Diagnostic accuracy of urine biomarkers for urinary tract infection in older women: a case-control study.

OBJECTIVES: Urinary tract infection (UTI) is common among older women. However, diagnosis is challenging because of frequent chronic lower urinary tract symptoms, cognitive impairment, and a high prevalence of asymptomatic bacteriuria (ASB). Current urine diagnostics lack specificity, leading to unnecessary treatment and antimicrobial resistance. This study aimed to evaluate the diagnostic accuracy of 12 urine biomarkers for diagnosing UTI in older women. METHODS: In this case-control study, cases were women ≥65 years with ≥2 new-onset lower urinary tract symptoms, pyuria, and one uropathogen ≥104 CFU/mL. Controls were asymptomatic and classified as ASB (one uropathogen ≥105 CFU/mL), negative culture, or mixed flora. Urine biomarker concentrations were measured through liquid chromatography-mass spectrometry and ELISA. Diagnostic accuracy parameters of individual biomarkers and a biomarker model were derived from receiver operating characteristic curves. RESULTS: We included 162 community-dwelling and institutionalized older women. Five urine inflammatory biomarkers demonstrated high discriminative ability (area under the curve ≥0.80): interleukin 6, azurocidin, neutrophil gelatinase-associated lipocalin, tissue inhibitor of metalloproteinases 2, and C-X-C motif chemokine 9. Azurocidin exhibited the highest diagnostic accuracy (sensitivity 86% [95% CI 75%-93%] and specificity 89% [95% CI 82%-94%] at 16.7 ng/mmol creatinine). A combined biomarker and pyuria model showed improved diagnostic accuracy in patients with UTI and ASB, compared with pyuria alone. DISCUSSION: We identified several urine biomarkers that accurately differentiated older women with UTI from asymptomatic women, including ASB. These findings represent a potential advancement towards improved diagnostics for UTI in older women and warrant validation in a diverse population.

Current Pyuria Cutoffs Promote Inappropriate Urinary Tract Infection Diagnosis in Older Women.

BACKGROUND: Pre-existing lower urinary tract symptoms (LUTS), cognitive impairment, and the high prevalence of asymptomatic bacteriuria (ASB) complicate the diagnosis of urinary tract infection (UTI) in older women. The presence of pyuria remains the cornerstone of UTI diagnosis. However, >90% of ASB patients have pyuria, prompting unnecessary treatment. We quantified pyuria by automated microscopy and flowcytometry to determine the diagnostic accuracy for UTI and to derive pyuria thresholds for UTI in older women. METHODS: Women ≥65 years with ≥2 new-onset LUTS and 1 uropathogen ≥104 colony-forming units (CFU)/mL were included in the UTI group. Controls were asymptomatic and classified as ASB (1 uropathogen ≥105 CFU/mL), negative culture, or mixed flora. Patients with an indwelling catheter or antimicrobial pretreatment were excluded. Leukocyte medians were compared and sensitivity-specificity pairs were derived from a receiver operating characteristic curve. RESULTS: We included 164 participants. UTI patients had higher median urinary leukocytes compared with control patients (microscopy: 900 vs 26 leukocytes/µL; flowcytometry: 1575 vs 23 leukocytes/µL; P < .001). Area under the curve was 0.93 for both methods. At a cutoff of 264 leukocytes/µL, sensitivity and specificity of microscopy were 88% (positive and negative likelihood ratio: 7.2 and 0.1, respectively). The commonly used cutoff of 10 leukocytes/µL had a poor specificity (36%) and a sensitivity of 100%. CONCLUSIONS: The degree of pyuria can help to distinguish UTI in older women from ASB and asymptomatic controls with pyuria. Current pyuria cutoffs are too low and promote inappropriate UTI diagnosis in older women. Clinical Trials Registration. International Clinical Trials Registry Platform: NL9477 (https://trialsearch.who.int/Trial2.aspx?TrialID=NL9477).

Moving average quality control of routine chemistry and hematology parameters - a toolbox for implementation.

