Paper-based sample-to-answer molecular diagnostic platform for point-of-care diagnostics.

PubMed

Choi, Jane Ru; Tang, Ruihua; Wang, ShuQi; Wan Abas, Wan Abu Bakar; Pingguan-Murphy, Belinda; Xu, Feng

2015-12-15

Nucleic acid testing (NAT), as a molecular diagnostic technique, including nucleic acid extraction, amplification and detection, plays a fundamental role in medical diagnosis for timely medical treatment. However, current NAT technologies require relatively high-end instrumentation, skilled personnel, and are time-consuming. These drawbacks mean conventional NAT becomes impractical in many resource-limited disease-endemic settings, leading to an urgent need to develop a fast and portable NAT diagnostic tool. Paper-based devices are typically robust, cost-effective and user-friendly, holding a great potential for NAT at the point of care. In view of the escalating demand for the low cost diagnostic devices, we highlight the beneficial use of paper as a platform for NAT, the current state of its development, and the existing challenges preventing its widespread use. We suggest a strategy involving integrating all three steps of NAT into one single paper-based sample-to-answer diagnostic device for rapid medical diagnostics in the near future. Copyright © 2015 Elsevier B.V. All rights reserved.

Antibody-controlled actuation of DNA-based molecular circuits.

PubMed

Engelen, Wouter; Meijer, Lenny H H; Somers, Bram; de Greef, Tom F A; Merkx, Maarten

2017-02-17

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody-epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Antibody-controlled actuation of DNA-based molecular circuits

NASA Astrophysics Data System (ADS)

Engelen, Wouter; Meijer, Lenny H. H.; Somers, Bram; de Greef, Tom F. A.; Merkx, Maarten

2017-02-01

DNA-based molecular circuits allow autonomous signal processing, but their actuation has relied mostly on RNA/DNA-based inputs, limiting their application in synthetic biology, biomedicine and molecular diagnostics. Here we introduce a generic method to translate the presence of an antibody into a unique DNA strand, enabling the use of antibodies as specific inputs for DNA-based molecular computing. Our approach, antibody-templated strand exchange (ATSE), uses the characteristic bivalent architecture of antibodies to promote DNA-strand exchange reactions both thermodynamically and kinetically. Detailed characterization of the ATSE reaction allowed the establishment of a comprehensive model that describes the kinetics and thermodynamics of ATSE as a function of toehold length, antibody-epitope affinity and concentration. ATSE enables the introduction of complex signal processing in antibody-based diagnostics, as demonstrated here by constructing molecular circuits for multiplex antibody detection, integration of multiple antibody inputs using logic gates and actuation of enzymes and DNAzymes for signal amplification.

Molecular malaria diagnostics: A systematic review and meta-analysis.

PubMed

Roth, Johanna M; Korevaar, Daniël A; Leeflang, Mariska M G; Mens, Pètra F

2016-01-01

Accurate diagnosis of malaria is essential for identification and subsequent treatment of the disease. Currently, microscopy and rapid diagnostic tests are the most commonly used diagnostics, next to treatment based on clinical signs only. These tests are easy to deploy, but have a relatively high detection limit. With declining prevalence in many areas, there is an increasing need for more sensitive diagnostics. Molecular tools may be a suitable alternative, although costs and technical requirements currently hamper their implementation in resource limited settings. A range of (near) point-of-care diagnostics is therefore under development, including simplifications in sample preparation, amplification and/or read-out of the test. Accuracy data, in combination with technical characteristics, are essential in determining which molecular test, if any, would be the most promising to be deployed. This review presents a comprehensive overview of the currently available molecular malaria diagnostics, ranging from well-known tests to platforms in early stages of evaluation, and systematically evaluates their published accuracy. No important difference in accuracy was found between the most commonly used PCR-based assays (conventional, nested and real-time PCR), with most of them having high sensitivity and specificity, implying that there are no reasons other than practical ones to choose one technique over the other. Loop-mediated isothermal amplification and other (novel) diagnostics appear to be highly accurate as well, with some offering potential to be used in resource-limited settings.

Hemlock woolly adelgid biological control: molecular methods to distinguish Laricobius nigrinus, L. rubidus, and their hybrids

Treesearch

Nathan P. Havill; Gina Davis; Joanne Klein; Adalgisa Caccone; Scott Salom

2011-01-01

Molecular diagnostics use DNA-based methods to assign unknown organisms to species. As such, they rely on a priori species designation by taxonomists and require validation with enough samples to capture the variation within species for accurately selecting diagnostic characters.

Molecular Diagnostic Yield of Chromosomal Microarray Analysis and Whole-Exome Sequencing in Children With Autism Spectrum Disorder.

PubMed

Tammimies, Kristiina; Marshall, Christian R; Walker, Susan; Kaur, Gaganjot; Thiruvahindrapuram, Bhooma; Lionel, Anath C; Yuen, Ryan K C; Uddin, Mohammed; Roberts, Wendy; Weksberg, Rosanna; Woodbury-Smith, Marc; Zwaigenbaum, Lonnie; Anagnostou, Evdokia; Wang, Zhuozhi; Wei, John; Howe, Jennifer L; Gazzellone, Matthew J; Lau, Lynette; Sung, Wilson W L; Whitten, Kathy; Vardy, Cathy; Crosbie, Victoria; Tsang, Brian; D'Abate, Lia; Tong, Winnie W L; Luscombe, Sandra; Doyle, Tyna; Carter, Melissa T; Szatmari, Peter; Stuckless, Susan; Merico, Daniele; Stavropoulos, Dimitri J; Scherer, Stephen W; Fernandez, Bridget A

2015-09-01

The use of genome-wide tests to provide molecular diagnosis for individuals with autism spectrum disorder (ASD) requires more study. To perform chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) in a heterogeneous group of children with ASD to determine the molecular diagnostic yield of these tests in a sample typical of a developmental pediatric clinic. The sample consisted of 258 consecutively ascertained unrelated children with ASD who underwent detailed assessments to define morphology scores based on the presence of major congenital abnormalities and minor physical anomalies. The children were recruited between 2008 and 2013 in Newfoundland and Labrador, Canada. The probands were stratified into 3 groups of increasing morphological severity: essential, equivocal, and complex (scores of 0-3, 4-5, and ≥6). All probands underwent CMA, with WES performed for 95 proband-parent trios. The overall molecular diagnostic yield for CMA and WES in a population-based ASD sample stratified in 3 phenotypic groups. Of 258 probands, 24 (9.3%, 95%CI, 6.1%-13.5%) received a molecular diagnosis from CMA and 8 of 95 (8.4%, 95%CI, 3.7%-15.9%) from WES. The yields were statistically different between the morphological groups. Among the children who underwent both CMA and WES testing, the estimated proportion with an identifiable genetic etiology was 15.8% (95%CI, 9.1%-24.7%; 15/95 children). This included 2 children who received molecular diagnoses from both tests. The combined yield was significantly higher in the complex group when compared with the essential group (pairwise comparison, P = .002). [table: see text]. Among a heterogeneous sample of children with ASD, the molecular diagnostic yields of CMA and WES were comparable, and the combined molecular diagnostic yield was higher in children with more complex morphological phenotypes in comparison with the children in the essential category. If replicated in additional populations, these findings may inform appropriate selection of molecular diagnostic testing for children affected by ASD.

Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays.

PubMed

Glais, Laurent; Jacquot, Emmanuel

2015-01-01

Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.

New molecular medicine: Diagnomics and pharmacogenomics

NASA Astrophysics Data System (ADS)

Kauffman, Michael G.

1999-04-01

Millennium Predictive Medicine (MPMx), a subsidiary of Millennium Pharmaceuticals, is focusing on the discovery and clinical validation of Diagnomic and Pharmacogenomic Tests which will replace many of the subjective elements of clinical decision making. Diagnomics are molecular diagnostic markers with prognostic and economic impact. While the majority of currently available diagnostics represent data points, Diagnomics allow patients and physicians to make scientifically based, individualized decisions about their disease and its therapy. Pharmacogenomics are diagnostics that specify the use or avoidance of specific therapeutics based on an individual genotype and/or disease subtype. MPMx uses the broad Millennium genomics, proteomics, and bioinformatics technologies in the analysis of human disease and drug response. These technologies permit global and unbiased approaches towards the elucidation of disease pathways and mechanisms at the molecular level. Germline or somatic mutations, RNA levels, or protein levels comprising these pathways and mechanisms are currently being evaluated as markers for disease predisposition, stage, aggressiveness, and likely drug response or drug toxicity. Diagnomic and Pharmacogenomic Tests are part of the new molecular medicine that is transforming clinical practice forma symptom/pathology-based art into a pre-symptom, mechanism- based science.

Diagnosis and therapy with CRISPR advanced CRISPR based tools for point of care diagnostics and early therapies.

PubMed

Uppada, Vanita; Gokara, Mahesh; Rasineni, Girish Kumar

2018-05-20

Molecular diagnostics is of critical importance to public health worldwide. It facilitates not only detection and characterization of diseases, but also monitors drug responses, assists in the identification of genetic modifiers and disease susceptibility. Based upon DNA variation, a wide range of molecular-based tests are available to assess/diagnose diseases. The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. As designing CRISPR-guided nucleases can be done easily and relatively fast, the CRISPR/Cas9 system has evolved as widely used DNA editing tool. This technique led to a large number of gene editing studies in variety of organisms. CRISPR/Cas9-mediated diagnosis and therapy has picked up pace due to specificity and accuracy of CRISPR. The aim is not only to identify specific pathogens, especially virus but also to repair disease-causing alleles by changing the DNA sequence at the exact location on the chromosome. At present, PCR-based molecular diagnostic testing predominates; however, alternative technologies aimed at reducing genome complexity without PCR are anticipated to gain momentum in the coming years. Furthermore, development of integrated chip devices should allow point-of-care testing and facilitate genetic readouts from single cells and molecules. Together with molecular based therapy CRISPR based diagnostic testing will be a revolution in modern health care settings. In this review, we emphasize on current developing diagnostic techniques based upon CRISPR Cas approach along with short insights on its therapeutic usage. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA barcodes and molecular diagnostics to distinguish an introduced and native Laricobius (Coleoptera: Derodontidae) species in eastern North America

Treesearch

G.A. Davis; N.P. Havill; Z.N. Adelman; A. Caccone; L.T. Kok; S.M. Salom

2011-01-01

Molecular diagnostics based on DNA barcodes can be powerful identification tools in the absence of distinctive morphological characters for distinguishing between closely related species. A specific example is distinguishing the endemic species Laricobius rubidus from Laricobius nigrinus, a biological control agent of hemlock...

Optical biosensor technologies for molecular diagnostics at the point-of-care

NASA Astrophysics Data System (ADS)

Schotter, Joerg; Schrittwieser, Stefan; Muellner, Paul; Melnik, Eva; Hainberger, Rainer; Koppitsch, Guenther; Schrank, Franz; Soulantika, Katerina; Lentijo-Mozo, Sergio; Pelaz, Beatriz; Parak, Wolfgang; Ludwig, Frank; Dieckhoff, Jan

2015-05-01

Label-free optical schemes for molecular biosensing hold a strong promise for point-of-care applications in medical research and diagnostics. Apart from diagnostic requirements in terms of sensitivity, specificity, and multiplexing capability, also other aspects such as ease of use and manufacturability have to be considered in order to pave the way to a practical implementation. We present integrated optical waveguide as well as magnetic nanoparticle based molecular biosensor concepts that address these aspects. The integrated optical waveguide devices are based on low-loss photonic wires made of silicon nitride deposited by a CMOS compatible plasma-enhanced chemical vapor deposition (PECVD) process that allows for backend integration of waveguides on optoelectronic CMOS chips. The molecular detection principle relies on evanescent wave sensing in the 0.85 μm wavelength regime by means of Mach-Zehnder interferometers, which enables on-chip integration of silicon photodiodes and, thus, the realization of system-on-chip solutions. Our nanoparticle-based approach is based on optical observation of the dynamic response of functionalized magneticcore/ noble-metal-shell nanorods (`nanoprobes') to an externally applied time-varying magnetic field. As target molecules specifically bind to the surface of the nanoprobes, the observed dynamics of the nanoprobes changes, and the concentration of target molecules in the sample solution can be quantified. This approach is suitable for dynamic real-time measurements and only requires minimal sample preparation, thus presenting a highly promising point-of-care diagnostic system. In this paper, we present a prototype of a diagnostic device suitable for highly automated sample analysis by our nanoparticle-based approach.

Molecular Diagnostics in Pathology: Time for a Next-Generation Pathologist?

PubMed

Fassan, Matteo

2018-03-01

- Comprehensive molecular investigations of mainstream carcinogenic processes have led to the use of effective molecular targeted agents in most cases of solid tumors in clinical settings. - To update readers regarding the evolving role of the pathologist in the therapeutic decision-making process and the introduction of next-generation technologies into pathology practice. - Current literature on the topic, primarily sourced from the PubMed (National Center for Biotechnology Information, Bethesda, Maryland) database, were reviewed. - Adequate evaluation of cytologic-based and tissue-based predictive diagnostic biomarkers largely depends on both proper pathologic characterization and customized processing of biospecimens. Moreover, increased requests for molecular testing have paralleled the recent, sharp decrease in tumor material to be analyzed-material that currently comprises cytology specimens or, at minimum, small biopsies in most cases of metastatic/advanced disease. Traditional diagnostic pathology has been completely revolutionized by the introduction of next-generation technologies, which provide multigene, targeted mutational profiling, even in the most complex of clinical cases. Combining traditional and molecular knowledge, pathologists integrate the morphological, clinical, and molecular dimensions of a disease, leading to a proper diagnosis and, therefore, the most-appropriate tailored therapy.

