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Two-phase porous media flow is important in many applications from drug delivery to groundwater diffusion and oil recovery and is of particular interest to biomedical diagnostic test developers using cellulose and nitrocellulose membranes with limited fluid sample volumes. This work presents a new two-phase porous media flow model based on the incompressible Navier-Stokes equation. The model aims to address the limitations of existing methods by incorporating a partial saturation distribution in porous media to account for limited fluid volumes. The basic parameters of the model are the pore size distribution and the contact angle. To validate the model, we solved five analytical solutions and compared them to corresponding experimental data. The experimentally measured penetration length data agreed with the model predictions, demonstrating model accuracy. Our findings suggest that this new two-phase porous media flow model can provide a valuable tool for researchers developing fluidic assays in paper and other porous media.
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Microfluidic techniques are widely applied in biomolecular analysis and disease diagnostic assays. While the volume of the sample that is directly used in such assays is often only femto-to microliters, the "dead volume" of solutions supplied in syringes and tubing can be much larger, even up to milliliters, increasing overall reagent use and making analysis significantly more expensive. To reduce the difficulty and cost, we designed a new chip using a low volume solution for analysis and applied it to obtain real-time data for protein-protein interaction measurements. The chip takes advantage of air/aqueous two-phase droplet flow, on-chip rapid mixing within milliseconds, and a droplet capture method, that ultimately requires only 2 µL of reagent solution. The interaction is analyzed by particle diffusometry, a nonintrusive and precise optical detection method to analyze the properties of microparticle diffusion in solution. Herein, we demonstrate on-chip characterization of human immunodeficiency virus p24 antibody-antigen protein binding kinetics imaged via fluorescence microscopy and analyzed by PD. The measured kon and koff are 1 × 106 M-1 s-1 and 3.3 × 10-4 s-1, respectively, and agree with independent measurement via biolayer interferometry and previously calculated p24-antibody binding kinetics. This new microfluidic chip and the protein-protein interaction analysis method can also be applied in other fields that require low-volume solutions to perform accurate measurement, analysis, and detection.
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Técnicas Analíticas Microfluídicas , Microfluídica , Humanos , Cinética , Difusión , Indicadores y Reactivos , Técnicas Analíticas Microfluídicas/métodosRESUMEN
A shift in the traditional technocentric view of medical device design to a human-centered one is needed to bridge existing translational gaps and improve health equity. To ensure the successful and equitable adoption of health technology innovations, engineers must think beyond the device and the direct end user and must seek a more holistic understanding of broader stakeholder needs and the intended context of use early in a design process. The objectives of this review article are (a) to provide rationale for the need to incorporate meaningful stakeholder analysis and contextual investigation in health technology development and biomedical engineering pedagogy, (b) to review existing frameworks and human- and equity-centered approaches to stakeholder engagement and contextual investigation for improved adoption of innovative technologies, and (c) to present case studyexamples of medical device design that apply these approaches to bridge the gaps between biomedical engineers and the contexts for which they are designing.
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Tecnología Biomédica , Diseño de Equipo , HumanosRESUMEN
The measurement and optimization of protein-protein interactions are critical in the design of biotherapeutics, biomolecular sensing elements, and functional protein-based biomaterials among other biomolecular sciences and engineering. Current gold standard assays require specifically designed core facilities, equipment, and expertise to implement the measurement, making it inconvenient for most labs unless implemented routinely. We developed a new method aiming at measuring protein binding kinetics based on microfluidics and particle diffusometry (PD), which only needs very general lab equipment, including a fluorescence microscope, a syringe pump, and a simple microchannel fabricated on a glass slide. Protein binding pairs are immobilized on two kinds of nanoparticles with different diameters using widely available conjugation chemistries. The two diluted particle suspensions are injected using a syringe pump into a Y-junction microchannel, where they bind and form particle complexes with increasing size, thereby decreasing particles' Brownian motion amplitude and diffusivity, which can be detected by PD. By taking images at a series of specific points along the microchannel, the particle diffusivity is measured at different time points after the introduction of protein-protein binding. These data are then used to quantify the protein binding kinetic constant. This label-free particle-based method is simple to operate and as accurate as the current gold standard. We demonstrate the feasibility of this accessible method by quantifying the streptavidin-biotin association constant (1.74 ± 0.51 × 107 M-1 s-1), which compares well with previously published results.
