic echinococcosis (CE) offers the opportunity of evaluating the prognosis of the infection as well as detecting relapse. This study aimed to evaluate the performance of the new multiepitope recombinant peptide (recDipol) antigen in the follow-up of CE patients treated by surgery or percutaneous aspiration injection respiration. Methods: A total of 137 blood samples from 28 patients were evaluated by IgG-ELISA method using recDipol and hydatid fluid (HF) antigens. The patients were simultaneously checked for recurrence by ultrasonography. Results: The seropositivity rate of the 28 patients varied considerably during the follow-up. When the first blood of the patients was evaluated, 4 (14.28%) were seronegative by HF-ELISA and 9 (32.14%) were recDipol-ELISA. During the entire follow-up, only 1 (3.5%) and 6 (21.4%) patients were seronegative by HF-ELISA and recDipol ELISA, respectively. Conclusion: We found that recDipol did not perform as expected in the follow-up due to the higher number of seronegative patients compared to HF-ELISA in the first blood and during the entire follow-up. Our results suggest that imaging methods are gold standards in the diagnosis and that, in parallel, longer-term patient follow-up is required with recombinant antigens that have an improved diagnostic performance.