Current and future molecular diagnostics for ocular infectious diseases.

PubMed

Doan, Thuy; Pinsky, Benjamin A

2016-11-01

Confirmation of ocular infections can pose great challenges to the clinician. A fundamental limitation is the small amounts of specimen that can be obtained from the eye. Molecular diagnostics can circumvent this limitation and have been shown to be more sensitive than conventional culture. The purpose of this review is to describe new molecular methods and to discuss the applications of next-generation sequencing-based approaches in the diagnosis of ocular infections. Efforts have focused on improving the sensitivity of pathogen detection using molecular methods. This review describes a new molecular target for Toxoplasma gondii-directed polymerase chain reaction assays. Molecular diagnostics for Chlamydia trachomatis and Acanthamoeba species are also discussed. Finally, we describe a hypothesis-free approach, metagenomic deep sequencing, which can detect DNA and RNA pathogens from a single specimen in one test. In some cases, this method can provide the geographic location and timing of the infection. Pathogen-directed PCRs have been powerful tools in the diagnosis of ocular infections for over 20 years. The use of next-generation sequencing-based approaches, when available, will further improve sensitivity of detection with the potential to improve patient care.

Plasmon-Based Colorimetric Nanosensors for Ultrasensitive Molecular Diagnostics.

PubMed

Tang, Longhua; Li, Jinghong

2017-07-28

Colorimetric detection of target analytes with high specificity and sensitivity is of fundamental importance to clinical and personalized point-of-care diagnostics. Because of their extraordinary optical properties, plasmonic nanomaterials have been introduced into colorimetric sensing systems, which provide significantly improved sensitivity in various biosensing applications. Here we review the recent progress on these plasmonic nanoparticles-based colorimetric nanosensors for ultrasensitive molecular diagnostics. According to their different colorimetric signal generation mechanisms, these plasmonic nanosensors are classified into two categories: (1) interparticle distance-dependent colorimetric assay based on target-induced forming cross-linking assembly/aggregate of plasmonic nanoparticles; and (2) size/morphology-dependent colorimetric assay by target-controlled growth/etching of the plasmonic nanoparticles. The sensing fundamentals and cutting-edge applications will be provided for each of them, particularly focusing on signal generation and/or amplification mechanisms that realize ultrasensitive molecular detection. Finally, we also discuss the challenge and give our future perspective in this emerging field.

A rational regulatory approach for positron emission tomography imaging probes: from "first in man" to NDA approval and reimbursement.

PubMed

Barrio, Jorge R; Marcus, Carol S; Hung, Joseph C; Keppler, Jennifer S

2004-01-01

We propose a new regulatory approach for positron emission tomography (PET) molecular imaging probes, essential tools in today's medicine. Even though the focus of this paper is on positron-emitting labeled probes, it is also justified to extend this proposed regulatory approach to other diagnostic nuclear medicine radiopharmaceuticals. Key aspects of this proposal include: (1) PET molecular imaging probes would be placed in a "no significant risk" category, similar to that category for devices in current Food and Drug Administration (FDA) regulations, based on overwhelming scientific evidence that demonstrates their faultless safety profile; (2) the FDA-sanctioned Radioactive Drug Research Committee (RDRC) will oversee all diagnostic research with these probes. The newly defined RDRC should approve "first in man" use; supervise a broader spectrum of diagnostic research protocols, including those looking to demonstrate initial efficacy, as well as multicenter clinical trials and the use of molecular imaging probes as a screening tool in drug discovery. The current investigational new drug (IND) mechanism is thus eliminated for these diagnostic probes; (3) when a molecular imaging probe has demonstrated diagnostic efficacy, FDA approval (i.e., NDA) will be sought. The review will be done by a newly constituted Radioactive Drug Advisory Committee (RDAC) composed of experts chosen by the professional societies, who would provide a binding assessment of the adequacy of the safety and efficacy data. When the RDAC recommends its diagnostic use on scientific and medical grounds, the molecular imaging probe becomes FDA approved. After a molecular imaging probe is approved for a diagnostic indication, the existing mechanism to seek reimbursement will be utilized; and (4) the FDA would retain its direct oversight function for traditional manufacturers engaged in commercial distribution of the approved diagnostic molecular imaging probes (i.e., under NDA) to monitor compliance with existing US Pharmacopeia (USP) requirements. With abbreviated and more appropriate regulations, new PET molecular imaging probes for diagnostic use would be then rapidly incorporated into the mainstream diagnostic medicine. Equally importantly, this approach would facilitate the use of molecular imaging in drug discovery and development, which would substantially reduce the costs and time required to bring new therapeutic drugs to market.

Study design requirements for RNA sequencing-based breast cancer diagnostics.

PubMed

Mer, Arvind Singh; Klevebring, Daniel; Grönberg, Henrik; Rantalainen, Mattias

2016-02-01

Sequencing-based molecular characterization of tumors provides information required for individualized cancer treatment. There are well-defined molecular subtypes of breast cancer that provide improved prognostication compared to routine biomarkers. However, molecular subtyping is not yet implemented in routine breast cancer care. Clinical translation is dependent on subtype prediction models providing high sensitivity and specificity. In this study we evaluate sample size and RNA-sequencing read requirements for breast cancer subtyping to facilitate rational design of translational studies. We applied subsampling to ascertain the effect of training sample size and the number of RNA sequencing reads on classification accuracy of molecular subtype and routine biomarker prediction models (unsupervised and supervised). Subtype classification accuracy improved with increasing sample size up to N = 750 (accuracy = 0.93), although with a modest improvement beyond N = 350 (accuracy = 0.92). Prediction of routine biomarkers achieved accuracy of 0.94 (ER) and 0.92 (Her2) at N = 200. Subtype classification improved with RNA-sequencing library size up to 5 million reads. Development of molecular subtyping models for cancer diagnostics requires well-designed studies. Sample size and the number of RNA sequencing reads directly influence accuracy of molecular subtyping. Results in this study provide key information for rational design of translational studies aiming to bring sequencing-based diagnostics to the clinic.

Development of a molecular diagnostic test for Retinitis Pigmentosa in the Japanese population.

PubMed

Maeda, Akiko; Yoshida, Akiko; Kawai, Kanako; Arai, Yuki; Akiba, Ryutaro; Inaba, Akira; Takagi, Seiji; Fujiki, Ryoji; Hirami, Yasuhiko; Kurimoto, Yasuo; Ohara, Osamu; Takahashi, Masayo

2018-05-21

Retinitis Pigmentosa (RP) is the most common form of inherited retinal dystrophy caused by different genetic variants. More than 60 causative genes have been identified to date. The establishment of cost-effective molecular diagnostic tests with high sensitivity and specificity can be beneficial for patients and clinicians. Here, we developed a clinical diagnostic test for RP in the Japanese population. Evaluation of diagnostic technology, Prospective, Clinical and experimental study. A panel of 39 genes reported to cause RP in Japanese patients was established. Next generation sequence (NGS) technology was applied for the analyses of 94 probands with RP and RP-related diseases. After interpretation of detected genetic variants, molecular diagnosis based on a study of the genetic variants and a clinical phenotype was made by a multidisciplinary team including clinicians, researchers and genetic counselors. NGS analyses found 14,343 variants from 94 probands. Among them, 189 variants in 83 probands (88.3% of all cases) were selected as pathogenic variants and 64 probands (68.1%) have variants which can cause diseases. After the deliberation of these 64 cases, molecular diagnosis was made in 43 probands (45.7%). The final molecular diagnostic rate with the current system combining supplemental Sanger sequencing was 47.9% (45 of 94 cases). The RP panel provides the significant advantage of detecting genetic variants with a high molecular diagnostic rate. This type of race-specific high-throughput genotyping allows us to conduct a cost-effective and clinically useful genetic diagnostic test.

The rapid evolution of molecular genetic diagnostics in neuromuscular diseases.

PubMed

Volk, Alexander E; Kubisch, Christian

2017-10-01

The development of massively parallel sequencing (MPS) has revolutionized molecular genetic diagnostics in monogenic disorders. The present review gives a brief overview of different MPS-based approaches used in clinical diagnostics of neuromuscular disorders (NMDs) and highlights their advantages and limitations. MPS-based approaches like gene panel sequencing, (whole) exome sequencing, (whole) genome sequencing, and RNA sequencing have been used to identify the genetic cause in NMDs. Although gene panel sequencing has evolved as a standard test for heterogeneous diseases, it is still debated, mainly because of financial issues and unsolved problems of variant interpretation, whether genome sequencing (and to a lesser extent also exome sequencing) of single patients can already be regarded as routine diagnostics. However, it has been shown that the inclusion of parents and additional family members often leads to a substantial increase in the diagnostic yield in exome-wide/genome-wide MPS approaches. In addition, MPS-based RNA sequencing just enters the research and diagnostic scene. Next-generation sequencing increasingly enables the detection of the genetic cause in highly heterogeneous diseases like NMDs in an efficient and affordable way. Gene panel sequencing and family-based exome sequencing have been proven as potent and cost-efficient diagnostic tools. Although clinical validation and interpretation of genome sequencing is still challenging, diagnostic RNA sequencing represents a promising tool to bypass some hurdles of diagnostics using genomic DNA.

The Effect of Molecular Diagnostics on the Treatment of Glioma.

PubMed

Bush, Nancy Ann Oberheim; Butowski, Nicholas

2017-04-01

This review summarizes the use of molecular diagnostics in glioma and its effect on the development of novel therapeutics and management decisions. Genomic and proteomic profiling of brain tumors has provided significant expansion of our understanding of oncogenesis, characterization, and prognostication of brain tumors. Molecular markers such as MGMT, EGFR, IDH, 1p19q, ATRX, TERT, FGFR-TACC, and BRAF are now being used to classify brain tumors as well as influence management decisions. Several of these markers are also being used as therapeutic targets. We review the use of several molecular diagnostics in gliomas and discuss their impact on drug development and clinical trial design. In the future, molecular characterization based on a specific genomic, proteomic as well as transcriptomes for bioformatics analysis will provide clinicians the ability to rationally select drugs with actionable targets for each patient.

Development of companion diagnostics

DOE PAGES

Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.; ...

2015-12-12

The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods asmore » companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. Lastly, the review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.« less

Development of companion diagnostics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.

The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods asmore » companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. Lastly, the review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic.« less

Development of Companion Diagnostics

PubMed Central

Mankoff, David A.; Edmonds, Christine E.; Farwell, Michael D.; Pryma, Daniel A.

2016-01-01

The goal of individualized and targeted treatment and precision medicine requires the assessment of potential therapeutic targets to direct treatment selection. The biomarkers used to direct precision medicine, often termed companion diagnostics, for highly targeted drugs have thus far been almost entirely based on in vitro assay of biopsy material. Molecular imaging companion diagnostics offer a number of features complementary to those from in vitro assay, including the ability to measure the heterogeneity of each patient’s cancer across the entire disease burden and to measure early changes in response to treatment. We discuss the use of molecular imaging methods as companion diagnostics for cancer therapy with the goal of predicting response to targeted therapy and measuring early (pharmacodynamic) response as an indication of whether the treatment has “hit” the target. We also discuss considerations for probe development for molecular imaging companion diagnostics, including both small-molecule probes and larger molecules such as labeled antibodies and related constructs. We then describe two examples where both predictive and pharmacodynamic molecular imaging markers have been tested in humans: endocrine therapy for breast cancer and human epidermal growth factor receptor type 2–targeted therapy. The review closes with a summary of the items needed to move molecular imaging companion diagnostics from early studies into multicenter trials and into the clinic. PMID:26687857

The role of molecular genetic analysis in the diagnosis of primary ciliary dyskinesia.

PubMed

Kim, Raymond H; A Hall, David; Cutz, Ernest; Knowles, Michael R; Nelligan, Kathleen A; Nykamp, Keith; Zariwala, Maimoona A; Dell, Sharon D

2014-03-01

Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder of motile cilia. The diagnosis of PCD has previously relied on ciliary analysis with transmission electron microscopy or video microscopy. However, patients with PCD may have normal ultrastructural appearance, and ciliary analysis has limited accessibility. Alternatively, PCD can be diagnosed by demonstrating biallelic mutations in known PCD genes. Genetic testing is emerging as a diagnostic tool to complement ciliary analysis where interpretation and access may delay diagnosis. To determine the diagnostic yield of genetic testing of patients with a confirmed or suspected diagnosis of PCD in a multiethnic urban center. Twenty-eight individuals with confirmed PCD on transmission electron microscopy of ciliary ultrastructure and 24 individuals with a probable diagnosis of PCD based on a classical PCD phenotype and low nasal nitric oxide had molecular analysis of 12 genes associated with PCD. Of 49 subjects who underwent ciliary biopsy, 28 (57%) were diagnosed with PCD through an ultrastructural defect. Of the 52 individuals who underwent molecular genetic analysis, 22 (42%) individuals had two mutations in known PCD genes. Twenty-four previously unreported mutations in known PCD genes were observed. Combining both diagnostic modalities of biopsy and molecular genetics, the diagnostic yield increased to 69% compared with 57% based on biopsy alone. The diagnosis of PCD is challenging and has traditionally relied on ciliary biopsy, which is unreliable as the sole criterion for a definitive diagnosis. Molecular genetic analysis can be used as a complementary test to increase the diagnostic yield.

Molecular imaging in neuroendocrine tumors: molecular uptake mechanisms and clinical results.

