Rapid diagnosis of infectious diseases and timely initiation of appropriate treatment are critical determinants that promote optimal clinical outcomes and general public health. Conventional in vitro diagnostics for infectious diseases are time-consuming and require centralized laboratories, experienced personnel and bulky equipment. Recent advances in biosensor technologies have potential to deliver point-of-care diagnostics that match or surpass conventional standards in regards to time, accuracy and cost. Broadly classified as either label-free or labeled, modern biosensors exploit micro- and nanofabrication technologies and diverse sensing strategies including optical, electrical and mechanical transducers. Despite clinical need, translation of biosensors from research laboratories to clinical applications has remained limited to a few notable examples, such as the glucose sensor. Challenges to be overcome include sample preparation, matrix effects and system integration. We review the advances of biosensors for infectious disease diagnostics and discuss the critical challenges that need to be overcome in order to implement integrated diagnostic biosensors in real world settings.
Current regimens for the detection and surveillance of bladder cancer are invasive and have suboptimal sensitivity. Here, we present a novel high-throughput sequencing (HTS) method for detection of urine tumor DNA (utDNA) called utDNA CAPP-Seq (uCAPP-Seq) and apply it to 67 healthy adults and 118 patients with early-stage bladder cancer who had urine collected either prior to treatment or during surveillance. Using this targeted sequencing approach, we detected a median of 6 mutations per patient with bladder cancer and observed surprisingly frequent mutations of the PLEKHS1 promoter (46%), suggesting these mutations represent a useful biomarker for detection of bladder cancer. We detected utDNA pretreatment in 93% of cases using a tumor mutationinformed approach and in 84% when blinded to tumor mutation status, with 96% to 100% specifi city. In the surveillance setting, we detected utDNA in 91% of patients who ultimately recurred, with utDNA detection preceding clinical progression in 92% of cases. uCAPP-Seq outperformed a commonly used ancillary test (UroVysion, P = 0.02) and cytology and cystoscopy combined (P ≤ 0.006), detecting 100% of bladder cancer cases detected by cytology and 82% that cytology missed. Our results indicate that uCAPP-Seq is a promising approach for early detection and surveillance of bladder cancer. SIGNIFICANCE: This study shows that utDNA can be detected using HTS with high sensitivity and specifi city in patients with early-stage bladder cancer and during post-treatment surveillance, signifi cantly outperforming standard diagnostic modalities and facilitating noninvasive detection, genotyping, and monitoring.
48 Background. Nucleic acid amplification tests (NAATs) are the primary means of 49 identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 50 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and 51 isolation resources and allow for rapid therapeutic interventions. 52 Methods. We evaluated the analytical and clinical performance characteristics of the Xpert ® 53 Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. 54 Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2, 55 other infectious coronavirus species including SARS-CoV, and 85 nasopharyngeal swab 56 specimens positive for other respiratory viruses including endemic human coronaviruses 57 (hCoVs). Clinical performance was assessed using 483 remnant upper and lower respiratory 58 specimens previously analyzed by standard of care (SOC) NAATs. 59 Results. The limit of detection of the Xpert test was 0.01 plaque forming units (PFU)/mL. 60 Other hCoVs, including Middle East Respiratory Syndrome coronavirus, were not detected by 61 the Xpert test. SARS-CoV, a closely related species in the Sarbecovirus subgenus, was 62 detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-63 specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 64 219/220 (99.5%) and the negative agreement was 250/261 (95.8%). A third tie-breaker 65 NAAT resolved all but three of the discordant results in favor the Xpert test. 66 Conclusions. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a 67 variety of upper and lower respiratory tract specimens. The high sensitivity and fast time to 68 results of approximately 45 minutes may impact patient management. 69 70 Laboratory diagnosis of infections caused by severe acute respiratory syndrome coronavirus 2 72 (SARS-CoV-2) is usually accomplished by performing nucleic acid amplification tests 73 (NAATs) on respiratory tract specimens. An antibody response is often not detected in the 74 first week to ten days of symptoms and antibody testing is therefore generally unhelpful for 75 acute diagnosis(1-3), with virus isolation in culture presenting significant biosafety risks. 76 Upper respiratory tract (URT) specimens such as nasopharyngeal swabs (NPS) and 77 oropharyngeal swabs (OPS) generally have high SARS-CoV-2 viral loads upon symptom 78 onset.(2, 4-6) URT specimens may also have detectable RNA during the pre-symptomatic 79 period(7), and pediatric patients who remain asymptomatic through the entire course of 80 on June 9, 2020 by guest http://jcm.asm.org/ Downloaded from 4 infection can persistently shed RNA in URT specimens for two weeks or longer.(4, 8) 81 Importantly, NPS may have higher viral loads than OPS.(6) Lower respiratory tract (LRT) 82 specimens including sputum(7, 9) and tracheal aspirates(10) (TA) are often positive for RNA 83 early in disease and remain positive longer than URT sources.(5) 84 NAATs are...
This study reports the use of microfluidics, which intrinsically has a large surface-to-volume ratio, toward rapid antimicrobial susceptibility testing at the point of care. By observing the growth of uropathogenic E. coli in gas permeable polymeric microchannels with different dimensions, we demonstrate that the large surface-to-volume ratio of microfluidic systems facilitates rapid growth of bacteria. For microchannels with 250 micrometer or less in depth, the effective oxygenation can sustain the growth of E. coli to over 10 9 cfu/ml without external agitation or oxygenation, which eliminates the requirement of bulky instrumentation and facilitates rapid bacterial growth for antimicrobial susceptibility testing at the point of care. The applicability of microfluidic rapid antimicrobial susceptibility testing is demonstrated in culture media and in urine with clinical bacterial isolates that have different antimicrobial resistance profiles. The antimicrobial resistance pattern can be determined as rapidly as 2 hours compared to days in standard clinical procedures facilitating diagnostics at the point of care.
