Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Fabrication of Multilayer Microfluidic Arrays for Passive, Efficient DNA Trapping and Profiling.

Trace amounts of cell-free DNA containing cancer-specific biomarkers can be found in blood plasma. Detection of these biomarkers holds tremendous potential for applications such as noninvasive cancer diagnostics and therapeutic monitoring. However, such DNA molecules are extremely rare, and a typical patient blood sample may only contain a few copies. Here we describe the fabrication and operation of a microfluidic device to efficiently trap single DNA molecules into chambers for detection of tumor-specific biomarkers through a passive, geometric manipulation strategy.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography (CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis.

Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution, enabling direct transfer from sample preparation to dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a coinfection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR and then perform dHRM to identify the two bacterial species.

Nanoarray Digital Polymerase Chain Reaction with High-Resolution Melt for Enabling Broad Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Test.

Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial samples in a timely manner. To address this need, a microfluidic and machine-learning-based platform that performs broad bacteria identification (ID) and rapid yet reliable antimicrobial susceptibility testing (AST) is developed. Specifically, this platform builds on "pheno-molecular AST", a strategy that transforms nucleic acid amplification tests (NAATs) into phenotypic AST through quantitative detection of bacterial genomic replication, and utilizes digital polymerase chain reaction (PCR) and digital high-resolution melt (HRM) to quantify and identify bacterial DNA molecules. Bacterial species are identified using integrated experiment-machine learning algorithm via HRM profiles. Digital DNA quantification allows for rapid growth measurement that reflects susceptibility profiles of each bacterial species within only 30 min of antibiotic exposure. As a demonstration, multiple bacterial species and their susceptibility profiles in a spiked-in polymicrobial urine specimen were correctly identified with a total turnaround time of ∼4 h. With further development and clinical validation, this platform holds the potential for improving clinical diagnostics and enabling targeted antibiotic treatments.

Highly Efficient Real-Time Droplet Analysis Platform for High-Throughput Interrogation of DNA Sequences by Melt.

Droplet microfluidic platforms have greatly enhanced the throughput and sensitivity of single-molecule and single-cell analyses. However, real-time analyses of individual droplets remain challenging. Most droplet microfluidic platforms have fundamental drawbacks that undermine their utility toward applications that rely on real-time monitoring to identify rare variants, such as bacterial persistence, drug discovery, antibody production, epigenetic biomarker analyses, etc. We present a platform for high-density droplet trapping and real-time analysis with 100% loading and trapping efficiency at a packing density of 110,000 droplets per in2. To demonstrate real-time analysis capabilities, we perform digital PCR and parallelized digital high-resolution melt curve acquisition on droplets to discriminate methylation levels of a tumor suppressor gene, CDO1, on a molecule-by-molecule basis. We hope that this platform, which is compatible with a large range of droplet sizes and generation technologies, may facilitate high-throughput real-time analyses on a molecule-by-molecule or cell-by-cell basis of heterogeneous populations.

Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation.

Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).

Facile profiling of molecular heterogeneity by microfluidic digital melt.

This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14ARF). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.

A Position Statement on Population Data Science: The Science of Data about People.

Information is increasingly digital, creating opportunities to respond to pressing issues about human populations using linked datasets that are large, complex, and diverse. The potential social and individual benefits that can come from data-intensive science are large, but raise challenges of balancing individual privacy and the public good, building appropriate socio-technical systems to support data-intensive science, and determining whether defining a new field of inquiry might help move those collective interests and activities forward. A combination of expert engagement, literature review, and iterative conversations led to our conclusion that defining the field of Population Data Science (challenge 3) will help address the other two challenges as well. We define Population Data Science succinctly as the science of data about people and note that it is related to but distinct from the fields of data science and informatics. A broader definition names four characteristics of: data use for positive impact on citizens and society; bringing together and analyzing data from multiple sources; finding population-level insights; and developing safe, privacy-sensitive and ethical infrastructure to support research. One implication of these characteristics is that few people possess all of the requisite knowledge and skills of Population Data Science, so this is by nature a multi-disciplinary field. Other implications include the need to advance various aspects of science, such as data linkage technology, various forms of analytics, and methods of public engagement. These implications are the beginnings of a research agenda for Population Data Science, which if approached as a collective field, can catalyze significant advances in our understanding of trends in society, health, and human behavior.

Assessing privacy risks in population health publications using a checklist-based approach.

