Improved Bacterial and Viral Recoveries from 'Complex' Samples using Electrophoretically Assisted Acoustic Focusing

Automated front-end sample preparation technologies can significantly enhance the sensitivity and reliability of biodetection assays [1]. We are developing advanced sample preparation technologies for biowarfare detection and medical point-of-care diagnostics using microfluidic systems with continuous sample processing capabilities. Here we report an electrophoretically assisted acoustic focusing technique to rapidly extract and enrich viral and bacterial loads from 'complex samples', applied in this case to human nasopharyngeal samples as well as simplified surrogates. The acoustic forces capture and remove large particles (> 2 {micro}m) such as host cells, debris, dust, and pollen from the sample. We simultaneously apply an electric field transverse to the flow direction to transport small ({le} 2 {micro}m), negatively-charged analytes into a separate purified recovery fluid using a modified H-filter configuration [Micronics US Patent 5,716,852]. Hunter and O'Brien combined transverse electrophoresis and acoustic focusing to measure the surface charge on large particles, [2] but to our knowledge, our work is the first demonstration combining these two techniques in a continuous flow device. Marina et al. demonstrated superimposed dielectrophoresis (DEP) and acoustic focusing for enhanced separations [3], but these devices have limited throughput due to the rapid decay of DEP forces. Both acoustic standing waves and electric fields exert significant forces over the entire fluid volume in microchannels, thus allowing channels with larger dimensions (> 100 {micro}m) and high throughputs (10-100 {micro}L/min) necessary to process real-world volumes (1 mL). Previous work demonstrated acoustic focusing of microbeads [4] and biological species [5] in various geometries. We experimentally characterized our device by determining the biological size-cutoff where acoustic radiation pressure forces no longer transport biological particles. Figure 1 shows images of E.Coli ({approx}1 {micro}m) and yeast ({approx}4-5 {micro}m) flowing in a microchannel (200 {micro}m deep, 500 {micro}m wide) at a flow rate of 10 {micro}L/min. The E.Coli does not focus in the acoustic field while the yeast focuses at the channel centerline. This result suggests the acoustic size-cutoff for biological particles in our device lies between 2 and 3 {micro}m. Transverse electrophoresis has been explored extensively in electric field flow fractionation [6] and isoelectric focusing devices [7]. We demonstrated transverse electrophoretic transport of a wide variety of negatively-charged species, including fluorophores, beads, viruses, E.Coli, and yeast. Figure 2 shows the electromigration of a fluorescently labeled RNA virus (MS2) from the lower half of the channel to the upper half region with continuous flow. We demonstrated the effectiveness of our electrophoretically assisted acoustic focusing device by separating virus-like particles (40 nm fluorescent beads, selected to aid in visualization) from a high background concentration of yeast contaminants (see Figure 3). Our device allows for the efficient recovery of virus into a pre-selected purified buffer while background contaminants are acoustically captured and removed. We also tested the device using clinical nasopharyngeal samples, both washes and lavages, and demonstrated removal of unknown particulates (>2 ?m size) from the sample. Our future research direction includes spiking known amounts of bacteria and viruses into clinical samples and performing quantitative off-chip analysis (real-time PCR and flow cytometry)

