Accurate Molecular Diagnostics Research Articles

Molecular epidemiology of brucellosis in Asia: insights from genotyping analyses.

Brucellosis infects humans and animals worldwide but is particularly prevalent in Asia. In many Asian countries, molecular diagnostic tools for accurate molecular diagnostics and molecular epidemiology are lacking. Nonetheless, some countries have conducted in-depth molecular epidemiological studies. The objective of this study was to reveal the genetic relationships, geographic origins, and distributions of Brucella strains across Asia for two primary species, B. abortus and B. melitensis. For this, we systematically searched genotyping data from published studies on the molecular epidemiology of Brucella species for both humans and livestock in Asia. We used data from multilocus sequence typing (MLST), multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing analysis of Brucella strains. We also analyzed the MLVA genotypes of 129 B. abortus isolates and 242 B. melitensis isolates with known origins in Asia from an online MLVA database using MLVA-11 data in minimum spanning trees and MLVA-16 data in neighbor-joining trees. We found that the B. melitensis East Mediterranean lineage is predominant across the continent, with only a small number of samples from the Africa and Americas lineages, and none from the West Mediterranean lineage. The "abortus C" genotype was the most common group of B. abortus in Asia, with limited genetic variation for this species. Several studies also reported that Near Eastern countries frequently encounter human brucellosis cases of B. abortus from genotypes 42 and 43. Our study highlights the inconsistent collection of genetic data for Brucella species across Asia and a need for more extensive sampling in most countries. Finally, a consistent nomenclature is necessary to define various groupings of strains within a lineage (i.e., clade) so uniform terminology should denote particular genetic groups that are understood by all researchers.

CRISPR: The frontier technology of next-generation RNA detection

Rapid and accurate molecular diagnostics are crucial for disease diagnosis and precision medicine. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins have emerged as highly effective tools for molecular diagnostics. Numerous nucleic acid detection instruments and biosensors utilizing the CRISPR/Cas system have been developed. The profiling activity of CRISPR/Cas effectors has facilitated the creation of instrument-free, sensitive, precise, and rapid nucleic acid diagnostics. This review summarizes recent advancements in CRISPR technology for RNA detection, focusing on the application of Cas12 and Cas13 systems in two scenarios: in combination with isothermal amplification technology and without amplification. It also explores the significant potential of CRISPR as a next-generation technology for RNA detection and anticipates future developments. The ongoing advancements in CRISPR are expected to enhance precision and convenience in RNA testing, impacting both biomedical research and public health practices.

Palm-Sized Lab-In-A-Magnetofluidic Tube Platform for Rapid and Sensitive Virus Detection.

Simple, sensitive, and accurate molecular diagnostics are critical for preventing rapid spread of infection and initiating early treatment of diseases. However, current molecular detection methods typically rely on extensive nucleic acid sample preparation and expensive instrumentation. Here, a simple, fully integrated, lab-in-a-magnetofluidic tube (LIAMT) platform is presented for "sample-to-result" molecular detection of virus. By leveraging magnetofluidic transport of micro/nano magnetic beads, the LIAMT device integrates viral lysis, nucleic acid extraction, isothermal amplification, and CRISPR detection within a single engineered microcentrifuge tube. To enable point-of-care molecular diagnostics, a palm-sized processor is developed for magnetofluidic separation, nucleic acid amplification, and visual fluorescence detection. The LIAMT platform is applied to detect SARS-CoV-2 and HIV viruses, achieving a detection sensitivity of 73.4 and 63.9 copiesµL-1, respectively. Its clinical utility is further demonstrated by detecting SARS-CoV-2 and HIV in clinical samples. This simple, affordable, and portable LIAMT platform holds promise for rapid and sensitive molecular diagnostics of infectious diseases at the point-of-care.

