Urine Tests for Diagnosis of Infectious Diseases and Antibiotic-Resistant Pathogens

The relation between disease and urine was recognized by physicians since the earliest civilization BC. Urine is considered an ideal diagnostic specimen for its noninvasive and easy method of collection. Urinalysis encompasses a wide range of tests, which includes a variety of chemical tests, urine microscopy, bacterial cultures, and molecular tests. Importantly, urine tests can diagnose patients with antibiotic-resistant urinary tract infections (UTI), directly from urine and/or bacterial culture. This chapter summarizes the most common urine tests in the infectious disease field, with a special focus on diagnosing UTI and characterizing their antibiotic resistant. In addition to describing the advantages and limitation of these tests, the chapter explores the promising emerging technologies and methods in this field. This chapter is beneficial for scientists and healthcare workers in the field

ADVANCED IMMUNOSENSING PLATFORMS FOR BIOMONITORING AND ENVIRONMENTAL MONITORING

Chemical analysis of complex sample matrix is significant towards the efficient monitoring of human and environmental health. In the area of environmental contamination control and disease diagnosis, traditional analytical approaches rely on the separation effects by chromatography, such as gas chromatography (GC) and liquid chromatography (LC). Although GC/LC coupled with various detectors are extensively employed in analytical settings as the mature technologies, the disadvantages are also obvious, including the dependence on large and bulky instruments, complex and time-consuming instrumental operation, inevitable sample pretreatment and high cost. These disadvantages greatly limit their application in rapid diagnosis of disease, on-site samples testing, and household preclinic diagnosis. Immunosensing approaches, however, are promising substitutes applicable to the above scenarios. Featured with extraordinary selectivity and accuracy of detection attributed to the specific antibody-antigen recognition, immunoassays (IAs) have attracted extensive interests in recent years. Nevertheless, the major challenges are to overcome the inherent defects of IAs, such as low sensitivity of detection and difficult to realize multi-target simultaneous detection, in the meantime, to develop high-performance portable detection platforms. In this dissertation, advanced immunosensing platforms based on the principle of enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) were systematically explored from the aspects of biorecognition, signal amplification, and signal quantification. Advanced functional nanomaterials, including metal-organic framework (MOF), graphene, carbon dot, mesoporous nanocomposite, etc., with unique physical or chemical properties were synthesized and employed as signal reporters in IAs for amplified sensitivity of detection. In addition, we designed and manufactured immunosensing platforms using 3D printing to achieve optimal integration of immunoassays, nanomaterials, and quantitative detection methods, thus the on-site, point-of-care, high-performance sample assessment was realized. Further, the extraordinary analytical performances of the immunosensing platforms were validated in practical applications for the detection of woodsmoke biomarkers in various human biofluids representing the exposure of human to contaminated air from wildfires, as well as the detection of environmental pesticide contaminants and the corresponding human metabolites in biofluids

Microfluidic smartphone quantitation of <i>Escherichia coli</i> in synthetic urine

In spite of the clinical need, there is a major gap in rapid diagnostics for identification and quantitation of E. coli and other pathogens, also regarded as the biggest bottleneck in the fight against the spread of antimicrobial resistant bacterial strains. This study reports for the first time an optical, smartphone-based microfluidic fluorescence sandwich immunoassay capable of quantifying E. coli in buffer and synthetic urine in less than 25 min without sample preparation nor concentration. A limit of detection (LoD) up to 240 CFU/mL, comensurate with cut-off for UTIs (103-105 CFUs/mL) was achieved. Replicas of full response curves performed with 100-107 CFUs/mL of E. coli K12 in synthetic urine yielded recovery values in the range 80-120%, assay reproducibility below 30% and precision below 20%, therefore similar to high-performance automated immunoassays. The unrivalled LoD was mainly linked to the 'open fluidics' nature of the 10-bore microfluidic strips used that enabled passing a large volume of sample through the microcapillaries coated with capture antibody. The new smartphone based test has the potential of being as a rapid, point-of-care test for rule-in of E. coli infections that are responsible for around 80% of UTIs, helping to stop the over-prescription of antibiotics and the monitoring of patients with other symptomatic communicable diseases caused by E. coli at global scale.</p

