Jacqueline C. Linnes

Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens

The 2009 Influenza A (H1N1) pandemic disproportionately affected the developing world and highlighted the key inadequacies of traditional diagnostic methods that make them unsuitable for use in resource-limited settings, from expensive equipment and infrastructure requirements to unacceptably long turnaround times. While rapid immunoassay diagnostic tests were much less costly and more context-appropriate, they suffered from drastically low sensitivities and high false negative rates. An accurate, sensitive, and specific molecular diagnostic that is also rapid, low-cost, and independent of laboratory infrastructure is needed for effective point-of-care detection and epidemiological control in these developing regions. We developed a paper-based assay that allows for the extraction and purification of RNA directly from human clinical nasopharyngeal specimens through a poly(ether sulfone) paper matrix, H1N1-specific in situ isothermal amplification directly within the same paper matrix, and immediate visual detection on lateral flow strips. The complete sample-to-answer assay can be performed at the point-of-care in just 45 min, without the need for expensive equipment or laboratory infrastructure, and it has a clinically relevant viral load detection limit of 10(6) copies/mL, offering a 10-fold improvement over current rapid immunoassays.

Paper-based molecular diagnostic for Chlamydia trachomatis.

Herein we show the development of a minimally instrumented paper-based molecular diagnostic for point of care detection of sexually transmitted infections caused by Chlamydia trachomatis. This new diagnostic platform incorporates cell lysis, isothermal nucleic acid amplification, and lateral flow visual detection using only a pressure source and heat block, eliminating the need for expensive laboratory equipment. This paper-based test can be performed in less than one hour and has a clinically relevant limit of detection that is 100x more sensitive than current rapid immunoassays used for chlamydia diagnosis.

Enabling the Development and Deployment of Next Generation Point-of-Care Diagnostics

A major goal of the 1st International Point-of-Care Diagnostic Workshop in Nairobi, Kenya was to provide a forum for open dialog concerning current challenges in, and potential solutions for, the development of the next generation of POC diagnostics. The focus was not solely on descriptions of new technologies in development but also included demonstrations of their use in a field setting. The resulting conversations identified a number of obstacles to the successful translation of prototypes into field-deployable tools. These obstacles superseded those typically encountered in research; changes must be implemented at the institutional and governmental level to enable equitable collaborations between Western and African partners, and proper funding mechanisms must be established to support these collaborations. Additionally, this workshop showcased emerging technologies for POC tests and fostered new partnerships between technology developers and African research laboratories. Equitable partnerships are critical for the successful implementation of new POC technology. The attendees agreed that the most effective methods to effect change require improved communication of needs, ideas and abilities, and a conduit for the sharing of experiences and information. We plan to implement many of the changes that are suggested here in our own research programs and to use future conferences and workshops to guide the development of both technologies and partnerships. Our successes and failures will serve as models for those scientists striving to develop technological and biomedical solutions to similar problems in global health.

Ventilated cookstoves associated with improvements in respiratory health-related quality of life in rural Bolivia

BACKGROUND Household air pollution (HAP) from combustion of biomass fuels worldwide is linked to asthma, respiratory infections and chronic pulmonary diseases. Implementation of ventilated cookstoves significantly reduces exposure to HAP. However, improvements in concurrent respiratory health-related quality of life (HRQoL) have not been previously evaluated with a standardized questionnaire. METHODS The association between woodsmoke exposure and respiratory HRQoL outcomes was evaluated using an intervention study in a rural community in Bolivia. Indoor carbon monoxide (CO) levels from traditional stoves and from cookstoves with chimneys were analyzed alongside interview results of women heads-of-households using the St. Georges Respiratory Questionnaire (SGRQ) in 2009 and 1-year post-intervention. RESULTS Pronounced improvements in respiratory HRQoL and significant reductions of household CO levels followed installation of ventilated cookstoves. Stove implementation yielded lower indoor CO values and correlated positively with improved SGRQ scores. CONCLUSIONS This is the first use of a standardized respiratory HRQoL assessment to determine the impact of ventilated cookstove implementation on reducing HAP. This preliminary study utilizes the SGRQ as a valuable tool enabling analysis of these health effects in relation to other respiratory disease states.

Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics

Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications.

Giant Extracellular Matrix Binding Protein Expression in Staphylococcus epidermidis is Regulated by Biofilm Formation and Osmotic Pressure

Staphylococcus epidermidis is an opportunistic bacterium that thrives as a commensal cutaneous organism and as a vascular pathogen. The S. epidermidis extracellular matrix binding protein (Embp) has been reported to be a virulence factor involved in colonization of medical device implants and subsequent biofilm formation. Here, we characterize the expression patterns of Embp in planktonic and biofilm cultures, as well as under high osmotic stresses that typify the commensal environment of the skin. Embp expression without osmotic stress was similar for planktonic and adherent cultures. Addition of osmotic stress via NaCl caused slight increases in embp expression in planktonic cultures. However, in adherent cultures a 100-fold increase in embp expression with NaCl versus controls occurred and coincided with altered biofilm morphology. Results suggest that the central role of Embp lies in commensal skin colonization, stabilizing the cell wall against osmotic stresses, rather than as a virulence factor promoting adhesion.

