Michael J. Pugia

Abstract: Timed urine collections are difficult to use in clinical practice owing to inaccurate collections making calculations of the 24-h albumin or protein excretion questionable. One of our goals was to assess the 'correction' of urinary albumin and (or) protein excretion by dividing these by either the creatinine concentration or the term, (specific gravity-1)x100(1). The 24-h creatinine excretion can be estimated based on the patients' gender, age and weight. We studied the influence of physiological extremes of hydration and exercise, and protein and creatinine excretion in patients with or suspected kidney disorders. Specimens were collected from healthy volunteers every 4 h during one 24-h period. We assayed the collections individually to give us an assessment of the variability of the analytes with time, and then reassayed them after combining them to give a 24-h urine. For all volunteers, the mean intra-individual CVs based on the 4-h collections expressed in mg/24 h were 80.0% for albumin and 96.5% for total protein (P0.2). The CVs were reduced by dividing the albumin or protein concentration by the creatinine concentration or by the term, (SG-1)x100. This gave a CV for mg albumin/g creatinine of 52% (P<0.1 vs. albumin mg/g creatinine); mg protein/g creatinine of 39% (P<0.05 vs. mg protein/g creatinine); mg albumin/[(SG-1)x100] of 49% (P<0.1 vs. albumin)/[(SG-1)x100]; and mg protein/[(SG-1)x100] of 37% (P<0. 05 vs. mg protein)/[(SG-1)x100]. For the 68 subjects in the study, the strongest correlation was between the creatinine concentrations and the 24-h urine volume: r=0.786, P<0.001. The correlation of (SG-1)x100 vs. the 24-h urine volume was: r=0.606, P<0.001; for (SG-1)x100 and the creatinine concentration, the correlation was: r=0.666, P<0.001. Compared to the volunteers, the albumin and protein excretion in mg/24 h were more variable in the patients. The same was true if the albumin or protein concentrations were divided by the creatinine concentration or by (SG-1)x100. Protein and albumin concentrations were lower in dilute urines. Dividing the albumin or protein concentrations by the creatinine concentration reduced the number of false negative protein and albumin results. Dividing the albumin or protein values in mg/24 h by (SG-1)x100 eliminated fewer false negatives. Albumin concentrations increased significantly after vigorous exercise. The increase was almost eliminated when the albumin result was divided by the creatinine concentration suggesting that a decreased urine flow and not increased glomerular permeability causes an increase of post-exercise albuminuria. The same was true for proteinuria. A dipstick test plus an optical strip reader that can measure urine protein, albumin, and creatinine and calculate the appropriate ratios provides a better screening test for albuminuria or proteinuria than one measuring only albumin or protein.

Abstract: Infectious disease, commonly caused by bacterial pathogens, is now the world's leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations.

Abstract: Inflammation is an important indicator of tissue injury. In the acute form, there is usually accumulation of fluids and plasma components in the affected tissues. Platelet activation and the appearance in blood of abnormally increased numbers of polymorphonucleocytes, lymphocytes, plasma cells and macrophages usually occur. Infectious disorders such as sepsis, meningitis, respiratory infection, urinary tract infection, viral infection, and bacterial infection usually induce an inflammatory response. Chronic inflammation is often associated with diabetes mellitus, acute myocardial infarction, coronary artery disease, kidney diseases, and certain auto-immune disorders, such as rheumatoid arthritis, organ failures and other disorders with an inflammatory component or etiology. The disorder may occur before inflammation is apparent. Markers of inflammation such as C-reactive protein (CRP) and urinary trypsin inhibitors have changed our appraisal of acute events such as myocardial infarction; the infarct may be a response to acute infection and (or) inflammation. We describe here the pathophysiology of an anti-inflammatory agent termed urinary trypsin inhibitor (uTi). It is an important anti-inflammatory substance that is present in urine, blood and all organs. We also describe the anti-inflammatory agent bikunin, a selective inhibitor of serine proteases. The latter are important in modulating inflammatory events and even shutting them down.