OBJECTIVE: Recent growth in the number of population health researchers accessing detailed datasets, either on their own computers or through virtual data centers, has the potential to increase privacy risks. In response, a checklist for identifying and reducing privacy risks in population health analysis outputs has been proposed for use by researchers themselves. In this study we explore the usability and reliability of such an approach by investigating whether different users identify the same privacy risks on applying the checklist to a sample of publications. METHODS: The checklist was applied to a sample of 100 academic population health publications distributed among 5 readers. Cohen's κ was used to measure interrater agreement. RESULTS: Of the 566 instances of statistical output types found in the 100 publications, the most frequently occurring were counts, summary statistics, plots, and model outputs. Application of the checklist identified 128 outputs (22.6%) with potential privacy concerns. Most of these were associated with the reporting of small counts. Among these identified outputs, the readers found no substantial actual privacy concerns when context was taken into account. Interrater agreement for identifying potential privacy concerns was generally good. CONCLUSION: This study has demonstrated that a checklist can be a reliable tool to assist researchers with anonymizing analysis outputs in population health research. This further suggests that such an approach may have the potential to be developed into a broadly applicable standard providing consistent confidentiality protection across multiple analyses of the same data.

Anonymization for outputs of population health and health services research conducted via an online data center.

OBJECTIVE: Online data centers (ODCs) are becoming increasingly popular for making health-related data available for research. Such centers provide good privacy protection during analysis by trusted researchers, but privacy concerns may still remain if the system outputs are not sufficiently anonymized. In this article, we propose a method for anonymizing analysis outputs from ODCs for publication in academic literature. METHODS: We use as a model system the Secure Unified Research Environment, an online computing system that allows researchers to access and analyze linked health-related data for approved studies in Australia. This model system suggests realistic assumptions for an ODC that, together with literature and practice reviews, inform our solution design. RESULTS: We propose a two-step approach to anonymizing analysis outputs from an ODC. A data preparation stage requires data custodians to apply some basic treatments to the dataset before making it available. A subsequent output anonymization stage requires researchers to use a checklist at the point of downloading analysis output. The checklist assists researchers with highlighting potential privacy concerns, then applying appropriate anonymization treatments. CONCLUSION: The checklist can be used more broadly in health care research, not just in ODCs. Ease of online publication as well as encouragement from journals to submit supplementary material are likely to increase both the volume and detail of analysis results publicly available, which in turn will increase the need for approaches such as the one suggested in this paper.

Individual privacy versus public good: protecting confidentiality in health research.

Health and medical data are increasingly being generated, collected, and stored in electronic form in healthcare facilities and administrative agencies. Such data hold a wealth of information vital to effective health policy development and evaluation, as well as to enhanced clinical care through evidence-based practice and safety and quality monitoring. These initiatives are aimed at improving individuals' health and well-being. Nevertheless, analyses of health data archives must be conducted in such a way that individuals' privacy is not compromised. One important aspect of protecting individuals' privacy is protecting the confidentiality of their data. It is the purpose of this paper to provide a review of a number of approaches to reducing disclosure risk when making data available for research, and to present a taxonomy for such approaches. Some of these methods are widely used, whereas others are still in development. It is important to have a range of methods available because there is also a range of data-use scenarios, and it is important to be able to choose between methods suited to differing scenarios. In practice, it is necessary to find a balance between allowing the use of health and medical data for research and protecting confidentiality. This balance is often presented as a trade-off between disclosure risk and data utility, because methods that reduce disclosure risk, in general, also reduce data utility.

Cost-effectiveness of an advance notification letter to increase colorectal cancer screening.

OBJECTIVES: The aim of this study is to evaluate the cost-effectiveness of a patient-direct mailed advance notification letter on participants of a National Bowel Cancer Screening Program (NBCSP) in Australia, which was launched in August 2006 and offers free fecal occult blood testing to all Australians turning 50, 55, or 65 years of age in any given year. METHODS: This study followed a hypothetical cohort of 50-year-old, 55-year-old, and 65-year-old patients undergoing fecal occult blood test (FOBT) screening through a decision analytic Markov model. The intervention compared two strategies: (i) advance letter, NBCSP, and FOBT compared with (ii) NBCSP and FOBT. The main outcome measures were life-years gained (LYG), quality-adjusted life-years (QALYs) gained and incremental cost-effectiveness ratio. RESULTS: An advance notification screening letter would yield an additional 54 per 100,000 colorectal cancer deaths avoided compared with no letter. The estimated cost-effectiveness was $3,976 per LYG and $6,976 per QALY gained. CONCLUSIONS: An advance notification letter in the NBCSP may have a significant impact on LYG and cancer deaths avoided. It is cost-effective and offers a feasible strategy that could be rolled out across other screening program at an acceptable cost.

Single nucleotide polymorphism array lesions, TET2, DNMT3A, ASXL1 and CBL mutations are present in systemic mastocytosis.

We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A) and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM). SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2 and the CBL gene family. Overall survival (OS) was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning TET2/KIT and DNMT3A. Mutations in TET2, DNMT3A, ASXL1 and CBL were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in EZH2 and IDH1/IDH2. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined TET2/DNMT3A/ASXL1 mutations independent of KIT (P = 0.04) and sole TET2 mutations (P<0.001). In conclusion, TET2, DNMT3A and ASXL1 mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Loss of heterozygosity in 7q myeloid disorders: clinical associations and genomic pathogenesis.