Adhesion of Staphylococcus epidermidis to biomaterials is inhibited by fibronectin and albumin.

Decades of contradictory results have obscured the exact role of adsorbed fibronectin in the adhesion of the bacterium, Staphylococcus epidermidis, to biomaterials. Here, the ability of adsorbed fibronectin (FN) or bovine serum albumin (BSA) to modulate S. epidermidis adhesion to various biomaterials is reported. FN or BSA was adsorbed in increasing surface densities up to saturated monolayer coverage onto various common biomaterials, including poly(ethylene terephthalate), fluorinated ethylene propylene, poly(ether urethane), silicone, and borosilicate glass. Despite the wide range of surface characteristics represented, adsorption isotherms varied only subtly between materials for the two proteins considered. S. epidermidis adhesion to the various protein-coated biomaterials was quantified in a static-fluid batch adhesion assay. Although slight differences in overall adherent cell numbers were observed between the various protein-coated substrata, all materials exhibited significant dose-dependent decreases in S. epidermidis adhesion with increasing adsorption of either protein (FN, BSA) to all surfaces. Results here indicate that S. epidermidis adhesion to FN-coated surfaces is not a specific adhesion (i.e., receptor: ligand) mediated process, as no significant difference in adhesion was found between FN- and BSA-coated materials. Rather, results indicate that increasing surface density of either FN or BSA actually inhibited S. epidermidis adhesion to all biomaterials examined.

Ultraviolet Germicidal Irradiation: Future Directions for Air Disinfection and Building Applications

Ultraviolet germicidal irradiation (UVGI) for air disinfection applications has relied on low‐pressure mercury vapor lamps for decades. New design requirements have generated the need for alternatives in some uses. This study describes the current state of UVGI technology and describes future directions for technology development, including the use of lamps produced from nontoxic materials and light‐emitting diode lamps. Important applications are discussed such as the use of ultraviolet germicidal lamps in developing countries, in heating, ventilating and air‐conditioning systems to improve energy efficiency and indoor air quality, and for whole room disinfection.

Thermally actuated wax valves for paper-fluidic diagnostics

The complexity of current paper-fluidic diagnostic devices is limited by their imperfect control of reagent incubation and delivery. Valves providing complete fluid obstruction and multiple actuation steps within a single paper-fluidic diagnostic would increase the range of diagnostic bioassays in these porous membranes. Here, we report the rapid fabrication of tunable wax-ink valves that are thermally actuated within porous membranes. Varying width bands of wax-ink were printed onto strips of nitrocellulose and cellulose membranes and characterized by their triggered obstruction and release of fluid wicking through the membranes. To demonstrate the utility of these wax-ink valves, we have transformed a traditional lateral flow immunoassay into a multi-step, semi-autonomous assay that enhances bacterial detection signal intensity 6-fold. The wax-ink valves are applicable to other paper fluidic assays by fully obstructing fluid flow for a sustained reaction time and are able to be actuated multiple times for sequential fluid delivery with minimal user involvement. These easily fabricated wax-ink valves are a versatile addition to the toolkit of fluidic control mechanisms required to develop more sensitive paper-based diagnostics.

Portable digital lock-in instrument to determine chemical constituents with single-color absorption measurements for Global Health Initiatives.

Innovations in international health require the use of state-of-the-art technology to enable clinical chemistry for diagnostics of bodily fluids. We propose the implementation of a portable and affordable lock-in amplifier-based instrument that employs digital technology to perform biochemical diagnostics on blood, urine, and other fluids. The digital instrument is composed of light source and optoelectronic sensor, lock-in detection electronics, microcontroller unit, and user interface components working with either power supply or batteries. The instrument performs lock-in detection provided that three conditions are met. First, the optoelectronic signal of interest needs be encoded in the envelope of an amplitude-modulated waveform. Second, the reference signal required in the demodulation channel has to be frequency and phase locked with respect to the optoelectronic carrier signal. Third, the reference signal should be conditioned appropriately. We present three approaches to condition the signal appropriately: high-pass filtering the reference signal, precise offset tuning the reference level by low-pass filtering, and by using a voltage divider network. We assess the performance of the lock-in instrument by comparing it to a benchmark device and by determining protein concentration with single-color absorption measurements. We validate the concentration values obtained with the proposed instrument using chemical concentration measurements. Finally, we demonstrate that accurate retrieval of phase information can be achieved by using the same instrument.