Abstract
Automated Diagnosis
High-Throughput and Automated Diagnosis of Antimicrobial Resistance Using a Cost-Effective Cellphone-Based Microplate Reader
Detecting antimicrobial resistance (i.e., antimicrobial susceptibility testing [AST]) is important to curb its increasing prevalence. Currently, antimicrobial resistance is insufficiently monitored because of technological challenges, high costs, and lack of professional training. In this report, the authors demonstrate an automated, convenient, and cost-effective cellphone-based 96-well microtiter plate reader able to carry out AST without trained diagnosticians.
The gold standard for AST involves trained experts examining visible growth under a light source from bacterial suspensions containing different concentration levels of antibiotics. The diagnostician also needs to determine if the bacterium is susceptible or resistant to better guide physician treatment decisions. With the technological advancement of smartphone devices, an optomechanical hardware attachment (custom 3D printed) is added to a regular Nokia Lumia 1020 mobile phone paired with a data processing server (PC or laptop). Previously, the authors used a similar setup to perform enzyme-linked immunosorbent assays for detecting mumps, measles, and herpes simplex virus from 96-well plates.
Following the insertion of the AST test plate, transmitted light from each well is captured with multiple exposures. These images are uploaded to a local or remote server to automatically quantify turbidity. The cellphone-based AST is then used to obtain highly accurate minimum inhibitory concentration determination and drug susceptibility interpretation using gram-negative Klebsiella pneumoniae. Following the setting of thresholds and blind testing, the authors achieve a well turbidity detection accuracy of 98.2%, inhibitory concentration accuracy of 96.1%, and drug susceptibility interpretation accuracy of 99.23% from 78 patient plates.
Critically, this automated mobile phone–based system exceeds the Food and Drug Administration (FDA) criteria for clinical susceptibility testing. Thus, by developing this mobile phone–based system, AST can be performed more regularly, in an accurate and cost-effective way. The mobile phone’s wireless connectivity and digital record saving are helpful features that further facilitate collection of drug-resistance profiles for tracking its global spread (Feng, S.; et al. Sci. Rep.
Performance of the GeneXpert Ebola Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study
Diagnosis of the devastating and contagious Ebola virus (EV) disease is heavily reliant on reverse transcription PCR (RT-PCR) assays. Conventional assays are highly cumbersome, requiring numerous steps to lyse samples, extract nucleic acids, and perform RT-PCR. Sample collection (due to its biohazardous nature) and multistep processing by skilled operators further hinder operation. Ideally, highly accurate and automated RT-PCR assay systems would greatly improve and simplify diagnostic capability. The Cepheid GeneXpert Ebola assay is an automated “sample-to-answer” system that integrates sample preparation, virus inactivation, nucleic acid amplification, and detection that is approved by the U.S. FDA and World Health Organization for EV diagnosis.
The Xpert Ebola assay consists of two targets for the glycoprotein and nucleoprotein genes of the EV. Furthermore, it consists of two internal controls: an exogenous sample processing control (to determine if amplification is inhibited) and a sample adequacy control that detects human genomic DNA (to confirm that cellular material is present and intact). The Cepheid GeneXpert Ebola assay is compared with a Trombley assay (used by the Public Health England field biocontainment laboratory). In total, 211 whole-blood specimens generate valid results for Xpert and Trombley. Of these, 22 of 22 specimens are positive for both tests. One hundred eight-one of 189 that test negative by the Trombley assay are similarly negative by Xpert, whereas the rest are Trombley negative, Xpert positive. These eight discordant samples were found to be from known EV patients (previously positive from Trombley assay) who received follow-up testing to monitor virus clearance. Thus, low-level virus counts potentially missed by the Trombley method can be missed by the GeneXpert assay.
In addition to whole-blood specimens, buccal swab (BS) samples were also collected with fresh and frozen samples tested. Despite some invalid results from the BS samples, Trombley and GeneXpert assays were in 100% agreement. The presence of discordant data between the GeneXpert and the Trombley assay (from whole blood) might suggest a lower limit of detection for the GeneXpert. However, an alternative explanation suggests that blood plasma may be a less optimal sample source compared with whole blood.
