Modular Pressure and Flow Rate-Balanced Microfluidic Serial Dilution Networks for Miniaturised Point-of-Care Diagnostic Platforms.

Fast, efficient and more importantly accurate serial dilution is a necessary requirement for most biochemical microfluidic-based quantitative diagnostic applications. Over the last two decades, a multitude of microfluidic devices has been proposed, each one demonstrating either a different type of dilution technique or complex system architecture based on various flow source and valving combinations. In this work, a novel serial dilution network architecture is demonstrated, implemented on two entirely different substrates for validation and performance characterisation. The single layer, stepwise serial diluter comprises an optimised microfluidic network, where identical dilution ratios per stage are ensured, either by applying equal pressure or equal flow rates at both inlets. The advantages of this serial diluter are twofold: Firstly, it is structured as a modular unit cell, simplifying the required fluid driving mechanism to a single source for both sample and buffer solution. Thus, this unit cell can be used as a fundamental microfluidic building block, forming multistage serial dilution cascades, once combined appropriately with itself or other similar unit cells. Secondly, the serial diluter can tolerate the inevitable flow source fluctuations, ensuring constant dilution ratios without the need to employ damping mechanisms, making it ideal for Point of Care (PoC) platforms. Proof-of-concept experiments with glucose have demonstrated good agreement between simulations and measurements, highlighting the validity of our serial diluter.

A Novel Microfluidic Point-of-Care Biosensor System on Printed Circuit Board for Cytokine Detection.

Point of Care (PoC) diagnostics have been the subject of considerable research over the last few decades driven by the pressure to detect diseases quickly and effectively and reduce healthcare costs. Herein, we demonstrate a novel, fully integrated, microfluidic amperometric enzyme-linked immunosorbent assay (ELISA) prototype using a commercial interferon gamma release assay (IGRA) as a model antibody binding system. Microfluidic assay chemistry was engineered to take place on Au-plated electrodes within an assay cell on a printed circuit board (PCB)-based biosensor system. The assay cell is linked to an electrochemical reporter cell comprising microfluidic architecture, Au working and counter electrodes and a Ag/AgCl reference electrode, all manufactured exclusively via standard commercial PCB fabrication processes. Assay chemistry has been optimised for microfluidic diffusion kinetics to function under continual flow. We characterised the electrode integrity of the developed platforms with reference to biological sampling and buffer composition and subsequently we demonstrated concentration-dependent measurements of H2O2 depletion as resolved by existing FDA-validated ELISA kits. Finally, we validated the assay technology in both buffer and serum and demonstrate limits of detection comparable to high-end commercial systems with the addition of full microfluidic assay architecture capable of returning diagnostic analyses in approximately eight minutes.

New considerations for colorectal cancer screening based on the demographic profile of colorectal cancer in a Greek population.

Colorectal cancer screening has long been recommended for middle age and older individuals. Recent evidence indicates increasing incidence and mortality among young adults. Therefore, the present study re-examined the current recommendations using an asymptomatic average-risk population screened by colonoscopy. A total of 716 participants of a wide age range were prospectively enrolled in an open-access endoscopic screening program based on self-referral. Comparisons between different age, gender and location groups, and receiver operating characteristic curves (ROC) curves for best age selection for detection of lesions were employed. Increased incidence of advanced lesions was observed in adults <50 years old. Although the polyp size was <1 cm in 85% of the cohort, a significant number of participants harbored advanced lesions. A disturbing incidence of lesions in women 30-49 years was located mainly in the left colon. One-third of the important pathology resides exclusively in the right colon. ROC curves demonstrated that with the current starting age of screening at 50 years, 92% of polyps and 95% of adenomas could be detected by colonoscopy, but a number of potential precancerous lesions will appear at an earlier age and therefore will be missed. The present study supported the notion that it is critical to reduce screening initiation below the currently accepted age of 50 years. Colonoscopy is a suitable method for addressing the increased prevalence of proximal lesions and the meticulous resection of smaller polyps.

