RESUMO
We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in <20 minutes at 49 °C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in <30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , L-Lactato Desidrogenase/genética , Staphylococcus aureus Resistente à Meticilina/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Infecções Estafilocócicas/diagnóstico , Proteínas de Bactérias/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , L-Lactato Desidrogenase/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/economia , Sistemas Automatizados de Assistência Junto ao Leito/economia , Infecções Estafilocócicas/microbiologia , Fatores de TempoRESUMO
Lateral flow devices are commonly used for many point-of-care (POC) applications in low-resource settings. However, they lack the sensitivity needed for many analytes relevant in the diagnosis of diseases. One approach to achieve higher sensitivity is signal amplification, which is commonly used in laboratory assays, but uses reagents that require refrigeration and inherently requires multiple assay steps not normally compatible with POC settings. Enzyme-based signal amplification, such as the one used in ELISA, could greatly improve the limit of detection if it were translated to a format compatible with POC requirements. A signal-amplified POC device not only requires the reagents to be stored in a stable form, but also requires automation of the multiple sequential steps of signal amplification protocols. Here, we describe a method for the long-term dry storage of ELISA reagents: horseradish peroxidase (HRP) conjugated antibody label and its colorimetric substrate diaminobenzidine (DAB). The HRP conjugate retained â¼80% enzymatic activity after dry storage at 45 °C for over 5 months. The DAB substrate was also stable at 45 °C and exhibited no detectable loss of activity over 3 months. These reagents were incorporated into a two-dimensional paper network (2DPN) device that automated the steps of ELISA for the detection of a malarial biomarker. These results demonstrate the potential of enzyme-based signal amplification for enhanced sensitivity in POC devices for low resource settings.
Assuntos
Antígenos de Protozoários/análise , Ensaio de Imunoadsorção Enzimática/instrumentação , Peroxidase do Rábano Silvestre/química , Imunoconjugados/química , Plasmodium falciparum/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Preservação Biológica , Proteínas de Protozoários/análise , 3,3'-Diaminobenzidina/metabolismo , Antígenos de Protozoários/imunologia , Dessecação , Estabilidade Enzimática , Desenho de Equipamento , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Imunoconjugados/imunologia , Indicadores e Reagentes , Limite de Detecção , Malária Falciparum/diagnóstico , Malária Falciparum/microbiologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologiaAssuntos
Procedimentos Cirúrgicos Cardíacos/métodos , Fator VIII/administração & dosagem , Cuidados Pré-Operatórios/métodos , Doenças de von Willebrand/sangue , Doenças de von Willebrand/cirurgia , Fator de von Willebrand/administração & dosagem , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Fator VIII/metabolismo , Humanos , Doenças de von Willebrand/diagnóstico , Fator de von Willebrand/metabolismoRESUMO
Background: The American Board of Medical Specialties definition of medical professionalism cites the need to acquire, maintain, and advance a value system serving the patients' and public's interests above self-interests.4 Medical professionalism is a one of the core physician competencies assessed by both the ACGME training program evaluation and the ABA certification process. However, a growing concern for the decline of professionalism and altruism in medicine resulted in increased publications on the matter, citing various potential sources for the issue. Methods: All residents and fellows (Focus Group 1) of the Anesthesiology Department of Montefiore Medical Center in Bronx, NY were invited to participate in a semi-structured interview via Zoom, held on two separate dates. A separate invitation was sent to the faculty of the department (Focus Group 2), held on one date. During the interview, guiding questions were provided by the 4 interviews to facilitate discussion. The interviewers, all members of the anesthesia faculty, took notes as the interviews progressed. The notes were reviewed for common themes as well as supporting and contradicting quotations. Results: A total of 23 residents and fellows and a total of 25 faculty members within the Anesthesiology department at Montefiore Medical Center were interviewed. Amongst the findings, common discussions concerned motivating and demotivating factors contributing to the professionalism and altruism exhibited by the residents and fellows when caring for critical COVID-19 patients during the height of the pandemic. It was widely regarded that patient improvement, community and team support, as well as intrinsic desire to help greatly motivated the team while continuous patient deterioration, uncertainty in staffing and treatment, and concerns for personal and family safety were sources of discouragement. Overall, faculty perceived an increased demonstration of altruism amongst residents and fellows. The statements made by the residents and fellows during their interviews supported this observation. Conclusions: The actions of the Montefiore Anesthesiology residents and fellows demonstrated that altruism and professionalism were readily available amongst physicians. Increased levels of empathy and responsibility contributed to a demonstration of professionalism that challenges previous views of a perceived decline of these attributes in the medical field. The findings of this study stress the importance of creating a curriculum and exercise that stress empathy-based care and altruism in order to improve resident satisfaction and decrease feelings of burnout. Additionally, curriculum additions to facilitate professionalism are proposed.
