Automatic detection of Cryptosporidium in optical microscopy images using YOLOv5x: a comparative study.

Here, a machine learning tool (YOLOv5) enables the detection of Cryptosporidium microorganisms using optical and phase contrast microscope images. The two databases were processed using 520 images (optical microscopy) and 1200 images (phase contrast microscopy). It used Python libraries to label, standardize the size, and crop the images to generate the input tensors to the YOLOv5 network (s, m, and l). It implemented two experiments using randomly initialized weights in optical and phase contrast microscope images. The other two experiments used the parameters for the best training time obtained before and after retraining the models. Metrics used to assess model accuracy were mean average accuracy, confusion matrix, and the F1 scores. All three metrics confirmed that the optimal model used the best epoch of optical imaging training and retraining with phase contrast imaging. Experiments with randomly initialized weights with optical imaging showed the lowest precision for Cryptosporidium detection. The most stable model was YOLOv5m, with the best results in all categories. However, the differences between all models are lower than 2%, and YOLOv5s is the best option for Cryptosporidium detection considering the differences in computational costs of the models.

The Potential Roles of Extracellular Vesicles as Biomarkers for Parkinson's Disease: A Systematic Review.

Parkinson's disease (PD) is a slowly progressive neurodegenerative disorder, characterized by the misfolding and aggregation of α-synuclein (α-syn) into Lewy bodies and the degeneration of dopaminergic neurons in the substantia nigra pars compacta. The urge for an early diagnosis biomarker comes from the fact that clinical manifestations of PD are estimated to appear once the substantia nigra has deteriorated and there has been a reduction of the dopamine levels from the striatum. Nowadays, extracellular vesicles (EVs) play an important role in the pathogenesis of neuro-degenerative diseases as PD. A systematic review dated August 2022 was carried out with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses with the aim to analyze the potential role of EVs as biomarkers for PD. From a total of 610 articles retrieved, 29 were eligible. This review discusses the role of EVs biochemistry and their cargo proteins, such as α-syn and DJ-1 among others, detected by a proteomic analysis as well as miRNAs and lncRNAs, as potential biomarkers that can be used to create standardized protocols for early PD diagnosis as well as to evaluate disease severity and progression.

Blastocystis Subtypes and Culture Characteristics of Isolates from Human Stools Related with the Presence of Gastrointestinal Symptoms: A Case-Control Study.

PURPOSE: To stablish if Blastocystis subtypes influences gastrointestinal symptoms. METHODS: Case-control study. We obtained sequencing for Blastocysts subtyping from 13 patients with gastrointestinal symptoms (diarrhea or abdominal pain) and 12 from individuals without symptoms. RESULTS: 12 sequences were from Subtype 2 and one from Subtype 3 in symptomatic individuals and nine samples were from Subtype 1, one from Subtype 2, and two from Subtype 3 in asymptomatic individuals. The prevalence of subtype 2 in symptomatic individuals was vastly different compared to the frequency in asymptomatic individuals (84.6% vs. 16.6%; OR 27.5 95% CI 3.2-233; Fisher exact test p = 0.0010201335). After in vitro culture, 22 isolates were obtained. Significant differences were observed for the 12 isolates from Subtype 2 that get a smaller number of total cells with dominant growth of vacuolar forms, compared with Subtypes 1 and 3, after eight days of culture. CONCLUSION: Our results suggest that gastrointestinal symptoms in Colombian individuals with Blastocystis infection depend on the infecting subtype with peculiar phenotypic characteristics in in vitro culture.

Detection of protozoa in water samples by formalin/ether concentration method.

Methods to detect protozoa in water samples are expensive and laborious. We evaluated the formalin/ether concentration method to detect Giardia sp., Cryptosporidium sp. and Toxoplasma in water. In order to test the properties of the method, we spiked water samples with different amounts of each protozoa (0, 10 and 50 cysts or oocysts) in a volume of 10 L of water. Immunofluorescence assay was used for detection of Giardia and Cryptosporidium. Toxoplasma oocysts were identified by morphology. The mean percent of recovery in 10 repetitions of the entire method, in 10 samples spiked with ten parasites and read by three different observers, were for Cryptosporidium 71.3 ± 12, for Giardia 63 ± 10 and for Toxoplasma 91.6 ± 9 and the relative standard deviation of the method was of 17.5, 17.2 and 9.8, respectively. Intraobserver variation as measured by intraclass correlation coefficient, was fair for Toxoplasma, moderate for Cryptosporidium and almost perfect for Giardia. The method was then applied in 77 samples of raw and drinkable water in three different plant of water treatment. Cryptosporidium was found in 28 of 77 samples (36%) and Giardia in 31 of 77 samples (40%). Theses results identified significant differences in treatment process to reduce the presence of Giardia and Cryptosporidium. In conclusion, the formalin ether method to concentrate protozoa in water is a new alternative for low resources countries, where is urgently need to monitor and follow the presence of theses protozoa in drinkable water.