PubMed

Koopmans, Klaas P; Neels, Oliver N; Kema, Ido P; Elsinga, Philip H; Links, Thera P; de Vries, Elisabeth G E; Jager, Pieter L

2009-09-01

Neuroendocrine tumors can originate almost everywhere in the body and consist of a great variety of subtypes. This paper focuses on molecular imaging methods using nuclear medicine techniques in neuroendocrine tumors, coupling molecular uptake mechanisms of radiotracers with clinical results. A non-systematic review is presented on receptor based and metabolic imaging methods. Receptor-based imaging covers the molecular backgrounds of somatostatin, vaso-intestinal peptide (VIP), bombesin and cholecystokinin (CCK) receptors and their link with nuclear imaging. Imaging methods based on specific metabolic properties include meta-iodo-benzylguanide (MIBG) and dimercapto-sulphuric acid (DMSA-V) scintigraphy as well as more modern positron emission tomography (PET)-based methods using radio-labeled analogues of amino acids, glucose, dihydroxyphenylalanine (DOPA), dopamine and tryptophan. Diagnostic sensitivities are presented for each imaging method and for each neuroendocrine tumor subtype. Finally, a Forest plot analysis of diagnostic performance is presented for each tumor type in order to provide a comprehensive overview for clinical use.

Sequencing-based breast cancer diagnostics as an alternative to routine biomarkers.

PubMed

Rantalainen, Mattias; Klevebring, Daniel; Lindberg, Johan; Ivansson, Emma; Rosin, Gustaf; Kis, Lorand; Celebioglu, Fuat; Fredriksson, Irma; Czene, Kamila; Frisell, Jan; Hartman, Johan; Bergh, Jonas; Grönberg, Henrik

2016-11-30

Sequencing-based breast cancer diagnostics have the potential to replace routine biomarkers and provide molecular characterization that enable personalized precision medicine. Here we investigate the concordance between sequencing-based and routine diagnostic biomarkers and to what extent tumor sequencing contributes clinically actionable information. We applied DNA- and RNA-sequencing to characterize tumors from 307 breast cancer patients with replication in up to 739 patients. We developed models to predict status of routine biomarkers (ER, HER2,Ki-67, histological grade) from sequencing data. Non-routine biomarkers, including mutations in BRCA1, BRCA2 and ERBB2(HER2), and additional clinically actionable somatic alterations were also investigated. Concordance with routine diagnostic biomarkers was high for ER status (AUC = 0.95;AUC(replication) = 0.97) and HER2 status (AUC = 0.97;AUC(replication) = 0.92). The transcriptomic grade model enabled classification of histological grade 1 and histological grade 3 tumors with high accuracy (AUC = 0.98;AUC(replication) = 0.94). Clinically actionable mutations in BRCA1, BRCA2 and ERBB2(HER2) were detected in 5.5% of patients, while 53% had genomic alterations matching ongoing or concluded breast cancer studies. Sequencing-based molecular profiling can be applied as an alternative to histopathology to determine ER and HER2 status, in addition to providing improved tumor grading and clinically actionable mutations and molecular subtypes. Our results suggest that sequencing-based breast cancer diagnostics in a near future can replace routine biomarkers.

Using molecular diagnostic testing to personalize the treatment of patients with gastrointestinal stromal tumors.

PubMed

Bannon, Amber E; Klug, Lillian R; Corless, Christopher L; Heinrich, Michael C

2017-05-01

The diagnosis and treatment of gastrointestinal stromal tumor (GIST) has emerged as a paradigm for modern cancer treatment ('precision medicine'), as it highlights the importance of matching molecular defects with specific therapies. Over the past two decades, the molecular classification and diagnostic work up of GIST has been radically transformed, accompanied by the development of molecular therapies for specific subgroups of GIST. This review summarizes the developments in the field of molecular diagnosis of GIST, particularly as they relate to optimizing medical therapy. Areas covered: Based on an extensive literature search of the molecular and clinical aspects of GIST, the authors review the most important developments in this field with an emphasis on the differential diagnosis of GIST including mutation testing, therapeutic implications of each molecular subtype, and emerging technologies relevant to the field. Expert commentary: The use of molecular diagnostics to classify GIST has been shown to be successful in optimizing patient treatment, but these methods remain under-utilized. In order to facilitate efficient and comprehensive molecular testing, the authors have developed a decision tree to aid clinicians.

Assessing value of innovative molecular diagnostic tests in the concept of predictive, preventive, and personalized medicine.

PubMed

Akhmetov, Ildar; Bubnov, Rostyslav V

2015-01-01

Molecular diagnostic tests drive the scientific and technological uplift in the field of predictive, preventive, and personalized medicine offering invaluable clinical and socioeconomic benefits to the key stakeholders. Although the results of diagnostic tests are immensely influential, molecular diagnostic tests (MDx) are still grudgingly reimbursed by payers and amount for less than 5 % of the overall healthcare costs. This paper aims at defining the value of molecular diagnostic test and outlining the most important components of "value" from miscellaneous assessment frameworks, which go beyond accuracy and feasibility and impact the clinical adoption, informing healthcare resource allocation decisions. The authors suggest that the industry should facilitate discussions with various stakeholders throughout the entire assessment process in order to arrive at a consensus about the depth of evidence required for positive marketing authorization or reimbursement decisions. In light of the evolving "value-based healthcare" delivery practices, it is also recommended to account for social and ethical parameters of value, since these are anticipated to become as critical for reimbursement decisions and test acceptance as economic and clinical criteria.

A WAO - ARIA - GA²LEN consensus document on molecular-based allergy diagnostics

PubMed Central

2013-01-01

Molecular-based allergy (MA) diagnostics is an approach used to map the allergen sensitization of a patient at a molecular level, using purified natural or recombinant allergenic molecules (allergen components) instead of allergen extracts. Since its introduction, MA diagnostics has increasingly entered routine care, with currently more than 130 allergenic molecules commercially available for in vitro specific IgE (sIgE) testing. MA diagnostics allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in three key aspects of allergy diagnosis: (1) resolving genuine versus cross-reactive sensitization in poly-sensitized patients, thereby improving the understanding of triggering allergens; (2) assessing, in selected cases, the risk of severe, systemic versus mild, local reactions in food allergy, thereby reducing unnecessary anxiety for the patient and the need for food challenge testing; and (3) identifying patients and triggering allergens for specific immunotherapy (SIT). Singleplex and multiplex measurement platforms are available for MA diagnostics. The Immuno-Solid phase Allergen Chip (ISAC) is the most comprehensive platform currently available, which involves a biochip technology to measure sIgE antibodies against more than one hundred allergenic molecules in a single assay. As the field of MA diagnostics advances, future work needs to focus on large-scale, population-based studies involving practical applications, elucidation and expansion of additional allergenic molecules, and support for appropriate test interpretation. With the rapidly expanding evidence-base for MA diagnosis, there is a need for allergists to keep abreast of the latest information. The aim of this consensus document is to provide a practical guide for the indications, determination, and interpretation of MA diagnostics for clinicians trained in allergology. PMID:24090398

A WAO - ARIA - GA²LEN consensus document on molecular-based allergy diagnostics.

PubMed

Canonica, Giorgio Walter; Ansotegui, Ignacio J; Pawankar, Ruby; Schmid-Grendelmeier, Peter; van Hage, Marianne; Baena-Cagnani, Carlos E; Melioli, Giovanni; Nunes, Carlos; Passalacqua, Giovanni; Rosenwasser, Lanny; Sampson, Hugh; Sastre, Joaquin; Bousquet, Jean; Zuberbier, Torsten

2013-10-03

Molecular-based allergy (MA) diagnostics is an approach used to map the allergen sensitization of a patient at a molecular level, using purified natural or recombinant allergenic molecules (allergen components) instead of allergen extracts. Since its introduction, MA diagnostics has increasingly entered routine care, with currently more than 130 allergenic molecules commercially available for in vitro specific IgE (sIgE) testing.MA diagnostics allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in three key aspects of allergy diagnosis: (1) resolving genuine versus cross-reactive sensitization in poly-sensitized patients, thereby improving the understanding of triggering allergens; (2) assessing, in selected cases, the risk of severe, systemic versus mild, local reactions in food allergy, thereby reducing unnecessary anxiety for the patient and the need for food challenge testing; and (3) identifying patients and triggering allergens for specific immunotherapy (SIT).Singleplex and multiplex measurement platforms are available for MA diagnostics. The Immuno-Solid phase Allergen Chip (ISAC) is the most comprehensive platform currently available, which involves a biochip technology to measure sIgE antibodies against more than one hundred allergenic molecules in a single assay. As the field of MA diagnostics advances, future work needs to focus on large-scale, population-based studies involving practical applications, elucidation and expansion of additional allergenic molecules, and support for appropriate test interpretation. With the rapidly expanding evidence-base for MA diagnosis, there is a need for allergists to keep abreast of the latest information. The aim of this consensus document is to provide a practical guide for the indications, determination, and interpretation of MA diagnostics for clinicians trained in allergology.

Molecular diagnosis and precision medicine in allergy management.

PubMed

Riccio, Anna Maria; De Ferrari, Laura; Chiappori, Alessandra; Ledda, Sabina; Passalacqua, Giovanni; Melioli, Giovanni; Canonica, Giorgio Walter

2016-11-01

Precision medicine (PM) can be defined as a structural model aimed at customizing healthcare, with medical decisions/products tailored on an individual patient at a highly detailed level. In this sense, allergy diagnostics based on molecular allergen components allows to accurately define the patient's IgE repertoire. The availability of highly specialized singleplexed and multiplexed platforms support allergists with an advanced diagnostic armamentarium. The therapeutic intervention, driven by the standard diagnostic approach, but further supported by these innovative tools may result, for instance, in a more appropriate prescription of allergen immunotherapy (AIT). Also, the phenotyping of patients, which may have relevant effects on the treatment strategy, could be take advantage by the molecular allergy diagnosis.

Can and should value-based pricing be applied to molecular diagnostics?

PubMed

Garau, Martina; Towse, Adrian; Garrison, Louis; Housman, Laura; Ossa, Diego

2013-01-01

Current pricing and reimbursement systems for diagnostics are not efficient. Prices for diagnostics are often driven by administrative practices and expected production cost. The purpose of the paper is to discuss how a value-based pricing framework being used to ensure efficient use and price of medicines could also be applied to diagnostics. Diagnostics not only facilitates health gain and cost savings, but also information to guide patients' decisions on interventions and their future 'behaviors'. For value assessment processes we recommend a two-part approach. Companion diagnostics introduced at the launch of the drug should be assessed through new drug assessment processes considering a broad range of value elements and a balanced analysis of diagnostic impacts. A separate diagnostic-dedicated committee using value-based pricing principles should review other diagnostics lying outside the companion diagnostics-and-drug 'at-launch' situation.

Recommendations for the use of molecular diagnostics in the diagnosis of allergic dis-eases.

PubMed

Villalta, D; Tonutti, E; Bizzaro, N; Brusca, I; Sargentini, V; Asero, R; Bilo, M B; Manzotti, G; Murzilli, F; Cecchi, L; Musarra, A

2018-03-01

The Study Group on Allergology of the Italian Society of Clinical Pathology and Laboratory Medicine (SIPMeL) and the Associazione Italiana degli Allergologi e Immunologi Territoriali e Ospedalieri (AAIITO) developed the present recommendations on the diagnosis of allergic diseases based on the use of molecular allergenic components, whose purpose is to provide the pathologists and the clinicians with information and algorithms enabling a proper use of this second-level diagnostics. Molecular diagnostics allows definition of the exact sensitization profile of the allergic patient. The methodology followed to develop these recommendations included an initial phase of discussion between all the components to integrate the knowledge derived from scientific evidence, a revision of the recommendations made by Italian and foreign experts, and the subsequent production of this document to be disseminated to all those who deal with allergy diagnostics.

Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design.

PubMed

Pilotte, Nils; Papaiakovou, Marina; Grant, Jessica R; Bierwert, Lou Ann; Llewellyn, Stacey; McCarthy, James S; Williams, Steven A

2016-03-01

The soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world's most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays. Utilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay. The utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

Benefits and challenges of molecular diagnostics for childhood tuberculosis.

PubMed

Gutierrez, Cristina

2016-12-01

Expanding tuberculosis (TB)-diagnostic services, including access to rapid tests, is a World Health Organization (WHO) strategy to accelerate progress toward ending TB. Faster and more sensitive molecular tests capable of diagnosing TB and drug-resistant TB have the technical capacity to address limitations associated with smears and cultures by increasing accuracy and shortening turnaround times as compared with those of these conventional laboratory methods. Nucleic acid amplification assays used to detect and analyze Mycobacterium tuberculosis (MTB)-complex nucleic acids can be used directly on specimens from patients suspected of having TB. Recently, several commercial molecular tests were developed to detect MTB and determine the drug resistance (DR) based on detection of specific genetic mutations conferring resistance. The first to be endorsed by the WHO was molecular line-probe assay technology. This test uses polymerase chain reaction (PCR) and reverse-hybridization methods to rapidly identify MTB and DR-related mutations simultaneously. More recently, the WHO endorsed Xpert MTB/RIF, Cepheid Inc, CA, USA, a fully automated assay used for TB diagnosis that relies upon PCR techniques for detection of TB and rifampicin resistance-related mutations. Other promising molecular TB assays for simplifying PCR-based testing protocols and increasing their accuracy are under development and evaluation. Although we lack a practical gold standard for the diagnosis of childhood TB, its bacteriological confirmation is always recommended to be sought whenever possible prior to a diagnostic decision being made. Conventional diagnostic laboratory TB tests are less efficient for children as compared with adults, because sufficient sputum samples are more difficult to collect from infants and young children, and their disease is often paucibacillary, resulting in smear-negative disease. These inherent challenges associated with childhood TB are due to immunological- and pathophysiological-response differences relative to those observed in adults. Several recent meta-analyses showed low sensitivity estimates of PCR-based TB assays for paucibacillary forms of TB (extrapulmonary TB and smear-negative pulmonary disease), which represent the vast majority of childhood TB cases. Despite the lack of evidence regarding use of the rapid molecular assays to identify TB and detect DR in children, and due to the clinical nature of childhood TB, TB-expert groups recommend including rapid methods for TB identification and DR detection in diagnostic algorithms for children suspected of both smear-positive and -negative pulmonary or extrapulmonary TB, both with or without human immunodeficiency virus (HIV)-coinfection, when combined with standard methods (including clinical, microbiological, and radiological assessment) for diagnosing active TB and conventional DR. Since 2011, the WHO has specifically recommended use of the Xpert MTB/RIF test as an initial diagnostic tool for children with suspected HIV-associated TB or multidrug-resistant TB based on successful treatment data related to adults. Implementation of the rapid molecular assays for rapid detection of TB and DR should occur in laboratories with proven capability to run molecular tests and where quality control systems are implemented. Molecular approaches should be more largely tested in children, given their status as the group in whom the diagnostic dilemma is most pronounced. These tests should also be included in specific childhood TB diagnostic algorithms adapted to the local/national context in combination with other strategies for improving diagnostics, including more effective specimen collection. Copyright © 2016.