OBJECTIVES: Moving average quality control (MA QC) is a patient-based real-time quality control system. Advantages compared to conventional periodic internal quality control (IQC) include absence of commutability problems and continuous monitoring of performance. We implemented MA QC for multiple routine hematology and chemistry parameters. We describe the evaluation process and provide practical tools to aid MA QC implementation. METHODS: Nine parameters (serum sodium, calcium, bicarbonate and free thyroxine, hemoglobin [Hb], mean corpuscular volume, mean corpuscular hemoglobin concentration [MCHC], reticulocyte count and erythrocyte sedimentation rate [ESR]) were chosen for initial consideration. Using data extractions from the laboratory information system (LIS; General Laboratory Information Management System), evaluation of usefulness and optimization of MA QC settings was performed using bias detection curves. After this, MA QC settings were incorporated in our LIS for further evaluation and implementation in routine care. RESULTS: Three out of nine parameters (Hb, ESR, and sodium) were excluded from MA QC implementation due to high variation and technical issues in the LIS. For the six remaining parameters, MA QC showed added value to IQC and was therefore implemented in the LIS. For three parameters a direct MA alarm work-up method was set up, including newly developed built-in features in the LIS. For the other parameters, we identified MA utilization beyond real-time monitoring. CONCLUSIONS: Implementation of MA QC has added value for our laboratory setting. Additional utilization beyond real-time QC monitoring was identified. We find MA QC especially useful for trend monitoring, detection of small shifts after maintenance and inter-analyzer comparisons.

Highly Specific Protein Identification by Immunoprecipitation-Mass Spectrometry Using Antifouling Microbeads.

A common method to study protein complexes is immunoprecipitation (IP), followed by mass spectrometry (thus labeled: IP-MS). IP-MS has been shown to be a powerful tool to identify protein-protein interactions. It is, however, often challenging to discriminate true protein interactors from contaminating ones. Here, we describe the preparation of antifouling azide-functionalized polymer-coated beads that can be equipped with an antibody of choice via click chemistry. We show the preparation of generic immunoprecipitation beads that target the green fluorescent protein (GFP) and show how they can be used in IP-MS experiments targeting two different GFP-fusion proteins. Our antifouling beads were able to efficiently identify relevant protein-protein interactions but with a strong reduction in unwanted nonspecific protein binding compared to commercial anti-GFP beads.

Systematic Comparison of Zwitterionic and Non-Zwitterionic Antifouling Polymer Brushes on a Bead-Based Platform.

Nonspecific adsorption of biomolecules to solid surfaces, a process called biofouling, is a major concern in many biomedical applications. Great effort has been made in the development of antifouling polymer coatings that are capable of repelling the nonspecific adsorption of proteins, cells, and micro-organisms. In this respect, we herein contribute to understanding the factors that determine which polymer brush results in the best antifouling coating. To this end, we compared five different monomers: two sulfobetaines, a carboxybetaine, a phosphocholine, and a hydroxyl acrylamide. The antifouling coatings were analyzed using our previously described bead-based method with flow cytometry as the read-out system. This method allows for the quick and automated analysis of thousands of beads per second, enabling fast analysis and good statistics. We report the first direct comparison made between a sulfobetaine with opposite charges separated by two and three methylene groups and a carboxybetaine bearing two separating methylene groups. It was concluded that both the distance between opposite charges and the nature of the anionic groups have a distinct effect on the antifouling performance. Phosphocholines and simple hydroxyl acrylamides are not often compared with the betaines. However, here we found that they perform equally well or even better, yielding the following overall antifouling ranking: HPMAA ≥ PCMA-2 ≈ CBMAA-2 > SBMAA-2 > SBMAA-3 ≫ nonmodified beads (HPMAA being the best).

Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry.

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

Direct Creation of Biopatterns via a Combination of Laser-Based Techniques and Click Chemistry.

In this paper, we present the immobilization of thiol-modified aptamers on alkyne- or alkene-terminated silicon nitride surfaces by laser-induced click chemistry reactions. The aptamers are printed onto the surface by laser-induced forward transfer (LIFT), which also induces the covalent bonding of the aptamers by thiol-ene or thiol-yne reactions that occur upon UV irradiation of the thiol-modified aptamers with ns laser pulses. This combination of LIFT and laser-induced click chemistry allows the creation of high-resolution patterns without the need for masks. Whereas the click chemistry already takes place during the printing process (single-step process) by the laser pulse used for the printing process, further irradiation of the LIFT-printed aptamers by laser pulses (two-step process) leads to a further increase in the immobilization efficiency.

Water-repairable zwitterionic polymer coatings for anti-biofouling surfaces.

We developed a strategy to prepare new types of zwitterionic polymer network (ZPN) coatings that display excellent self-healing and anti-biofouling properties. Upon mechanical damage (scratching), the coatings can be easily repaired within 1 min by immersion in water, and this self-healing property can even be achieved for nanoscale-thick coatings. Both the mechanical properties and the anti-biofouling characteristics are repaired after healing.

Efficient and Tunable Three-Dimensional Functionalization of Fully Zwitterionic Antifouling Surface Coatings.