Detection by PCR of pathogenic protozoa in raw and drinkable water samples in Colombia.

We evaluated the presence of DNA of Giardia, Toxoplasma, and Cryptosporidium by PCR, and of Giardia and Cryptosporidium genera by immunofluorescence antibody test (IFAT), in water samples, before, during, and after plant treatment for drinkable water. We applied this method in 38 samples of 10 l of water taken from each of the water treatment steps and in 8 samples taken at home (only for Toxoplasma PCR) in Quindio region in Colombia. There were 8 positive samples for Cryptosporidium parvum (21 %), 4 for Cryptosporidium hominis (10.5 %), 27 for Toxoplasma gondii (58.6 %), 2 for Giardia duodenalis assemblage A (5.2 %), and 5 for G. duodenalis assemblage B (13.1 %). By IFAT, 23 % were positive for Giardia and 21 % for Cryptosporidium. An almost perfect agreement was found between IFAT and combined results of PCR, by Kappa composite proportion analysis. PCR positive samples were significantly more frequent in untreated raw water for C. parvum (p = 0.02). High mean of fecal coliforms, high pH values, and low mean of chlorine residuals were strongly correlated with PCR positivity for G. duodenalis assemblage B. High pH value was correlated with PCR positivity for C. parvum. Phylogenetic analysis of DNA sequences was possible, showing water and human clinical sequences for Toxoplasma within the same phylogenetic group for B1 repeated sequence. PCR assay is complementary to IFAT assay for monitoring of protozoa in raw and drinkable water, enabling species identification and to look for phylogenetic analysis in protozoa from human and environmental sources.

Effect of fingolimod (FTY720) on cerebral blood flow, platelet function and macular thickness in healthy volunteers.

AIM: Fingolimod, a sphingosine 1-phosphate receptor modulator, is the first oral disease modifying therapy approved for the treatment of relapsing multiple sclerosis. The aim of this double-blind, placebo-controlled study was to evaluate the effect of fingolimod on cerebral blood flow, platelet function and macular thickness in healthy volunteers. METHODS: The study included 88 healthy volunteers who received fingolimod 0.5 mg or 1.25 mg or matched placebo over a period of 4 weeks. Transcranial colour coded sonography was performed to measure mean blood flow velocities, the platelet function was measured by the PFA-100® assay using a collagen/epinephrine cartridge and macular thickness was measured using optical coherence tomography. An assessment of non-inferiority of fingolimod vs. placebo was performed against a reference value (20% of the overall baseline value). RESULTS: All 88 randomized participants completed the study. At day 28 compared with baseline value, for 0.5 mg, 1.25 mg and placebo treatments, the mean middle cerebral artery blood flow velocity decreased by 4, 1 and 3.7 cm s(-1), respectively. The platelet function analyzer closure time increase was not significant (7.8, 7.5 and 10.4 s, respectively). The mean percentage change in the central foveal thickness from baseline for both eyes was below 3% for all groups. The safety profile of fingolimod in this study was found consistent with the previous reports. CONCLUSIONS: In healthy volunteers, the changes seen with both fingolimod doses were found to be within normal variability, non-inferior and comparable with those observed with placebo for all the pharmacodynamic parameters assessed.