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Microfluídica , Nanopartículas , Estreptavidina/química , Biotina/química , Cinética , Nanopartículas/química , Tamaño de la PartículaRESUMEN
Quantitation of therapeutic monoclonal antibodies (mAbs) in human serum could ensure that patients have adequate levels of mAbs for effective treatment. This research describes the use of affinity, glass-fiber membranes in a 96-well-plate format for rapid (<5 min) quantitation of the therapeutic mAb trastuzumab and a mAb against the SARS-CoV-2 spike protein. Adsorption of a poly(acrylic acid)-containing film in membrane pores and activation of the -COOH groups in the film enable covalent-linking of affinity peptides or proteins to the membrane. Passage of mAb-containing serum through the affinity membrane results in mAb capture within 1 min. Subsequent rinsing, binding of a secondary antibody conjugated to a fluorophore, and a second rinse yield mAb-concentration-dependent fluorescence intensities in the wells. Calibration curves established from analyses on different days have low variability and allow determination of mAb levels in separately prepared samples with an average error <10%, although errors in single-replicate measurements may reach 40%. The assays can occur in diluted serum with physiologically relevant mAb concentrations, as well as in undiluted serum. Thus, the combination of 96-well plates containing affinity membranes, a microplate reader, and a simple vacuum manifold affords convenient mAb quantitation in <5 min.
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COVID-19 , SARS-CoV-2 , Afinidad de Anticuerpos , Humanos , Glicoproteína de la Espiga del Coronavirus , TrastuzumabRESUMEN
BACKGROUND: The COVID-19 pandemic thrust people living with HIV (PLWH) and HIV/AIDS service organizations into an environment ripe with uncertainty. This study examined Indiana HIV/AIDS service provider perceptions of how COVID-19 affected the overall health and access to care of their clients, and how the organizations prepared for, adapted, and responded to the needs of PLWH during the pandemic. METHODS: Guided by the socioecological model, fifteen semi-structured interviews were conducted with ten different HIV/AIDS service organizations across the state of Indiana. RESULTS: Despite the profound disruptions experienced by HIV programs, HIV/AIDS service organizations responded quickly to the challenges posed by the COVID-19 pandemic through myriad innovative strategies, largely informed by prior experiences with the HIV epidemic. CONCLUSIONS: The lessons provided by HIV/AIDS service organizations are invaluable to informing future pandemic response for PLWH. Service delivery innovations in response to the COVID-19 crisis may provide insights to improve HIV care continuity strategies for vulnerable populations far beyond the pandemic.
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Síndrome de Inmunodeficiencia Adquirida , COVID-19 , Infecciones por VIH , Síndrome de Inmunodeficiencia Adquirida/epidemiología , COVID-19/epidemiología , Infecciones por VIH/epidemiología , Infecciones por VIH/terapia , Humanos , Indiana/epidemiología , Pandemias , SARS-CoV-2RESUMEN
BACKGROUND: Globally, there are over 200 million cases of malaria annually and over 400,000 deaths. Early and accurate detection of low-density parasitaemia and asymptomatic individuals is key to achieving the World Health Organization (WHO) 2030 sustainable development goals of reducing malaria-related deaths by 90% and eradication in 35 countries. Current rapid diagnostic tests are neither sensitive nor specific enough to detect the low parasite concentrations in the blood of asymptomatic individuals. METHODS: Here, an imaging-based sensing technique, particle diffusometry (PD), is combined with loop mediated isothermal amplification (LAMP) on a smartphone-enabled device to detect low levels of parasitaemia often associated with asymptomatic malaria. After amplification, PD quantifies the Brownian motion of fluorescent nanoparticles in the solution during a 30 s video taken on the phone. The resulting diffusion coefficient is used to detect the presence of Plasmodium DNA amplicons. The coefficients of known negative samples are compared to positive samples using a one-way ANOVA post-hoc Dunnett's test for confirmation of amplification. RESULTS: As few as 3 parasite/µL of blood was detectable in 45 min without DNA extraction. Plasmodium falciparum parasites were detected from asymptomatic individuals' whole blood samples with 89% sensitivity and 100% specificity when compared to quantitative polymerase chain reaction (qPCR). CONCLUSIONS: PD-LAMP is of value for the detection of low density parasitaemia especially in areas where trained personnel may be scarce. The demonstration of this smartphone biosensor paired with the sensitivity of LAMP provides a proof of concept to achieve widespread asymptomatic malaria testing at the point of care.
