Combining deep learning and droplet microfluidics for rapid and label-free antimicrobial susceptibility testing of colistin.

Efficient tools for rapid antibiotic susceptibility testing (AST) are crucial for appropriate use of antibiotics, especially colistin, which is now often considered a last resort therapy with extremely drug resistant Gram-negative bacteria. Here, we developed a rapid, easy and miniaturized colistin susceptibility assay based on microfluidics, which allows for culture and high-throughput analysis of bacterial samples. Specifically, a simple microfluidic platform that can easily be operated was designed to encapsulate bacteria in nanoliter droplets and perform a fast and automated bacterial growth detection in 2 h, using standardized samples. Direct bright-field imaging of compartmentalized samples proved to be a faster and more accurate detection method as compared to fluorescence-based analysis. A deep learning powered approach was implemented for the sensitive detection of the growth of several strains in droplets. The DropDeepL AST method (Droplet and Deep learning-based method for AST) developed here allowed the determination of the colistin susceptibility profiles of 21 fast-growing Enterobacterales (E. coli and K. pneumoniae), including clinical isolates with different resistance mechanisms, showing 100 % categorical agreement with the reference broth microdilution (BMD) method performed simultaneously. Direct AST of bacteria in urine samples on chip also provided accurate results in 2 h, without the need of complex sample preparation procedures. This method can easily be implemented in clinical microbiology laboratories, and has the potential to be adapted to a variety of antibiotics, especially for last-line antibiotics to optimize treatment of patients infected with multi-drug resistant strains.

BRCA1 and RAD51C promotor methylation in human resectable pancreatic adenocarcinoma.

BACKGROUND: Homozygous Recombination Deficiency (HRD) is associated with sensitivity to PARP-inhibitors (PARPi) in different cancer types. In pancreatic adenocarcinoma (PA) the main cause of HRD is BRCA1/2 germline mutation and patients with mutations in BRCA1/2 may benefit from PARPi. Recently other mechanisms leading to HRD were described in different cancer types, including gene mutations and epigenetic changes such as promoter hypermethylation. In PA, BRCA1 promoter hypermethylation, a known mechanism of gene silencing, was recently described. However, results are discordant between North American studies (0.7% of PA) and Asian ones (up to 60% of PA) and the association with HRD is not clear. METHODS: Here, we developed 2 quantifications methods to explore BRCA1 and RAD51C promoter methylation in a series of 121 Formalin Fixed-Paraffin-Embedded (FFPE) specimens from resected PA without neoadjuvant treatment. The methylation-specific PCR was done with 2 different methods after DNA bisulfite conversion: a digital droplet PCR, and a PCR followed by capillary electrophoresis, to score the methylated / non methylated ratios in tumor samples. Methods were validated for specificity and sensibility using 100, 20, 10, 5 and 0% methylated commercial DNA for fragment analysis with a detection cutoff of 5-10%. Limit of blank was defined as 5 dropplets/20µL for RAD51C and 1 dropplet/20µL for BRCA1 for ddPCR. Samples were reviewed by a pathologist, macrodissected before DNA extraction to obtain 50-60% of tumoral cells. DNAs were treated for bisulfite conversion and analyzed using both methods in parallel to known positive and negative controls in each run. RESULTS AND CONCLUSION: No methylation at BRCA1 or RAD51C was found in this series of PA suggesting that HRD gene promoter methylation is a rare event in European patients.

Circulating tumor DNA is a prognostic marker of tumor recurrence in stage II and III colorectal cancer: multicentric, prospective cohort study (ALGECOLS).

BACKGROUND: In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence. METHOD: The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3-4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy. RESULTS: Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71-7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32-7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1-6 months) (adjusted HR = 5, 95% CI 1.9-12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study. CONCLUSION: This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques.

The Microbiome Meets Nanotechnology: Opportunities and Challenges in Developing New Diagnostic Devices.

Monitoring of the human microbiome is an emerging area of diagnostics for personalized medicine. Here, the potential of different nanomaterials and nanobiosensing technologies is reviewed for the development of novel diagnostic devices for the detection and measurement of microbiome-related biomarkers. Moreover, the current and future landscape of microbiome-based diagnostics is defined by exploring the advantages and disadvantages of current nanotechnology-based approaches, especially in the context of developing point-of-care (PoC) devices that would meet the international guidelines known as REASSURED (Real-time connectivity; Ease of specimen collection; Affordability; Sensitivity; Specificity; User-friendliness; Rapid & robust operation; Equipment-free; and Deliverability). Finally, the strategies of the latest international scientific consortia working in this field are analyzed, the current microbiome diagnostics market are reported and the principal ethical, legal, and societal issues related to microbiome R&D and innovation are discussed.

Facile Production of Large-Area Cell Arrays Using Surface-Assembled Microdroplets.

Techniques that enable the spatial arrangement of living cells into defined patterns are broadly applicable to tissue engineering, drug screening, and cell-cell investigations. Achieving large-scale patterning with single-cell resolution while minimizing cell stress/damage is, however, technically challenging using existing methods. Here, a facile and highly scalable technique for the rational design of reconfigurable arrays of cells is reported. Specifically, microdroplets of cell suspensions are assembled using stretchable surface-chemical patterns which, following incubation, yield ordered arrays of cells. The microdroplets are generated using a microfluidic-based aerosol spray nozzle that enables control of the volume/size of the droplets delivered to the surface. Assembly of the cell-loaded microdroplets is achieved via mechanically induced coalescence using substrates with engineered surface-wettability patterns based on extracellular matrices. Robust cell proliferation inside the patterned areas is demonstrated using standard culture techniques. By combining the scalability of aerosol-based delivery and microdroplet surface assembly with user-defined chemical patterns of controlled functionality, the technique reported here provides an innovative methodology for the scalable generation of large-area cell arrays with flexible geometries and tunable resolution.