In summary, even though limitations and considerations are discussed in this study (instrument handling, biosafety, sample collection, etc.), it demonstrates excellent field laboratory performance overall, giving accurate and rapid diagnosis of EV (Semper, A. E.; et al. PLoS Med.
Fully-Automated, Chemiluminescence IgA and IgG Anti-Tissue Transglutaminase (tTG) Antibodies Serum Assays for the Screening of Celiac Disease
Celiac disease (CD) is an immune-mediated enteropathy caused by gluten ingestion in subjects who are genetically predisposed. As dietary gluten triggers this condition, a gluten-free diet is essential to avoid chronic disease complications such as osteoporosis, impaired splenic function, neurologic disorders, infertility, and malignancies. While histologic confirmation of mucosal damage is needed to obtain definitive diagnosis, serologic testing has recently gained in its importance. The presence of anti-endomysium (EMA) antibodies is highly specific (98%–100%) for CD diagnosis but is based on fluorescent microscopy. This is time-consuming, is relatively expensive, has operator variability, and requires trained laboratory personnel. Other types of antibodies such as anti-transglutaminase (tTG) IgA and IgG have also been suggested as possible diagnostic CD biomarkers. The aim of this study is to evaluate a chemiluminescent immunoassay for IgA and IgG anti-tTG antibodies in comparison with anti-EMA among a general population of unselected CD patients.
Of the 727 samples, 701 show perfect agreement of negative results among anti-tTG IgA, anti-tTG IgG, and EMA antibodies. Although there are several discordant results between the different diagnostic modalities, no sample is positive for EMA but negative for anti-tTG antibodies. Those obtaining agreement between anti-tTG IgA and EMA antibodies are diagnosed with CD. However, the few discordant studies can be explained by borderline expression of the biomarkers. As a result, these individuals were considered affected by CD rather than given full diagnosis status. Whereas IgA serologic testing for CD has been performed before, this study is performed on an unselected general population including adults and children. Importantly, no sample is positive for anti-EMA antibodies and negative for anti-tTG antibodies, which suggests it does not have a poorer diagnostic capability. Some studies also suggest that anti-EMA has a lower sensitivity compared with anti-tTG IgA antibodies. Thus, a novel chemiluminescence assay for measuring anti-tTG antibodies demonstrates good agreement with anti-EMA detection. This facilitates less monitoring and detection (both of which are highly time-consuming), is more standardized, and does not require trained personnel (Daves, M.; et al. J. Immunol. Methods.
Targeted DNA Sequencing and In Situ Mutation Analysis Using Mobile Phone Microscopy
To improve diagnostic accuracy, molecular diagnostics can complement morphology-based diagnosis. Because of assay and infrastructure requirements, molecular diagnosis is typically outsourced to specialized labs and centers that have limited availability in many resource-scarce locations. Digital pathology is a trend that bridges this gap, allowing remote assessment of samples and data, bridging limitations in pathologist availability. Advances in mobile phones facilitate this trend with improved connectivity, imaging, detection, and computational capabilities.
The authors demonstrate how a cost-effective and compact microscope integrated on a mobile phone can be used for targeted DNA sequencing and in situ point mutation analysis that integrate molecular analysis with tumor tissue morphology. The mobile phone microscope exhibits a 4-log dynamic range (1 fM–10 pM), demonstrating its utility to image and analyze individual rolling circle–amplified single molecules. To evaluate if the mobile phone microscope is suitable for next-generation sequencing (NGS) applications, it is used to read sequencing-by-ligation reactions of rolling circle amplification products (RCP) from microscope slides.
The mobile phone microscope successfully detects species from a mixture of 1:1000 mutant:wild-type. The mutation detection sensitivity (<1%) is comparable with U.S. FDA–approved KRAS PCR techniques that are further validated on cell lines and colon tissue biopsies. The mobile phone microscope is further used for in situ analysis to identify KRAS mutant cells. This involves padlock probes to target the most prevalent single base mutations on codon 12 and 13. To automate quantification of RCP species, a machine-learning–based image analysis algorithm is developed to recognize and count RCPs that yielded similar results to regular microscopy. The mobile phone microscope technique is then used to detect A549 cells bearing the KRAS mutation spiked into wild-type onco-DG1 cells at a ratio of 1:100 and 1:1000. At the 1:1000 ratio, the padlock probes express a significantly higher detection rate compared with cells bearing the wild-type KRAS genotype. Finally, the mobile phone microscope is applied to clinical samples (n = 6) and is found to generate equivalent performance to NGS molecular diagnosis.