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Enfermedades Asintomáticas/epidemiología , Pruebas Diagnósticas de Rutina/métodos , Malaria Falciparum/diagnóstico , Malaria Vivax/diagnóstico , Parasitemia/diagnóstico , Sistemas de Atención de Punto/normas , Teléfono Inteligente/estadística & datos numéricos , Niño , Preescolar , Humanos , Lactante , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Plasmodium falciparum/aislamiento & purificación , Plasmodium vivax/aislamiento & purificación , UgandaRESUMEN
Diabetes prevalence within the global population has nearly doubled since 1980, with the most rapid growth occurring in low- and middle-income countries. Diabetes management in resource-limited settings such as Haiti presents many challenges, including the storage of insulin. Despite a lack of published data on insulin thermostability, storage at 2-8°C or at room temperature (25°C) is recommended. In Haiti, access to refrigeration and thereby proper insulin storage is severely limited. Commercial storage devices such as the FRIO cooling wallet are cost-prohibitive and not available locally, and alternatives such as small clay pots are fragile and nonportable. Here, we designed and tested the cooling efficacy of a homemade wallet made of acrylate polymer beads and a hand-sewn cotton pouch compared to a FRIO wallet and a clay pot. All studies were conducted over a ten-day period at the Kay Mackenson Clinic in Montrouis, Haiti. Temperature and humidity values were continuously collected using wireless monitors placed inside each device, and hourly ambient temperature and humidity values were manually recorded. Evaporative cooling efficacy was calculated using collected data. The homemade wallet and FRIO cooling wallet demonstrated comparable cooling efficacy with an average of 71% and 73%, respectively. The clay pot demonstrated significantly decreased efficacy with an average of 27% (p < 0.05). The homemade insulin wallet is a promising alternative for the storage of insulin in low-resource settings without the financial and physical barriers of commercial and locally sourced devices. Additionally, this wallet could be readily adapted for the storage of other perishable medical supplies in low-income countries.
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Insulina , Refrigeración/instrumentación , Agua/fisiología , Frío , Almacenaje de Medicamentos/métodos , Diseño de Equipo , Haití , Humanos , Humedad , Insulina/uso terapéutico , Microesferas , Refrigeración/métodos , Temperatura , Agua/químicaRESUMEN
The demand for wearable and point-of-care devices has led to an increase in electrochemical sensor development to measure an ever-increasing array of biological molecules. In order to move from the benchtop to truly portable devices, the development of new biosensors requires miniaturized instrumentation capable of making highly sensitive amperometric measurements. To meet this demand, we have developed KickStat, a miniaturized potentiostat that combines the small size of the integrated Texas Instruments LMP91000 potentiostat chip (Texas Instruments, Dallas, TX, USA) with the processing power of the ARM Cortex-M0+ SAMD21 microcontroller (Microchip Technology, Chandler, AZ, USA) on a custom-designed 21.6 mm by 20.3 mm circuit board. By incorporating onboard signal processing via the SAMD21, we achieve 1 mV voltage increment resolution and an instrumental limit of detection of 4.5 nA in a coin-sized form factor. This elegant engineering solution allows for high-resolution electrochemical analysis without requiring extensive circuitry. We measured the faradaic current of an anti-cocaine aptamer using cyclic voltammetry and square wave voltammetry and demonstrated that KickStat's response was within 0.6% of a high-end benchtop potentiostat. To further support others in electrochemical biosensors development, we have made KickStat's design and firmware available in an online GitHub repository.