Abstract: We developed a dye-binding method for albumin in urine based on bis (3',3"-diiodo4'4"-dihydroxy-5'5"-dinitrophenyl)-3,4,5,6-tetrabr omosulfonphthalein (DIDNTB), a dye that has a higher chemical sensitivity and specificity for albumin when compared to two other commonly used dyes. We prepared urine dipsticks with DIDNTB and certain other compounds to prevent "nonspecific" binding to the dipstick matrix. The detection limit for albumin with DIDNTB as the dye is about 10 mg/L. The extent of dye binding to proteins and other compounds was studied using ultracentrifugation and a selectively permeable membrane that permitted the passage of free but not bound dye; we believe this method is superior to photometric titration. The affinity of the dyes for albumin was found to be pH dependent with stronger binding at pH 1.8 than at pH 7.0. At pH 1.8, DIDNTB had a ca.10-fold greater binding coefficient to albumin when compared to the widely used dyes, tetrabromophenol blue (CI 4430-25-5) or bromophenol blue (CI 115-39-9). We developed a system that minimized nonspecific binding by the dye through the use of polymethyl vinyl ethers and bis-(heptapropylene glycol) carbonate. DIDNTB showed a greater chemical specificity for albumin when compared to most other proteins. The new albumin dipsticks are resistant to many potential interferences at substantial concentrations, making the dipsticks suitable to screen for albuminuria.

Abstract: We describe a new dip- and read dipstick that detects urine albumin at concentrations of 10 mg/l and above and urine creatinine at concentrations of 300 mg/l and above The albumin assay is based on a high-affinity, dye-binding technique while the creatinine assay is based on the peroxidase-like activity of copper creatinine complexes With these two-test dipsticks, urines from normal adults supplemented with albumin and creatinine were correctly identified to within +/- 15% of the expected value for both analytes; the between-day coefficients of variation ranged from 71% to 161% We tested 275 patients' unmodified urines by the Bayer and Boehringer Mannheim Micral-Test albumin dipsticks and for albumin with the Beckman Array on the same specimens We also analyzed 42 selected urines from the group of 275 for albumin by another quantitative immunochemical method and by electrophoresis plus a total protein method to estimate the albumin concentration The quantitative immunochemical methods appear to underestimate the urine albumin concentrations; in these 42 urines measured as negative, ie, or = 16 mg/l at an 80% rate At a cutoff of 20 mg/l, the rate increased to 87% We also determined the urinary albumin/creatinine ratios on the 275 patients using the Bayer two-pad dipstick and found agreement 84% of the time with the same ratio obtained from a quantitative immunochemical method for albumin and a rate-Jaffe method for creatinine; an albumin/creatinine ratio (mg/g) of 30 was used as the discrimination point Albumin stability studies performed on the Beckman Array patients with six fresh urines showed small but consistent decreases at -20 degrees C but not at 4 degrees C after one month of storage The albumin in contrived urines, as estimated by electrophoreses/total protein and by the dipsticks did not change at these storage conditions Boric acid at 1 g/l as a urine preservative had no effect on the measurement of albumin by any of the methods described here nor of the assay of creatinine Other urinary proteins present at abnormal excretion rates did not interfere with the Bayer albumin dipstick Abnormal concentrations of bilirubin, citrate, creatine, ascorbic acid, albumin, hemoglobin and myoglobin in urine did not interfere with the creatinine dipstick measurements The first four of the above did not affect the Bayer dipstick results for albumin

Abstract: Introduction: Chronic kidney disease as a public health problem. Chronic kidney disease is a worldwide public health problem. In the United States, there is a rising incidence and prevalence of kidney failure, with poor outcomes and high cost. There is an even higher prevalence of earlier stages of chronic kidney disease. Increasing evidence, accrued in the past decades, indicates that the adverse outcomes of chronic kidney disease, such as kidney failure, cardiovascular disease, and premature death, can be prevented or delayed. Earlier stages of chronic kidney disease can be detected through laboratory testing. Treatment of earlier stages of chronic kidney disease is effective in slowing the progression toward kidney failure. Initiation of treatment for cardiovascular risk factors at earlier stages of chronic kidney disease should be effective in reducing cardiovascular disease events both before and after the onset of kidney failure. Unfortunately, chronic kidney disease is "under-diagnosed" and "under-treated" in the United States, resulting in lost opportunities for prevention. One reason is the lack of agreement on a definition