With the mobile phone microscope, the authors demonstrate a cost-effective means to integrate molecular diagnosis in pathology and increase its accessibility. Its potential applications are very broad because it effectively transfers diagnosis from the point of care to the point of expertise (Kühnemund, M.; et al. Nat. Commun.
Nanomedicine
Precise Diagnosis in Different Scenarios Using Photoacoustic and Fluorescence Imaging with Dual-Modality Nanoparticles
As a means to improve diagnostic accuracy in clinical diagnosis, a dual-modality nanoparticle is designed by Peng et al. Although photoacoustic imaging (PAI) is highly advantageous for a number of reasons (e.g., better imaging depth, weaker scattering effects, etc.), fluorescence molecular imaging (FMI) has a wider range of molecular probes and dyes and is ahead in terms of its clinical translation degree. However, FMI has a limited imaging depth with high photon scattering in soft tissues. The combined FMI-PAI approach involves a gold nanorod (AuNR) core coated with an indocyanine green (ICG)–loaded silica shell (
Under increasing concentrations of
This study demonstrates the effectiveness of multimodal imaging-based nanoparticle diagnostic systems. Using dual modalities leverages the advantages of each. For example, FMI would allow a rapid diagnosis of limb ischemia, whereas PAI could facilitate classification into severe or mild (Peng, D.; et al. Nanoscale
Local MicroRNA Delivery Targets Palladin and Prevents Metastatic Breast Cancer
Metastasis is the primary cause of mortality for breast cancer. It is a multistep process consisting of cells undergoing epithelial-mesenchymal transition before the cells leave the primary tumor site, infiltrate blood and lymphatic vessels, and seed distant metastases. Targeting specific steps of this highly orchestrated process potentially yields novel antimetastatic cancer therapy strategies. Using breast cancer bioinformatics analysis, 20 single-nucleotide polymorphisms (SNPs) are implicated in gene-binding activity. Focusing on the SNP with the highest (>43%) minor allele frequency (largest population effect), the SNP-rs1071738 is found to be involved in the binding between miR-96, miR-182, and the PALLD (palladin) gene.
Further investigation suggests that palladin expression regulation might influence a number of pathways involved in cell motility. Using luciferase assay studies, miR-96/182 down-regulates palladin in HeLa and HEK-293 T cells with complementary C alleles, although its expression is not affected by alternate G alleles. Evidence from invasive and noninvasive breast cancer cell lines further validates this notion that palladin expression is controlled by miR-96/182 through the C allele variants.
The authors then hypothesize that the mutation could be repaired using complementary engineered miRNA. Using functional assays such as wound closure and transwell invasion studies, miR-96/182 are found to down-regulate palladin expression and inhibit migration and invasion in breast cancer cells. Delivering a local dosage of miR-96/182 to reduce systemic side effects in the subject, oligonucleotides are bound to gold nanoparticles contained within dendrimer-dextran hydrogels for sustained release and delivered to the tumor site. This approach was previously shown to be successful in sustaining miRNA delivery for anticancer therapy. In addition to miRNA therapy, cisplatin is added to the hydrogel platform, which intercalates with miRNA species and provides synergistic suppression of the breast cancer tumor. CREKA (Cys-Arg-Glu-Lys-Ala) peptides are incorporated to improve homing to the tumor and improve cellular uptake. In the orthotopic mouse model, the addition of the drug with miRNA-loaded gold nanoparticles is found to be critical in reducing tumor volume. Harvested samples confirm that the respective miRNA expression levels are enhanced and palladin expression down-regulates as intended. Because of the metastatic potential to the lungs, liver, and brain, these organs are then assessed using micro–computed tomography and fluorescence imaging. Strikingly, the number of lung metastases is reduced when treated with nanoparticles bearing miR-96/182. Fluorescence imaging also shows decreased secondary tumor metastasis size in the lung, liver, and brain. Interestingly, it also prevents spleen enlargement, an indication of the advanced state of disease.