Molecular Testing for miRNA, mRNA, and DNA on Fine-Needle Aspiration Improves the Preoperative Diagnosis of Thyroid Nodules With Indeterminate Cytology.

PubMed

Labourier, Emmanuel; Shifrin, Alexander; Busseniers, Anne E; Lupo, Mark A; Manganelli, Monique L; Andruss, Bernard; Wylie, Dennis; Beaudenon-Huibregtse, Sylvie

2015-07-01

Molecular testing for oncogenic mutations or gene expression in fine-needle aspirations (FNAs) from thyroid nodules with indeterminate cytology identifies a subset of benign or malignant lesions with high predictive value. This study aimed to evaluate a novel diagnostic algorithm combining mutation detection and miRNA expression to improve the diagnostic yield of molecular cytology. Surgical specimens and preoperative FNAs (n = 638) were tested for 17 validated gene alterations using the miRInform Thyroid test and with a 10-miRNA gene expression classifier generating positive (malignant) or negative (benign) results. Cross-sectional sampling of thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) cytology (n = 109) was conducted at 12 endocrinology centers across the United States. Qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Mutations were detected in 69% of nodules with malignant outcome. Among mutation-negative specimens, miRNA testing correctly identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73-97%) and 85% (95% CI, 75-92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85-98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative to mRNA-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. Multiplatform testing for DNA, mRNA, and miRNA can accurately classify benign and malignant thyroid nodules, increase the diagnostic yield of molecular cytology, and further improve the preoperative risk-based management of benign nodules with AUS/FLUS or FN/SFN cytology.

A review of current and future molecular diagnostic tests for use in the microbiology laboratory.

PubMed

Jannes, Geert; De Vos, Daniel

2006-01-01

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined only a single analyte. Recent improvements in detection technologies have paved the way for the development of multiparameter assays using macroarrays or micro-arrays, while the introduction of closed-tube real-time polymerase chain reaction systems has resulted in the development of rapid microbial diagnostics with a reduced contamination risk. The use of these new molecular technologies is not restricted to detection and identification of microbial pathogens but also can be used for genotyping, allowing one to determine antibiotic resistance or to perform microbial fingerprinting.

Molecular diagnostics for low resource settings

NASA Astrophysics Data System (ADS)

Weigl, Bernhard H.

2010-03-01

As traditional high quality diagnostic laboratories are not widely available or affordable in developing country health care settings, microfluidics-based point-of-care diagnostics may be able to address the need to perform complex assays in under-resourced areas. Many instrument-based as well as non-instrumented microfluidic prototype diagnostics are currently being developed. In addition to various engineering challenges, the greatest remaining issue is the search for truly low-cost disposable manufacturing methods. Diagnostics for global health, and specifically microfluidics and molecular-based low resource diagnostics, have become a very active research area over the last five years, thanks in part to new funding that became available from the Bill and Melinda Gates Foundation, the National Institutes of Health, and other sources. This has led to a number of interesting prototype devices that are now in advanced development or clinical validation. These devices include disposables and instruments that perform multiplexed PCR-based lab-on-a-chips for enteric, febrile, and vaginal diseases, as well as immunoassays for diseases such as malaria, HIV, and various sexually transmitted diseases. More recently, instrument-free diagnostic disposables based on isothermal nucleic acid amplification have been developed as well. Regardless of platform, however, the search for truly low-cost manufacturing methods that would result in cost of goods per disposable of around US1/unit at volume remains a big challenge. This talk will give an overview over existing platform development efforts as well as present some original research in this area at PATH.

Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone

PubMed Central

Im, Hyungsoon; Castro, Cesar M.; Shao, Huilin; Liong, Monty; Song, Jun; Pathania, Divya; Fexon, Lioubov; Min, Changwook; Avila-Wallace, Maria; Zurkiya, Omar; Rho, Junsung; Magaoay, Brady; Tambouret, Rosemary H.; Pivovarov, Misha; Weissleder, Ralph; Lee, Hakho

2015-01-01

The widespread distribution of smartphones, with their integrated sensors and communication capabilities, makes them an ideal platform for point-of-care (POC) diagnosis, especially in resource-limited settings. Molecular diagnostics, however, have been difficult to implement in smartphones. We herein report a diffraction-based approach that enables molecular and cellular diagnostics. The D3 (digital diffraction diagnosis) system uses microbeads to generate unique diffraction patterns which can be acquired by smartphones and processed by a remote server. We applied the D3 platform to screen for precancerous or cancerous cells in cervical specimens and to detect human papillomavirus (HPV) DNA. The D3 assay generated readouts within 45 min and showed excellent agreement with gold-standard pathology or HPV testing, respectively. This approach could have favorable global health applications where medical access is limited or when pathology bottlenecks challenge prompt diagnostic readouts. PMID:25870273

Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone.

PubMed

Im, Hyungsoon; Castro, Cesar M; Shao, Huilin; Liong, Monty; Song, Jun; Pathania, Divya; Fexon, Lioubov; Min, Changwook; Avila-Wallace, Maria; Zurkiya, Omar; Rho, Junsung; Magaoay, Brady; Tambouret, Rosemary H; Pivovarov, Misha; Weissleder, Ralph; Lee, Hakho

2015-05-05

The widespread distribution of smartphones, with their integrated sensors and communication capabilities, makes them an ideal platform for point-of-care (POC) diagnosis, especially in resource-limited settings. Molecular diagnostics, however, have been difficult to implement in smartphones. We herein report a diffraction-based approach that enables molecular and cellular diagnostics. The D3 (digital diffraction diagnosis) system uses microbeads to generate unique diffraction patterns which can be acquired by smartphones and processed by a remote server. We applied the D3 platform to screen for precancerous or cancerous cells in cervical specimens and to detect human papillomavirus (HPV) DNA. The D3 assay generated readouts within 45 min and showed excellent agreement with gold-standard pathology or HPV testing, respectively. This approach could have favorable global health applications where medical access is limited or when pathology bottlenecks challenge prompt diagnostic readouts.

Application of Immunohistochemistry and Molecular Diagnostics to Clinically Relevant Problems in Endometrial Cancer Bojana Djordjevic, Shannon Westin, Russell R. Broaddus

PubMed Central

Djordjevic, Bojana; Westin, Shannon; Broaddus, Russell R.

2012-01-01

Synopsis A number of different clinical scenarios are presented in which lab-based analyses beyond the usual diagnosis based on light microscopic examination of H&E stained slides – immunohistochemistry and PCR-based assays such as sequencing, mutation testing, microsatellite instability analysis, and determination of MLH1 methylation - are most helpful for guiding diagnosis and treatment of endometrial cancer. The central goal of this information is to provide a practical guide of key current and emerging issues in diagnostic endometrial cancer pathology that require the use of ancillary laboratory techniques, such as immunohistochemistry and molecular testing. The authors present the common diagnostic problems in endometrial carcinoma pathology, types of endometrial carcinoma, description of tissue testing and markers, pathological features, and targeted therapy. PMID:23687522

Development and validation of risk models and molecular diagnostics to permit personalized management of cancer.

PubMed

Pu, Xia; Ye, Yuanqing; Wu, Xifeng

2014-01-01

Despite the advances made in cancer management over the past few decades, improvements in cancer diagnosis and prognosis are still poor, highlighting the need for individualized strategies. Toward this goal, risk prediction models and molecular diagnostic tools have been developed, tailoring each step of risk assessment from diagnosis to treatment and clinical outcomes based on the individual's clinical, epidemiological, and molecular profiles. These approaches hold increasing promise for delivering a new paradigm to maximize the efficiency of cancer surveillance and efficacy of treatment. However, they require stringent study design, methodology development, comprehensive assessment of biomarkers and risk factors, and extensive validation to ensure their overall usefulness for clinical translation. In the current study, the authors conducted a systematic review using breast cancer as an example and provide general guidelines for risk prediction models and molecular diagnostic tools, including development, assessment, and validation. © 2013 American Cancer Society.

Molecular Methods and Platforms for Infectious Diseases Testing

PubMed Central

Emmadi, Rajyasree; Boonyaratanakornkit, Jerry B.; Selvarangan, Rangaraj; Shyamala, Venkatakrishna; Zimmer, Barbara L.; Williams, Laurina; Bryant, Bonita; Schutzbank, Ted; Schoonmaker, Michele M.; Amos Wilson, Jean A.; Hall, Leslie; Pancholi, Preeti; Bernard, Kathryn

2011-01-01

The superior sensitivity and specificity associated with the use of molecular assays has greatly improved the field of infectious disease diagnostics by providing clinicians with results that are both accurate and rapidly obtained. Herein, we review molecularly based infectious disease diagnostic tests that are Food and Drug Administration approved or cleared and commercially available in the United States as of December 31, 2010. We describe specific assays and their performance, as stated in the Food and Drug Administration's Summary of Safety and Effectiveness Data or the Office of In Vitro Diagnostic Device Evaluation and Safety's decision summaries, product inserts, or peer-reviewed literature. We summarize indications for testing, limitations, and challenges related to implementation in a clinical laboratory setting for a wide variety of common pathogens. The information presented in this review will be particularly useful for laboratories that plan to implement or expand their molecular offerings in the near term. PMID:21871973

[Role of contemporary pathological diagnostics in the personalized treatment of cancer].

PubMed

Tímár, József

2013-03-01

Due to the developments of pathology in the past decades (immunohistochemistry and molecular pathology) classification of cancers changed fundamentally, laying a ground for personalized management of cancer patients. Our picture of cancer is more complex today, identifying the genetic basis of the morphological variants. On the other hand, this picture has a much higher resolution enabling us to subclassify similar histological cancer types based on molecular markers. This redefined classification of cancers helps us to better predict the possible biological behavior of the disease and/or the therapeutic sensitivity, opening the way toward a more personalized treatment of this disease. The redefined molecular classification of cancer may affect the universal application of treatment protocols. To achieve this goal molecular diagnostics must be an integral and reimbursed part of the routine pathological diagnostics. On the other hand, it is time to extend the multidisciplinary team with molecular pathologist to improve the decision making process of the management of cancer patients.

Dielectrophoresis-Based Sample Handling in General-Purpose Programmable Diagnostic Instruments

PubMed Central

Gascoyne, Peter R. C.; Vykoukal, Jody V.

2009-01-01

As the molecular origins of disease are better understood, the need for affordable, rapid, and automated technologies that enable microscale molecular diagnostics has become apparent. Widespread use of microsystems that perform sample preparation and molecular analysis could ensure that the benefits of new biomedical discoveries are realized by a maximum number of people, even those in environments lacking any infrastructure. While progress has been made in developing miniaturized diagnostic systems, samples are generally processed off-device using labor-intensive and time-consuming traditional sample preparation methods. We present the concept of an integrated programmable general-purpose sample analysis processor (GSAP) architecture where raw samples are routed to separation and analysis functional blocks contained within a single device. Several dielectrophoresis-based methods that could serve as the foundation for building GSAP functional blocks are reviewed including methods for cell and particle sorting, cell focusing, cell ac impedance analysis, cell lysis, and the manipulation of molecules and reagent droplets. PMID:19684877

Lymph Node Metastases Optical Molecular Diagnostic and Radiation Therapy

DTIC Science & Technology

2017-03-01

because most imaging is based upon structures and not molecular functions. The one tool commonly used for metastases imaging is nuclear medicine...imaging, which is that micro metastases cannot be visualized at a relevant stage., largely because most imaging is based upon structures and not...evaluate the limits on structural , metabolic and immunologic probes for molecular imaging, and (4) to complete studies on metastatic breast cancer

The impact of commercial rapid respiratory virus diagnostic tests on patient outcomes and health system utilization.

PubMed

Ko, Fiona; Drews, Steven J

2017-10-01

Acute respiratory tract infections due to influenza A/B and respiratory syncytial virus (RSV) are major causes of morbidity and mortality globally. Rapid tests for detection of these pathogens include antigen detection point of care tests (POC) and newer easy to use molecular tests. From experience, these assays improve both laboratory workflow and assay interpretation issues. However, the question of the benefits of using rapid test technology compared to routine laboratory testing for respiratory viral pathogens is still often asked. Areas covered: Specifically, this review aims to; 1) identify clinical/patient indicators that can be measured prior to and following the implementation of rapid diagnostic test for influenza and RSV, 2) provide multiple perspectives on the extent of impact of a rapid diagnostic test, including direct and indirect outcomes, and 3) identify the technological advancements in the development of rapid testing, demonstrating a timeline that transitions from antigen-based assays to molecular assays. Expert commentary: Key benefits to the use of either antigen-based or molecular rapid tests for patient care, patient flow within institutions, as well as laboratory utilization are identified. Due to improved test characteristics, the authors feel that rapid molecular tests have greater benefits than antigen-based detection methods.