Actively targeted and dual-stimuli-responsive branch-shaped system for simultaneous microRNAs imaging in living cells

Designing a multifunctional nucleic acid platform with the ability to identify multiple biomarkers is crucial for accurate molecular diagnostics. Here we reported a dual-target branch-shaped nucleic acid probing (DBNP) system and its targeted delivery for simultaneous analysis of intracellular microRNAs (miRNAs). The system consisted of a cross-shaped DNA scaffold equipped with four hairpin strands and an aptamer strand. The four hairpin strands constituted two catalytic hairpin assembly (CHA) reactions, enabling the system to simultaneously recognize and amplify two kinds of miRNAs. We demonstrated that the cross-shaped DNA scaffold structurally offered a spatial-confinement effect, well-known in increasing the intramolecular collision of the probes and accelerating the CHA reactions, which allows us to resolve multiplex miRNAs profiling with high efficiency by using DBNP system. In addition, because of the inherent character of aptamer capable of specifically binding with cell-membrane receptor, targeted delivery of DBNP system into the unique receptor-positive cells can be realized to detect intracellular miRNAs. The dual miRNA-mediated stimuli-response, operating in parallel with a receptor-specific recognition of target cells, synergistically enhances the accuracy of cell discrimination and cancer diagnosis. This innovative approach holds great promise for advancing the development of cutting-edge signal amplification technologies and biosensors utilized in cancer diagnostics.

B-237 Analytical and Clinical Performance Evaluation of the Panther Fusion® GI Parasite Assay (In Development)

Abstract Background Acute diarrhea from gastrointestinal (GI) infections is the leading cause of outpatient visits, hospitalizations, and loss of quality of life, with an estimated global impact of 500 million illnesses and 230 000 deaths annually. Most GI infections from bacteria, viruses, and parasites present similar symptoms, but successful treatment is dependent on accurate pathogen identification. For identification of parasites, microscopic testing is often used, but is laborious and often results in inconclusive or inaccurate diagnoses. Clinicians now rely on rapid and accurate molecular diagnostics to correctly identify the causative organism, which leads to optimal infection control and appropriate treatment. The Panther Fusion (PF) GI Parasite Assay is a multiplex qualitative real-time PCR assay that addresses the need for accurate and sensitive identification and detection of the most common parasites that are known to cause gastroenteritis: Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Methods Stool specimens collected in Cary-Blair (CB) are transferred to the AptimaTM Multitest collection tube (Hologic). Multitest tubes are loaded on Panther Fusion where nucleic acid isolation, amplification, and detection are performed. Analytical sensitivity was assessed using cell suspension of Cryptosporidium, Giardia or E. histolytica spiked in negative CB Stool (CBS) matrix and the limit of detection (LoD) was determined using probit. Since Cyclospora cayetanensis cannot be cultured, in-vitro transcribed RNA (IVT) was spiked in CBS matrix for LoD evaluation. Inclusivity was evaluated using cell suspensions for 20 Giardia and 15 E. histolytica strains spiked in CBS matrix. Cryptosporidium inclusivity was evaluated with synthetic nucleic acid. Specificity was evaluated by spiking cells suspensions or synthetic nucleic acid of various microbes in CBS in the presence or absence of GI targets. For clinical performance, 119 archived frozen stool specimens in CB and 40 contrived specimens were tested on Film Array GI Panel (BioFire) and PF GI Parasite Assay and results were compared. Results The LoD for Cryptosporidium, Giardia and E. histolytica was determined to be < 1 cell/mL in the Aptima Multitest tube. For C. cayetanensis, the LoD was found to be 1032 copies/mL. The PF GI Parasite Assay detected 20 strains of Giardia lamblia and 15 E. histolytica strains. In-silico and initial testing of synthetic nucleic acid demonstrated detection of 10 Cryptosporidium species. No cross-reactivity to closely related organisms or other microorganisms commonly found in the gastrointestinal tract was observed, except for cross-reactivity to Entamoeba nuttalli, which rarely infects humans. In testing of clinical remnant specimens, the PF GI Parasite Assay had a 97% PPA (35/36) for Cryptosporidium, 100% PPA (58/58) for Giardia lamblia, 100% PPA (42/42) for E. histolytica (included 40 contrived specimens) and 100% PPA (8/8) for Cyclospora with the Film Array GI Panel. Conclusion The PF GI Parasite Assay demonstrated excellent analytical and clinical performance in the detection and differentiation of Cryptosporidium, Entamoeba histolytica, Giardia lamblia and Cyclospora cayetanensis. Together with its companion Panther Fusion assays that detect bacterial and viral GI pathogens (also in development), this assay will provide a sensitive and accurate molecular tool for the automated detection of parasites in human stool.

Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.

DNA polymerases are essential for nucleic acid synthesis, cloning, sequencingand molecular diagnostics technologies. Conditional intein splicing is a powerful tool for controlling enzyme reactions. We have engineered a thermal switch into thermostable DNA polymerases from two structurally distinct polymerase families by inserting a thermally activated intein domain into a surface loop that is integral to the polymerase active site, thereby blocking DNA or RNA template access. The fusion proteins are inactive, but retain their structures, such that the intein excises during a heat pulse delivered at 70-80°C to generate spliced, active polymerases. This straightforward thermal activation step provides a highly effective, one-component 'hot-start' control of PCR reactions that enables accurate target amplification by minimizing unwanted by-products generated by off-target reactions. In one engineered enzyme, derived from Thermus aquaticus DNA polymerase, both DNA polymerase and reverse transcriptase activities are controlled by the intein, enabling single-reagent amplification of DNA and RNA under hot-start conditions. This engineered polymerase provides high-sensitivity detection for molecular diagnostics applications, amplifying 5-6 copies of the tested DNA and RNA targets with>95% certainty. The design principles used to engineer the inteins can be readily applied to construct other conditionally activated nucleic acid processing enzymes.

Toward Rapid and Accurate Molecular Diagnostics at Home.

The global outbreaks of infectious diseases have significantly driven an imperative demand for rapid and accurate molecular diagnostics. Nucleic acid amplification tests (NAATs) feature high sensitivity and high specificity; however, the labor-intensive sample preparation and nucleic acid amplification steps remain challenging in order to carry out rapid and precision molecular diagnostics at home. This review discusses the advances and challenges of automatic solutions of sample preparation integrated with on-chip nucleic acid amplification for effective and accurate molecular diagnostics at home. The sample preparation methods of whole blood, urine, saliva/nasal swab, and stool on chip are examined. Then, the repurposable integrated sample preparation on a chip using various biological samples is investigated. Finally, the on-chip NAATs that can be integrated with automated sample preparation are evaluated. The user-friendly approaches with combined sample preparation and NAATs can be the game changers for next-generation rapid and precision home diagnostics.

Digital Protein Detection in Bulk Solutions.

Quick and accurate molecular diagnostics in protein detection can greatly benefit medicine in disease diagnosis and lead to positive patient outcomes. However, specialized equipment used in clinical laboratories often comes with trade-offs between operation and function serving a single role for very specific needs. For example, to achieve high analytical sensitivity and specificity, instruments such as high-performance liquid chromatography and/or liquid chromatography–mass spectrometry use a complex instrument design and require thorough training of the users. On the other hand, simple tests such as protein detection in urinary tract infection using dip-stick assays provide very quick results but suffer from poor analytical sensitivity. Here, we present an application study for the 3D particle counter technology, which is based on optical confocal detection in order to scan large sample volumes (0.5–3 mL) in glass cuvettes, that aims to close the gap between analytical sensitivity and turnover assay time and simplify protein detection by adopting bead-based immunoassays. Combining the 3D particle counter technology with bead-based immunoassays, a subpicomolar limit of detection—ranging from 119 to 346 fM—was achieved within 3.5-hour assay time for recombinant mouse interleukin 6 detection. As an alternative instrument to a flow cytometer, the 3D particle counter takes advantages of bead-based immunoassays and provides unique accessibility and flexibility for users.

Multiplexed reverse-transcriptase quantitative polymerase chain reaction using plasmonic nanoparticles for point-of-care COVID-19 diagnosis.

Quantitative polymerase chain reaction (qPCR) offers the capabilities of real-time monitoring of amplified products, fast detection, and quantitation of infectious units, but poses technical hurdles for point-of-care miniaturization compared with end-point polymerase chain reaction. Here we demonstrate plasmonic thermocycling, in which rapid heating of the solution is achieved via infrared excitation of nanoparticles, successfully performing reverse-transcriptase qPCR (RT-qPCR) in a reaction vessel containing polymerase chain reaction chemistry, fluorescent probes and plasmonic nanoparticles. The method could rapidly detect SARS-CoV-2 RNA from human saliva and nasal specimens with 100% sensitivity and 100% specificity, as well as two distinct SARS-CoV-2 variants. The use of small optical components for both thermocycling and multiplexed fluorescence monitoring renders the instrument amenable to point-of-care use. Overall, this study demonstrates that plasmonic nanoparticles with compact optics can be used to achieve real-time and multiplexed RT-qPCR on clinical specimens, towards the goal of rapid and accurate molecular clinical diagnostics in decentralized settings.