Having knowledge of SNP-rs1071738, the authors formulate a novel therapy. miRs 96 and 182 are combined with cisplatin to achieve local and specific delivery for breast cancer. This leads to substantial reductions in tumor growth and metastasis in an orthotopic breast cancer model. As palladin is implicated in other cancers, such as renal cell carcinoma as well as tumors in the lung, stomach, colon, and pancreas, this platform may see greater applicability. The authors propose using this local, specific therapy shortly after malignancy is confirmed, prior to surgical intervention, to improve clinical outcomes. This could be done by reducing primary tumor mass and reducing/preventing metastasis (Gilam, A.; et al. Nat. Commun.
Stem Cell Therapy and Manufacturing
Bulk Cell Density and Wnt/TGFbeta Signaling Regulate Mesendodermal Patterning of Human Pluripotent Stem Cells
Due to their replicative and maturation potential, pluripotent stem cells (PSCs) are a well-characterized, generally unlimited cell source that are amenable to large-scale production of cell progeny. To fulfill their therapeutic potential, well-defined and reproducible culture conditions are required to direct immature progenitors into the desired phenotype.
Directing the maturation of PSC aggregates in culture recapitulates a number of aspects of gastrulation. Interestingly, varying the size of PSC aggregates using bulk cell density (BCD) in conjunction with supplementing the GSK3 inhibitor Chir99021 (CHIR) mimics aspects of primitive streak (PS)-like development in vitro. Even during the initial 24 hours, different BCD conditions result in distinct cell fates reminiscent of various PS lineages.
This report highlights the importance of controlling BCD conditions to ensure desired lineage specification and process reproducibility during early stages of differentiation. Using gene reporter NKX2.5-GFP labeled hPSCs, the authors show that distinct WNT and BMP signals are required for cardiomyogenesis in aggregated PSCs. Several combinations of high BCD concentration even generate secreted factors that delay PS specification. Thus, these results allow the authors to propose a model using BCD, CHIR, and time to generate various definitive endoderm, cardiac mesoderm, and presomitic mesoderm lineages. It also gives a plausible and mechanistic explanation for how CHIR concentration alone, with BCD and time modulation, can lead to different progeny arising.
Understanding the earliest stages of mesendodermal differentiation in hPSCs promotes better experimental control of culture processes and facilitates rational translation of culture conditions. This study provides a novel mechanistic and predictive understanding of the earliest stages of mesendodermal priming of hPSCs and promotes better experimental control of these processes to manufacture hPSCs on larger scales for therapy (Kempf, H;. et al. Nat. Commun.
Periodic Harvesting of Embryonic Stem Cells from a Hollow-Fiber Membrane–Based Four-Compartment Bioreactor
As a good manufacturing practice–enabling technology, bioreactor culture is starting to gain prominence to scale up PSCs production. Meanwhile, the limitations of two-dimensional petri dish/flask culture (e.g., its laboriousness, discontinuous media exchange, and impracticality for handling larger cell numbers) are becoming evident.
Some concerns involving 3D culture include shear stress caused by agitation, preadaptation culture, and toxic metabolite accumulation. Knospel et al. introduce a hollow-fiber bioreactor with continuous perfusion, metabolite monitoring, and oxygenation features. This design minimizes shear stress and allows increased cell densities and constant monitoring of culture parameters. Given that the PSC phenotype is highly sensitive to cell density and colony size, enzymatic-mechanical features are integrated to perform sterile harvesting.
Following 3 days of PSC expansion, the bioreactor is circulated with a trypsin/EDTA solution with cell detachment enhanced by mechanical agitation. Media are then introduced to flush out detached single PSCs. The cells collected from different harvests typically show >95% or higher viability with a reasonable doubling time compared with control cultures. The harvested cells also do not show any signs of microbiological contamination and expressed a similar gene profile compared with control 2D culture. Monitoring of metabolic parameters in situ also suggests that the different cell harvests do not display markedly different cell metabolism, suggesting that the harvesting procedure does not inflict injury on cells. Thus, such a hollow fiber–based bioreactor system with monitoring and harvesting features represents an innovative and feasible method to harvest PSCs for large-scale pharmacologic and therapeutic applications (Knöspel, F.; et al. Biotechnol. Progress.