NCCN Oncology Research Program's Investigator Steering Committee and NCCN Best Practices Committee Molecular Profiling Surveys.

PubMed

Kurzrock, Razelle; Colevas, A Dimitrios; Olszanski, Anthony; Akerley, Wallace; Arteaga, Carlos L; Carson, William E; Clark, Jeffrey W; DiPersio, John F; Ettinger, David S; Morgan, Robert J; Schwartzberg, Lee S; Venook, Alan P; Gocke, Christopher D; Tait, Jonathan; Stewart, F Marc

2015-11-01

With advances such as next-generation sequencing (NGS) increasing understanding of the basis of cancer and its response to treatment, NCCN believes it is important to understand how molecular profiling/diagnostic testing is being performed and used at NCCN Member Institutions and their community affiliates. The NCCN Oncology Research Program's Investigator Steering Committee and the NCCN Best Practices Committee gathered baseline information on the use of cancer-related molecular testing at NCCN Member Institutions and community members of the NCCN Affiliate Research Consortium through 2 separate surveys distributed in December 2013 and September 2014, respectively. A total of 24 NCCN Member Institutions and 8 affiliate sites provided quantitative and qualitative data. In the context of these surveys, "molecular profiling/diagnostics" was defined as a panel of at least 10 genes examined as a diagnostic DNA test in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory. Results indicated that molecular profiling/diagnostics are used at 100% of survey respondents' institutions to make patient care decisions. However, challenges relating to reimbursement, lack of data regarding actionable targets and targeted therapies, and access to drugs on or off clinical trials were cited as barriers to integration of molecular profiling into patient care. Frameworks for using molecular diagnostic results based on levels of evidence, alongside continued research into the predictive value of biomarkers and targeted therapies, are recommended to advance understanding of the role of genomic biomarkers. Greater evidence and consensus regarding the clinical and cost-effectiveness of molecular profiling may lead to broader insurance coverage and increased integration into patient care. Copyright © 2015 by the National Comprehensive Cancer Network.

Laboratory Information Systems in Molecular Diagnostics: Why Molecular Diagnostics Data are Different.

PubMed

Lee, Roy E; Henricks, Walter H; Sirintrapun, Sahussapont J

2016-03-01

Molecular diagnostic testing presents new challenges to information management that are yet to be sufficiently addressed by currently available information systems for the molecular laboratory. These challenges relate to unique aspects of molecular genetic testing: molecular test ordering, informed consent issues, diverse specimen types that encompass the full breadth of specimens handled by traditional anatomic and clinical pathology information systems, data structures and data elements specific to molecular testing, varied testing workflows and protocols, diverse instrument outputs, unique needs and requirements of molecular test reporting, and nuances related to the dissemination of molecular pathology test reports. By satisfactorily addressing these needs in molecular test data management, a laboratory information system designed for the unique needs of molecular diagnostics presents a compelling reason to migrate away from the current paper and spreadsheet information management that many molecular laboratories currently use. This paper reviews the issues and challenges of information management in the molecular diagnostics laboratory.

[Molecular Genetics as Best Evidence in Glioma Diagnostics].

PubMed

Masui, Kenta; Komori, Takashi

2016-03-01

The development of a genomic landscape of gliomas has led to the internally consistent, molecularly-based classifiers. However, development of a biologically insightful classification to guide therapy is still ongoing. Further, tumors are heterogeneous, and they change and adapt in response to drugs. The challenge of developing molecular classifiers that provide meaningful ways to stratify patients for therapy remains a major challenge for the field. Therefore, by incorporating molecular markers into the new World Health Organization (WHO) classification of tumors of the central nervous system, the traditional principle of diagnosis based on histologic criteria will be replaced by a multilayered approach combining histologic features and molecular information in an "integrated diagnosis", to define tumor entities as narrowly as possible. We herein review the current status of diagnostic molecular markers for gliomas, focusing on IDH mutation, ATRX mutation, 1p/19q co-deletion, and TERT promoter mutation in adult tumors, as well as BRAF and H3F3A aberrations in pediatric gliomas, the combination of which will be a promising endeavor to render molecular genetics as a best evidence in the glioma diagnositics.

Promise and pitfalls of molecular markers of thyroid nodules

PubMed Central

Jadhav, S.; Lila, Anurag; Bandgar, Tushar; Shah, Nalini

2012-01-01

Thyroid nodules are common in the general population with a prevalence of 5-7% The initial evaluation of thyroid nodules commonly involves thyroid function tests, an ultrasound (USG) and fine needle aspiration biopsy (FNAB). The optimal management of patients with thyroid nodules with indeterminate cytology is plagued by the lack of highly sensitive and specific diagnostic modalities In this article we attempt to review the available literature on the molecular markers which are increasingly being studied for their diagnostic utility in assessing thyroid nodules. The various molecular markers consist of gene mutations, gene re arrangements, RNA based assays and immunohistochemical markers. The molecular markers definitely would help to optimise the management of such patients. PMID:23565369

Nucleic Acid-Based Approaches for Detection of Viral Hepatitis

PubMed Central

Behzadi, Payam; Ranjbar, Reza; Alavian, Seyed Moayed

2014-01-01

Context: To determining suitable nucleic acid diagnostics for individual viral hepatitis agent, an extensive search using related keywords was done in major medical library and data were collected, categorized, and summarized in different sections. Results: Various types of molecular biology tools can be used to detect and quantify viral genomic elements and analyze the sequences. These molecular assays are proper technologies for rapidly detecting viral agents with high accuracy, high sensitivity, and high specificity. Nonetheless, the application of each diagnostic method is completely dependent on viral agent. Conclusions: Despite rapidity, automation, accuracy, cost-effectiveness, high sensitivity, and high specificity of molecular techniques, each type of molecular technology has its own advantages and disadvantages. PMID:25789132

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

NASA Astrophysics Data System (ADS)

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Synthetic biology devices for in vitro and in vivo diagnostics.

PubMed

Slomovic, Shimyn; Pardee, Keith; Collins, James J

2015-11-24

There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms.

Synthetic biology devices for in vitro and in vivo diagnostics

PubMed Central

Slomovic, Shimyn; Pardee, Keith; Collins, James J.

2015-01-01

There is a growing need to enhance our capabilities in medical and environmental diagnostics. Synthetic biologists have begun to focus their biomolecular engineering approaches toward this goal, offering promising results that could lead to the development of new classes of inexpensive, rapidly deployable diagnostics. Many conventional diagnostics rely on antibody-based platforms that, although exquisitely sensitive, are slow and costly to generate and cannot readily confront rapidly emerging pathogens or be applied to orphan diseases. Synthetic biology, with its rational and short design-to-production cycles, has the potential to overcome many of these limitations. Synthetic biology devices, such as engineered gene circuits, bring new capabilities to molecular diagnostics, expanding the molecular detection palette, creating dynamic sensors, and untethering reactions from laboratory equipment. The field is also beginning to move toward in vivo diagnostics, which could provide near real-time surveillance of multiple pathological conditions. Here, we describe current efforts in synthetic biology, focusing on the translation of promising technologies into pragmatic diagnostic tools and platforms. PMID:26598662

EMQN best practice guidelines for the molecular genetic testing and reporting of chromosome 11p15 imprinting disorders: Silver–Russell and Beckwith–Wiedemann syndrome

PubMed Central

Eggermann, Katja; Bliek, Jet; Brioude, Frédéric; Algar, Elizabeth; Buiting, Karin; Russo, Silvia; Tümer, Zeynep; Monk, David; Moore, Gudrun; Antoniadi, Thalia; Macdonald, Fiona; Netchine, Irène; Lombardi, Paolo; Soellner, Lukas; Begemann, Matthias; Prawitt, Dirk; Maher, Eamonn R; Mannens, Marcel; Riccio, Andrea; Weksberg, Rosanna; Lapunzina, Pablo; Grønskov, Karen; Mackay, Deborah JG; Eggermann, Thomas

2016-01-01

Molecular genetic testing for the 11p15-associated imprinting disorders Silver–Russell and Beckwith–Wiedemann syndrome (SRS, BWS) is challenging because of the molecular heterogeneity and complexity of the affected imprinted regions. With the growing knowledge on the molecular basis of these disorders and the demand for molecular testing, it turned out that there is an urgent need for a standardized molecular diagnostic testing and reporting strategy. Based on the results from the first external pilot quality assessment schemes organized by the European Molecular Quality Network (EMQN) in 2014 and in context with activities of the European Network of Imprinting Disorders (EUCID.net) towards a consensus in diagnostics and management of SRS and BWS, best practice guidelines have now been developed. Members of institutions working in the field of SRS and BWS diagnostics were invited to comment, and in the light of their feedback amendments were made. The final document was ratified in the course of an EMQN best practice guideline meeting and is in accordance with the general SRS and BWS consensus guidelines, which are in preparation. These guidelines are based on the knowledge acquired from peer-reviewed and published data, as well as observations of the authors in their practice. However, these guidelines can only provide a snapshot of current knowledge at the time of manuscript submission and readers are advised to keep up with the literature. PMID:27165005

A review of Theileria diagnostics and epidemiology

PubMed Central

Mans, Ben J.; Pienaar, Ronel; Latif, Abdalla A.

2015-01-01

An extensive range of serological and molecular diagnostic assays exist for most of the economically important Theileira species such as T. annulata, T. equi, T. lestoquardi, T. parva, T. uilenbergi and other more benign species. Diagnostics of Theileria is considered with regard to sensitivity and specificity of current molecular and serological assays and their use in epidemiology. In the case of serological assays, cross-reactivity of genetically closely related species reduces the use of the gold standard indirect fluorescent antibody test (IFAT). Development of antigen-specific assays does not necessarily address this problem, since closely related species will potentially have similar antigens. Even so, serological assays remain an important line of enquiry in epidemiological surveys. Molecular based assays have exploded in the last decade with significant improvements in sensitivity and specificity. In this review, the current interpretation of what constitute a species in Theileria and its impact on accurate molecular diagnostics is considered. Most molecular assays based on conventional or real-time PCR technology have proven to be on standard with regard to analytical sensitivity. However, consideration of the limits of detection in regard to total blood volume of an animal indicates that most assays may only detect >400,000 parasites/L blood. Even so, natural parasitaemia distribution in carrier-state animals seems to be above this limit of detection, suggesting that most molecular assays should be able to detect the majority of infected individuals under endemic conditions. The potential for false-negative results can, however, only be assessed within the biological context of the parasite within its vertebrate host, i.e. parasitaemia range in the carrier-state that will support infection of the vector and subsequent transmission. PMID:25830110

Intra-tumor heterogeneity in breast cancer has limited impact on transcriptomic-based molecular profiling.

PubMed

Karthik, Govindasamy-Muralidharan; Rantalainen, Mattias; Stålhammar, Gustav; Lövrot, John; Ullah, Ikram; Alkodsi, Amjad; Ma, Ran; Wedlund, Lena; Lindberg, Johan; Frisell, Jan; Bergh, Jonas; Hartman, Johan

2017-11-29

Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.

[Molecular and immunohistochemical diagnostics in melanoma].

PubMed

Schilling, B; Griewank, K G

2016-07-01

To provide appropriate therapy and follow-up to patients with malignant melanoma, proper diagnostics are of critical importance. Targeted therapy of advanced melanoma is based on the molecular genetic analyses of tumor tissue. In addition, sequencing of genes and other genetic approaches can provide insight into the origin of melanocytic tumors and can aid in distinguishing benign from malignant lesions. In this regard, spizoid neoplasms remain a challenging entity. Aside from genetic analyses of tumor tissue, immunohistochemistry remains an essential tool in melanoma diagnostics and TNM classification. With new immunotherapies being approved for advanced melanoma, immunohistochemistry to determine PD-L1 expression has gained clinical interest. While PD-L1 expression is associated with response to PD-1 blockade, a substantial number of patients without PD-L1 expression can still experience tumor remission upon treatment. In this review, current and future developments in melanoma diagnostics with regard to molecular genetics and immunohistochemistry are summarized. The utilization of such analyses in clinical decision making is also discussed.

The development, evaluation and performance of molecular diagnostics for detection of Mycobacterium tuberculosis.

PubMed

Bates, Matthew; Zumla, Alimuddin

2016-01-01

The unique pathogenesis of tuberculosis (TB) poses several barriers to the development of accurate diagnostics: a) the establishment of life-long latency by Mycobacterium tuberculosis (M.tb) after primary infection confounds the development of classical antibody or antigen based assays; b) our poor understanding of the molecular pathways that influence progression from latent to active disease; c) the intracellular nature of M.tb infection in tissues means that M.tb and/or its components, are not readily detectable in peripheral specimens; and d) the variable presence of M.tb bacilli in specimens from patients with extrapulmonary TB or children. The literature on the current portfolio of molecular diagnostics tests for TB is reviewed here and the developmental pipeline is summarized. Also reviewed are data from recently published operational research on the GeneXpert MTB/RIF assay and discussed are the lessons that can be taken forward for the design of studies to evaluate the impact of TB diagnostics.

Small business development for molecular diagnostics.

PubMed

Anagostou, Anthanasia; Liotta, Lance A

2012-01-01

Molecular profiling, which is the application of molecular diagnostics technology to tissue and blood -specimens, is an integral element in the new era of molecular medicine and individualized therapy. Molecular diagnostics is a fertile ground for small business development because it can generate products that meet immediate demands in the health-care sector: (a) Detection of disease risk, or early-stage disease, with a higher specificity and sensitivity compared to previous testing methods, and (b) "Companion diagnostics" for stratifying patients to receive a treatment choice optimized to their individual disease. This chapter reviews the promise and challenges of business development in this field. Guidelines are provided for the creation of a business model and the generation of a marketing plan around a candidate molecular diagnostic product. Steps to commercialization are outlined using existing molecular diagnostics companies as learning examples.