and classification of stages in the progression of chronic kidney disease. A clinically applicable classification would be based on laboratory evaluation of the severity of kidney disease, association of level of kidney function with complications, and stratification of risks for loss of kidney function and development of cardiovascular disease. Charge to the K/DOQI work group on chronic kidney disease. In 2000, the National Kidney Foundation (NKF) Kidney Disease Outcome Quality Initiative (K/DOQI) Advisory Board approved development of clinical practice guidelines to define chronic kidney disease and to classify stages in the progression of chronic kidney disease. The Work Group charged with developing the guidelines consisted of experts in nephrology, pediatric nephrology, epidemiology, laboratory medicine, nutrition, social work, gerontology, and family medicine. An Evidence Review Team, consisting of nephrologists and methodologists, was responsible for assembling the evidence. Defining chronic kidney disease and classifying the stages of severity would provide a common language for communication among providers, patients and their families, investigators, and policy-makers and a framework for developing a public health approach to affect care and improve outcomes of chronic kidney disease. A uniform terminology would permit: 1. More reliable estimates of the prevalence of earlier stages of disease and of the population at increased risk for development of chronic kidney disease 2. Recommendations for laboratory testing to detect earlier stages and progression to later stages 3. Associations of stages with clinical manifestations of disease 4. Evaluation of factors associated with a high risk of progression from one stage to the next or of development of other adverse outcomes 5. Evaluation of treatments to slow progression or prevent other adverse outcomes. Clinical practice guidelines, clinical performance measures, and continuous quality improvement efforts could then be directed to stages of chronic kidney disease. The Work Group did not specifically address evaluation and treatment for chronic kidney disease. However, this guideline contains brief reference to diagnosis and clinical interventions and can serve as a "road map" linking other clinical practice guidelines and pointing out where other guidelines need to be developed. Eventually, K/DOQI will include interventional guidelines. The first three of these, on bone disease, dyslipidemia, and blood pressure management are currently under development. Other guidelines on cardiovascular disease in dialysis patients and kidney biopsy will be initiated in the Winter of 2001. This report contains a summary of background information available at the time the Work Group began its deliberations, the 15 guidelines and the accompanying rationale, suggestions for clinical performance measures, a clinical approach to chronic kidney disease using these guidelines, and appendices to describe methods for the review of evidence. The guidelines are based on a systematic review of the literature and the consensus of the Work Group. The guidelines have been reviewed by the K/DOQI Advisory Board, a large number of professional organizations and societies, selected experts, and interested members of the public and have been approved by the Board of Directors of the NKF. Framework. The Work Group defined "chronic kidney disease" to include conditions that affect the kidney, with the potential to cause either progressive loss of kidney function or complications resulting from decreased kidney function. Chronic kidney disease was thus defined as the presence of kidney damage or decreased level of kidney function for three months or more, irrespective of diagnosis. The target population includes individuals with chronic kidney disease or at increased risk of developing chronic kidney disease. The majority of topics focus on adults (age ≥18 years). Many of the same principles apply to children as well. In particular, the classification of stages of disease and principles of diagnostic testing are similar. A subcommittee of the Work Group examined issues related to children and participated in development of the first six guidelines of the present document. However, there are sufficient differences between adults and children in the association of GFR with signs and symptoms of uremia and in stratification of risk for adverse outcomes that these latter issues are addressed only for adults. A separate set of guidelines for children will have to be developed by a later Work Group. The target audience includes a wide range of individuals: those who have or are at increased risk of developing chronic kidney disease (the target population) and their families; health care professionals caring for the target population; manufacturers of instruments and diagnostic laboratories performing measurements of kidney function; agencies and institutions planning, providing or paying