Hybrid point-of-care devices for visual detection of biomarkers and drugs

Early diagnostics is a crucial part of clinical practice offering a rapid and convenient way to investigate and quantify the presence of key biomarkers related to specific pathologies and increasing the chance of successful treatments. In this regard, point-of-care testing (POCT) shows several advantages enabling simple and rapid analyses, allowing for real-time results, and permitting home testing. Metallic nanoparticles (NPs), like gold NPs (AuNPs), can be beneficially integrated into POC devices thanks to their tunable plasmonic properties which provide a naked-eye read-out. Moreover, the high sensitivity of NPs enables the detection of biomarkers in non-invasive fluids where the concentrations are typically low. These biofluids, like saliva and urine, are functionally equivalent to serum in reflecting the physiological state of the body, whilst they are easier to handle, collect, and store. In this thesis, I first reported the design and development of a colorimetric strategy based on the morphological change of multibranched plasmonic AuNPs, aimed at detecting glucose in saliva. The sensing approach relied on a target-induced reshaping process which involves the oxidation of the NP tips and the transformation into a spherical shape, characterized by a naked-eye detectable blue-to-pink color change. The platform proved to be beneficial in the early and non-invasive diagnosis of hyperglycemia. The successful technological transfer on a solid substrate paved the way for the realization of a dipstick prototype for home testing. Then, the strategy was adapted to other biomarkers, leading to the development of a multiplexing test for the simultaneous detection of three salivary analytes (cholesterol, glucose, and lactate). This multiplexing assay enabled to save reagents, costs, and time, whilst increasing the overall clinical value of the test. Exploiting the microfluidics applied on a paper sheet, I realized a monolithic and fully integrated POC device, through a low-cost and fast CO2 laser cutter. The platform showed excellent selectivity and multiplexing ability, with negligible interferences. The second part of my thesis was focused on the development of POC devices for the detection of anticancer drug contaminations in water solutions and urine samples. Antiblastic agents have revealed high toxicity for the exposed healthcare workers who prepare and administer these drugs in occupational environments. Hence, continuous monitoring is highly required, and POCT shows tremendous potential in this context. With this aim, I realized a lateral-flow (LF) device for the assessment of doxorubicin contamination, using the fluorescent properties of the drug for naked-eye detection. The pharmacological recognition of the DNA probe was exploited to overcome the lack of anti-doxorubicin antibodies. The highly sensitive strategy was successfully adapted to a real urine sample, without resorting to complex pretreatment procedures. Then, I developed a competitive LF device for the detection of methotrexate (MTX). AuNPs were employed as the label molecules and the pharmacological competition of folic acid and MTX for the capture enzyme was exploited as the recognition mechanism, instead of costly antibodies. Despite the sensitivity requires further improvements, the strategy showed fast and reliable results, demonstrating a high potential for workers’ safety control

The Development of a Primer Payload with Microparticles for UTI Pathogen Identification Using Polythymidine- Modified LAMP Primers in Droplet LAMP

Nucleic acid amplification tests (NAATs) are among the diagnostic tests with the highest sensitivity and specificity. However, they are more complex to develop than other diagnostic tests such as biochemical tests and lateral flow immunoassay tests. Polymerase chain reaction (PCR) is the gold standard for NAATs. PCR requires thermal cycling to achieve clonal amplification of the target pathogen DNA for diagnosis. Thermal cycling poses a challenge in the development of PCR diagnostics for point-of-care (POC) settings. Loop-mediated isothermal amplification (LAMP) offers an isothermal method for NAATs diagnostics. The advancement of the microfluidics field significantly enhances the development of LAMP diagnostics devices for POC testing. Another challenge with NAATs, is the limitation in the development of multiplex NAATs. Multiplexing however, occupies an important role in the efforts to address the antimicrobial resistance global crisis. Multiplexing will help to provide more thorough and complete diagnostics of infections, and enable doctors to prescribe the most effective antibiotics to the patients. This will help slow the emergence of antibiotic resistant pathogens. We are currently in a period of discovery void, with regards to antibiotics discovery. At this rate, more pathogens are becoming resistant to the antibiotics that we have, faster than we are developing new classes of antibiotics. According to the World Health Organization (WHO) interagency coordination group on AMR report to the secretary general of the United Nations, by 2050, there will be 10 million annual deaths globally, as a result of AMR-related events. There will also be $55 billion productivity losses globally due to AMR. In addition, there will be a total of$ 1 trillion in healthcare costs, and 28 million people will be living in poverty, as a result of the economic impact of uncontrolled AMR. Another area where multiplex diagnostics play a crucial role is infection control in the era of epidemics and pandemics. The increasing prevailing frequency of global pandemics stresses the need for the development of highly accurate and decentralized POC diagnostics. Over the last ten years, there have been more than 30 epidemics and pandemics around the world, including SARS-CoV-2, Monkey pox, India black fungus, Dengue fever, Measles, Zika, Avian influenza, Influenza A and Ebola. With advancing technology and international commerce and relations, we are now more connected than ever. This means that if there are no developments to make molecular tests more accessible at the POC, the future waves of epidemics and pandemics will have faster spread, further reach and more devastating impacts on the lives of the 8 billion people on our planet. We have developed a diagnostic method for executing droplet microfluidics LAMP via a microparticle primer payload mechanism and have demonstrated it with urinary tract infection (UTI) pathogens. With inspiration from overhang PCR and RNA-Seq, we engineered LAMP primers with 5’ polythymidine (PolyT) oligonucleotide (PolyT is placed in the middle of the Forward inner primers and Backward inner primers). The PolyT sequence is recognized by a biotinylated capture oligonucleotide engineered with a polyadenylated (PolyA) polynucleotide on the 3’ end. The streptavidin-coated microparticles functionalized with the PolyA oligonucleotide and PolyT primers, capture their specific target DNA and deliver the cargo into emulsion droplets of LAMP reagents for amplification. This platform provides the ability to multiplex by coding specific pathogen target DNA with different fluorescent signatures of the microparticles