Challenges in the Histopathologic Diagnosis of Brain Tumors: An Institutional Experience in a Series of Cases

Abstract Background Central nervous system (CNS) tumors are relatively rare. However, brain tumors are one of the leading causes of cancer-related morbidity and mortality. Accurate histopathologic diagnosis and molecular diagnostics are critical for managing these patients. Histopathology plays a vital role in diagnosis, but clinical and radiological information is also crucial while evaluating brain tumors. Materials and Methods A cross-sectional observational study was performed for a period of 1 year in the pathology department of a tertiary hospital. All the brain biopsies sent for histopathological analysis were analyzed, and among which five brain biopsy tissue posing the diagnostic dilemma in conventional histopathology were included in the study. Immunohistochemistry was performed wherever necessary. Results During the study period, we encountered 32 cases of brain biopsy. Five cases posing diagnostic challenges in histopathological diagnosis were included in the study. Expert opinions of neuropathologists were taken in all of these cases. We reviewed the cases for the diagnostic difficulties. A review of all these cases has impacted further treatment. Conclusion In this case series, we present five CNS tumors with a diagnostic dilemma in histopathological diagnosis where clinicoradiological correlation, immunohistochemistry, and expert opinion helped in the definitive diagnosis revealing the importance of a multidisciplinary approach for the better management of patients.

The deployment of mobile diagnostic laboratories for Ebola virus disease diagnostics in Sierra Leone and Guinea.

BackgroundEbola virus emerged in West Africa in December 2013. The ease of mobility, porous borders, and lack of public health infrastructure led to the largest Ebola virus disease (EVD) outbreak to date.InterventionThe 2013 EVD outbreak signalled the need for laboratory diagnostic capabilities in areas without strong public health systems. As part of the United States’ Department of Defense response, MRIGlobal was contracted to design, fabricate, equip, deploy, and operate two mobile diagnostic laboratories (MDLs). The first laboratory analysed blood samples from patients in an adjacent Ebola Treatment Centre (ETC) and buccal swabs from the deceased in the community in Moyamba, Sierra Leone. The second laboratory was deployed to support an ETC in Conakry, Guinea. The Department of Defense provided real-time quantitative reverse transcription polymerase chain reaction assays that were deployed and validated on-site.Lessons LearntPrompt and accurate molecular diagnostics reduced sample turn-around times from over 24 h to under 4 h. Experienced laboratory staff tested up to 110 samples per day and on-site engineering proved necessary for MDL setup and operation. As the Ebola response slowed, the sustainment of the MDLs’ operations was prioritised, including staff training and the transition of the MDLs to local governments. Training programmes for local staff were prepared in Sierra Leone and Guinea.RecommendationsThe MRIGlobal MDL team significantly contributed to establishing increased laboratory capacity during the EVD outbreak in West Africa. Using the MDLs for molecular diagnosis is highly recommended until more sustainable solutions can be provided.

Ultrafast and Real-Time Nanoplasmonic On-Chip Polymerase Chain Reaction for Rapid and Quantitative Molecular Diagnostics.

Advent and fast spread of pandemic diseases draw worldwide attention to rapid, prompt, and accurate molecular diagnostics with technical development of ultrafast polymerase chain reaction (PCR). Microfluidic on-chip PCR platforms provide highly efficient and small-volume bioassay for point-of-care diagnostic applications. Here we report ultrafast, real-time, and on-chip nanoplasmonic PCR for rapid and quantitative molecular diagnostics at point-of-care level. The plasmofluidic PCR chip comprises glass nanopillar arrays with Au nanoislands and gas-permeable microfluidic channels, which contain reaction microchamber arrays, a precharged vacuum cell, and a vapor barrier. The on-chip configuration allows both spontaneous sample loading and microbubble-free PCR reaction during which the plasmonic nanopillar arrays result in ultrafast photothermal cycling. After rapid sample loading less than 3 min, two-step PCR results for 40 cycles show rapid amplification in 264 s for lambda-DNA, and 306 s for plasmids expressing SARS-CoV-2 envelope protein. In addition, the in situ cyclic real-time quantification of amplicons clearly demonstrates the amplification efficiencies of more than 91%. This PCR platform can provide rapid point-of-care molecular diagnostics in helping slow the fast-spreading pandemic.