To enhance the sensitivity and selectivity of surface-based (bio)sensors, it is of crucial importance to diminish background signals that arise from the nonspecific binding of biomolecules, so-called biofouling. Zwitterionic polymer brushes have been shown to be excellent antifouling materials. However, for sensing purposes, antifouling does not suffice but needs to be combined with the possibility to efficiently modify the brush with recognition units. So far this has been achieved only at the expense of either antifouling properties or binding capacity. Herein we present a conceptually new approach by integrating both characteristics into a single tailor-made monomer: a novel sulfobetaine-based zwitterionic monomer equipped with a clickable azide moiety. Copolymerization of this monomer with a well-established standard sulfobetaine monomer results in highly antifouling surface coatings with a large yet tunable number of clickable groups present throughout the entire brush. Subsequent functionalization of the azido brushes via widely used strain-promoted alkyne azide click reactions yields fully zwitterionic 3D-functionalized coatings with a recognition unit of choice that can be tailored for any specific application. Here we show a proof of principle with biotin-functionalized brushes on Si3N4 that combine excellent antifouling properties with specific avidin binding from a protein mixture. The signal-to-noise ratio is significantly improved over that of traditional chain-end modification of sulfobetaine polymer brushes, even if the azide content is lowered to 1%. This therefore offers a viable approach to the development of biosensors with greatly enhanced performance on any surface.

Quality Control of Trichinella Testing at the Slaughterhouse Laboratory: Evaluation of the Use of a 400-Micrometer-Mesh-Size Sieve in the Magnetic Stirrer Method.

The performance of a 400-µm-mesh-size sieve (sieve400) has not previously been compared with that of a 180-µm-mesh-size sieve (sieve180). Using pork samples spiked with 0 to 10 Trichinella muscle larvae and an artificial digestion method, sieve performance was evaluated for control of Trichinella in meat-producing animals. The use of a sieve400 resulted in 12% lower larval counts, 147% more debris, and 28% longer counting times compared with the use of a sieve180. Although no false-negative results were obtained, prolonged counting times with the sieve400 may have an impact on performance in a high-throughput environment such as a slaughterhouse laboratory. Based on our results, the sieve180 remains the sieve of choice for Trichinella control in meat in slaughterhouse laboratories, according to the European Union reference method (European Commission regulation 2075/2005). Furthermore, the results of the present study contribute to the discussion of harmonization of meat inspection requirements among countries.

A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2.

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2.

Mono-fluorinated alkyne-derived SAMs on oxide-free Si(111) surfaces: preparation, characterization and tuning of the Si workfunction.

Organic monolayers derived from ω-fluoro-1-alkynes of varying carbon chain lengths (C(10)-C(18)) were prepared on Si(111) surfaces, resulting in changes of the physical and electronic properties of the surface. Analysis of the monolayers using XPS, Infrared Reflection Absorption Spectroscopy, ellipsometry and static water contact angle measurements provided information regarding the monolayer thickness, the tilt angle, and the surface coverage. Additionally, PCFF molecular mechanics studies were used to obtain information on the optimal packing density and the layer thickness, which were compared to the experimentally found data. From the results, it can be concluded that the monolayers derived from longer chain lengths are more ordered, possess a lower tilt angle, and have a higher surface coverage than monolayers derived from shorter chains. We also demonstrate that by substitution of an H by F atom in the terminal group, it is possible to controllably modify the surface potential and energy barrier for charge transport in a full metal/monolayer-semiconductor (MOMS) junction.

Effect of processed and fermented soyabeans on net absorption in enterotoxigenic Escherichia coli-infected piglet small intestine.

Infectious diarrhoea is a major problem in both children and piglets. Infection of enterotoxigenic Escherichia coli (ETEC) results in fluid secretion and electrolyte losses in the small intestine. In the present study the effect of processed and fermented soyabean products on net absorption during ETEC infection was investigated. Soyabean was processed into an autoclaved, a cooked and a mould-fermented (tempeh) product. The soyabean products were pre-digested and the effect of the products on net absorption in the small intestine of piglets was studied. Pairs of small-intestinal segments, one non-infected and the other ETEC-infected, were perfused simultaneously with the different products during 8 h. Net absorption of fluid, DM, Na, chloride, K and total solutes was determined. Net fluid absorption was highest for cooked soyabean followed by autoclaved soyabean and tempeh as a result of the osmolality of these products. In ETEC-infected segments, cooked soyabean and tempeh showed minor fluid losses (27 (SE 23) and 43 (SE 20) microl/cm(2), respectively) compared with the saline control (260 (SE 23) microl/cm(2)). Tempeh resulted in a high uptake of solutes. Processed soyabean products, particularly cooked soyabean and tempeh, are beneficial in maintaining fluid balance during ETEC infection. Additionally, tempeh showed high DM and total solute absorption. Therefore, particularly, tempeh may be beneficial in the case of post-weaning diarrhoea in piglets and possibly in children as well.