Separation of Yeast Cells from MS2 Viruses Using Acoustic Radiation Force

We report a rapid and robust separation of Saccharomyces cerevisiae and MS2 bacteriophage using acoustic focusing in a microfluidic device. A piezoelectric transducer (PZT) generates acoustic standing waves in the microchannel. These standing waves induce acoustic radiation force fields that direct microparticles towards the nodes (i.e., pressure minima) or the anti-nodes (i.e., pressure maxima) of the standing waves depending on the relative compressidensity between the particle and the suspending liquid.[1] For particles larger than 2 {micro}m, the transverse velocities generated by these force fields enable continuous, high throughput separation. Extensive work in the last decade [2-4] has demonstrated acoustic focusing for manipulating microparticles or biological samples in microfluidic devices. This prior work has primarily focused on experimental realization of acoustic focusing without modeling or with limited one-dimensional modeling estimates. We recently developed a finite element modeling tool to predict the two-dimensional acoustic radiation force field perpendicular to the flow direction in microfluidic devices.[1] Here we compare results from this model with experimental parametric studies including variations of the PZT driving frequencies and voltages as well as various particle sizes and compressidensities. These experimental parametric studies also provide insight into the development of an adjustable 'virtual' pore-size filter as well as optimal operating conditions for various microparticle sizes. Figure 1 shows a typical experimental acoustic focusing result for microparticles (diameter = 2.0 {micro}m) in a 500 {micro}m wide by 200 {micro}m deep microchannel. In this case, the PZT driving frequency and voltage are, respectively, 1.459 MHz and 6.6 V. The microparticles tightly focus (full width half maximum (FWHM) {approx}30 {micro}m) less than 30 s after the initiation of the acoustic field. We simulated the same geometry and operating conditions for comparison. The surface plot in Figure 2 illustrates the two-dimensional pressure field orthogonal to the flow direction (x-direction) from the simulation. The superimposed vector plot shows the acoustic radiation force in this plane. The dark regions and the light regions respectively represent the nodes and anti-nodes of the acoustic pressure field. The corresponding force field predicts acoustic focusing at the center of the microchannel, which is confirmed by the experimental results shown in Figure 1. We demonstrated the separation of Saccharomyces cerevisiae (typical cell size of 4-6 {micro}m depending on the cell growth stage, measured using a Coulter counter) and MS2 bacteriophage (typical diameter {approx}30 nm [5]) using acoustic focusing (Figure 3). A mixture of S. cerevisiae and MS2 labeled with Ribogreen was prepared and injected into one inlet of the microchip (i.e., half of the microchannel was filled with the sample). We varied driving voltages from 1.96 to 4.76 V, while fixing the driving frequency at 1.459 MHz and flow rate at 20 {micro}l/min. The acoustic radiation force did not affect the MS2 viruses, and their concentration profile remained unchanged. Increased driving voltages enhanced the acoustic focusing of the yeast cells thereby achieving good separation. We are able to achieve yields of > 80% and sample purities of > 90% in this continuous-flow sample preparation device

Clinical Validation of Integrated Nucleic Acid and Protein Detection on an Electrochemical Biosensor Array for Urinary Tract Infection Diagnosis

BACKGROUND: Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management. METHODOLOGY/PRINCIPAL FINDINGS: The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both). CONCLUSION/SIGNIFICANCE: We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics

No Association between Fish Intake and Depression in over 15,000 Older Adults from Seven Low and Middle Income Countries–The 10/66 Study

Background: Evidence on the association between fish consumption and depression is inconsistent and virtually nonexistent from low-and middle-income countries. Using a standard protocol, we aim to assess the association of fish consumption and late-life depression in seven low-and middle-income countries. Methodology/Findings: We used cross-sectional data from the 10/66 cohort study and applied two diagnostic criteria for late-life depression to assess the association between categories of weekly fish consumption and depression according to ICD-10 and the EURO-D depression symptoms scale scores, adjusting for relevant confounders. All-catchment area surveys were carried out in Cuba, Dominican Republic, Venezuela, Peru, Mexico, China, and India, and over 15,000 community-dwelling older adults (65+) were sampled. Using Poisson models the adjusted association between categories of fish consumption and ICD-10 depression was positive in India (p for trend = 0.001), inverse in Peru (p = 0.025), and not significant in all other countries. We found a linear inverse association between fish consumption categories and EURO-D scores only in Cuba (p for trend = 0.039) and China (p&lt;0.001); associations were not significant in all other countries. Between-country heterogeneity was marked for both ICD-10 (I-2&gt;61%) and EURO-D criteria (I-2&gt;66%). Conclusions: The associations of fish consumption with depression in large samples of older adults varied markedly across countries and by depression diagnosis and were explained by socio-demographic and lifestyle variables. Experimental studies in these settings are needed to confirm our findings.Multidisciplinary SciencesSCI(E)SSCI0ARTICLE6null

Ageing and dementia in low and middle income countries - Using research to engage with public and policy makers

Abstract While two thirds of the 24 million people with dementia worldwide live in low and middle income countries, very little research has been conducted to support policy making in these regions. Among the non-communicable diseases, dementia (in common with other chronic NCDs linked more to long-term disability than to mortality) has been relatively under-prioritized. International agreements, plans and policy guidelines have called for an end to ageist discrimination and a focus upon reducing disadvantage arising from poverty and the consequences of ill health. Social protection, access to good quality age-appropriate healthcare and addressing the problem of disability are all key issues. However, as yet, little progress has been made in addressing these concerns. In this review we outline the current international policy agenda for older individuals, and its specific relevance to those with dementia and other disabling non-communicable diseases. We consider the potential for epidemiological research to raise awareness, refine the policy agenda, and promote action, using the example of the dissemination strategy developed by the 10/66 Dementia Research Group