Hormonal and metabolic effects of morning or evening dosing of the dipeptidyl peptidase IV inhibitor vildagliptin in patients with type 2 diabetes.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Vildagliptin is an orally active, potent inhibitor of dipeptidyl peptidase IV and was developed for the treatment of type 2 diabetes. In clinical trials, once or twice daily dosing with vildagliptin (up to 100 mg day(-1)) has been shown to reduce endogenous glucose production and fasting plasma glucose in patients with type 2 diabetes. The comparative efficacy of vildagliptin under a morning vs. evening dosing regimen has not previously been determined. WHAT THIS STUDY ADDS: Once daily dosing with vildagliptin 100 mg for 28 days improved glycaemic control in patients with type 2 diabetes independent of whether vildagliptin was administered in the morning or evening. Morning or evening dosing with vildagliptin had similar effects on 24 h glycaemic control and plasma concentrations of the hormones insulin, glucagon and glucagon-like peptide 1. AIM: This randomized, double-blind, crossover study compared post-prandial hormonal and metabolic effects of vildagliptin, (an oral, potent, selective inhibitor of dipeptidyl peptidase IV [DPP-4]) administered morning or evening in patients with type 2 diabetes. METHODS: Forty-eight patients were randomized to once daily vildagliptin 100 mg administered before breakfast or before dinner for 28 days then crossed over to the other dosing regimen. Blood was sampled frequently after each dose at baseline (day -1) and on days 28 and 56 to assess pharmacodynamic parameters. RESULTS: Vildagliptin inhibited DPP-4 activity (>80% for 15.5 h post-dose), and increased active glucagon-like peptide-1 compared with placebo. Both regimens reduced total glucose exposure compared with placebo (area under the 0-24 h effect-time curve [AUE(0,24 h)]: morning -467 mg dl(-1) h, P= 0.014; evening -574 mg dl(-1) h, P= 0.003) with no difference between regimens (P= 0.430). Reductions in daytime glucose exposure (AUE(0,10.5 h)) were similar between regimens. Reduction in night-time exposure (AUE(10.5,24 h) was greater with evening than morning dosing (-336 vs.-218 mg dl(-1) h, P= 0.192). Only evening dosing significantly reduced fasting plasma glucose (-13 mg dl(-1), P= 0.032) compared with placebo. Insulin exposure was greater with evening dosing (evening 407 microU ml(-1) h; morning 354 microU ml(-1) h, P= 0.050). CONCLUSIONS: Both morning and evening dosing of once daily vildagliptin 100 mg significantly reduced post-prandial glucose in patients with type 2 diabetes; only evening dosing significantly decreased fasting plasma glucose. Although evening dosing with vildagliptin 100 mg tended to decrease night-time glucose exposure more than morning dosing, both regimens were equally effective in reducing 24 h mean glucose exposure (AUE(0,24 h)) in patients with type 2 diabetes.

Absorption, distribution, metabolism, and elimination of the direct renin inhibitor aliskiren in healthy volunteers.

Aliskiren (2(S),4(S),5(S),7(S)-N-(2-carbamoyl-2-methylpropyl)-5-amino-4-hydroxy-2,7-diisopropyl-8-[4-methoxy-3-(3-methoxypropoxy)phenyl]-octanamid hemifumarate) is the first in a new class of orally active, nonpeptide direct renin inhibitors developed for the treatment of hypertension. The absorption, distribution, metabolism, and excretion of [(14)C]aliskiren were investigated in four healthy male subjects after administration of a single 300-mg oral dose in an aqueous solution. Plasma radioactivity and aliskiren concentration measurements and complete urine and feces collections were made for 168 h postdose. Peak plasma levels of aliskiren (C(max)) were achieved between 2 and 5 h postdose. Unchanged aliskiren represented the principal circulating species in plasma, accounting for 81% of total plasma radioactivity (AUC(0-infinity)), and indicating very low exposure to metabolites. Terminal half-lives for radioactivity and aliskiren in plasma were 49 h and 44 h, respectively. Dose recovery over 168 h was nearly complete (91.5% of dose); excretion occurred almost completely via the fecal route (90.9%), with only 0.6% recovered in the urine. Unabsorbed drug accounted for a large dose proportion recovered in feces in unchanged form. Based on results from this and from previous studies, the absorbed fraction of aliskiren can be estimated to approximately 5% of dose. The absorbed dose was partly eliminated unchanged via the hepatobiliary route. Oxidized metabolites in excreta accounted for at least 1.3% of the radioactive dose. The major metabolic pathways for aliskiren were O-demethylation at the phenyl-propoxy side chain or 3-methoxy-propoxy group, with further oxidation to the carboxylic acid derivative.

Divergent pathways of gene expression are activated by the RAGE ligands S100b and AGE-BSA.

Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers a variety of proinflammatory responses. However, our previous work revealed that RAGE-binding AGEs free of endotoxin were incapable of inducing vascular cell adhesion molecule-1 (VCAM-1) or tumor necrosis factor-alpha (TNF-alpha) expression. Thus, the objective of this study was to clarify the role of AGEs in cell activation through gene expression profiling using both in vitro and in vivo model systems. Endothelial cells treated with AGE-BSA, previously shown to bind RAGE with high affinity, did not show gene expression changes indicative of an inflammatory response. In contrast, the alternate RAGE ligand, S100b, triggered an increase in endothelial mRNA expression of a variety of immune-related genes. The effects of AGEs were studied in vivo using healthy mice exposed to two different treatment conditions: 1) intravenous injection of a single dose of model AGEs or 2) four intraperitoneal injections of model AGEs (once per day). In both cases, the liver was extracted for gene expression profiling. Both of the short-term AGE treatments resulted in a moderate increase in liver mRNA levels for genes involved in macrophage-based clearance/detoxification of foreign agents. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation.

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics.

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.