for the health care needs of the target population; and investigators studying chronic kidney disease. There will be only brief reference to clinical interventions, sufficient to provide a basis for other clinical practice guidelines relevant to the evaluation and management of chronic kidney disease. Subsequent K/DOQI clinical practice guidelines will be based on the framework developed here. Definition of chronic kidney disease. Why "Kidney"? The word "kidney" is of Middle English origin and is immediately understood by patients, their families, providers, health care professionals, and the lay public of native English speakers. On the other hand, "renal" and "nephrology," derived from Latin and Greek roots, respectively, commonly require interpretation and explanation. The Work Group and the NKF are committed to communicating in language that can be widely understood, hence the preferential use of "kidney" throughout these guidelines. The term "End-Stage Renal Disease" (ESRD) has been retained because of its administrative usage in the United States referring to patients treated by dialysis or transplantation, irrespective of their level of kidney function. Why Develop a New Classification? Currently, there is no uniform classification of the stages of chronic kidney disease. A review of textbooks and journal articles clearly demonstrates ambiguity and overlap in the meaning of current terms. The Work Group concluded that uniform definitions of terms and stages would improve communication between patients and providers, enhance public education, and promote dissemination of research results. In addition, it was believed that uniform definitions would enhance conduct of clinical research. Why Base a New Classification System on Severity of Disease? Adverse outcomes of kidney disease are based on the level of kidney function and risk of loss of function in the future. Chronic kidney disease tends to worsen over time. Therefore, the risk of adverse outcomes increases over time with disease severity. Many disciplines in medicine, including related specialties of hypertension, cardiovascular disease, diabetes, and transplantation, have adopted classification systems based on severity to guide clinical interventions, research, and professional and public education. Such a model is essential for any public health approach to disease. Why Classify Severity as the Level of GFR? The level of glomerular filtration rate (GFR) is widely accepted as the best overall measure of kidney function in health and disease. Providers and patients are familiar with the concept that "the kidney is like a filter." GFR is the best measure of the kidneys' ability to filter blood. In addition, expressing the level of kidney function on a continuous scale allows development of patient and public education programs that encourage individuals to "Know your number!" The term "GFR" is not intuitively evident to anyone. Rather, it is a learned term, which allows the ultimate expression of the complex functions of the kidney in one single numerical expression. Conversely, numbers are an intuitive concept and easily understandable by everyone.

Abstract: This communication describes a simple method for patterning paper to create well-defined, millimeter-sized channels, comprising hydrophilic paper bounded by hydrophobic polymer. We believe that this type of patterned paper will become the basis for low-cost, portable, and technically simple multiplexed bioassays. We demonstrate this capability by the simultaneous detection of glucose and protein in 5 μL of urine. The assay system is small, disposable, easy to use (and carry), and requires no external equipment, reagents, or power sources. We believe this kind of system is attractive for uses in less-industrialized countries, in the field, or as an inexpensive alternative to more advanced technologies already used in clinical settings.[1-4] The analysis of biological fluids is necessary for monitoring the health of populations,[2] but these measurements are difficult to implement in remote regions such as those found in less-industrialized countries, in emergency situations, or in home health-care settings.[3] Conventional laboratory instruments provide quantitative measurements of biological samples, but they are unsuitable for these situations since they are large, expensive, and require trained personnel and considerable volumes of biological samples.