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As the capabilities of low-resource field testing have begun to expand to incorporate more complex diagnostic technologies, many of these devices remain tethered to large heaters requiring relatively high-power inputs. Highly efficient microheaters would enable miniaturization of devices for more economic and effective heating with high temperatures and sustained incubation. This work reports the development and application of resistive microheaters printed with nanosilver ink for improved methods of automated sample heating in paper-based point-of-care (POC) and in-field diagnostics. Resistance is easily predicted, and shapes can be altered to fit space and heat-transfer needs, sustained and discrete heating of precise regions are possible. Here, we demonstrate both isothermal nucleic acid amplification at 65 °C and bacterial culture at 37 °C using our microheaters. Printed nanosilver microheaters are easily integrated into reactions that require low-power battery heating, can sustain heating for 16-hour incubations, and cost between 0.17 and 0.58 US dollars each. Further, the microheaters are reusable, stable over 6 months, and can be wetted without degradation or reduction in conductivity. These versatile printed microheaters enable thermal control for a variety of low power heating applications.
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Sensitive and specific detection of pathogens via nucleic acid amplification is currently constrained to laboratory settings and portable equipment with costly fluorescent detectors. Nucleic acid-detecting lateral flow immunoassay strips (LFIAs) offer a low-cost visual transduction strategy at points of need. Unfortunately, these LFIAs frequently detect amplification byproducts that can yield spurious results which can only be deciphered through statistical analysis. We integrated customizable strand displacement probes into standard loop mediated isothermal amplification (LAMP) assays to prevent byproduct capture on commercial LFIAs. We find that combining strand displacement with LAMP (SD-LAMP) yields LFIA test band intensities that can be unequivocally interpreted by human subjects without additional instrumentation, thereby alleviating the need for a portable reader's analysis. Using SD-LAMP, we capture target amplicons on commercially available LFIAs from as few as 3.5 Vibrio cholerae and 2â¯750 Escherichia coli bacteria without false positive or false negative interpretation. Moreover, we demonstrate that LFIA capture of SD-LAMP products remain specific even in the presence of complex sample matrixes, providing a significant step toward reliable instrument-free pathogen detection outside of laboratories.
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Escherichia coli/aislamiento & purificación , Inmunoensayo , Técnicas de Amplificación de Ácido Nucleico , Vibrio cholerae/aislamiento & purificación , Células Cultivadas , Escherichia coli/citología , Humanos , Vibrio cholerae/citologíaRESUMEN
Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.
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Dispositivos Laboratorio en un Chip , Neisseria gonorrhoeae/aislamiento & purificación , Papel , Pruebas en el Punto de Atención , ADN Bacteriano/genética , Humanos , Neisseria gonorrhoeae/genéticaRESUMEN
Analytical characterization of DNA microviscosity provides critical biophysical insights into nuclear crowding, nucleic acid based pharmaceutical development, and nucleic acid based biosensor device design. However, most viscosity characterization methods require large sample volumes and destructive testing. In contrast, particle diffusometry permits in situ analysis of DNA microviscosity with short measurement times (8 s) using small volumes (<3 µL) which are compatible with DNA preparatory procedures. This unconventional biosensing approach involves measuring the change in sample viscosity using image processing and correlation-based algorithms. Particle diffusometry requires only a fluorescence microscope equipped with a charge-coupled device (CCD) camera and is a nondestructive measurement method. We use particle diffusometry to characterize the effect of DNA topology, length, and concentration on solution viscosity. In addition, we use particle diffusometry to detect the amplification of DNA from Staphylococcus aureus and Klebsiella pneumoniae, two pathogens commonly related to neonatal sepsis. Successful characterization of pathogen amplification with particle diffusometry provides a new opportunity to apply viscosity characterization toward downstream applications in nucleic acid based pathogen detection.
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Técnicas Biosensibles , ADN Bacteriano/análisis , Klebsiella pneumoniae/química , Staphylococcus aureus/química , Difusión , Tamaño de la Partícula , Propiedades de Superficie , ViscosidadRESUMEN
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.