Alarming endoscopic data in young and older asymptomatic people: Results of an open access, unlimited age colonoscopic screening for colorectal cancer.

There is a lack of a national organized screening program for colorectal cancer in Greece, and asymptomatic detection is usually the result of individual decisions. The collection of epidemiologic endoscopic data from a population of interest would therefore provide valuable information for future treatment guidance, especially during periods of economic austerity. The current cross-sectional study included 380 asymptomatic, average risk individuals undergoing screening colonoscopy for the first time, during the period of one year in a tertiary public hospital in Athens. Descriptive and analytic epidemiologic data were analyzed. The prevalence of adenomas and advanced lesions were compared between the younger and older cohort, and a regression model was applied for risk evaluation. The mean age of participants was 63 years, and 53% were male. A significant proportion of patients presented with polyps (51.5%) and 25% of them had lesions in the proximal colon. The prevalence of adenomas and advanced adenomas was 29.5 and 11.8%, respectively. Similar high prevalence rates of lesions were identified in the cohort of individuals <50 years of age and the older cohort (>50 years of age). Regression models identified age, number and size of polyps as the major risk factors for the detection of adenomas. The increase of advanced lesions in the older and younger cohort requires confirmation by larger studies. Overall, the results of the present study indicate the requirement for a well-organized screening colonoscopy program starting from as early as 40 years of age. This program may confer an additional endoscopic burden with socioeconomic consequences in a country with limited health resources.

Role of microglia in the dissemination of Zika virus from mother to fetal brain.

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as "Trojan horses," contributing to ZIKV fetal brain dissemination and congenital brain defects.

High-performance PCB-based capillary pumps for affordable point-of-care diagnostics.

Capillary pumps are integral components of passive microfluidic devices. They can displace precise volumes of liquid, avoiding the need for external active components, providing a solution for sample preparation modules in Point-of-Care (PoC) diagnostic platforms. In this work, we describe a variety of high-performance capillary pump designs, suitable for the Lab-on-Printed-Circuit-Board technology (LoPCB). Pumps are fabricated entirely on Printed Circuit Board (PCB) substrates via commercially available manufacturing processes. We demonstrate the concept of LoPCB technology and detail the fabrication method of different architectures of PCB-based capillary pumps. The capillary pumps are combined with microfluidic channels of various hydraulic resistances and characterised experimentally for different micropillar shapes and minimum feature size. Their performance in terms of flow rate is reported. Due to the superhydrophilic properties of oxygen plasma treated FR-4 PCB substrate, the capillary pump flow rates are much higher (138 µL/min, for devices comprising micropillar arrays without preceding microchannel) than comparable devices based on glass, silicon or polymers. Finally, we comment on the technology's prospects, such as incorporating more complicated microfluidic networks that can be tailored for assays.

An Assay System for Point-of-Care Diagnosis of Tuberculosis using Commercially Manufactured PCB Technology.

Rapid advances in clinical technologies, detection sensitivity and analytical throughput have delivered a significant expansion in our knowledge of prognostic and diagnostic biomarkers in many common infectious diseases, such as Tuberculosis (TB). During the last decade, a significant number of approaches to TB diagnosis have been attempted at Point-of-Care (PoC), exploiting a large variation of techniques and materials. In this work, we describe an electronics-based Enzyme-Linked ImmunoSorbent Assay (eELISA), using a Lab-on-a-Printed Circuit Board (LoPCB) approach, for TB diagnosis based on cytokine detection. The test relies upon an electrochemical (amperometric) assay, comprising a high-precision bioinstrumentation board and amperometric sensors, produced exclusively using standard PCB manufacturing processes. Electrochemical detection uses standard Au and Ag electrodes together with a bespoke, low-power, multichannel, portable data-acquisition system. We demonstrate high-performance assay chemistry performed at microfluidic volumes on Au pads directly at the PCB surface with improved limit of detection (~10 pg/mL) over standard colorimetric ELISA methods. The assay has also been implemented in plasma, showing the utility of the system for medical applications. This work is a significant step towards the development of a low-cost, portable, high-precision diagnostic and monitoring technology, which once combined with appropriate PCB-based microfluidic networks will provide complete LoPCB platforms.