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The lateral flow test has become the standard bioassay format in low-resource settings because it is rapid, easy to use, and low in cost, uses reagents stored in dry form, and is equipment-free. However, lateral flow tests are often limited to a single chemical delivery step and not capable of the multistep processing characteristic of high performance laboratory-based assays. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions; this allows incorporation of multistep chemical processing, while still retaining the advantages of conventional lateral flow tests. Here, we demonstrate this format for an easy-to-use, signal-amplified sandwich format immunoassay for the malaria protein PfHRP2. The card contains reagents stored in dry form such that the user need only add sample and water. The multiple flows in the device are activated in a single user step of folding the card closed; the configuration of the paper network automatically delivers the appropriate volumes of (i) sample plus antibody conjugated to a gold particle label, (ii) a rinse buffer, and (iii) a signal amplification reagent to the capture region. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.
Assuntos
Antígenos de Protozoários/análise , Imunoensaio , Malária/metabolismo , Papel , Ouro/química , Malária/diagnóstico , Malária/parasitologia , Malária/patologia , Plasmodium falciparum/isolamento & purificação , Plasmodium falciparum/metabolismo , Sistemas Automatizados de Assistência Junto ao Leito , Proteínas de Protozoários/análiseRESUMO
Aim and Objectives: The aim and objective of the study were to evaluate the immunohistochemical expression of proliferative markers, Ki67, and MCM2 in oral epithelial dysplasia (OED) and oral squamous cell carcinoma (OSCC), to compare the relationship of their staining patterns, and to look for correlation between them, if any. Materials and Methods: Thirty archival paraffin-embedded tissue blocks of previously diagnosed cases of OED, OSCC each, and 10 normal oral mucosa were used in the study. Immunohistochemical staining for MCM2 and Ki67 markers was done and the slides were individually evaluated for MCM2 and Ki67 expression, with immunopositivity determined on the basis of dark brown staining of the nucleus. The number of positively stained nuclei was counted in 10 representative areas and the data were charted and statistically analyzed. Results: The overall mean expression of both the proteins increased progressively from normal mucosa to OED to OSCC. In normal mucosa, all positively stained nuclei were seen in the basal compartment of the epithelium, while in dysplastic cases, expression was seen toward the surface of squamous epithelium. In OSCC, the frequency of expression of MCM2 and Ki-67 proteins showed an inverse correlation with the degree of tumor differentiation. In well-differentiated cases, the positivity of either marker was restricted to the outermost layer of the tumor cells. In moderately differentiated cases, an expression of Ki-67 was more diffuse in inner layers, whereas the MCM2 antigen was found to be more intense and diffuse in both the inner and outer layers. Whereas in poorly differentiated SCC, positive expression was seen in most of the tumor cells, the mean expression of MCM2 was found to be higher than that of Ki67 in all cases. Conclusion: MCM2, as a proliferation marker, is superior to Ki67 as it indicates the capacity of proliferation and the ability of DNA replication of a cell.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Humanos , Hiperplasia , Antígeno Ki-67/metabolismo , Componente 2 do Complexo de Manutenção de Minicromossomo , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e PescoçoRESUMO
Point-of-care diagnostic assays that are rapid, easy-to-use, and low-cost are needed for use in low-resource settings; the lateral flow test has become the standard bioassay format in such settings because it meets those criteria. However, for a number of analytes, conventional lateral flow tests lack the sensitivity needed to have clinical utility. To address this limitation, we are developing a paper network platform that extends the conventional lateral flow test to two dimensions. The two-dimensional structures allow incorporation of multistep processes for improved sensitivity, while still retaining the positive aspects of conventional lateral flow tests. Here we create an easy-to-use, signal-amplified immunoassay based on a modified commercial strip test for human chorionic gonadotropin, the hormone used to detect pregnancy, and demonstrate an improved limit of detection compared to a conventional lateral flow assay. These results highlight the potential of the paper network platform to enhance access to high-quality diagnostic capabilities in low-resource settings in the developed and developing worlds.