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Técnicas Genéticas , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/diagnóstico , ARN/genética , Técnicas Genéticas/economía , Técnicas Genéticas/instrumentación , Técnicas Genéticas/normas , Humanos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Límite de Detección , Papel , Sistemas de Atención de Punto , ARN/químicaRESUMEN
BACKGROUND: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed. RESULTS: We developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/µL of saliva within 30 min without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples. SIGNIFICANCE: IACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices.
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Pandemias , Sistemas de Atención de Punto , Humanos , Reproducibilidad de los Resultados , Pruebas en el Punto de Atención , SARS-CoV-2/genética , ARN RibosómicoRESUMEN
Background/Objective: The utilization of rapid HIV tests has been effective at reducing transmission rates in high-risk populations by allowing individuals to receive diagnosis in as little as one minute and begin treatment. However, no current rapid tests can detect HIV immediately after infection in the acute HIV infection (AHI) phase, when the virus is at its most infectious, and instead require a waiting period of up to 90 days after exposure. Rapid HIV tests to detect AHI are currently under development. Investigation of stakeholder perspectives and context-specific needs are critical to ensure successful translation of novel AHI tests. The objectives of this study were to 1) understand context-specific factors such as barriers to HIV testing in Indiana, a state with one of 48 prioritized counties for HIV elimination; 2) assess the acceptability of a novel rapid AHI test, and 3) identify key implementation considerations for such a device, including ideal end-users. Methods: Semi-structured in-depth interviews were conducted with staff (n = 14) and clients (n = 5) of Indiana-based organizations that conduct HIV testing, including syringe service programs. Utilizing human-centered design frameworks, interview guides were developed and tailored to each participant group to understand their experiences with HIV testing, perspectives on a novel rapid AHI test in development, and preferences for self-testing versus testing by a community health worker (CHW) or a peer recovery coach. Thematic analysis was conducted to identify major themes, including barriers to HIV testing and perceived benefits and concerns of the proposed AHI test. Results: Overall acceptability for a novel AHI rapid test was high with a greater preference for CHW/Peerled testing. While self-testing was not a preferred modality, it was still seen as a potential tool to reach and address key barriers among high-risk individuals. Key considerations for implementation emphasized accuracy, cost-effectiveness, ease of use, ensuring access to counseling, education, and navigation to care while maintaining a human element to self-testing. Conclusion: Stakeholder engagement is meaningfully informing the design, development, and implementation of rapid AHI testing in order to facilitate adoption among populations at high-risk for HIV.
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Introduction: Approximately 1.5 million neonatal deaths occur among premature and small (low birthweight or small-for gestational age) neonates annually, with a disproportionate amount of this mortality occurring in low- and middle-income countries (LMICs). Hypothermia, the inability of newborns to regulate their body temperature, is common among prematurely born and small babies, and often underlies high rates of mortality in this population. In high-resource settings, incubators and radiant warmers are the gold standard for hypothermia, but this equipment is often scarce in LMICs. Kangaroo Mother Care/Skin-to-skin care (KMC/STS) is an evidence-based intervention that has been targeted for scale-up among premature and small neonates. However, KMC/STS requires hours of daily contact between a neonate and an able adult caregiver, leaving little time for the caregiver to care for themselves. To address this, we created a novel self-warming biomedical device, NeoWarm, to augment KMC/STS. The present study aimed to validate the safety and efficacy of NeoWarm. Methods: Sixteen, 0-to-5-day-old piglets were used as an animal model due to similarities in their thermoregulatory capabilities, circulatory systems, and approximate skin composition to human neonates. The piglets were placed in an engineered cooling box to drop their core temperature below 36.5°C, the World Health Organizations definition of hypothermia for human neonates. The piglets were then warmed in NeoWarm (n = 6) or placed in the ambient 17.8°C ± 0.6°C lab environment (n = 5) as a control to assess the efficacy of NeoWarm in regulating their core body temperature. Results: All 6 piglets placed in NeoWarm recovered from hypothermia, while none of the 5 piglets in the ambient environment recovered. The piglets warmed in NeoWarm reached a significantly higher core body temperature (39.2°C ± 0.4°C, n = 6) than the piglets that were warmed in the ambient environment (37.9°C ± 0.4°C, n = 5) (p < 0.001). No piglet in the NeoWarm group suffered signs of burns or skin abrasions. Discussion: Our results in this pilot study indicate that NeoWarm can safely and effectively warm hypothermic piglets to a normal core body temperature and, with additional validation, shows promise for potential use among human premature and small neonates.