Challenges of liver cancer: Future emerging tools in imaging and urinary biomarkers.

© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.Chronic liver disease has become a global health problem as a result of the increasing incidence of viral hepatitis, obesity and alcohol misuse. Over the past three decades, in the United Kingdom alone, deaths from chronic liver disease have increased both in men and in women. Currently, 2.5% of deaths worldwide are attributed to liver disease and projected figures suggest a doubling in hospitalisation and associated mortality by 2020. Chronic liver diseases vary for clinical manifestations and natural history, with some individuals having relatively indolent disease and others with a rapidly progressive course. About 30% of patients affected by hepatitis C has a progressive disease and develop cirrhosis over a 20 years period from the infection, usually 5-10 years after initial medical presentation. The aim of the current therapeutic strategies is preventing the progression from hepatitis to fibrosis and subsequently, cirrhosis. Hepatic steatosis is a risk factor for chronic liver disease and is affecting about the half of patients who abuse alcohol. Moreover non-alcoholic fatty liver disease is part of the metabolic syndrome, associated with obesity, hypertension, type ? diabetes mellitus and dyslipidaemia, and a subgroup of patients develops non-alcoholic steatohepatitis and fibrosis with subsequent cirrhosis. The strengths and pitfalls of liver biopsy are discussed and a variety of new techniques to assess liver damage from transient elastography to experimental techniques, such as in vitro urinary nuclear magnetic resonance spectroscopy. Some of the techniques and tests described are already suitable for more widespread clinical application, as is the case with ultrasound-based liver diagnostics, but others, such as urinary metabonomics, requires a period of critical evaluation or development to take them from the research arena to clinical practice

MicroRNA detection on microsensor arrays by SPR imaging measurements with enzymatic signal enhancement.

We investigated sequence-specific and simultaneous microRNA (miRNA) detections by surface plasmon resonance (SPR) imaging measurements on SPR chips possessing an Au spot array modified with probe DNAs based on a miRNA-detection-selective SPR signal amplification method. MiRNAs were detected with the detection limit of the attomole level by SPR imaging measurements for different miRNA concentrations on a single chip. SPR signals were enhanced based on a combination process of sequence-specific hybridization of the miRNA to the probe DNAs, extension reaction of polyadenine (poly(A)) tails by poly(A) polymerase, binding of a ternary complex of T30-biotin/horseradish peroxidase (HRP)-biotin/streptavidin to the poly(A) tails, and the oxidation reaction of tetramethylbenzidine (TMB) on the HRP by providing a blue precipitate on the surface. This process sequence-specifically and dramatically amplified the SPR signals. This is a simple, cost-effective, and feasible signal amplification method based on the organic compound TMB instead of metal nanoparticles

Recent Advancements in the Technologies Detecting Food Spoiling Agents

To match the current life-style, there is a huge demand and market for the processed food whose manufacturing requires multiple steps. The mounting demand increases the pressure on the producers and the regulatory bodies to provide sensitive, facile, and cost-effective methods to safeguard consumers’ health. In the multistep process of food processing, there are several chances that the food-spoiling microbes or contaminants could enter the supply chain. In this contest, there is a dire necessity to comprehend, implement, and monitor the levels of contaminants by utilizing various available methods, such as single-cell droplet microfluidic system, DNA biosensor, nanobiosensor, smartphone-based biosensor, aptasensor, and DNA microarray-based methods. The current review focuses on the advancements in these methods for the detection of food-borne contaminants and pathogens