A diagnostic LAMP assay for the destructive grapevine insect pest, phylloxera (Daktulosphaira vitifoliae)

Grape phylloxera (Daktulosphaira vitifoliae) is a destructive insect pest of grapevines that is highly invasive worldwide, despite strict biosecurity containment measures in place at farm and regional levels. Current phylloxera identification by visual inspection and laboratory-based molecular methods is time-consuming and costly. More rapid and cost-effective methods for identification of this pest would benefit industry, growers, and biosecurity services. Loop mediated isothermal amplification (LAMP) is a new portable technology available for rapid and accurate in-field molecular diagnostics. This study outlines the development of a new LAMP assay to enable the identification of phylloxera specimens. New LAMP primers were developed to specifically amplify phylloxera mitochondrial DNA (5′-COI), which we have shown is effective as a DNA barcode for identification of phylloxera, using LAMP technology. Positive LAMP reactions, containing phylloxera DNA, amplified in less than twelve minutes with an anneal derivative temperature of approximately 79 °C to 80 °C compared to a newly designed synthetic DNA (gBlock) fragment which had an anneal derivative temperature of 82 °C. No LAMP amplification was detected in any of the non-target species tested, i.e. no false-positive identification resulted for these species. We also successfully optimised a non-destructive DNA extraction procedure, HotSHOT “HS6”, for use in the field on phylloxera adults, nymphs and eggs, to retain physical specimens. DNA extracted using this method was also suitable for species and genotype molecular identification methods, such as DNA barcoding, qPCR and microsatellite genotyping. The new LAMP assay provides a novel visual molecular tool for accurate diagnostics of phylloxera in the laboratory and field.

Enhancing the performance of paper-based electrochemical impedance spectroscopy nanobiosensors: An experimental approach

Accurate, rapid, and low-cost molecular diagnostics is essential in managing outbreaks of infectious diseases, such as the pandemic of coronavirus disease 2019 (COVID-19). Accordingly, microfluidic paper-based analytical devices (μPADs) have emerged as promising diagnostic tools. Among the extensive efforts to improve the performance and usability of diagnostic tools, biosensing mechanisms based on electrochemical impedance spectroscopy (EIS) have shown great promise because of their label-free operation and high sensitivity. However, the method to improve EIS biosensing on μPADs is less explored. Here, we present an experimental approach to enhancing the performance of paper-based EIS biosensors featuring zinc oxide nanowires (ZnO NWs) directly grown on working electrodes (WEs). Through a comparison of different EIS settings and an examination of ZnO-NW effects on EIS measurements, we show that ZnO-NW-enhanced WEs function reliably with Faradaic processes utilizing iron-based electron mediators. We calibrate paper-based EIS biosensors with different morphologies of ZnO NWs and achieve a low limit of detection (0.4 pg ml−1) in detecting p24 antigen as a marker for human immunodeficiency virus (HIV). Through microscopic imaging and electrochemical characterization, we reveal that the morphological and the electrochemical surface areas of ZnO-NW-enhanced WEs indicate the sensitivities and sensing ranges of the EIS nanobiosensors. Finally, we report that the EIS nanobiosensors are capable of differentiating the concentrations (blank, 10 ng ml−1, 100 ng ml−1, and 1 μg ml−1) of IgG antibody (CR3022) to SARS-CoV-2 in human serum samples, demonstrating the efficacy of these devices for COVID-19 diagnosis. This work provides a methodology for the rational design of high-performance EIS μPADs and has the potential to facilitate diagnosis in pandemics.

