RESUMO
A microfluidic based system was developed for automated online method for the rapid detection and monitoring of drinking water contamination utilising microbial Adrenosine-5'-Triphosphate (ATP) as a bacterial indicator. The system comprises a polymethyl methacrylate based microfluidic cartridge inserted into an enclosure incorporating the functions of fluid storage and delivery, lysis steps and real-time detection. Design, integration and operation of the resulting automated system are reported, including the lysis method, the design of the mixing circuit, the choices of flow rate, temperature and reagent amount. Calibration curves of both total and free ATP were demonstrated to be highly linear over a range from 2.5-5000â¯pg/mL with the limit of detection being lower than 2.5â¯pg/mL of total ATP. The system was trialled in a lab study with different types of water, with lysis efficiency being found to be strongly dependent upon water type. Further development is required before online implementation.
Assuntos
Trifosfato de Adenosina/análise , Bactérias/isolamento & purificação , Água Potável/microbiologia , Microfluídica/métodos , Qualidade da Água , Contagem de Colônia MicrobianaRESUMO
Giardia duodenalis is a ubiquitous flagellated protozoan parasite known to cause giardiasis throughout the world. Potential transmission vehicles for this zoonotic parasite are both water and food sources. As such consumption of water contaminated by feces, or food sources washed in contaminated water containing parasite cysts, may result in outbreaks. This creates local public health risks which can potentially cause widespread infection and long-term post-infection sequelae. This paper provides an up-to-date overview of G. duodenalis assemblages, sub-assemblages, hosts and locations identified. It also summarizes knowledge of potential infection/transmission routes covering water, food, person-to-person infection and zoonotic transmission from livestock and companion animals. Public health implications focused within the UK, based on epidemiological data, are discussed and recommendations for essential Giardia developments are highlighted.
Assuntos
Giardia lamblia/fisiologia , Giardíase , Animais , Giardíase/diagnóstico , Giardíase/epidemiologia , Giardíase/transmissão , Giardíase/veterinária , Humanos , Saúde Pública , Fatores de Risco , Reino UnidoRESUMO
Continuous and reliable monitoring of water sources for human consumption is imperative for public health. For protozoa, which cannot be multiplied efficiently in laboratory settings, concentration and recovery steps are key to a successful detection procedure. Recently, the use of megasonic energy was demonstrated to recover Cryptosporidium from commonly used water industry filtration procedures, forming thereby a basis for a simplified and cost effective method of elution of pathogens. In this article, we report the benefits of incorporating megasonic sonication into the current methodologies of Giardia duodenalis elution from an internationally approved filtration and elution system used within the water industry, the Filta-Max®. Megasonic energy assisted elution has many benefits over current methods since a smaller final volume of eluent allows removal of time-consuming centrifugation steps and reduces manual involvement resulting in a potentially more consistent and more cost-effective method. We also show that megasonic sonication of G. duodenalis cysts provides the option of a less damaging elution method compared to the standard Filta-Max® operation, although the elution from filter matrices is not currently fully optimised. A notable decrease in recovery of damaged cysts was observed in megasonic processed samples, potentially increasing the abilities of further genetic identification options upon isolation of the parasite from a filter sample. This work paves the way for the development of a fully automated and more cost-effective elution method of Giardia from water samples.
Assuntos
Água Potável/parasitologia , Giardia lamblia/isolamento & purificação , Giardíase/prevenção & controle , Sonicação/instrumentação , Doenças Transmitidas pela Água/prevenção & controle , Centrifugação/instrumentação , Água Potável/normas , Monitoramento Ambiental/economia , Monitoramento Ambiental/instrumentação , Filtração/instrumentação , Giardia lamblia/patogenicidade , Humanos , Sonicação/economia , Som , Microbiologia da Água/normasRESUMO
At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.
Assuntos
Criptosporidiose/diagnóstico , Cryptosporidium parvum/fisiologia , Giardia lamblia/fisiologia , Giardíase/diagnóstico , Técnicas Analíticas Microfluídicas , Oocistos/fisiologia , Animais , Células Cultivadas , Diagnóstico Diferencial , Impedância Elétrica , Citometria de Fluxo , Humanos , Sensibilidade e Especificidade , Análise de Célula Única , Especificidade da EspécieRESUMO
Inertial focusing is a microfluidic based separation and concentration technology that has expanded rapidly in the last few years. Throughput is high compared to other microfluidic approaches although sample volumes have typically remained in the millilitre range. Here we present a strategy for achieving rapid high volume processing with stacked and cascaded inertial focusing systems, allowing for separation and concentration of particles with a large size range, demonstrated here from 30 µm-300 µm. The system is based on curved channels, in a novel toroidal configuration and a stack of 20 devices has been shown to operate at 1 L/min. Recirculation allows for efficient removal of large particles whereas a cascading strategy enables sequential removal of particles down to a final stage where the target particle size can be concentrated. The demonstration of curved stacked channels operating in a cascaded manner allows for high throughput applications, potentially replacing filtration in applications such as environmental monitoring, industrial cleaning processes, biomedical and bioprocessing and many more.
RESUMO
Microfluidics has shown rapid growth in both research and development and offers significant commercialisation potential for biomedical and diagnostic applications in particular. However, there is a lack of awareness of microfluidics outside the field of study, and few dedicated educational programmes are available. While many topics incorporate microfluidics teaching, reported initiatives in the literature have not yet taken a problem based learning (PBL) approach to the delivery of practical sessions. The educational approaches already reported typically focus upon production and testing of pre-determined device designs for specific applications, using a "recipe" style of lab teaching. Here, we report on a newly designed lab section of a microfluidic teaching component utilising problem based learning (PBL) to involve the students in all aspects of design, manufacture, and performance characterisation of microfluidic solutions. Details of the lab design and development are given enabling others to replicate the lab structure described here or use it as a basis for the design of similar PBL microfluidics teaching labs. A key focus of the work has been the evaluation of the student experience, and the results of a survey indicate a high degree of student satisfaction and skills development due to the PBL approach.