Biomedical analysis of formalin-fixed, paraffin-embedded tissue samples: The Holy Grail for molecular diagnostics.

PubMed

Donczo, Boglarka; Guttman, Andras

2018-06-05

More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field. Copyright © 2018. Published by Elsevier B.V.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

PubMed Central

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies. PMID:28094784

Enabling Equal Access to Molecular Diagnostics: What Are the Implications for Policy and Health Technology Assessment?

PubMed

Plun-Favreau, Juliette; Immonen-Charalambous, Kaisa; Steuten, Lotte; Strootker, Anja; Rouzier, Roman; Horgan, Denis; Lawler, Mark

2016-01-01

Molecular diagnostics can offer important benefits to patients and are a key enabler of the integration of personalised medicine into health care systems. However, despite their promise, few molecular diagnostics are embedded into clinical practice (especially in Europe) and access to these technologies remains unequal across countries and sometimes even within individual countries. If research translation and the regulatory environments have proven to be more challenging than expected, reimbursement and value assessment remain the main barriers to providing patients with equal access to molecular diagnostics. Unclear or non-existent reimbursement pathways, together with the lack of clear evidence requirements, have led to significant delays in the assessment of molecular diagnostics technologies in certain countries. Additionally, the lack of dedicated diagnostics budgets and the siloed nature of resource allocation within certain health care systems have significantly delayed diagnostics commissioning. This article will consider the perspectives of different stakeholders (patients, health care payers, health care professionals, and manufacturers) on the provision of a research-enabled, patient-focused molecular diagnostics platform that supports optimal patient care. Through the discussion of specific case studies, and building on the experience from countries that have successfully integrated molecular diagnostics into clinical practice, this article will discuss the necessary evolutions in policy and health technology assessment to ensure that patients can have equal access to appropriate molecular diagnostics. © 2016 S. Karger AG, Basel.

European Thyroid Association Guidelines regarding Thyroid Nodule Molecular Fine-Needle Aspiration Cytology Diagnostics.

PubMed

Paschke, Ralf; Cantara, Silvia; Crescenzi, Anna; Jarzab, Barbara; Musholt, Thomas J; Sobrinho Simoes, Manuel

2017-07-01

Molecular fine-needle aspiration (FNA) cytology diagnostics has the potential to address the inherent limitation of FNA cytology which is an indeterminate (atypia of undetermined significance/follicular lesion of undetermined significance follicular neoplasm) cytology. Because of the emerging role of molecular FNA cytology diagnostics, the European Thyroid Association convened a panel of international experts to review methodological aspects, indications, results, and limitations of molecular FNA cytology diagnostics. The panel reviewed the evidence for the diagnostic value of mutation panel assessment (including at least BRAF , NRAS , HRAS , KRAS , PAX8/PPARG , RET/PTC ) of targeted next generation sequencing and of a microarray gene expression classifier (GEC) test in the diagnostic assessment of an indeterminate cytology thyroid nodule. Moreover, possible surgical consequences of molecular FNA diagnostic results of thyroid nodules and the evidence that analysis of a molecular FNA diagnostic panel of somatic mutations or a microarray GEC test can alter the follow-up were reviewed. Molecular tests may help clinicians to drive patient care and the surgical decision if the analysis is performed in specialized laboratories. These molecular tests require standardization of performance characteristics and appropriate calibration as well as analytic validation before clinical interpretation.

European Thyroid Association Guidelines regarding Thyroid Nodule Molecular Fine-Needle Aspiration Cytology Diagnostics

PubMed Central

Paschke, Ralf; Cantara, Silvia; Crescenzi, Anna; Jarzab, Barbara; Musholt, Thomas J.; Sobrinho Simoes, Manuel

2017-01-01

Molecular fine-needle aspiration (FNA) cytology diagnostics has the potential to address the inherent limitation of FNA cytology which is an indeterminate (atypia of undetermined significance/follicular lesion of undetermined significance follicular neoplasm) cytology. Because of the emerging role of molecular FNA cytology diagnostics, the European Thyroid Association convened a panel of international experts to review methodological aspects, indications, results, and limitations of molecular FNA cytology diagnostics. The panel reviewed the evidence for the diagnostic value of mutation panel assessment (including at least BRAF, NRAS, HRAS, KRAS, PAX8/PPARG, RET/PTC) of targeted next generation sequencing and of a microarray gene expression classifier (GEC) test in the diagnostic assessment of an indeterminate cytology thyroid nodule. Moreover, possible surgical consequences of molecular FNA diagnostic results of thyroid nodules and the evidence that analysis of a molecular FNA diagnostic panel of somatic mutations or a microarray GEC test can alter the follow-up were reviewed. Molecular tests may help clinicians to drive patient care and the surgical decision if the analysis is performed in specialized laboratories. These molecular tests require standardization of performance characteristics and appropriate calibration as well as analytic validation before clinical interpretation. PMID:28785538

Patterns of diagnostic marker assessment in adult diffuse glioma: a survey of the European Confederation of Neuropathological Societies (Euro-CNS)

PubMed Central

Woehrer, Adelheid; Kristensen, Bjarne W.; Vital, Anne; Hainfellner, Johannes A.

2017-01-01

The 2016 update of the WHO classification has introduced an integrated diagnostic approach that incorporates both tumor morphology and molecular information. This conceptual change has far-reaching implications, especially for neuropathologists who are in the forefront of translating molecular markers to routine diagnostic use. Adult diffuse glioma is a prototypic example for a group of tumors that underwent substantial regrouping, and it represents a major workload for surgical neuropathologists. Hence, we conducted a survey among members of the European Confederation of Neuropathological Societies (Euro-CNS) in order to assess 1) the extent to which molecular markers have already been incorporated in glioma diagnoses, 2) which molecular techniques are in daily use, and 3) to set a baseline for future surveys in this field. Based on 130 responses from participants across 40 nations neuropathologists uniformly rate molecular marker testing as highly relevant and already incorporate molecular information in their diagnostic assessments. At the same time however, the survey documents substantial differences in access to crucial biomarkers and molecular techniques across geographic regions and within individual countries. Concerns are raised concerning the validity of test assays with MGMT, 1p19q, and ATRX; being perceived as most problematic. Neuropathologists advocate the need for international harmonization of standards and consensus guidelines, and the majority is willing to actively engage in interlaboratory trials aiming at quality control (Figure 1). PMID:27966427

[Advances of Molecular Diagnostic Techniques Application in Clinical Diagnosis.

PubMed

Ying, Bin-Wu

2016-11-01

Over the past 20 years,clinical molecular diagnostic technology has made rapid development,and became the most promising field in clinical laboratory medicine.In particular,with the development of genomics,clinical molecular diagnostic methods will reveal the nature of clinical diseases in a deeper level,thus guiding the clinical diagnosis and treatments.Many molecular diagnostic projects have been routinely applied in clinical works.This paper reviews the advances on application of clinical diagnostic techniques in infectious disease,tumor and genetic disorders,including nucleic acid amplification,biochip,next-generation sequencing,and automation molecular system,and so on.

The Development of a Post-Baccalaureate Certificate Program in Molecular Diagnostics

PubMed Central

Williams, Gail S.; Brown, Judith D.; Keagle, Martha B.

2000-01-01

A post-baccalaureate certificate program in diagnostic molecular sciences was created in 1995 by the Diagnostic Genetic Sciences Program in the School of Allied Health at the University of Connecticut. The required on-campus lecture and laboratory courses include basic laboratory techniques, health care issues, cell biology, immunology, human genetics, research, management, and molecular diagnostic techniques and laboratory in molecular diagnostics. These courses precede a 6-month, full-time practicum at an affiliated full-service molecular laboratory. The practicum includes amplification and blotting methods, a research project, and a choice of specialized electives including DNA sequencing, mutagenesis, in situ hybridization methods, or molecular diagnostic applications in microbiology. Graduates of the program are immediately eligible to sit for the National Credentialing Agency examination in molecular biology to obtain the credential Clinical Laboratory Specialist in Molecular Biology (CLSp(MB). This description of the University of Connecticut program may assist other laboratory science programs in creating similar curricula. PMID:11232107

A diagnostic algorithm for atypical spitzoid tumors: guidelines for immunohistochemical and molecular assessment.

PubMed

Cho-Vega, Jeong Hee

2016-07-01

Atypical spitzoid tumors are a morphologically diverse group of rare melanocytic lesions most frequently seen in children and young adults. As atypical spitzoid tumors bear striking resemblance to Spitz nevus and spitzoid melanomas clinically and histopathologically, it is crucial to determine its malignant potential and predict its clinical behavior. To date, many researchers have attempted to differentiate atypical spitzoid tumors from unequivocal melanomas based on morphological, immonohistochemical, and molecular diagnostic differences. A diagnostic algorithm is proposed here to assess the malignant potential of atypical spitzoid tumors by using a combination of immunohistochemical and cytogenetic/molecular tests. Together with classical morphological evaluation, this algorithm includes a set of immunohistochemistry assays (p16(Ink4a), a dual-color Ki67/MART-1, and HMB45), fluorescence in situ hybridization (FISH) with five probes (6p25, 8q24, 11q13, CEN9, and 9p21), and an array-based comparative genomic hybridization. This review discusses details of the algorithm, the rationale of each test used in the algorithm, and utility of this algorithm in routine dermatopathology practice. This algorithmic approach will provide a comprehensive diagnostic tool that complements conventional histological criteria and will significantly contribute to improve the diagnosis and prediction of the clinical behavior of atypical spitzoid tumors.

New Approaches to Sepsis: Molecular Diagnostics and Biomarkers

PubMed Central

Bauer, Michael; Riedemann, Niels C.; Hartog, Christiane S.

2012-01-01

Summary: Sepsis is among the most common causes of death in hospitals. It arises from the host response to infection. Currently, diagnosis relies on nonspecific physiological criteria and culture-based pathogen detection. This results in diagnostic uncertainty, therapeutic delays, the mis- and overuse of antibiotics, and the failure to identify patients who might benefit from immunomodulatory therapies. There is a need for new sepsis biomarkers that can aid in therapeutic decision making and add information about screening, diagnosis, risk stratification, and monitoring of the response to therapy. The host response involves hundreds of mediators and single molecules, many of which have been proposed as biomarkers. It is, however, unlikely that one single biomarker is able to satisfy all the needs and expectations for sepsis research and management. Among biomarkers that are measurable by assays approved for clinical use, procalcitonin (PCT) has shown some usefulness as an infection marker and for antibiotic stewardship. Other possible new approaches consist of molecular strategies to improve pathogen detection and molecular diagnostics and prognostics based on transcriptomic, proteomic, or metabolic profiling. Novel approaches to sepsis promise to transform sepsis from a physiologic syndrome into a group of distinct biochemical disorders and help in the development of better diagnostic tools and effective adjunctive sepsis therapies. PMID:23034322

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative.

PubMed

Loibner, Martina; Buzina, Walter; Viertler, Christian; Groelz, Daniel; Hausleitner, Anja; Siaulyte, Gintare; Kufferath, Iris; Kölli, Bettina; Zatloukal, Kurt

2016-01-01

Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

The impact of new trends in POCTs for companion diagnostics, non-invasive testing and molecular diagnostics.

PubMed

Huckle, David

2015-06-01

Point-of-care diagnostics have been slowly developing over several decades and have taken on a new importance in current healthcare delivery for both diagnostics and development of new drugs. Molecular diagnostics have become a key driver of technology change and opened up new areas in companion diagnostics for use alongside pharmaceuticals and in new clinical approaches such as non-invasive testing. Future areas involving smartphone and other information technology advances, together with new developments in molecular biology, microfluidics and surface chemistry are adding to advances in the market. The focus for point-of-care tests with molecular diagnostic technologies is focused on advancing effective applications.

Point of Injury Sampling Technology for Battlefield Molecular Diagnostics

DTIC Science & Technology

2011-11-14

Injury" Sampling Technology for Battlefield Molecular Diagnostics November 14, 2011 Sponsored by Defense Advanced Research Projects Agency (DOD...Date of Contract: April 25, 2011 Short Title of Work: "Point of Injury" Sampling Technology for Battlefield Molecular Diagnostics " Contract...PHASE I FINAL REPORT: Point of Injury, Sampling Technology for Battlefield Molecular Diagnostics . W31P4Q-11-C-0222 (UNCLASSIFIED) P.I: Bernardo

Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL.

PubMed

Deans, Zandra C; Costa, Jose Luis; Cree, Ian; Dequeker, Els; Edsjö, Anders; Henderson, Shirley; Hummel, Michael; Ligtenberg, Marjolijn Jl; Loddo, Marco; Machado, Jose Carlos; Marchetti, Antonio; Marquis, Katherine; Mason, Joanne; Normanno, Nicola; Rouleau, Etienne; Schuuring, Ed; Snelson, Keeda-Marie; Thunnissen, Erik; Tops, Bastiaan; Williams, Gareth; van Krieken, Han; Hall, Jacqueline A

2017-01-01

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.

Recombinase polymerase amplification: Emergence as a critical molecular technology for rapid, low-resource diagnostics.