Cell-free Chromatin Immunoprecipitation (cfChIP) from blood plasma can determine gene-expression in tumors from non-small-cell lung cancer patients

ObjectivesLung cancer is the leading cause of cancer related death worldwide. Accurate molecular diagnostics from a tumor biopsy is paramount for correct diagnosis, treatment strategy, and prediction of outcome. However, a tumor biopsy can be misleading due to tumor heterogeneity and consecutive biopsies are rarely achievable. Importantly, tumor-specific genetic information concerning mutations and translocations, can also be obtained from liquid biopsies, e.g. blood plasma, containing cell-free DNA (cfDNA) with both systemic and tumor origin. Tumor-specific gene-expression information can also be determined from liquid biopsies using cfDNA methylation and cell-free RNA analyses. However, supplementary methodologies that can determine gene-expression patterns in lung tumors from liquid biopsies could also have diagnostic impact. Materials and methodsWe here present the method cell-free chromatin Immunoprecipitation (cfChIP), which for genes having high expression specifically in the tumor, can determine such gene-expression from blood plasma. In cfChIP cell-free nucleosomes modified with histone H3 lysine 36 tri-methylation (H3K36me3), a mark quantitatively correlated with the transcription of the underlying gene, are isolated, and associated cfDNA quantified. ResultsWe demonstrate that cfChIP from lung cancer patient blood plasma can successfully quantify the level of H3K36me3 associated with circulating cell-free nucleosomes and thereby quantify the transcriptional level of genes associated with these nucleosomes. Moreover, as a proof-of-principle we show that in blood plasma from 14 lung cancer patients, H3K36me3 cfChIP can replicate the expected higher expression of KRT6 in lung squamous cell carcinoma relative to adenocarcinoma. ConclusionThis work shows that for genes with a high expression specifically in tumor, cfChIP can determine this gene-expression pattern from blood plasma. cfChIP is a method that determine gene-expression at the transcriptional level and accordingly can supplement cfDNA methylation and cell-free RNA analyses.

Associations of genetic markers of the sympathoadrenal system and endothelial dysfunction with coronary heart disease on the example of a small Shoriya population

Aim . To establish associations of polymorphisms of candidate genes coding for the components of the endothelial (NOS3, MTHFR) and sympathoadrenal systems (ADRB1, ADRA2B) with coronary heart disease (CHD) in the cohort of the small population of Mountain Shoriya. Material and methods . A clinical and epidemiological study was conducted in the areas of Mountain Shoriya (Orton, Ust-Kabyrza, Sheregesh, Tashtagol). Analysis of risk factors included recording the age, sex, body mass index, waist circumference, presence/absence of arterial hypertension (AH), smoking, and the presence of disorders of lipid and carbohydrate metabolism. CHD diagnosis was made by three epidemiological criteria: coding of the electrocardiogram according to the Minnesota code, Rose questionnaire, and a history of myocardial infarction. The patient underwent molecular genetic testing. DNA isolation from blood was carried out by the method of phenol-chloroform extraction. Statistical processing was carried out using the programs STATISTICA 6.1 (StatSoft Inc., USA) and SNPStats. Results . The prevalence of CHD in the Shoriya cohort was 9,7%. The results of our study established the relationship of genetic markers of the sympathoadrenal and endothelial systems with coronary atherosclerosis in the indigenous population of Shoriya. The high risk of atherosclerotic heart disease was associated with the 4b/4a genotype of the NOS3 gene (OR 2,57 95% CI (1,12-5,86), p=0,026) according to the dominant mode of inheritance. The relationship of the T/T genotype of the MTHFR gene with CHD by recessive inheritance was established (OR 13,28; 95% CI (1,79-98,40), p=0,024). Conclusion . Due to genetic research, there is a real opportunity not only to carry out accurate molecular diagnostics, but also to determine an underlying risk for a disease. Detection of genetic disposition to CHD can be carried out long before the onset of clinical symptoms, which can effectively prevent its development.

Exploring predictive biomarkers from clinical genome-wide association studies via multidimensional hierarchical mixture models.

Although the detection of predictive biomarkers is of particular importance for the development of accurate molecular diagnostics, conventional statistical analyses based on gene-by-treatment interaction tests lack sufficient statistical power for this purpose, especially in large-scale clinical genome-wide studies that require an adjustment for multiplicity of a huge number of tests. Here we demonstrate an alternative efficient multi-subgroup screening method using multidimensional hierarchical mixture models developed to overcome this issue, with application to stroke and breast cancer randomized clinical trials with genomic data. We show that estimated effect size distributions of single nucleotide polymorphisms (SNPs) associated with outcomes, which could provide clues for exploring predictive biomarkers, optimizing individualized treatments, and understanding biological mechanisms of diseases. Furthermore, using this method we detected three new SNPs that are associated with blood homocysteine levels, which are strongly associated with the risk of stroke. We also detected six new SNPs that are associated with progression-free survival in breast cancer patients.