[2] Other bioassay platforms provide alternatives to more expensive instruments,[5-7] but the need remains for a platform that uses small volumes of sample and that is sufficiently inexpensive to be used widely for measuring samples from large populations. We believe that paper may serve as a particularly convenient platform for running bioassays in the remote situations locations. As a prototype for a mthod we believe to be particularly promosing, we patterned photoresist onto chromatography paper to form defined areas of hydrophilic paper separated by hydrophobic lines or “walls”; these patterns provide spatial control of biological fluids and enable fluid transport, without pumping, due to capillary action in the millimeter-sized channels produced. This method for patterning paper makes it possible to run multiple diagnostic assays on one strip of paper, while still using only small volumes of a single sample. In a fully developed technology, patterned photoresist would be replaced by an appropriate printing technology, but patterning paper with photoresist is: i) convenient for prototyping these devices, and ii) a useful new micropatterning technology in its own right. We patterned chromatography paper with SU-8 2010 photoresist as shown in Figure 1a and as described below: we soaked a 7.5-cm diameter piece of chromatography paper in 2 mL of SU-8 2010 for 30 s, spun it at 2000 rpm for 30 s, and then baked it at 95 °C for 5 min to remove the cyclopentanone in the SU-8 formula. We then exposed the photoresist and paper to 405 nm UV light (50 mW/cm2) for 10 s through a photo-mask (CAD/Art Services, Inc.) that was aligned using a mask aligner (OL-2 Mask Aligner, AB-M, Inc). After exposure, we baked the paper a second time at 95 °C for 5 min to cross-link the exposed portions of the resist. The unpolymerized photoresist was removed by soaking the paper in propylene glycol monomethyl ether acetate (PGMEA) (5 min), and by washing the pattern with propan-2-ol (3 × 10 mL). The paper was more hydrophobic after it was patterned, presumably due to residual resist bound to the paper, so we exposed the entire surface to an oxygen plasma for 10 s at 600 millitorr (SPI Plasma-Prep II, Structure Probe, Inc) to increase the hydrophilicity of the paper (Figures 2a and 2b). Figure 1 Chromatography paper patterned with photoresist. The darker lines are cured photoresist; the lighter areas are unexposed paper. (a) Patterned paper after absorbing 5 μL of Waterman red ink by capillary action. The central channel absorbs the sample ... Figure 2 Assays contaminated with (a) dirt, (b) plant pollen, and (c) graphite powder. The pictures were taken before and after running an artificial urine solution that contained 550 mM glucose and 75 μM BSA. The particulates do not move up the channels ... The patterned paper can be derivatized for biological assays by adding appropriate reagents to the test areas (Figures 1b and ​and2b).2b). In this communication, we demonstrate the method by detecting glucose and protein,[8] but the surface should be suitable for measuring many other analytes as well.[7] The glucose assay is based on the enzymatic oxidation of iodide to iodine,[9] where a color change from clear to brown is associated with the presence of glucose.[10] The protein assay is based on the color change of tetrabromophenol blue (TBPB) when it ionizes and binds to proteins;[11] a positive result in this case is indicated by a color change from yellow to blue. For the glucose assay, we spotted 0.3 μL of a 0.6 M solution of potassium iodide, followed by 0.3 μL of a 1:5 horseradish peroxidase/glucose oxidase solution (15 units of protein per mL of solution). For the protein assay, we spotted 0.3 μL of a 250-mM citrate buffer (pH 1.8) in a well separate from the glucose assay, and then layered 0.3 μL of a 3.3 mM solution of tetrabromophenol blue (TBPB) in 95% ethanol over the citrate buffer. The spotted reagents were allowed to air dry at room temperature. This pre-loaded paper gave consistent results for the protein assay regardless of storage temperature and time (when stored for 15 d both at 0 °C and at 23 °C, wrapped in aluminum foil). The glucose assay was sensitive to storage conditions, and showed decreased signal for assays run 24 h after spotting the reagents (when stored at 23 °C); when stored at 0 °C, however, the glucose assay was as sensitive after day 15 as it was on day 1. We measured artificial samples of glucose and protein in clinically relevant ranges (2.5-50 mM for glucose and 0.38-7.5 μM for bovine serum albumin (BSA))[12, 13] by dipping the bottom of each test strip in 5 μL of a pre-made test solution (Figure 2d). The fluid filled the entire pattern within ca. one minute, but the assays required 10-11 min for the paper to dry and for the color to fully develop.