PubMed

James, Ameh; Macdonald, Joanne

2015-01-01

Isothermal molecular diagnostics are bridging the technology gap between traditional diagnostics and polymerase chain reaction-based methods. These new techniques enable timely and accurate testing, especially in settings where there is a lack of infrastructure to support polymerase chain reaction facilities. Despite this, there is a significant lack of uptake of these technologies in developing countries where they are highly needed. Among these novel isothermal technologies, recombinase polymerase amplification (RPA) holds particular potential for use in developing countries. This rapid nucleic acid amplification approach is fast, highly sensitive and specific, and amenable to countries with a high burden of infectious diseases. Implementation of RPA technology in developing countries is critically required to assess limitations and potentials of the diagnosis of infectious disease, and may help identify impediments that prevent adoption of new molecular technologies in low resource- and low skill settings. This review focuses on approaching diagnosis of infectious disease with RPA.

Mass spectrometry-based metabolomics: applications to biomarker and metabolic pathway research.

PubMed

Zhang, Aihua; Sun, Hui; Yan, Guangli; Wang, Ping; Wang, Xijun

2016-01-01

Mass spectrometry-based metabolomics has become increasingly popular in molecular medicine. High-definition mass spectrometry (MS), coupled with pattern recognition methods, have been carried out to obtain comprehensive metabolite profiling and metabolic pathway of large biological datasets. This sets the scene for a new and powerful diagnostic approach. Analysis of the key metabolites in body fluids has become an important part of improving disease diagnosis. With technological advances in analytical techniques, the ability to measure low-molecular-weight metabolites in bio-samples provides a powerful platform for identifying metabolites that are uniquely correlated with a specific human disease. MS-based metabolomics can lead to enhanced understanding of disease mechanisms and to new diagnostic markers and has a strong potential to contribute to improving early diagnosis of diseases. This review will highlight the importance and benefit with certain characteristic examples of MS-metabolomics for identifying metabolic pathways and metabolites that accurately screen for potential diagnostic biomarkers of diseases. Copyright © 2015 John Wiley & Sons, Ltd.

Challenging the Cancer Molecular Stratification Dogma: Intratumoral Heterogeneity Undermines Consensus Molecular Subtypes and Potential Diagnostic Value in Colorectal Cancer.

PubMed

Dunne, Philip D; McArt, Darragh G; Bradley, Conor A; O'Reilly, Paul G; Barrett, Helen L; Cummins, Robert; O'Grady, Tony; Arthur, Ken; Loughrey, Maurice B; Allen, Wendy L; McDade, Simon S; Waugh, David J; Hamilton, Peter W; Longley, Daniel B; Kay, Elaine W; Johnston, Patrick G; Lawler, Mark; Salto-Tellez, Manuel; Van Schaeybroeck, Sandra

2016-08-15

A number of independent gene expression profiling studies have identified transcriptional subtypes in colorectal cancer with potential diagnostic utility, culminating in publication of a colorectal cancer Consensus Molecular Subtype classification. The worst prognostic subtype has been defined by genes associated with stem-like biology. Recently, it has been shown that the majority of genes associated with this poor prognostic group are stromal derived. We investigated the potential for tumor misclassification into multiple diagnostic subgroups based on tumoral region sampled. We performed multiregion tissue RNA extraction/transcriptomic analysis using colorectal-specific arrays on invasive front, central tumor, and lymph node regions selected from tissue samples from 25 colorectal cancer patients. We identified a consensus 30-gene list, which represents the intratumoral heterogeneity within a cohort of primary colorectal cancer tumors. Using a series of online datasets, we showed that this gene list displays prognostic potential HR = 2.914 (confidence interval 0.9286-9.162) in stage II/III colorectal cancer patients, but in addition, we demonstrated that these genes are stromal derived, challenging the assumption that poor prognosis tumors with stem-like biology have undergone a widespread epithelial-mesenchymal transition. Most importantly, we showed that patients can be simultaneously classified into multiple diagnostically relevant subgroups based purely on the tumoral region analyzed. Gene expression profiles derived from the nonmalignant stromal region can influence assignment of colorectal cancer transcriptional subtypes, questioning the current molecular classification dogma and highlighting the need to consider pathology sampling region and degree of stromal infiltration when employing transcription-based classifiers to underpin clinical decision making in colorectal cancer. Clin Cancer Res; 22(16); 4095-104. ©2016 AACRSee related commentary by Morris and Kopetz, p. 3989. ©2016 American Association for Cancer Research.

Role of Proteomics in the Development of Personalized Medicine.

PubMed

Jain, Kewal K

2016-01-01

Advances in proteomic technologies have made import contribution to the development of personalized medicine by facilitating detection of protein biomarkers, proteomics-based molecular diagnostics, as well as protein biochips and pharmacoproteomics. Application of nanobiotechnology in proteomics, nanoproteomics, has further enhanced applications in personalized medicine. Proteomics-based molecular diagnostics will have an important role in the diagnosis of certain conditions and understanding the pathomechanism of disease. Proteomics will be a good bridge between diagnostics and therapeutics; the integration of these will be important for advancing personalized medicine. Use of proteomic biomarkers and combination of pharmacoproteomics with pharmacogenomics will enable stratification of clinical trials and improve monitoring of patients for development of personalized therapies. Proteomics is an important component of several interacting technologies used for development of personalized medicine, which is depicted graphically. Finally, cancer is a good example of applications of proteomic technologies for personalized management of cancer. © 2016 Elsevier Inc. All rights reserved.

DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

2017-02-01

Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

[Diagnosis, prognosis, and prediction of non-small cell lung cancer. Importance of morphology, immunohistochemistry and molecular pathology].

PubMed

Warth, A

2015-11-01

Tumor diagnostics are based on histomorphology, immunohistochemistry and molecular pathological analysis of mutations, translocations and amplifications which are of diagnostic, prognostic and/or predictive value. In recent decades only histomorphology was used to classify lung cancer as either small (SCLC) or non-small cell lung cancer (NSCLC), although NSCLC was further subdivided in different entities; however, as no specific therapy options were available classification of specific subtypes was not clinically meaningful. This fundamentally changed with the discovery of specific molecular alterations in adenocarcinoma (ADC), e.g. mutations in KRAS, EGFR and BRAF or translocations of the ALK and ROS1 gene loci, which now form the basis of targeted therapies and have led to a significantly improved patient outcome. The diagnostic, prognostic and predictive value of imaging, morphological, immunohistochemical and molecular characteristics as well as their interaction were systematically assessed in a large cohort with available clinical data including patient survival. Specific and sensitive diagnostic markers and marker panels were defined and diagnostic test algorithms for predictive biomarker assessment were optimized. It was demonstrated that the semi-quantitative assessment of ADC growth patterns is a stage-independent predictor of survival and is reproducibly applicable in the routine setting. Specific histomorphological characteristics correlated with computed tomography (CT) imaging features and thus allowed an improved interdisciplinary classification, especially in the preoperative or palliative setting. Moreover, specific molecular characteristics, for example BRAF mutations and the proliferation index (Ki-67) were identified as clinically relevant prognosticators. Comprehensive clinical, morphological, immunohistochemical and molecular assessment of NSCLCs allow an optimized patient stratification. Respective algorithms now form the backbone of the 2015 lung cancer World Health Organization (WHO) classification.

’Point of Injury’ Sampling Technology for Battlefield Molecular Diagnostics

DTIC Science & Technology

2012-03-17

Injury" Sampling Technology for Battlefield Molecular Diagnostics March 17,2012 Sponsored by Defense Advanced Research Projects Agency (DOD) Defense...Contract: April 25, 2011 Short Title of Work: "Point of Injury" Sampling Technology for Battlefield Molecular Diagnostics " Contract Expiration Date...SBIR PHASE I OPTION REPORT: Point of Injury, Sampling Technology for Battlefield Molecular Diagnostics . W31P4Q-1 l-C-0222 (UNCLASSIFIED) P.I

The diagnosis of chronic endometritis in infertile asymptomatic women: a comparative study of histology, microbial cultures, hysteroscopy, and molecular microbiology.

PubMed

Moreno, Inmaculada; Cicinelli, Ettore; Garcia-Grau, Iolanda; Gonzalez-Monfort, Marta; Bau, Davide; Vilella, Felipe; De Ziegler, Dominique; Resta, Leonardo; Valbuena, Diana; Simon, Carlos

2018-06-01

Chronic endometritis is a persistent inflammation of the endometrial mucosa caused by bacterial pathogens such as Enterobacteriaceae, Enterococcus, Streptococcus, Staphylococcus, Mycoplasma, and Ureaplasma. Although chronic endometritis can be asymptomatic, it is found in up to 40% of infertile patients and is responsible for repeated implantation failure and recurrent miscarriage. Diagnosis of chronic endometritis is based on hysteroscopy of the uterine cavity, endometrial biopsy with plasma cells being identified histologically, while specific treatment is determined based on microbial culture. However, not all microorganisms implicated are easily or readily culturable needing a turnaround time of up to 1 week. We sought to develop a molecular diagnostic tool for chronic endometritis based on real-time polymerase chain reaction equivalent to using the 3 classic methods together, overcoming the bias of using any of them alone. Endometrial samples from patients assessed for chronic endometritis (n = 113) using at least 1 or several conventional diagnostic methods namely histology, hysteroscopy, and/or microbial culture, were blindly evaluated by real-time polymerase chain reaction for the presence of 9 chronic endometritis pathogens: Chlamydia trachomatis, Enterococcus, Escherichia coli, Gardnerella vaginalis, Klebsiella pneumoniae, Mycoplasma hominis, Neisseria gonorrhoeae, Staphylococcus, and Streptococcus. The sensitivity and specificity of the molecular analysis vs the classic diagnostic techniques were compared in the 65 patients assessed by all 3 recognized classic methods. The molecular method showed concordant results with histological diagnosis in 30 samples (14 double positive and 16 double negative) with a matching accuracy of 46.15%. Concordance of molecular and hysteroscopic diagnosis was observed in 38 samples (37 double positive and 1 double negative), with an accuracy of 58.46%. When the molecular method was compared to microbial culture, concordance was present in 37 samples (22 double positive and 15 double negative), a matching rate of 56.92%. When cases of potential contamination and/or noncultivable bacteria were considered, the accuracy increased to 66.15%. Of these 65 patients, only 27 patients had consistent histological + hysteroscopic diagnosis, revealing 58.64% of nonconcordant results. Only 13 of 65 patients (20%) had consistent histology + hysteroscopy + microbial culture results. In these cases, the molecular microbiology matched in 10 cases showing a diagnostic accuracy of 76.92%. Interestingly, the molecular microbiology confirmed over half of the isolated pathogens and provided additional detection of nonculturable microorganisms. These results were confirmed by the microbiome assessed by next-generation sequencing. In the endometrial samples with concordant histology + hysteroscopy + microbial culture results, the molecular microbiology diagnosis demonstrates 75% sensitivity, 100% specificity, 100% positive and 25% negative predictive values, and 0% false-positive and 25% false-negative rates. The molecular microbiology method describe herein is a fast and inexpensive diagnostic tool that allows for the identification of culturable and nonculturable endometrial pathogens associated with chronic endometritis. The results obtained were similar to all 3 classic diagnostic methods together with a degree of concordance of 76.92% providing an opportunity to improve the clinical management of infertile patients with a risk of experiencing this ghost endometrial pathology. Copyright © 2018 Elsevier Inc. All rights reserved.

Invasive pulmonary aspergillosis: current diagnostic methodologies and a new molecular approach.

PubMed

Moura, S; Cerqueira, L; Almeida, A

2018-05-13

The fungus Aspergillus fumigatus is the main pathogenic agent responsible for invasive pulmonary aspergillosis. Immunocompromised patients are more likely to develop this pathology due to a decrease in the immune system's defense capacity. Despite of the low occurrence of invasive pulmonary aspergillosis, this pathology presents high rates of mortality, mostly due to late and unspecific diagnosis. Currently, the diagnostic methods used to detect this fungal infection are conventional mycological examination (direct microscopic examination, histological examination, and culture), imaging, non-culture-based tests for the detection of galactomannan, β(1,3)-glucan and an extracellular glycoprotein, and molecular tests based on PCR. However, most of these methods do not detect the species A. fumigatus; they only allow the identification of genus Aspergillus. The development of more specific detection methods is of extreme importance. Fluorescent in situ hybridization-based molecular methods can be a good alternative to achieve this purpose. In this review, it is intended to point out that most of the methods used for the diagnosis of invasive pulmonary aspergillosis do not allow to detect the fungus at the species level and that fluorescence in situ hybridization-based molecular method will be a promising approach in the A. fumigatus detection.

Undergraduate chemistry students' conceptions of atomic structure, molecular structure and chemical bonding

NASA Astrophysics Data System (ADS)

Campbell, Erin Roberts

The process of chemical education should facilitate students' construction of meaningful conceptual structures about the concepts and processes of chemistry. It is evident, however, that students at all levels possess concepts that are inconsistent with currently accepted scientific views. The purpose of this study was to examine undergraduate chemistry students' conceptions of atomic structure, chemical bonding and molecular structure. A diagnostic instrument to evaluate students' conceptions of atomic and molecular structure was developed by the researcher. The instrument incorporated multiple-choice items and reasoned explanations based upon relevant literature and a categorical summarization of student responses (Treagust, 1988, 1995). A covalent bonding and molecular structure diagnostic instrument developed by Peterson and Treagust (1989) was also employed. The ex post facto portion of the study examined the conceptual understanding of undergraduate chemistry students using descriptive statistics to summarize the results obtained from the diagnostic instruments. In addition to the descriptive portion of the study, a total score for each student was calculated based on the combination of correct and incorrect choices made for each item. A comparison of scores obtained on the diagnostic instruments by the upper and lower classes of undergraduate students was made using a t-Test. This study also examined an axiomatic assumption that an understanding of atomic structure is important in understanding bonding and molecular structure. A Pearson Correlation Coefficient, ṟ, was calculated to provide a measure of the strength of this association. Additionally, this study gathered information regarding expectations of undergraduate chemistry students' understanding held by the chemical community. Two questionnaires were developed with items based upon the propositional knowledge statements used in the development of the diagnostic instruments. Subgroups of items from the questionnaires were formed from the combination of items found to measure different aspects of a specific topic area using a reliability analysis. Average scores for the subgroups were compared to results obtained by students on the diagnostic instrument targeting the same topic area. There were no significant differences of the scores on both of the diagnostic instruments between the levels of undergraduate chemistry students. There were, however, significant differences on certain items of the diagnostic instruments between upper and lower class students. Additionally, misconceptions were identified within all levels of these undergraduate students that corresponded to previous results reported in the literature. A significant relationship was found to exist between the scores obtained on the two diagnostic instruments, as well as strong correlations between specific items and the total scores of the instruments. Response to the expectations questionnaires revealed no differences between the chemical industry and chemical academia, but did provide information concerning the chemical community's expectations of undergraduate chemistry students. Results indicate that undergraduate students majoring in chemistry have conceptions that are inconsistent with currently accepted scientific views. The findings also support the hypothesis that an understanding of the general structure of the atom and the roles played by electrons in molecular bonding and structure is important to an understanding of chemical properties and behavior.