Digital Microfluidics for Nucleic Acid Amplification

Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

A sample-to-answer, real-time convective polymerase chain reaction system for point-of-care diagnostics

Timely and accurate molecular diagnostics at the point-of-care (POC) level is critical to global health. To this end, we propose a handheld convective-flow real-time polymerase chain reaction (PCR) system capable of direct sample-to-answer genetic analysis for the first time. Such a system mainly consists of a magnetic bead-assisted photothermolysis sample preparation, a closed-loop convective PCR reactor, and a wireless video camera-based real-time fluorescence detection. The sample preparation exploits the dual functionality of vancomycin-modified magnetic beads (VMBs) for bacteria enrichment and photothermal conversion, enabling cell pre-concentration and lysis to be finished in less than 3min. On the presented system, convective thermocycling is driven by a single-heater thermal gradient, and its amplification is monitored in real-time, with an analysis speed of less than 25min, a dynamic linear range from 106 to 101 copies/µL and a detection sensitivity of as little as 1 copies/µL. Additionally, the proposed PCR system is self-contained with a control electronics, pocket-size and battery-powered, providing a low-cost genetic analysis in a portable format. Therefore, we believe that this integrated system may become a potential candidate for fast, accurate and affordable POC molecular diagnostics.

Molecular sequencing and gene fusion detection in non-small cell lung cancer (NSCLC) patients: Impact of co-existing alterations.

e23103 Background: There is a growing need for accurate molecular diagnostics for proper targeted therapeutics selection in NSCLC patients (pts). We assess whether next generation techniques increase the identification of alterations in a prospective cohort of NSCLC pts, and evaluate prognostic value of different molecular alterations. Methods: Next generation sequencing (Amplicon-seq, 60 genes) and NanoString Gene fusion (20 genes) testing were conducted in NSCLC pts treated in a single center from November 2014 to May 2016. Overall survival (OS) according to the molecular profile was analysed by Kaplan-Meier method and Cox models. Results: Molecular tests were performed in 73advanced NSCLC pts.Median age was 54 year, 50% were women. Histology: 73% adenocarcinoma; 7% squamous, 20% others (Neuroendocrine, NOS). Tests were performed in biopsies from metastatic sites in 55% of pts.These techniques identified molecular alterations in 89% of cases: TP53 mutation (mut) 37/73 (51%); KRASmut 21/73 (29%); EGFRmut 11/73 (15%) ; CDKN2mut 7/73 (9%); BRAFmut (pG469R), ERBB2mut and METexon14mut in (1/73) 1.3%, respectively; ALKfusion (fus) 2/73 (2.7%); ROS1fus 1/73 (1.3%) , and RET fus 3/73 (4%). Concomitant mutations were identified in 32% of pts mainly with TP53 (TP53/EGFR: (5/11) 45% and TP53/KRAS: (7/21) 33%). Matched targeted treatment was offered in 17/73 (23%), mostly tyrosine kinase inhibitors (TKI); anti-EGFR in 11/17 (65%) and ALK/ROS TKI in 3/17 (17.5%). Three pts (17.5%) received anti-MET or anti-HER2 TKI. OS in this whole population treated with target therapies (n = 17) was 45.7 months (m) (CI95% 26.8-NA). The KRASmut group had median OS of 10 m (CI95% 7.5-NA), while the remaining population with wild type (WT) for all driver alterations had median OS of 23 m (CI95% 20-NA), (HR 1.71 for KRASmut vs WT pts, p = 0.18). The presence of coexisting TP53mut did not negatively impact on OS in the cohorts with EGFRmut and KRASmut (p > 0.1) in a multivariate model. Conclusions: Combined next generation techniques for molecular profile in NSCLC pts identifies molecular alterations that have clinically relevant impact in survival. Coexisting TP53mut alteration shows no detrimental effect in OS.