Loss of heterozygosity affecting chromosome 7q is common in acute myeloid leukemia and myelodysplastic syndromes, pointing toward the essential role of this region in disease phenotype and clonal evolution. The higher resolution offered by recently developed genomic platforms may be used to establish more precise clinical correlations and identify specific target genes. We analyzed a series of patients with myeloid disorders using recent genomic technologies (1458 by single-nucleotide polymorphism arrays [SNP-A], 226 by next-generation sequencing, and 183 by expression microarrays). Using SNP-A, we identified chromosome 7q loss of heterozygosity segments in 161 of 1458 patients (11%); 26% of chronic myelomonocytic leukemia patients harbored 7q uniparental disomy, of which 41% had a homozygous EZH2 mutation. In addition, we describe an SNP-A-isolated deletion 7 hypocellular myelodysplastic syndrome subset, with a high rate of progression. Using direct and parallel sequencing, we found no recurrent mutations in typically large deletion 7q and monosomy 7 patients. In contrast, we detected a markedly decreased expression of genes included in our SNP-A defined minimally deleted regions. Although a 2-hit model is present in most patients with 7q uniparental disomy and a myeloproliferative phenotype, haplodeficient expression of defined regions of 7q may underlie pathogenesis in patients with deletions and predominant dysplastic features.

Cytogenetic and molecular predictors of response in patients with myeloid malignancies without del[5q] treated with lenalidomide.

BACKGROUND: While lenalidomide (LEN) shows high efficacy in myelodysplastic syndromes (MDS) with del[5q], responses can be also seen in patients presenting without del[5q]. We hypothesized that improved detection of chromosomal abnormalities with new karyotyping tools may better predict response to LEN. DESIGN AND METHODS: We have studied clinical, molecular and cytogenetic features of 42 patients with MDS, myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes and secondary acute myeloid leukemia (sAML) without del[5q] by metaphase cytogenetics (MC) who underwent therapy with LEN. RESULTS: Fluorescence in situ hybridization (FISH) or single nucleotide polymorphism array (SNP-A)-based karyotyping marginally increased the diagnostic yield over MC, detecting 2/42 (4.8%) additional cases with del[5q], one of whom were responded to LEN. Responses were more often observed in patients with a normal karyotype by MC (60% vs abnormal MC; 17%, p = .08) and those with gain of chromosome 8 material by either of all 3 karyotyping methods (83% vs all other chromosomal abnormalities; 44% p = .11). However, 5 out of those 6 patients received combined LEN/AZA therapy and it may also suggest those with gain of chromosome 8 material respond well to AZA. The addition of FISH or SNP-A did not improve the predictive value of normal cytogenetics by MC. Mutational analysis of TET2, UTX, CBL, EZH2, ASXL1, TP53, RAS, IDH1/2, and DNMT-3A was performed on 21 of 41 patients, and revealed 13 mutations in 11 patients, but did not show any molecular markers of responsiveness to LEN. CONCLUSIONS: Normal karyotype and gain of chromosome 8 material was predictive of response to LEN in non-del[5q] patients with myeloid malignancies.

Topography, clinical, and genomic correlates of 5q myeloid malignancies revisited.

PURPOSE: Interstitial deletions of chromosome 5q are common in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), pointing toward the pathogenic role of this region in disease phenotype and clonal evolution. The higher level of resolution of single-nucleotide polymorphism array (SNP-A) karyotyping may be used to find cryptic abnormalities and to precisely define the topographic features of the genomic lesions, allowing for more accurate clinical correlations. PATIENTS AND METHODS: We analyzed high-density SNP-A karyotyping at diagnosis for a cohort of 1,155 clinically well-annotated patients with malignant myeloid disorders. results: We identified chromosome 5q deletions in 142 (12%) of 1,155 patients and uniparental disomy segments (UPD) in four (0.35%) of 1,155 patients. Patients with deletions involving the centromeric and telomeric extremes of 5q have a more aggressive disease phenotype and additional chromosomal lesions. Lesions not involving the centromeric or telomeric extremes of 5q are not exclusive to 5q- syndrome but can be associated with other less aggressive forms of MDS. In addition, larger 5q deletions are associated with either del(17p) or UPD17p. In 31 of 33 patients with del(5q) AML, either a deletion involving the centromeric and/or telomeric regions or heterozygous mutations in NPM1 or MAML1 located in 5q35 were present. CONCLUSION: Our results suggest that the extent of the affected region on 5q determines clinical characteristics that can be further modified by heterozygous mutations present in the telomeric extreme.