[14] In all cases, we observed color changes corresponding roughly in intensity to the amount of glucose and protein in the test samples, where the lowest concentrations define the lower limits to which these assays can be used (Figure 2e). For comparison, commercially-available dipsticks detect glucose at concentrations as low as 5 mM[7, 9] and protein as low as 0.75 μM;[6, 15] these limits indicate that these paper-based assays are comparable in sensitivity to commercial dipstick assays. Our assay format also allows for the measurement of multiple analytes. This paper-based assay is suitable for measuring multiple samples in parallel and in a relatively short period of time. For example, in one trial, one researcher was able to run 20 different samples (all with 550 mM glucose and 75 μM BSA) within 7.5 min (followed by another 10.5 min for the color to fully develop). An 18-min assay of this type—one capable of measuring two analytes in 20 different sample—may be efficient enough to use in high-throughput screens of larger sample pools. In the field, samples will not be measured under sterile conditions, and dust and dirt may contaminate the assays. The combination of paper and capillary action provides a mechanism for separating particulates from a biological fluid. As a demonstration, we purposely contaminated the artificial urine samples with quantities of dirt, plant pollen, and graphite powder at levels higher than we might expect to see in the samples in the field. These particulates do not move up the channels under the action of capillary wicking, and do not interfere with the assay (Figure 3). Paper strips have been used in biomedical assays for decades because they offer an inexpensive platform for colorimetric chemical testing.[1] Patterned paper has characteristics that lead to miniaturized assays that run by capillary action (e.g., without external pumping), with small volumes of fluids. These methods suggest a path for the development of simple, inexpensive, and portable diagnostic assays that may be useful in remote settings, and in particular, in less-industrialized countries where simple assays are becoming increasingly important for detecting disease and monitoring health,[16, 17], for environmental monitoring, in veterinary and agricultural practice and for other applications.

Abstract: This article describes a prototype system for quantifying bioassays and for exchanging the results of the assays digitally with physicians located off-site. The system uses paper-based microfluidic devices for running multiple assays simultaneously, camera phones or portable scanners for digitizing the intensity of color associated with each colorimetric assay, and established communications infrastructure for transferring the digital information from the assay site to an off-site laboratory for analysis by a trained medical professional; the diagnosis then can be returned directly to the healthcare provider in the field. The microfluidic devices were fabricated in paper using photolithography and were functionalized with reagents for colorimetric assays. The results of the assays were quantified by comparing the intensities of the color developed in each assay with those of calibration curves. An example of this system quantified clinically relevant concentrations of glucose and protein in artificial uri...

Abstract: Despite the fact that we live in an era of advanced and innovative technologies for elucidating underlying mechanisms of diseases and molecularly designing new drugs, infectious diseases continue to be one of the greatest health challenges worldwide. The main drawbacks for conventional antimicrobial agents are the development of multiple drug resistance and adverse side effects. Drug resistance enforces high dose administration of antibiotics, often generating intolerable toxicity, development of new antibiotics, and requests for significant economic, labor, and time investments. Recently, nontraditional antibiotic agents have been of tremendous interest in overcoming resistance that is developed by several pathogenic microorganisms against most of the commonly used antibiotics. Especially, several classes of antimicrobial nanoparticles (NPs) and nanosized carriers for antibiotics delivery have proven their effectiveness for treating infectious diseases, including antibiotics resistant ones, in vitro as well as in animal models. This review summarizes emerging efforts in combating against infectious diseases, particularly using antimicrobial NPs and antibiotics delivery systems as new tools to tackle the current challenges in treating infectious diseases.

Abstract: This article describes a method for fabricating 3D microfluidic devices by stacking layers of patterned paper and double-sided adhesive tape. Paper-based 3D microfluidic devices have capabilities in microfluidics that are difficult to achieve using conventional open-channel microsystems made from glass or polymers. In particular, 3D paper-based devices wick fluids and distribute microliter volumes of samples from single inlet points into arrays of detection zones (with numbers up to thousands). This capability makes it possible to carry out a range of new analytical protocols simply and inexpensively (all on a piece of paper) without external pumps. We demonstrate a prototype 3D device that tests 4 different samples for up to 4 different analytes and displays the results of the assays in a side-by-side configuration for easy comparison. Three-dimensional paper-based microfluidic devices are especially appropriate for use in distributed healthcare in the developing world and in environmental monitoring and water analysis.