Quality Cell Therapy Manufacturing by Design
Transplanting live cells as therapeutic agents is the basis for treating a wide range of diseases. To date, successful translation of laboratory scale experiments to reliable manufacturing processes is currently hampered by the biological complexity of the live cell product. In this review, the authors propose that quality-by-design (QbD) principles need to be adopted to achieve successful cell manufacturing.
QbD integrates scientific knowledge and risk analysis during manufacturing process development. The QbD framework is already used by regulatory bodies to identify the underlying causes of drug manufacturing failures. Its concepts are discussed in application to four types of cell therapeutic products (CTPs): PSC-derived cardiomyocytes, hematopoietic stem and progenitor cells (HSPCs), mesenchymal stem/stromal cells (MSCs), and chimeric antigen receptor (CAR)-T cells. Despite the exciting clinical progress demonstrated, significant manufacturing challenges are still present. Using QbD as a rational framework allows cells to become a routine therapeutic option. Not addressing these challenges results in an inability to produce cell products efficaciously and cost-effectively at the required scale, thus hindering the emergence of regenerative medicine.
QbD is a thorough framework encompassing product and process description, characterization, design, monitoring, and iterative improvement. It begins by establishing product quality attributes such as quality target product profile (QTPP), identifying attributes that influence product safety and efficacy, processing parameters that influence these, and implementing a control strategy to maintain the parameters to ensure that product quality and process are validated. The production process is then monitored and modified iteratively. Achieving QTPP involves defining identity, potency, and purity, of which each CTP is therapy dependent.
Identity is typically demonstrated through the presence or absence of cell surface marker proteins that correlate with certain functional activity. This can miss the variation of expression during the manufacturing process or become altered during changes to the process. For HSPCs, the identity of these surface markers is more certain; however, this may be less defined for MSCs. In the case of CAR-T cells, the chimeric antigen receptor and the correct subset of T-cell markers must be recognized.
Potency is not only is the correct set of surface markers necessary. The cells must demonstrate function using appropriate in vitro assays. One such example is electrophysiology assays for PSC-derived cardiomyocytes showing the ability to rhythmically contract. Often, potency is also assessed as the stemness of MSCs (maturation potential), which would be erroneous because MSC therapies act through paracrine factor secretion.
Impurities in CTPs can include undesirable cell types, culture contamination, ancillary materials, and particulates. For cardiomyocyte therapy, noncardiac cell types do not electromagnetically couple to heart tissue, which makes the presence of undesired cell phenotypes potentially detrimental for therapy. Using PSCs as the starting source can also lead to the inadvertent inclusion of residual undifferentiated PSCs that may cause teratoma formation. Other issues of purity include immune complications from double umbilical cord HSPC transplantations, heterogeneous MSCs, and off-target effects of genetic engineering for CAR-T cells. Microorganism contamination (e.g., bacteria, fungi, mycoplasma, and endotoxin) as well as other ancillary materials (e.g., noncell particulates, bioactive chemicals, and pH indicators) are other factors to watch.
Following the establishment of QTPP, critical quality attributes are established through risk assessment to prioritize the cell product attributes for designing the cell manufacturing process. A critical process is next established by choosing temperature, pH, dissolved O2, agitation, feeding strategy, metabolite levels, and other process parameters. For highly biologically complex CTPs, many process parameters have been shown to inadvertently influence cell phenotype. Stirring features of bioreactors, pH, metabolites, O2, and so forth have all been demonstrated to strongly influence stem cell phenotype. Thus, a design space that defines the boundaries of normal operation should be implemented to optimize the performance of the bioprocess. Systems modeling and design of experiments are also tools that can be used to define the process control strategy. The CTP manufacturing process then undergoes ongoing monitoring to become iteratively validated.
Compared with current laboratory culture, industrializing CTP manufacturing via QbD appears highly challenging. Furthermore, downstream processes (e.g., collection, storage, and delivery) are not discussed in this review. Despite this, knowledge is advancing in the understanding of how cells interact with the environment, bioreactor culture systems, molecular basis of cells, and metabolism. Implementation of QbD for cost-effective CTP development will be critical to translate experimental CTPs into widely available medicines (Lipsitz, Y.; et al. Nat. Biotechnol.