RESUMO
We report on an inertial focussing based microfluidics technology for concentrating waterborne protozoa, achieving a 96% recovery rate of Cryptosporidium parvum and 86% for Giardia lamblia at a throughput (mL/min) capable of replacing centrifugation. The approach can easily be extended to other parasites and also bacteria.
Assuntos
Cryptosporidium parvum/isolamento & purificação , Giardia lamblia/isolamento & purificação , Técnicas Analíticas Microfluídicas/métodos , Água/parasitologia , Engenharia Biomédica/métodos , Centrifugação , Cryptosporidium parvum/patogenicidade , Desenho de Equipamento , Giardia lamblia/patogenicidade , MicrofluídicaRESUMO
Deterministic lateral displacement (DLD), a hydrodynamic, microfluidic technology, was first reported by Huang et al. in 2004 to separate particles on the basis of size in continuous flow with a resolution of down to 10 nm. For 10 years, DLD has been extensively studied, employed and modified by researchers in terms of theory, design, microfabrication and application to develop newer, faster and more efficient tools for separation of millimetre, micrometre and even sub-micrometre sized particles. To extend the range of potential applications, the specific arrangement of geometric features in DLD has also been adapted and/or coupled with external forces (e.g. acoustic, electric, gravitational) to separate particles on the basis of other properties than size such as the shape, deformability and dielectric properties of particles. Furthermore, investigations into DLD performance where inertial and non-Newtonian effects are present have been conducted. However, the evolvement and application of DLD has not yet been reviewed. In this paper, we collate many interesting publications to provide a comprehensive review of the development and diversity of this technology but also provide scope for future direction and detail the fundamentals for those wishing to design such devices for the first time.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Modelos Teóricos , Células Sanguíneas , Separação Celular , Células Cultivadas , Humanos , Tamanho da Partícula , PressãoRESUMO
Functional nonsynonymous single-nucleotide polymorphisms (nsSNPs) of folate metabolism genes can influence the methylation of tumour suppressor genes, thereby potentially impacting on tumour behaviour. To investigate whether such polymorphisms influence lung cancer survival, we genotyped 14 nsSNPs mapping to methylene-tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR); DNA methyltransferase (DNMT2), methylenetetrahydrofolate dehydrogenase (MTHFD1) and methenyltetrahydrofolate synthetase (MTHFS) in 619 Caucasian women with incident disease, 465 with non-small cell (NSCLC) and 154 with small cell lung cancer (SCLC). The most significant association detected was with MTHFS Thr202Ala, with carriers of variant alleles having a worse prognosis (hazard ratio (HR)=1.49; 95% confidence interval: 1.14-1.94). Associations were also detected between overall survival (OS) in SCLC and homozygosity for MTHFR 222Val (HR=1.92; 1.03-3.58) and between OS from NSCLC and MTRR 175Leu carrier status (HR=1.36; 1.06-1.75). While there is evidence that variation in the folate metabolism genes may influence prognosis from lung cancer, current data are insufficiently robust to distinguish individual patient outcome.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Pequenas/genética , Enzimas/genética , Ácido Fólico/metabolismo , Neoplasias Pulmonares/genética , Redes e Vias Metabólicas/genética , Feminino , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , PrognósticoRESUMO
The Gly388Arg polymorphism in the fibroblast growth factor receptor 4 (FGFR4) gene has been reported to influence prognosis in a wide variety of cancer types. To determine whether Gly388Arg is a marker for lung cancer prognosis, we genotyped 619 lung cancer patients with incident disease and examined the relationship between genotype and overall survival. While we employed a comprehensive set of statistical tests, including those sensitive to the detection of differences in early survival, our data provide little evidence to support the tenet that the FGFR4 Gly388Arg polymorphism is a clinically useful marker for lung cancer prognosis.
Assuntos
Substituição de Aminoácidos , Neoplasias Pulmonares/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Arginina , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/terapia , Terapia Combinada , Glicina , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Pessoa de Meia-Idade , Estadiamento de Neoplasias/mortalidade , Polimorfismo de Nucleotídeo Único , Prognóstico , Análise de SobrevidaRESUMO
Thalidomide is reported to suppress levels of several cytokines, angiogenic and growth factors including TNF-alpha, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6). The resulting anti-angiogenic, immunomodulatory and growth suppressive effects form the rationale for investigating thalidomide in the treatment of malignancies. We have evaluated the use of high-dose oral thalidomide (600 mg daily) in patients with renal carcinoma. 25 patients (all men; median age, 51 years; range 34-76 years) with advanced measurable renal carcinoma, who had either progressed on or were not suitable for immunotherapy, received thalidomide in an escalating schedule up to a maximum dose of 600 mg daily. Treatment continued until disease progression or unacceptable toxicity were encountered. 22 patients were assessable for response. 2 patients showed partial responses (9%; 95% CI: 1-29), 7 (32%; 95% CI: 14-55) had stable disease for more than 6 months and a further 5 (23%; 95% CI: 8-45) had stable disease for between 3 and 6 months. We also measured levels of TNF-alpha, bFGF, VEGF, IL-6 and IL-12 before and during treatment. In patients with SD > or = 3 months or an objective response, a statistically significant decrease in serum TNF-alpha levels was demonstrated (P = 0.05). The commonest toxicities were lethargy (> or = grade II, 10 patients), constipation (> or = grade II, 11 patients) and neuropathy (> or = grade II, 5 patients). Toxicities were of sufficient clinical significance for use of a lower and well tolerated dose of 400 mg in currently accruing studies.