Assuntos
Imunoensaio/métodos , Papel , Gonadotropina Coriônica/análise , Feminino , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , GravidezAssuntos
Ponte Cardiopulmonar/efeitos adversos , Implante de Prótese de Valva Cardíaca/efeitos adversos , Complicações Intraoperatórias/diagnóstico por imagem , Insuficiência da Valva Mitral/diagnóstico por imagem , Insuficiência da Valva Mitral/cirurgia , Adulto , Feminino , Humanos , Complicações Intraoperatórias/etiologia , Valva Mitral/diagnóstico por imagem , Valva Mitral/cirurgiaRESUMO
From March to June of 2020, Montefiore Medical Center faced one of the most acute surges in hospital admissions and critical illness ever experienced in the United States due to the severe acute respiratory syndrome coronavirus 2 pandemic. The pandemic had not yet spread to most of the country, and there was a relative deficit of knowledge regarding treatments, prognosis, and prevention of the virus, making this experience relatively unique and challenging. As part of a surge plan, our institution converted nonclinical spaces, such as conference rooms, to inpatient care settings and placed elective surgeries on hold to free up resources. A central deployment office suspended anesthesiology resident rotations and instead assigned them to intensive care settings based on need. For the Montefiore Medical Center Department of Anesthesiology, preserving its academic mission and commitment to Graduate Medical Education was essential. Adaptations included changing the residency rotation structure to biweekly, converting didactics online, ensuring adequate case numbers for graduating residents, actively pursuing wellness interventions, and prioritizing the safety of the residents caring for patients with coronavirus disease 2019 (COVID-19). In this brief report, the authors discuss solutions devised to maintain the quality of anesthesiology resident education and training as much as possible during the COVID-19 surge.
RESUMO
The novel SARS-CoV-2 pandemic starting in 2019 profoundly changed the world, and thousands of residents of New York City were affected, leading to one of the most acute surges in regional hospital capacity. As the largest academic medical center in the Bronx, Montefiore Medical Center was immediately impacted, and the entire hospital was mobilized to address the needs of its community. In this article, we describe our experiences as a large academic anesthesiology department during this pandemic. Our goals were to maximize our staff's expertise, maintain our commitment to wellness and safety, and preserve the quality of patient care. Lessons learned include the importance of critical care training presence and leadership, the challenges of converting an ambulatory surgery center to an intensive care unit (ICU), and the management of effective communication. Lastly, we provide suggestions for institutions facing an acute surge, or subsequent waves of COVID-19, based on a single center's experiences.
Assuntos
Centros Médicos Acadêmicos/tendências , Anestesiologia/tendências , COVID-19/epidemiologia , Cuidados Críticos/tendências , Reestruturação Hospitalar/tendências , Admissão e Escalonamento de Pessoal/tendências , Centros Médicos Acadêmicos/normas , Anestesiologia/normas , COVID-19/terapia , Cuidados Críticos/normas , Pessoal de Saúde/normas , Pessoal de Saúde/tendências , Reestruturação Hospitalar/normas , Humanos , Cidade de Nova Iorque , Pandemias , Admissão e Escalonamento de Pessoal/normasRESUMO
Probing the interaction of biological systems with radio frequencies holds great promise for research and drug screening applications. While a common assumption is that biological systems do not operate at radio frequencies, we find that currents due to ion transport through channels and pores in cell membranes are in the pA to nA range. These values translate via the average current I = ne/tau(d) = nef to frequencies in the range of 1 MHz-1 GHz, where n is the average number of ions transported and tau(d) is the dwell time of the ions in the channel. It is thus desirable to have circuitry available which facilitates radio frequency spectroscopy of ion transport. This will yield real-time in vitro information on ion channel operation. Here we present measurements on the local interaction of a radio frequency signal with single ion channels and pores. We find radio frequency rectification and pumping on the channels and pores embedded in suspended bilipid membranes, recorded in direct current measurements. This electromagnetic modulation can be used to probe the dynamics of ion channel conformational changes.