Covalent Bonding of Thermoplastics to Rubbers for Printable, Reel-to-Reel Processing in Soft Robotics and Microfluidics.

The lamination of mechanically stiff structures to elastic materials is prevalent in biological systems and popular in many emerging synthetic systems, such as soft robotics, microfluidics, stretchable electronics, and pop-up assemblies. The disparate mechanical and chemical properties of these materials have made it challenging to develop universal synthetic procedures capable of reliably adhering to these classes of materials together. Herein, a simple and scalable procedure is described that is capable of covalently laminating a variety of commodity ("off-the-shelf") thermoplastic sheets to silicone rubber films. When combined with laser printing, the nonbonding sites can be "printed" onto the thermoplastic sheets, enabling the direct fabrication of microfluidic systems for actuation and liquid handling applications. The versatility of this approach in generating thin, multifunctional laminates is demonstrated through the fabrication of milliscale soft actuators and grippers with hinged articulation and microfluidic channels with built-in optical filtering and pressure-dependent geometries. This method of fabrication offers several advantages, including technical simplicity, process scalability, design versatility, and material diversity. The concepts and strategies presented herein are broadly applicable to the soft robotics, microfluidics, and advanced and additive manufacturing communities where hybrid rubber/plastic structures are prevalent.

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS: We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS: Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS: These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

[Digital PCR compartmentalization II. Contribution for the quantitative detection of circulating tumor DNA]. / PCR digitale en micro-compartiments - II. Apport pour la détection quantitative d'ADN tumoral circulant.

Genetic markers are now widely used in the clinics, particularly in cancer patient management. Indeed, these tumor markers can help in the diagnosis and prognosis of the disease, and provide valuable information for treatment orientation in the context of personalized medicine. The presence of circulating cell-free tumor DNA (cftDNA) and thus of tumor markers in the blood can be considered to partly avoid the use of solid biopsies. The use of blood samples, as liquid biopsies, is less invasive and described as more representative of tumor heterogeneity. However, cftDNA can be found in blood in low proportion that can vary according to the nature and the progression of the tumor. For these reasons, the use of highly sensitive, specific and ideally quantitative methods for its detection are required. These requirements constituted until recently a technological limit, which now can be overcome thanks to digital PCR. This technology could now become a very efficient and non-invasive tool in oncology, complementary to conventional diagnostic techniques.

[Digital PCR compartmentalization I. Single-molecule detection of rare mutations]. / PCR digitale en micro-compartiments - I. Détection sensible de séquences d'acides nucléiques rares.

Polymerase chain reaction based techniques have been widely used in laboratory settings. Several applications in oncology, virology or prenatal diagnosis require highly sensitive detection methods, which cannot be achieved with conventional techniques. Digital PCR (dPCR) was developed from the association of PCR and limiting dilution procedures. It is based on the compartmentalization of DNA molecules in small volumes. Controlling the size and the content of each compartment is crucial to obtain a high sensitivity with a single molecule resolution. Microfluidics offers promising tools to isolate DNA fragments such as microdroplets, microchambers or microwells with volumes ranging from few picoliters to nanoliters. The review provides an overview of recent developments of microfluidics dPCR platforms and how this technology can influence the management of cancer patients.

Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy.

PURPOSE: KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis. EXPERIMENTAL DESIGN: From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction. RESULTS: In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors. CONCLUSIONS: This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool.

Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two.

A three dimensional thermoplastic microfluidic chip for robust cell capture and high resolution imaging.

We present a low cost microfluidic chip integrating 3D micro-chambers for the capture and the analysis of cells. This device has a simple design and a small footprint. It allows the implementation of standard biological protocols in a chip format with low volume consumption. The manufacturing process relies on hot-embossing of cyclo olefin copolymer, allowing the development of a low cost and robust device. A 3D design of microchannels was used to induce high flow velocity contrasts in the device and provide a selective immobilization. In narrow distribution channels, the liquid velocity induces a shear stress that overcomes adhesion forces and prevents cell immobilization or clogging. In large 3D chambers, the liquid velocity drops down below the threshold for cell attachment. The devices can be operated in a large range of input pressures and can even be handled manually using simple syringe or micropipette. Even at high flow injection rates, the 3D structures protect the captured cell from shear stress. To validate the performances of our device, we implemented immuno-fluorescence labeling and Fluorescence in Situ Hybridization (FISH) analysis on cancer cell lines and on a patient pleural effusion sample. FISH is a Food and Drug Administration approved cancer diagnostic technique that provides quantitative information about gene and chromosome aberration at the single cell level. It is usually considered as a long and fastidious test in medical diagnosis. This process can be easily implanted in our platform, and high resolution fluorescence imaging can be performed with reduced time and computer intensiveness. These results demonstrate the potential of this chip as a low cost, robust, and versatile tool adapted to complex and demanding protocols for medical diagnosis.

New non-covalent strategies for stable surface treatment of thermoplastic chips.

In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.