Clarithromycin-based regimens are commonly used as a first-line therapy for Helicobacter pylori-positive patients; however, resistance to clarithromycin has led to treatment failures. The aim of this study was to evaluate the feasibility of using stool samples to detect the presence of H. pylori DNA while concurrently detecting mutations associated with resistance to clarithromycin. For this purpose, total DNA was extracted from 294 raw stool specimens from H. pylori-positive and -negative patients. TaqMan real-time PCR amplification was used to detect the presence of H. pylori as well as to predict the phenotype of the organism and the related outcome for patients treated with clarithromycin. Clarithromycin resistance was determined upon analysis of the PCR result. Patients were also tested by a urea breath test and were subjected to esophagogastroduodenoscopy, followed by histology, culture, and a rapid urease test, in order to obtain a consensus patient infection status. Of 294 total stool samples, 227 were deemed true positive. The sensitivity of H. pylori detection by PCR was 93.8%. Of 213 true-positive samples that were sequenced, 36.2% showed point mutations associated with clarithromycin resistance (A2142C, A2142G, A2143G). The final correlation of the mutant genotypes as determined by sequencing with the eradication of infection was 86%. We found that Helicobacter pylori DNA can be detected in human stool specimens with high sensitivity and can therefore be used to determine the presence of the bacterium without obtaining a biopsy sample. Moreover, genotypic resistance to clarithromycin can be predicted without obtaining a biopsy sample, facilitating the choice of the right therapeutic approach.

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources.

We studied hemolytic activity of gold nanoparticles added to the whole blood (ex vivo) and of nanoparticles coated and not coated with plasma components on erythrocytes in hypotonic medium (osmotic hemolysis) in vitro. Gold nanoparticles did not stimulate erythrocyte hemolysis after 4-h incubation with the whole blood ex vivo. Hemolysis tended to increase in the presence of small gold nanoparticles (5, 10, 20 nm) at the maximum concentration of 20 μM (by gold content) used in our study in comparison with the control. This tendency was detected during the 1st hour of the nanoparticles incubation with blood. Gold nanoparticles in the used concentrations (up to 20 μM of gold) coated with plasma components after preincubation with autologous plasma and nanoparticles without coating caused no osmotic hemolysis of erythrocytes in vitro.

The dynamics of changes in albumin transport function during hypochlorite-induced oxidation of isolated albumin in blood plasma and serum was studied with a fluorescent probe K-35. Binding of the probe K-35 to albumin was characterized by effective concentration of albumin. Oxidative modification of proteins was evaluated by the content of carbonyl products of protein oxidation and bityrosine fluorescent products. Oxidation with hypochlorite was accompanied by a decrease in the effective concentration of albumin in albumin, diluted plasma, and serum and accumulation of carbonyl products of protein oxidation and bityrosine fluorescent products. The decrease in the effective concentration of albumin during oxidation with hypochlorite can be explained by oxidative damage to albumin binding sites. Oxidative modification of probe K-35 binding sites with hypochlorite contributes to a decrease in effective concentration of albumin under pathological conditions.

It has become critical to detect Helicobacter pylori (H. pylori) infection due to the link to gastric cancer with some strains. These strains are also increasing in resistance to antibiotics with clarithromycin leading the way as the first line treatment. Resistance to clarithromycin has been shown to correlate with the A2142G, A2142C, and A2143G mutations on the rrl gene. In the last few decades, non-invasive specimens, such as stool, have been a reliable alternate to gastric biopsy for immunoassay tests. More recently, it has been proven feasible for stool to be used in molecular based tests. Many of the core laboratories in the United States need a high throughput sample preparation to run this test. Here, a high throughput assay is compared to a previously published manual sample prep H. pylori molecular based assay. Using the Magna Pure 96 (Roche), at least 96 stool species and 96 biopsy specimens can be tested in an 8-hour shift of a clinical lab. The high throughput sample prep had a positive percent agreement (PPA) of 87% compared to the manual sample prep using the same testing configuration. The genotype predictions from the high throughput assay matched genotype predictions from the manual sample prep with the same stool sample 92% of the time. A concordance rate of 89% was observed with genotype predictions from the high throughput assay of the same patient stool and biopsy. In stool samples from the high throughput assay, there was 100% concordance between the quantitative polymerase chain reaction (qPCR)-derived genomic prediction and DNA sequencing data. The high throughput workflow can get more patients tested faster in addition to detection of mutations associated with clarithromycin resistance.

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The dynamics of albumin transport function was studied during metal-catalyzed oxidation of albumin in diluted blood plasma from healthy donors and in the solution of purified albumin using fluorescent probe K-35. The changes were compared with the dynamics of free radical oxidation markers. For oxidation, different concentrations of Cu(2+), Fe(2+), Fe(3+) ions as well as EDTA and H(2)O(2) were used. Oxidative modification of proteins was assessed by carbonyl and bityrosine fluorescent products. Oxidation of plasma lipids was assessed by the levels of TBA-reactive products. It was found that oxidation markedly decreased effective concentration of albumin characterizing albumin binding capacity, and leads to accumulation of carbonyl products of protein oxidation, bityrosine fluorescent products in proteins, and TBA-active products of lipid oxidation. It was hypothesized that reduced effective concentration of albumin is related to impairment of its binding sites and/or accumulation of free-radical oxidation products filling the binding sites of albumin.

An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae.