Water-stable perovskite-on-polymer fluorescent microspheres for simultaneous monitoring of pH, urea, and urease.

Perovskite materials have attracted attention due to their excellent optical and electrical properties; however, their unsatisfactory stability limits their application in biochemical detection. In this paper, CsPbBr3 perovskite quantum dots were successfully encapsulated in poly(styrene/acrylamide) microspheres, using a swelling-shrinking method. The manufactured perovskite microspheres (PDPS composites) not only maintained strong photoluminescence (PL) stability but also demonstrated great water solubility. Additionally, a real-time pH monitoring platform was constructed based on the prepared PDPS composites and dopamine, and the system showed a good linear relationship in a pH range of 4-12. Furthermore, urea could be hydrolyzed to produce hydroxyl groups, thereby increasing the pH of the solution. Therefore, this system was then extended for urea and urease detection. As a result, the detection limits of urea and urease were recorded as 1.67 µM and 2.1 mU/mL, respectively. This development provides an interesting demonstration of the expanding list of applications of perovskite materials.

Fatores de virulência e susceptibilidade a antifúngicos de Cryptococcus Spp / Virulence factors and susceptibility to Cryptococcus Spp. antifungals

Criptococose é uma doença grave que afeta tanto imunocomprometidos quanto imunocompetentes, com isso analisar a virulência é fundamental para novas terapêuticas. Objetivo: Analisar a capacidade de virulência e susceptibilidade aos antifúngicos de Cryptococcus spp. isolados de líquor de pacientes de hospital do norte do Paraná. Métodos: A partir de dois isolados clínicos C. neoformans e C. gattii, realizou-se a confirmação da identificação. Para a virulência, avaliou-se o tamanho da cápsula, capacidade de sobrevivência após exposição a neutrófilos, produção de melanina e urease. No antifungigrama por difusão em disco utilizou-se: anfotericina B, cetoconazol, voriconazol, itraconazol e miconazol. Resultados: C. gattii destaca-se por maior desenvolvimento da cápsula além da melhor capacidade de sobreviver a fagocitose em relação ao C. neoformans. No antifungigrama, ambos os isolados se apresentam sensíveis às drogas estudadas. Conclusão: Esses achados contribuem para a compreensão das diferentes patogêneses entre C. gattii e C. neoformans.

Cryptococcosis is a serious disease that can affect both immunocompromised and immunocompetent individuals, thus the virulence analysis is fundamental for the development of new treatments. Objective: To analyze the virulence and susceptibility of Cryptococcus spp. isolated from cerebrospinal fluid of patients from a hospital in the north of Paraná. Methods: From two clinical isolates, C. neoformans and C. gattii were confirmed and identified. For virulence, capsule size, survival capacity after exposure to neutrophils, melanin production and urease were evaluated. In the disc-diffusion method, the following antifungals were used: amphotericin B, ketoconazole, voriconazole, itraconazole and miconazole Results: It was observed that C. gattii presents greater results for development of the capsule beside presenting the best ability to survive phagocytosis in relation to C. neoformans. In the disc-diffusion method, both isolates presented sensitivity to the studied drugs. Conclusion: These findings contribute to the understanding of the different pathogens between C. gattii and C. neoformans.

Urease-immobilized magnetic microparticles in urine sample preparation for metabolomic analysis by gas chromatography-mass spectrometry.

Urea, as an end product of protein metabolism and an abundant polar compound, significantly complicates the metabolomic analysis of urine by GC-MS. We developed a sample preparation method removing urea from urine samples prior the GC-MS analysis. The method based on urease immobilized on magnetic microparticles was compared with the others that are conventionally used (liquid-liquid extraction, free urease protocol), and samples without any treatment. To study the impact of sample preparation approaches on the quality of analytical data, we employed comprehensive metabolomic analysis (using both GC-MS and LC-MS/MS platforms) of standard material based on human urine. Multivariate statistical analysis has shown that immobilized urease treatment provides similar results to a free urease approach. However, significant alterations in the profiles of metabolites were observed in the samples without any treatment and after the extraction. Compared to other approaches that were tested, the immobilization of urease on microparticles reduces both the number of artifacts and the variability of the metabolites (average CV of extraction 19.7%, no treatment 11.4%, free urease 5.0%, and immobilized urease 2.5%). The method that was developed was applied in a GC-MS metabolomic experiment of glutaric aciduria type I, where both known diagnostically important biomarkers and unknowns, as the most discriminating compounds, were found.