This study reports a hybrid electrokinetic technique for label-free manipulation of pathogenic bacteria in biological samples toward medical diagnostic applications. While most electrokinetic techniques only function in low-conductivity buffers, hybrid electrokinetics enables effective operation in high-conductivity samples, such as physiological fluids (~1 S m−1). The hybrid electrokinetic technique combines short-range electrophoresis and dielectrophoresis, and long-range AC electrothermal flow to improve its effectiveness. The major technical hurdle of electrode instability for manipulating high conductivity samples is tackled by using a Ti–Au–Ti sandwich electrode and a 3-parallel-electrode configuration is designed for continuous isolation of bacteria. The device operates directly with biological samples including urine and buffy coats. We show that pathogenic bacteria and biowarfare agents can be concentrated for over 3 orders of magnitude using hybrid electrokinetics.
Urine is the most abundant and easily accessible of all body fluids and provides an ideal route for non-invasive diagnosis of human diseases, particularly of the urinary tract. Electrochemical biosensors are well suited for urinary diagnostics due to their excellent sensitivity, low cost, and ability to detect a wide variety of target molecules including nucleic acids and protein biomarkers. We report the development of an electrochemical immunosensor for direct detection of the urinary tract infection (UTI) biomarker lactoferrin from infected clinical samples. An electrochemical biosensor array with alkanethiolate self-assembled monolayer (SAM) was used. Electrochemical impedance spectroscopy was used to characterize the mixed SAM, consisted of 11-mercaptoundecanoic acid and 6-mercapto-1-hexanol. A sandwich amperometric immunoassay was developed for detection of lactoferrin from urine, with a detection limit of 145 pg/ml. We validated lactoferrin as a biomarker of pyuria (presence of white blood cells in urine), an important hallmark of UTI, in 111 patient-derived urine samples. Finally, we demonstrated multiplex detection of urinary pathogens and lactoferrin through simultaneous detection of bacterial nucleic acid (16S rRNA) and host immune-response protein (lactoferrin) on a single sensor array. Our results represent first integrated sensor platform capable of quantitative pathogen identification and measurement of host immune response, potentially providing clinical diagnosis that is not only more expeditious but more informative than the current standard.
Microfluidics holds great promise to revolutionize various areas of biological engineering, such as single cell analysis, environmental monitoring, regenerative medicine, and point-of-care diagnostics. Despite the fact that intensive efforts have been devoted into the field in the past decades, microfluidics has not yet been adopted widely. It is increasingly realized that an effective system integration strategy that is low cost and broadly applicable to various biological engineering situations is required to fully realize the potential of microfluidics. In this article, we review several promising system integration approaches for microfluidics and discuss their advantages, limitations, and applications. Future advancements of these microfluidic strategies will lead toward translational lab-on-a-chip systems for a wide spectrum of biological engineering applications.
E lectrothermal flow is a promising technique in microfluidic manipulation toward laboratory automation applications, such as clinical diagnostics and high-throughput drug screening. Despite the potential of electrothermal flow in biomedical applications, relatively little is known about electrothermal manipulation of highly conductive samples, such as physiological fluids and buffer solutions. In this study, the characteristics and challenges of electrothermal manipulation of fluid samples with different conductivities were investigated systematically. Electrothermal flow was shown to create fluid motion for samples with a wide range of conductivity when the driving frequency was greater than 100 kHz. For samples with low conductivities (below 1 S/m), the characteristics of the electrothermal fluid motions were in quantitative agreement with the theory. For samples with high conductivities (greater than 1 S/m), the fluid motion appeared to deviate from the model as a result of potential electrochemical reactions and other electrothermal effects. These effects should be taken into consideration for electrothermal manipulation of biological samples with high conductivities. This study will provide insights in designing microfluidic devices for electrokinetic manipulation of biological samples toward laboratory automation applications in the future. ( JALA 2010;15:426-32) INTRODUCTIONThe development of automated microfluidic systems poses great promises for a variety of medical diagnostic applications. 1e3 Although extensive research efforts have been devoted to integrate various transduction mechanisms, including optical, inertial, interfacial, and electrochemical sensing, the transducers often require sample preparation components for handling clinical samples. 4e7 The implementation of the sample preparation modules, which critically determines the overall performance of the system, can often be cumbersome, labor intensive, and time consuming, and represents a major challenge for laboratory automation. 8,9 Among numerous microfluidic techniques, alternating current (AC) electrokinetics is one of the most promising approaches for addressing this fundamental hurdle in laboratory automation. 10e12 AC electrokinetics is especially effective in the micro and nano domains and can be easily integrated with other microfluidic components. Furthermore, combinations of different electrokinetic phenomena allow fundamental fluidic operations, including concentration, separation, mixing, and pumping, to be performed in the same platform. 13e15 Electrokinetics has also been applied in various mechanobiological applications. 16,17 All these features render electrokinetics one of the most promising approaches for developing fully integrated microfluidic diagnostic systems for laboratory automation. 18,19 Most electrokinetic techniques, such as dielectrophoresis and AC electroosmosis, are effective only in low-conductivity fluids. Electrothermal flow, on the other hand, is effective in fluids that have a wide range of cond...
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