Assuntos
Canais Iônicos/fisiologia , Bicamadas Lipídicas , Modelos Biológicos , Ondas de Rádio , Alameticina/metabolismo , Membrana Celular/fisiologia , Campos Eletromagnéticos , Proteínas Fúngicas/metabolismo , Proteínas Hemolisinas/metabolismoRESUMO
Introduction: ATTEMPTS to enforce optimization practices for operating room (OR) efficiency are often interpreted as a "pressure for production" which threatens patient safety. The aim of this study is to assess if and how improvements in OR efficiency affect patient safety and thus the quality of care. Methods: In an attempt to optimize OR efficiency, a new OR management approach "Integrated Practice Improvement Solutions" (IPIS) was developed at the Weiler Division of Montefiore Medical Center in 2011. IPIS is a flexible managerial system based on elements of multiple practice improvement methodologies incorporated into an open source framework. It was implemented in 2012. The data presented covers the period from 2012 through 2014 when the system was temporarily discontinued due to administrative restructuring. Data from 2011 was used as a baseline. The impact of IPIS on patient safety and quality of care was assessed based on quality improvement and patient safety (QIPS) Committee reports covering the same period of time. Results: IPIS implementation resulted in an increase in surgical workload by an average of 10.7%, an increase in OR and anesthesia revenues by 18.5% and 6.9%, respectively, and decreases in turnover time by 15% and overtime for the anesthesia staff by 26%. Based on QIPS reports, the total number of complications potentially attributable to "production pressure" was 0.25%, 0.2% and 0.16% in 2012, 2013 and 2014, respectively compared to 0.21% in 2011 (p = 0.56). Conclusions: Gradual implementation of a methodologically structured improvement in OR efficiency has no negative impact on patient safety and quality of care.
Assuntos
Eficiência Organizacional , Salas Cirúrgicas , Benchmarking , Humanos , Melhoria de QualidadeRESUMO
OBJECTIVE: Sleep deprivation has a negative effect on neurocognitive performance. The King-Devick test (KDT), which tests speed and accuracy of number-reading, requires integrity of saccades, visual processing, and cognition. This study investigated effects of sleep deprivation in on-call residents using KDT. METHODS: A prospective cohort study was conducted among 80 residents. KDT was performed at the beginning and end of an overnight call shift for the residents in the experimental group. A control group was tested at the beginning of 2 consecutive day shifts. Estimates of hours of sleep, Karolinska Sleepiness Scale (KSS)(1â¯=â¯extremely alert, 9â¯=â¯extremely sleepy), and time and accuracy of KDT were recorded. RESULTS: 42 residents were tested before and after overnight call shifts and 38 served as controls. Change in test time differed between the groups, with the experimental group performing 0.54(SDâ¯=â¯4.0) seconds slower after their night on call and the control group performing 2.32(SDâ¯=â¯3.0) seconds faster on the second day, pâ¯<â¯0.001. This difference was larger in surgical compared to medical residents. CONCLUSIONS: Sleep deprivation was inversely correlated with neurocognitive performance as measured by KDT, with more effect on surgical than medical residents. Further research could investigate whether this test could help determine fatigue level and ability to continue working after a long shift.
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Movimentos Oculares/fisiologia , Fadiga/diagnóstico , Internato e Residência , Doenças Profissionais/diagnóstico , Privação do Sono/diagnóstico , Adulto , Cognição/fisiologia , Medições dos Movimentos Oculares , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Estudos ProspectivosRESUMO
Successful long-term storage of a "smart dust" device integrating biomolecular motors and complex protein assemblies has been demonstrated using freezing or lyophilization, which implies that fabrication and application can be separated even for complex bionanodevices.
Assuntos
Microtúbulos , Nanotecnologia/instrumentação , Animais , Biotina/química , Química Encefálica , Soluções Tampão , Bovinos , Drosophila melanogaster/química , Corantes Fluorescentes/química , Liofilização , Congelamento , Concentração de Íons de Hidrogênio , Cinesinas/química , Microtúbulos/química , Microtúbulos/fisiologia , Fungos Mitospóricos/química , Nanotecnologia/métodos , Rodaminas/química , Fatores de Tempo , Tubulina (Proteína)/químicaRESUMO
The combination of inorganic semiconductor nanocrystals, also called quantum dots (QDs), with biological materials has recently attracted considerable interest since the QDs can be used as superior fluorescent labels. Here, we report on CdSe QD initiated current bursts in lipid bilayer membranes on application of a bias voltage. The current bursts observed resemble those produced by the peptaibol class of antibiotics such as alamethicin and trichorzins. The current fluctuations are dependent on the bias voltage and on the concentration of the QD applied to the membrane. Our data suggest that QDs with intrinsic dipole moments similar to alamethicin can be controlled by an external electric field, which creates a torque resulting in the insertion into the lipid membrane.
Assuntos
Coloides/química , Eletroquímica/métodos , Eletroporação/métodos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/efeitos da radiação , Fluidez de Membrana/efeitos da radiação , Potenciais da Membrana/efeitos da radiação , Pontos Quânticos , Coloides/efeitos da radiação , Condutividade Elétrica , Campos Eletromagnéticos , Membranas Artificiais , Permeabilidade , PorosidadeRESUMO
Given the improved survival in patients with corrected dextro-transposition of great arteries (D-TGA), it has evolved into an adult congenital heart defect. It is important to understand the management and complications observed in this population that eventually progresses to systemic ventricular failure requiring cardiac transplantation. Our case focuses on the rapid right ventricle (RV) deterioration of a patient with corrected D-TGA following a surgical procedure requiring systemic support. With such patients awaiting heart transplantation, there are limited assist devices available for RV support and no right ventricular assist device is approved for destination therapy yet. Current indications for implantation of the HeartWare ventricular assist device (HVAD) are limited by the Food and Drug Administration (FDA) to the left ventricle support as a bridge to transplantation. However, its use in the United States for right-sided support has rarely been described for adult congenital defects. In this case, a HeartWare assist device was used to provide systemic support as a bridge to cardiac transplant. The size and implantation design of the HVAD makes it a promising option for patients with this challenging patient population and RV failure as a late complication.
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Coração Auxiliar , Transposição dos Grandes Vasos/cirurgia , Disfunção Ventricular Direita/cirurgia , Adulto , Desenho de Equipamento , Transplante de Coração , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Transposição dos Grandes Vasos/fisiopatologia , Disfunção Ventricular Direita/fisiopatologiaRESUMO
Lateral flow tests (LFTs) are an ingenious format for rapid and easy-to-use diagnostics, but they are fundamentally limited to assay chemistries that can be reduced to a single chemical step. In contrast, most laboratory diagnostic assays rely on multiple timed steps carried out by a human or a machine. Here, we use dissolvable sugar applied to paper to create programmable flow delays and present a paper network topology that uses these time delays to program automated multi-step fluidic protocols. Solutions of sucrose at different concentrations (10-70% of saturation) were added to paper strips and dried to create fluidic time delays spanning minutes to nearly an hour. A simple folding card format employing sugar delays was shown to automate a four-step fluidic process initiated by a single user activation step (folding the card); this device was used to perform a signal-amplified sandwich immunoassay for a diagnostic biomarker for malaria. The cards are capable of automating multi-step assay protocols normally used in laboratories, but in a rapid, low-cost, and easy-to-use format.
Assuntos
Técnicas de Laboratório Clínico/instrumentação , Técnicas de Laboratório Clínico/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Papel , Colódio/química , Desenho de Equipamento , Sistemas Automatizados de Assistência Junto ao Leito , Solubilidade , Sacarose/química , Fatores de TempoRESUMO
This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays.
RESUMO
In many low resource settings multiple diseases are endemic. There is a need for appropriate multi-analyte diagnostics capable of differentiating between diseases that cause similar clinical symptoms. The work presented here was part of a larger effort to develop a microfluidic point-of-care system, the DxBox, for sample-to-result differential diagnosis of infections that present with high rapid-onset fever. Here we describe a platform that detects disease-specific antigens and IgM antibodies. The disposable microfluidic cards are based on a flow-through membrane immunoassay carried out on porous nitrocellulose, which provides rapid diffusion for short assay times and a high surface area for visual detection of colored assay spots. Fluid motion and on-card valves were driven by a pneumatic system and we present designs for using pneumatic control to carry out assay functions. Pneumatic actuation, while having the potential advantage of inexpensive and robust hardware, introduced bubbles that interfered with fluidic control and affected assay results. The cards performed all sample preparation steps including plasma filtration from whole blood, sample and reagent aliquoting for the two parallel assays, sample dilution, and IgG removal for the IgM assays. We demonstrated the system for detection of the malarial pfHRPII antigen (spiked) and IgM antibodies to Salmonella Typhi LPS (patient plasma samples). All reagents were stored on card in dry form; only the sample and buffer were required to run the tests. Here we detail the development of this platform and discuss its strengths and weaknesses.