High Efficacy of Rapid Urine Test for Diagnosis of Helicobacter pylori Infection in Thai People

Background: Accurate diagnosis of Helicobacter pylori (H. pylori) infection plays an important role in further effective treatment. Rapid urine test (RAPIRUN) is a test developed for qualitative detection of urine H. Pylori antibody and use for determine the sensitivity, specificity and accuracy. However, the test needs validation in Thai population before using in clinical practice. Objective: This study aimed to compare performance of different diagnostic tests on H. pylori detection in Thai population. Methods: Total of 94 patients with dyspepsia who referred to Thammasat University Hospital, Pathumthani, Thailand, between December 2012 and April 2013 were enrolled in this study. All patients underwent gastroscopy. Then, 3 biopsies at antrum were taken for H. pylori diagnosis. including rapid urease test (Pronto Dry, Eisai, Thailand), H. pylori culture, and histopathology. Urine samples were also collected at the same time for rapid urine test (RAPIRUN H. pylori Antibody, Otsuka Pharmaceutical Co., Ltd.). Patients were diagnosed with H. pylori-positive if their culture or rapid urease tests plus histopathology yielded positive results. Results: Total of 29 patients (30.9%) were infected with H. pylori. Prevalence of H. pylori infection by rapid urease test, histopathology, culture and rapid urine test were 25.5%, 28.7%, 29.8%, and 32.9% respectively. We observed that rapid urease test, histopathology, culture, and rapid urine test had sensitivity of 82.8%, 93.1%, 93.1% and 86.2%; specificity of 100%, 100%, 100%, and 90.8%; and accuracy of 95.7%, 97.9%, 97.9%, and 89.4%, respectively. Conclusion: Rapid urine test (RAPIRUN) provided a reliable result for diagnosis of H. pylori infection. Furthermore, this rapid urine test demonstrated high accuracy, reliable, safe handle and easy to use. We suggested rapid urine test for diagnosis of H. pylori infection in Thai population since we found it less invasive and with higher reliable efficacy.

Ultrafast and Ultrasensitive Naked-Eye Detection of Urease-Positive Bacteria with Plasmonic Nanosensors.

Identifying the pathogen responsible for an infection is a requirement in order to personalize antimicrobial treatments. Detecting bacterial enzymes, such as proteases, lipases, and oxidoreductases, is a winning approach for detecting pathogens at the point of care. In this Article, a new method for detecting urease-producing bacteria rapidly and at ultralow concentrations is reported. In this method, longsome bacteriological culture steps are substituted for a 10 min capture procedure with positively charged magnetic beads. The presence of urease-positive bacteria on the particles is then queried with a plasmonic signal generation step that generates blue- or red-colored nanoparticle suspensions upon addition of the enzyme substrate. These colorimetric signals, which can be easily identified by eye, are generated by the NH3-dependent assembly of gold nanoparticles in the presence of bovine serum albumin (BSA). The proposed method can detect Proteus mirabilis with a limit of detection of 101 cells mL-1, with a total assay time of 40 min, even in the presence of a large excess of urease-negative bacteria ( Pseudomonas aeruginosa). Furthermore, it does not require bulky equipment, and it can detect P. mirabilis at clinically relevant concentrations within minutes, making it suitable for detecting urease-positive pathogens at the point of care.

A liquid crystal based method for detection of urease activity and heavy metal ions by using stimulus-responsive surfactant-encapsulated phosphotungstate clusters.

A liquid crystal (LC) based method is described for the sensitive determination of the activity of urease and of heavy metal ions which acts as inhibitors. Stimulus-responsive surfactant-encapsulated phosphotungstate clusters (SECs) were fabricated and deposited onto octadecyltrichlorosilane-coated glass. A copper TEM grid filled with LCs was placed on the substrate to construct the LC optical cell. Upon addition of water to the LC interface, the optical appearance of LCs on the glass undergoes a bright-to-dark shift due to an orientational transition of the LCs from a planar to a homeotropic state. However, the LCs display a bright appearance if they are pretreated with an aqueous solution containing urea and urease. This is caused by the disassemby of the SECs from the glass surface due to an increase of the pH value that is induced by the enzymatic hydrolysis of urea by urease. The method is highly sensitive and can detect urease activities as low as 0.03 mU/mL. It can also be applied to the determination of heavy metal ions which exert an inhibitory effect on the activity of urease. For example, Cu(II) can be quantified via urease inhibition in 1 nM concentration. Graphical abstract Schematic presentation of a liquid crystal-based sensor for detection of urease and heavy metal ions by using stimulus-responsive surfactant-encapsulated phosphotungstate clusters.

Chromoendoscopy with red phenol in the diagnosis of Helicobacter pylori infection.

An analytic study to validate a diagnostic test was carried out at the Institute of Gastroenterology in Havana, Cuba in adult patients of both sexes in whom chromoendoscopy was carried out with red phenol at 0.1% over the gastric mucosa for the detection of Helicobacter pylori infection between November 2008 and December 2010. The staining with red phenol at 0.1% is included in the invasive tests for the diagnosis of Helicobacter pylori infection and of the reactive techniques. The sensibility of red phenol dye in the diagnosis of Helicobacter pylori infection in the patients studied was of 72.6% with a confidence interval (C.I.) of 95% (64.9 to 79.2%) and a specificity of 75.5% C.I. 95% (61.9 to 85.4%). The positive predictive value was of 89.8% C.I. 95% (83.1 to 94.1%) and the negative predictive value of 48.1% C.I. 95% (37.3 to 59.0%). The proportion of false positives was of 24.5% C.I. 95% (14.6 to 38.1%)and the proportion of false negatives was of 27.4% C.I. 95% (20.8 to 35.1%). The diagnostic accuracy of the dye on the patients studied was 73.3% C.I. 95% (66.7 to 79.0%). The diagnostic odds ratio was 8.17 C.I. 95% (3.88 to 17.23), the J Youden ratio of 0.5 and the Kappa coefficient of 0.40 C.I. 95% (0.27 to 0.54). The staining dye with red phenol at 0.1% resulted in a useful method in the diagnosis of Helicobacter pylori infection in the gastric mucosa, it can be applied in our environment and has multiple advantages (topographic localization, avoids contamination and fast and immediate reading).

An evaluation of a new in-house serum and urine ELISA test for detection of Helicobacter pylori infection in Thai population.

OBJECTIVE: Non-invasive tests play significant roles in the test-and-treat approach of Helicobacter pylori management. The detection of Helicobacter pylori antibodies in urine and serum is an easy and inexpensive way to diagnose this infection. In the present study, the authors developed an in-house serum and urine ELISA tests for H. pylori antibodies and evaluated their performance in a Thai population. MATERIAL AND METHOD: One hundred thirty eight dyspeptic patients were recruited. All subjects underwent upper endoscopy and one antral biopsy was obtained for rapid urease test, which was used as a standard reference. Urine and serum samples were collected before the procedure to run in-house ELISA test. RESULTS: Thirty (22%) subjects were positive for the rapid urease test and 108 (78%) were negative. Urine and serum optical density were significantly lower in the urease negative group (p = 0.011 and p < 0.001 respectively), while there were no differences in age, gender, or endoscopic findings between the two groups. Sensitivity, specificity, negative predictive value, positive predictive value, and accuracy of urine and serum ELISA tests were 72% vs. 96.3%, 63.5% vs. 627%, 89.6% vs. 98.5%, 33.3% vs. 40.6%, and 64.5% vs. 69.8% respectively. CONCLUSION: In-house serum ELISA test for H. pylori antibodies yielded a very good sensitivity with acceptable specificity, whereas urine ELISA was unable to produce satisfactory sensitivity or specificity

[Laboratory methods of predicting primary and recurrent nephrolithiasis]. / Laboratornye metody prognozirovaniia pervichnogo i retsidivnogo kamneobrazovaniia v pochkakh..

Concentrations of Na, K, Ca ions, oxalic and uric acids, total Ca, P as well as urease activity, the ability to form crystals, speed of crystallization, crystal chemical composition, pH stability were evaluated in the urine from 40 urolithiasis patients and 40 patients with recurrent nephroliths. It was found that fast or moderate crystallization, high urease activity and ability to form crystals, unstable urine pH indicate a high risk of both primary and recurrent nephrolith formation.

[Urinary urease activity in urolithiasis patients]. / Ureaznaia aktivnost' mochi u bol'nykh mochekamennoi bolezn'iu.

Urease activity (UA) and the ability of the urine to form crystals (AFC) were assessed in the urine from 149 urolithiasis patients and 11 healthy controls. Patients with primary uroliths had high UA and AFC in 17-18% of the cases, in recurrent urolithiasis this percentage was 29-35%. The speed of phosphate and oxalate crystals formation as well as formation of renal calculus correlated with UA and UA levels. Thus, UA and AFC may be employed as criteria in prognostication of the duration of phosphate and oxalate lithogenesis.

Urease-induced crystallizations of calcium phosphate and magnesium ammonium phosphate in synthetic urine and human urine.

An aggregometer technique was used to study urease-induced crystallizations in synthetic urine and human urine from healthy subjects and patients with chronic spinal cord injuries. The two different phases of crystallization, calcium phosphate and magnesium ammonium phosphate, were easily evaluated with a single assay using this technique. The crystallization of calcium phosphate and magnesium ammonium phosphate varied markedly among the different urine specimens after incubation with urease. The turbidity curves from human urine were divided into four patterns. We assumed that the variations in the patterns of the turbidity curves appeared to be mainly due to differences in the composition of the urine and in the original pH, and that the calcium and magnesium concentrations were very important in the urinary constituents.

The possible value of ascorbic acid as a prophylactic agent for urinary tract infection.