Equity in the delivery of community healthcare to older people: findings from 10/66 Dementia Research Group cross-sectional surveys in Latin America, China, India and Nigeria

<p>Abstract</p> <p>Background</p> <p>To describe patterns of recent health service utilisation, and consequent out-of-pocket expenses among older people in countries with low and middle incomes, and to assess the equity with which services are accessed and delivered.</p> <p>Methods</p> <p>17,944 people aged 65 years and over were assessed in one-phase population-based cross-sectional surveys in geographically-defined catchment areas in nine countries - urban and rural sites in China, India, Mexico and Peru, urban sites in Cuba, Dominican Republic, Puerto Rico and Venezuela, and a rural site in Nigeria. The main outcome was use of community health care services in the past 3 months. Independent associations were estimated with indicators of need (dementia, depression, physical impairments), predisposing factors (age, sex, and education), and enabling factors (household assets, pension receipt and health insurance) using Poisson regression to generate prevalence ratios and fixed effects meta-analysis to combine them.</p> <p>Results</p> <p>The proportion using healthcare services varied from 6% to 82% among sites. Number of physical impairments (pooled prevalence ratio 1.37, 95% CI 1.26-1.49) and ICD-10 depressive episode (pooled PR 1.21, 95% CI 1.07-1.38) were associated with service use, but dementia was inversely associated (pooled PR 0.93, 95% CI 0.90-0.97). Other correlates were female sex, higher education, more household assets, receiving a pension, and health insurance. Standardisation for age, sex, physical impairments, depression and dementia did not explain variation in service use. There was a strong borderline significant ecological correlation between the proportion of consultations requiring out-of-pocket costs and the prevalence of health service use (r = -0.50, p = 0.09).</p> <p>Conclusions</p> <p>While there was little evidence of ageism, inequity was apparent in the independent enabling effects of education and health insurance cover, the latter particularly in sites where out-of-pocket expenses were common, and private health insurance an important component of healthcare financing. Variation in service use among sites was most plausibly accounted for by stark differences in the extent of out-of-pocket expenses, and the ability of older people and their families to afford them. Health systems that finance medical services through out-of-pocket payments risk excluding the poorest older people, those without a secure regular income, and the uninsured.</p

A study protocol for the evaluation of occupational mutagenic/carcinogenic risks in subjects exposed to antineoplastic drugs: a multicentric project

<p>Abstract</p> <p>Background</p> <p>Some industrial hygiene studies have assessed occupational exposure to antineoplastic drugs; other epidemiological investigations have detected various toxicological effects in exposure groups labeled with the job title. In no research has the same population been studied both environmentally and epidemiologically. The protocol of the epidemiological study presented here uses an integrated environmental and biological monitoring approach. The aim is to assess in hospital nurses preparing and/or administering therapy to cancer patients the current level of occupational exposure to antineoplastic drugs, DNA and chromosome damage as cancer predictive effects, and the association between the two.</p> <p>Methods/Design</p> <p>About 80 healthy non-smoking female nurses, who job it is to prepare or handle antineoplastic drugs, and a reference group of about 80 healthy non-smoking female nurses not occupationally exposed to chemicals will be examined simultaneously in a cross-sectional study. All the workers will be recruited from five hospitals in northern and central Italy after their informed consent has been obtained.</p> <p>Evaluation of surface contamination and dermal exposure to antineoplastic drugs will be assessed by determining cyclophosphamide on selected surfaces (wipes) and on the exposed nurses' clothes (pads). The concentration of unmetabolized cyclophosphamide as a biomarker of internal dose will be measured in end-shift urine samples from exposed nurses.</p> <p>Biomarkers of effect and susceptibility will be assessed in exposed and unexposed nurses: urinary concentration of 8-hydroxy-2-deoxyguanosine; DNA damage detected using the single-cell microgel electrophoresis (comet) assay in peripheral white blood cells; micronuclei and chromosome aberrations in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e. glutathione <it>S</it>-transferases) will also be analysed.</p> <p>Using standardized questionnaires, occupational exposure will be determined in exposed nurses only, whereas potential confounders (medicine consumption, lifestyle habits, diet and other non-occupational exposures) will be assessed in both groups of hospital workers.</p> <p>Statistical analysis will be performed to ascertain the association between occupational exposure to antineoplastic drugs and biomarkers of DNA and chromosome damage, after taking into account the effects of individual genetic susceptibility, and the presence of confounding exposures.</p> <p>Discussion</p> <p>The findings of the study will be useful in updating prevention procedures for handling antineoplastic drugs.</p