A Global Genomic Characterization of Nairoviruses Identifies Nine Discrete Genogroups with Distinctive Structural Characteristics and Host-Vector Associations.

Nairoviruses are primarily tick-borne bunyaviruses, some of which are known to cause mild-to-severe febrile illness in humans or livestock. We describe the genome sequences of 11 poorly characterized nairoviruses that have ecological associations with either birds (Farallon, Punta Salinas, Sapphire II, Zirqa, Avalon, Clo Mor, Taggert, and Abu Hammad viruses), rodents (Qalyub and Bandia viruses), or camels (Dera Ghazi Khan virus). Global phylogenetic analyses of proteins encoded in the L, M, and S RNA segments of these and 20 other available nairovirus genomes identified nine well-supported genogroups (Nairobi sheep disease, Thiafora, Sakhalin, Keterah, Qalyub, Kasokero, Dera Ghazi Khan, Hughes, and Tamdy). Genogroup-specific structural variations were evident, particularly in the M segment encoding a polyprotein from which virion envelope glycoproteins (Gn and Gc) are generated by proteolytic processing. Structural variations include the extension, abbreviation, or absence sequences encoding an O-glycosylated mucin-like protein in the N-terminal domain, distinctive patterns of conserved cysteine residues in the GP38-like domain, insertion of sequences encoding a double-membrane-spanning protein (NSm) between the Gn and Gc domains, and the presence of an alternative long open reading frame encoding a viroporin-like transmembrane protein (Gx). We also observed strong genogroup-specific associations with categories of hosts and tick vectors.

Complete Genome Sequences of Five Zika Virus Isolates.

Zika virus is an emerging human pathogen of great concern due to putative links to microcephaly and Guillain-Barre syndrome. Here, we report the complete genomes, including the 5' and 3' untranslated regions, of five Zika virus isolates, one from the Asian lineage and four from the African lineage.

A method of alphavirus replicon particle titration based on expression of functional replicase/transcriptase.

Alphavirus replicon particles are being exploited for a variety of purposes both in vitro as gene expression vectors, and in vivo as vaccines or gene therapy vectors. There is a need for a simple and universal method of titration of replicon particles that is independent of expression of the foreign protein. We devised a method that uses modified vaccinia virus Ankara (MVA) as an indicator virus, to deliver a Venezuelan equine encephalitis virus (VEE) defective helper RNA encoding green fluorescent protein (GFP). Co-infection of cells with the MVA-based indicator and Venezuelan equine encephalitis virus replicon particles (VRP) results in expression of the GFP gene. VRP titer is readily determined by counting fluorescent cells.

Identification of dengue fever cases in Houston, Texas, with evidence of autochthonous transmission between 2003 and 2005.

Houston, Texas, maintains an environment conducive to dengue virus (DENV) emergence; however, surveillance is passive and diagnostic testing is not readily available. To determine if DENV is present in the area, we tested 3768 clinical specimens (2138 cerebrospinal fluid [CSF] and 1630 serum) collected from patients with suspected mosquito-borne viral disease between 2003 and 2005. We identified 47 immunoglobulin M (IgM)-positive dengue cases, including two cases that were positive for viral RNA in serum for dengue serotype 2. The majority of cases did not report any history of travel outside the Houston area prior to symptom onset. The epidemic curve suggests an outbreak occurred in 2003 with continued low-level transmission in 2004 and 2005. Chart abstractions were completed for 42 of the 47 cases; 57% were diagnosed with meningitis and/or encephalitis, and 43% met the case definition for dengue fever. Two of the 47 cases were fatal, including one with illness compatible with dengue shock syndrome. Our results support local transmission of DENV during the study period. These findings heighten the need for dengue surveillance in the southern United States.