The effect of ascorbic acid on urine pH was studied in spinal cord injury patients. Their urine was not colonized by urease positive microorganisms. The study was designed to compare the baseline urine pH value and the urine pH value after the administration of placebo or ascorbic acid 500 mg/6 h. The diet and medical treatment were not controlled. A significant decrease in urine pH value was not obtained. There was no clinical benefit from the use of ascorbic acid.

Continuous beds. Their applicability for immobilization of proteins.

An epoxy-activated continuous bed can be prepared for immobilization of proteins in a simple, rapid, and cost-effective way. The concentration of epoxy groups on the continuous bed was as high as 600 mumol/mL compressed bed (compression of the bed decreases the peak broadening). Human transferrin, human serum albumin and particularly urease were employed as model proteins. The immobilization of urease was virtually completed within 1 h in 1 M potassium phosphate, pH 7.4. The binding capacity was 97 mg of urease/mL compressed bed. This bed is of clinical interest, since it is inexpensive to prepare and permits reproducible enzymatic determination of urea in serum and urine (the chromatographic step is finished within 1-2 min).

Citrate and urease-induced crystallization in synthetic and human urine.

The effects of citrate on the different phases of urease-induced crystallization were studied using Coulter counter techniques and optical microscopy. Citrate increased urine pH and markedly delayed the initiation of the crystallization (nucleation) in both human and synthetic urine. In synthetic urine, particle aggregation and especially particle growth were delayed and inhibited by citrate. In human urine, aggregation was distinctly inhibited by citrate. It appears that the susceptibility of urine to form crystals in the presence of urease activity is influenced by its citrate concentration.

Influence of Escherichia coli on urease-induced crystallisation in human urine.

Urease was added to urines inoculated with Escherichia coli 24 hours earlier and to control urines not inoculated with E. coli. The inoculation did not change the concentration of the measured urine components. The urease-induced ammonium ion production and pH increase was reduced in E. coli-inoculated urines compared to control urines. This suggests that E. coli can inhibit urease. The precipitation of both phosphate and magnesium on glass rods inserted in the urine was reduced with 40-50% in the E. coli-inoculated urines. The results demonstrate that E. coli can influence urease-induced crystallisation.

Inhibition of urease activity by dipeptidyl hydroxamic acids.

A series of dipeptidyl hydroxamic acids (H-X-Gly-NHOH: X = amino acid residues) was synthesized, and the inhibitory activity against Jack bean and Proteus mirabilis ureases [EC 3.5.1.5] was examined. A number of H-X-Gly-NHOH inhibited Jack bean urease with an I50 of the order of 10(-6) M and inhibited Proteus mirabilis urease with an I50 of the order of 10(-5) M. The inhibition against Jack bean urease was more potent than that with the corresponding aminoacyl hydroxamic acids (H-X-NHOH).

The influence of pH and urine composition on urease enzymatic activity in human urine.

It is reasonable to assume that the rate of pH increase in urine induced by urease-producing microorganisms is one of the factors which determine whether crystallisation with subsequent stone formation will occur or not. To evaluate how the time needed to increase urine pH varies between different urine samples and how it depends on urine composition, a standardised amount of urease was added to different human urine samples. The incubations were performed in a pH-stat. This allowed simultaneous study of how urease enzymatic activity depends on urine pH and how it varies between different urines. The enzymatic activity was found to be negatively correlated to urine pH and to vary between different urines. The rate of the pH increase varied markedly between different urines. Small pH increases depended on the native urine pH and urease enzymatic activity. Higher pH increases up to the levels of phosphate crystallisation depended more on urine phosphate, the major urine buffer. The results presented show that urine composition influences the urease-induced pH increase. This might have clinical implications.

E. coli and urease-induced crystallisation in urine.

The effects of urine preinoculation with E. coli for 20 h on urease activities in urine have been studied in synthetic as well as human urine. The E. coli preinoculation increased pH in both synthetic and human urine. Urease enzymatic activity was enhanced in E. coli-preinoculated synthetic urine. The intraluminal urease-induced precipitation was increased in E. coli-preinoculated synthetic urine. The precipitation on glass rods, which more closely reflects crystal growth and aggregation, was reduced. The process of urease-induced crystallisation thus appears to be influenced by E. coli. In human urine, the effects of E. coli preinoculation were less uniform but a significantly increased urease-induced precipitation after E. coli preinoculation could be reproduced in human urine also.

[Detection of ureolytic bacteria in urine in urinary calculi]. / Nachweis harnstoffspaltender Keime im Urin bei Urolithiasis.

Detection of ureolytic bacteria in the urine of stone patients: using a very sensitive and selective indicator medium we tested the urine of 308 stone patients for ureolytic bacteria. Urease-producing bacteria were found in 41 patients. In the urine of 13 of these patients we found more than 10(4) bacteria/ml and in 28 patients 10(4) or less. 75% of the patients with a positive urease test had infection stones. We believe, that the test for urease is a convenient and necessary completion of the bacteriologic-diagnostic measures.