Prevalence, Distribution, and Impact of Mild Cognitive Impairment in Latin America, China, and India: A 10/66 Population-Based Study

A set of cross-sectional surveys carried out in Cuba, Dominican Republic, Peru, Mexico, Venezuela, Puerto Rico, China, and India reveal the prevalence and between-country variation in mild cognitive impairment at a population level

"The contribution of chronic diseases to the prevalence of dependence among older people in Latin America, China and India: a 10/66 Dementia Research Group population-based survey"

<p>Abstract</p> <p>Background</p> <p>The number of older people is set to increase dramatically worldwide. Demographic changes are likely to result in the rise of age-related chronic diseases which largely contribute to years lived with a disability and future dependence. However dependence is much less studied although intrinsically linked to disability. We investigated the prevalence and correlates of dependence among older people from middle income countries.</p> <p>Methods</p> <p>A one-phase cross-sectional survey was carried out at 11 sites in seven countries (urban sites in Cuba, Venezuela, and Dominican Republic, urban and rural sites in Peru, Mexico, China and India). All those aged 65 years and over living in geographically defined catchment areas were eligible. In all, 15,022 interviews were completed with an informant interview for each participant. The full 10/66 Dementia Research Group survey protocol was applied, including ascertainment of depression, dementia, physical impairments and self-reported diagnoses. Dependence was interviewer-rated based on a key informant's responses to a set of open-ended questions on the participant's needs for care. We estimated the prevalence of dependence and the independent contribution of underlying health conditions. Site-specific prevalence ratios were meta-analysed, and population attributable prevalence fractions (PAPF) calculated.</p> <p>Results</p> <p>The prevalence of dependence increased with age at all sites, with a tendency for the prevalence to be lower in men than in women. Age-standardised prevalence was lower in all sites than in the USA. Other than in rural China, dementia made the largest independent contribution to dependence, with a median PAPF of 34% (range 23%-59%). Other substantial contributors were limb impairment (9%, 1%-46%), stroke (8%, 2%-17%), and depression (8%, 1%-27%).</p> <p>Conclusion</p> <p>The demographic and health transitions will lead to large and rapid increases in the numbers of dependent older people particularly in middle income countries (MIC). The prevention and control of chronic neurological and neuropsychiatric diseases and the development of long-term care policies and plans should be urgent priorities.</p

The protocols for the 10/66 dementia research group population-based research programme

BACKGROUND: Latin America, China and India are experiencing unprecedentedly rapid demographic ageing with an increasing number of people with dementia. The 10/66 Dementia Research Group's title refers to the 66% of people with dementia that live in developing countries and the less than one tenth of population-based research carried out in those settings. This paper describes the protocols for the 10/66 population-based and intervention studies that aim to redress this imbalance. METHODS/DESIGN: Cross-sectional comprehensive one phase surveys have been conducted of all residents aged 65 and over of geographically defined catchment areas in ten low and middle income countries (India, China, Nigeria, Cuba, Dominican Republic, Brazil, Venezuela, Mexico, Peru and Argentina), with a sample size of between 1000 and 3000 (generally 2000). Each of the studies uses the same core minimum data set with cross-culturally validated assessments (dementia diagnosis and subtypes, mental disorders, physical health, anthropometry, demographics, extensive non communicable disease risk factor questionnaires, disability/functioning, health service utilisation, care arrangements and caregiver strain). Nested within the population based studies is a randomised controlled trial of a caregiver intervention for people with dementia and their families (ISRCTN41039907; ISRCTN41062011; ISRCTN95135433; ISRCTN66355402; ISRCTN93378627; ISRCTN94921815). A follow up of 2.5 to 3.5 years will be conducted in 7 countries (China, Cuba, Dominican Republic, Venezuela, Mexico, Peru and Argentina) to assess risk factors for incident dementia, stroke and all cause and cause-specific mortality; verbal autopsy will be used to identify causes of death. DISCUSSION: The 10/66 DRG baseline population-based studies are nearly complete. The incidence phase will be completed in 2009. All investigators are committed to establish an anonymised file sharing archive with monitored public access. Our aim is to create an evidence base to empower advocacy, raise awareness about dementia, and ensure that the health and social care needs of older people are anticipated and met