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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.
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Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Staphylococcus epidermidis/metabolismo , Pared Celular/genética , Pared Celular/metabolismo , Matriz Extracelular/metabolismo , Presión Osmótica , Unión Proteica , Staphylococcus epidermidis/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
From HIV and influenza to emerging pathogens like COVID-19, each new infectious disease outbreak has highlighted the need for massively-scalable testing that can be performed outside centralized laboratory settings at the point-of-care (POC) in order to prevent, track, and monitor endemic and pandemic threats. Nucleic acid amplification tests (NAATs) are highly sensitive and can be developed and scaled within weeks while protein-based rapid tests require months for production. Combining NAATs with paper-based detection platforms are promising due to the manufacturability, scalability, and simplicity of each of these components. Typically, paper-based NAATs consist of three sequential steps: sample collection and preparation, amplification of DNA or RNA from pathogens of interest, and detection. However, these exist within a larger ecosystem of sample collection and interpretation workflow, usability, and manufacturability which can be vastly perturbed during a pandemic emergence. This review aims to explore the challenges of paper-based NAATs covering sample-to-answer procedures along with three main types of clinical samples; blood, urine, and saliva, as well as broader operational, scale up, and regulatory aspects of device development and implementation. To fill the technological gaps in paper-based NAATs, a sample-in-result-out system that incorporates the integrated sample collection, sample preparation, and integrated internal amplification control while also balancing needs of users and manufacturability upfront in the early design process is required.
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COVID-19 , Ácidos Nucleicos , Humanos , Sistemas de Atención de Punto , Pandemias , EcosistemaRESUMEN
Background: The COVID-19 pandemic has led to a rise in point-of-care (POC) and home-based tests, but concerns over usability, accuracy, and effectiveness have arisen. The incorporation of internal amplification controls (IACs), essential control for translational POC diagnostics, could mitigate false-negative and false-positive results due to sample matrix interference or inhibition. Although emerging POC nucleic acid amplification tests (NAATs) for detecting SARS-CoV-2 show impressive analytical sensitivity in the lab, the assessment of clinical accuracy with IACs is often overlooked. In some cases, the IACs were run spatially, complicating assay workflow. Therefore, the multiplex assay for pathogen and IAC is needed. Results: We developed a one-pot duplex reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for saliva samples, a non-invasive and simple collected specimen for POC NAATs. The ORF1ab gene of SARS-CoV-2 was used as a target and a human 18S ribosomal RNA in human saliva was employed as an IAC to ensure clinical reliability of the RT-LAMP assay. The optimized assay could detect SARS-CoV-2 viral particles down to 100 copies/µL of saliva within 30 minutes without RNA extraction. The duplex RT-LAMP for SARS-CoV-2 and IAC is successfully amplified in the same reaction without cross-reactivity. The valid results were easily visualized in triple-line lateral flow immunoassay, in which two lines (flow control and IAC lines) represent valid negative results and three lines (flow control, IAC, and test line) represent valid positive results. This duplex assay demonstrated a clinical sensitivity of 95%, specificity of 100%, and accuracy of 96% in 30 clinical saliva samples. Significance: IACs play a crucial role in ensuring user confidence with respect to the accuracy and reliability of at-home and POC molecular diagnostics. We demonstrated the multiplex capability of SARS-COV-2 and human18S ribosomal RNA RT-LAMP without complicating assay design. This generic platform can be extended in a similar manner to include human18S ribosomal RNA IACs into different clinical sample matrices.