Evaluation of the Effect of Diagnostic Molecular Testing on the Surgical Decision-Making Process for Patients With Thyroid Nodules.

PubMed

Noureldine, Salem I; Najafian, Alireza; Aragon Han, Patricia; Olson, Matthew T; Genther, Dane J; Schneider, Eric B; Prescott, Jason D; Agrawal, Nishant; Mathur, Aarti; Zeiger, Martha A; Tufano, Ralph P

2016-07-01

Diagnostic molecular testing is used in the workup of thyroid nodules. While these tests appear to be promising in more definitively assigning a risk of malignancy, their effect on surgical decision making has yet to be demonstrated. To investigate the effect of diagnostic molecular profiling of thyroid nodules on the surgical decision-making process. A surgical management algorithm was developed and published after peer review that incorporated individual Bethesda System for Reporting Thyroid Cytopathology classifications with clinical, laboratory, and radiological results. This algorithm was created to formalize the decision-making process selected herein in managing patients with thyroid nodules. Between April 1, 2014, and March 31, 2015, a prospective study of patients who had undergone diagnostic molecular testing of a thyroid nodule before being seen for surgical consultation was performed. The recommended management undertaken by the surgeon was then prospectively compared with the corresponding one in the algorithm. Patients with thyroid nodules who did not undergo molecular testing and were seen for surgical consultation during the same period served as a control group. All pertinent treatment options were presented to each patient, and any deviation from the algorithm was recorded prospectively. To evaluate the appropriateness of any change (deviation) in management, the surgical histopathology diagnosis was correlated with the surgery performed. The study cohort comprised 140 patients who underwent molecular testing. Their mean (SD) age was 50.3 (14.6) years, and 75.0% (105 of 140) were female. Over a 1-year period, 20.3% (140 of 688) had undergone diagnostic molecular testing before surgical consultation, and 79.7% (548 of 688) had not undergone molecular testing. The surgical management deviated from the treatment algorithm in 12.9% (18 of 140) with molecular testing and in 10.2% (56 of 548) without molecular testing (P = .37). In the group with molecular testing, the surgical management plan of only 7.9% (11 of 140) was altered as a result of the molecular test. All but 1 of those patients were found to be overtreated relative to the surgical histopathology analysis. Molecular testing did not significantly affect the surgical decision-making process in this study. Among patients whose treatment was altered based on these markers, there was evidence of overtreatment.

Emerging technologies in point-of-care molecular diagnostics for resource-limited settings.

PubMed

Peeling, Rosanna W; McNerney, Ruth

2014-06-01

Emerging molecular technologies to diagnose infectious diseases at the point at which care is delivered have the potential to save many lives in developing countries where access to laboratories is poor. Molecular tests are needed to improve the specificity of syndromic management, monitor progress towards disease elimination and screen for asymptomatic infections with the goal of interrupting disease transmission and preventing long-term sequelae. In simplifying laboratory-based molecular assays for use at point-of-care, there are inevitable compromises between cost, ease of use and test performance. Despite significant technological advances, many challenges remain for the development of molecular diagnostics for resource-limited settings. There needs to be more advocacy for these technologies to be applied to infectious diseases, increased efforts to lower the barriers to market entry through streamlined and harmonized regulatory approaches, faster policy development for adoption of new technologies and novel financing mechanisms to enable countries to scale up implementation.

Culture-independent diagnostic testing: have we opened Pandora's box for good?

PubMed

Janda, J Michael; Abbott, Sharon A

2014-11-01

The ability to accurately and quickly identify microbial agents associated with infectious diseases has been a longstanding and continuous goal of diagnostic microbiology laboratories. Over the course of several decades, technology and testing methodologies in this field have gradually evolved from traditional- or classic-based culture and identification approaches to antigen capture systems and more molecular-oriented applications. Recently, these molecular-based applications have signaled a new era in clinical diagnostic microbiology with the commercial introduction of culture-independent diagnostic testing (CIDT) systems. The first major commercial venture into the CIDT arena involves the detection of acute bacterial gastroenteritis. Several commercial products are now on the market globally with at least 4 Food and Drug Administration approved since January of 2013. These new systems offer the direct detection of a variety of enteropathogens quickly without the need for traditional culture. In Greek mythology, Pandora opened a "jar" or "box" out of curiosity thereby releasing all of humanity's evils most notably diseases and plagues according to Hesiod's Theogony. While not ill-intentioned the only thing left in the box was Hope. Copyright © 2014 Elsevier Inc. All rights reserved.

Evolution of pollen morphology in Loranthaceae

PubMed Central

Grímsson, Friðgeir; Grimm, Guido W.; Zetter, Reinhard

2018-01-01

Abstract Earlier studies indicate a strong correlation of pollen morphology and ultrastructure with taxonomy in Loranthaceae. Using high-resolution light microscopy and scanning electron microscopy imaging of the same pollen grains, we document pollen types of 35 genera including 15 studied for the first time. Using a molecular phylogenetic framework based on currently available sequence data with good genus-coverage, we reconstruct trends in the evolution of Loranthaceae pollen and pinpoint traits of high diagnostic value, partly confirming earlier intuitive hypotheses based on morphological observations. We find that pollen morphology in Loranthaceae is strongly linked to phylogenetic relationships. Some pollen types are diagnostic for discrete genera or evolutionary lineages, opening the avenue to recruit dispersed fossil pollen as age constraints for dated phylogenies and as independent data for testing biogeographic scenarios; so far based exclusively on modern-day data. Correspondences and discrepancies between palynological and molecular data and current taxonomic/systematic concepts are identified and suggestions made for future palynological and molecular investigations of Loranthaceae. PMID:29386990

Surveillance at the molecular level: Developing an integrated network for detecting variation in avian influenza viruses in Indonesia.

PubMed

Hartaningsih, Nining; Wibawa, Hendra; Pudjiatmoko; Rasa, Fadjar Sumping Tjatur; Irianingsih, Sri Handayani; Dharmawan, Rama; Azhar, Muhammad; Siregar, Elly Sawitri; McGrane, James; Wong, Frank; Selleck, Paul; Allen, John; Broz, Ivano; Torchetti, Mia Kim; Dauphin, Gwenaelle; Claes, Filip; Sastraningrat, Wiryadi; Durr, Peter A

2015-06-01

Since 2006, Indonesia has used vaccination as the principal means of control of H5N1-HPAI. During this time, the virus has undergone gradual antigenic drift, which has necessitated changes in seed strains for vaccine production and associated modifications to diagnostic antigens. In order to improve the system of monitoring such viral evolution, the Government of Indonesia, with the assistance of FAO/OFFLU, has developed an innovative network whereby H5N1 isolates are antigenically and genetically characterised. This molecular surveillance network ("Influenza Virus Monitoring" or "IVM") is based on the regional network of veterinary diagnostic laboratories, and is supported by a web-based data management system ("IVM Online"). The example of the Indonesian IVM network has relevance for other countries seeking to establish laboratory networks for the molecular surveillance of avian influenza and other pathogens. Copyright © 2015 Elsevier B.V. All rights reserved.

Advances in point-of-care technologies for molecular diagnostics.

PubMed

Zarei, Mohammad

2017-12-15

Advances in miniaturization, nanotechnology, and microfluidics, along with developments in cloud-connected point-of-care (POC) diagnostics technologies are pushing the frontiers of POC devices toward low-cost, user-friendly, and enhanced sensitivity molecular-level diagnostics. The combination of various bio-sensing platforms within smartphone-integrated electronic readers provides accurate on-site and on-time diagnostics based on various types of chemical and biological targets. Further, 3D printing technology shows a huge potential toward fabrication and improving the performance of POC devices. Integration of skin-like flexible sensors with wireless communication technology creates a unique opportunity for continuous, real-time monitoring of patients for both preventative healthcare and during disease outbreaks. Here, we review recent developments and advances in POC technologies and describe how these advances enhance the performance of POC platforms. Also, this review describes challenges, directions, and future trends on application of emerging technologies in POC diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Current and future molecular diagnostics in colorectal cancer and colorectal adenoma.

PubMed

Tsang, Andy Hin-Fung; Cheng, Ka-Ho; Wong, Apple Siu-Ping; Ng, Simon Siu-Man; Ma, Brigette Buig-Yue; Chan, Charles Ming-Lok; Tsui, Nancy Bo-Yin; Chan, Lawrence Wing-Chi; Yung, Benjamin Yat-Ming; Wong, Sze-Chuen Cesar

2014-04-14

Colorectal cancer (CRC) is one of the most prevalent cancers in developed countries. On the other hand, CRC is also one of the most curable cancers if it is detected in early stages through regular colonoscopy or sigmoidoscopy. Since CRC develops slowly from precancerous lesions, early detection can reduce both the incidence and mortality of the disease. Fecal occult blood test is a widely used non-invasive screening tool for CRC. Although fecal occult blood test is simple and cost-effective in screening CRC, there is room for improvement in terms of the accuracy of the test. Genetic dysregulations have been found to play an important role in CRC development. With better understanding of the molecular basis of CRC, there is a growing expectation on the development of diagnostic tests based on more sensitive and specific molecular markers and those tests may provide a breakthrough to the limitations of current screening tests for CRC. In this review, the molecular basis of CRC development, the characteristics and applications of different non-invasive molecular biomarkers, as well as the technologies available for the detection were discussed. This review intended to provide a summary on the current and future molecular diagnostics in CRC and its pre-malignant state, colorectal adenoma.

Current and future molecular diagnostics in colorectal cancer and colorectal adenoma

PubMed Central

Tsang, Andy Hin-Fung; Cheng, Ka-Ho; Wong, Apple Siu-Ping; Ng, Simon Siu-Man; Ma, Brigette Buig-Yue; Chan, Charles Ming-Lok; Tsui, Nancy Bo-Yin; Chan, Lawrence Wing-Chi; Yung, Benjamin Yat-Ming; Wong, Sze-Chuen Cesar

2014-01-01

Colorectal cancer (CRC) is one of the most prevalent cancers in developed countries. On the other hand, CRC is also one of the most curable cancers if it is detected in early stages through regular colonoscopy or sigmoidoscopy. Since CRC develops slowly from precancerous lesions, early detection can reduce both the incidence and mortality of the disease. Fecal occult blood test is a widely used non-invasive screening tool for CRC. Although fecal occult blood test is simple and cost-effective in screening CRC, there is room for improvement in terms of the accuracy of the test. Genetic dysregulations have been found to play an important role in CRC development. With better understanding of the molecular basis of CRC, there is a growing expectation on the development of diagnostic tests based on more sensitive and specific molecular markers and those tests may provide a breakthrough to the limitations of current screening tests for CRC. In this review, the molecular basis of CRC development, the characteristics and applications of different non-invasive molecular biomarkers, as well as the technologies available for the detection were discussed. This review intended to provide a summary on the current and future molecular diagnostics in CRC and its pre-malignant state, colorectal adenoma. PMID:24744577

Severe childhood asthma and allergy to furry animals: refined assessment using molecular-based allergy diagnostics.

PubMed

Konradsen, Jon R; Nordlund, Björn; Onell, Annica; Borres, Magnus P; Grönlund, Hans; Hedlin, Gunilla

2014-03-01

Allergy to cats and dogs and polysensitization towards these animals are associated with severe childhood asthma. Molecular-based allergy diagnostics offers new opportunities for improved characterization and has been suggested to be particularly useful in patients with polysensitization and/or severe asthma. The aim was to use extract- and molecular-based allergy diagnostics to compare patterns of IgE sensitization towards aeroallergens in children with problematic severe and controlled asthma. Children with a positive ImmunoCAP towards any furry animal (cat, dog or horse) were recruited from a Nationwide Swedish study on severe childhood asthma. Severe (n = 37, age 13 years) and controlled (n = 28, age 14 years) asthmatics underwent assessment of allergic sensitization by ImmunoCap (kUA /l) and immunosolid-phase allergen chip (ISAC). In addition, Asthma Control Test, spirometry and a methacholine challenge were performed. Children with severe asthma had lower asthma control (p < 0.001) and FEV1 (p = 0.001) and more bronchial hyper-responsiveness (p = 0.008) in spite of high doses of inhaled steroids (≥800 μg budesonide). Children with severe asthma displayed higher levels of IgE antibodies towards cat (17 vs. 3.9, p = 0.027), dog (3.8 vs. 1.2, p = 0.012) and horse (7.4 vs. 0.7, p = 0.014). Sensitization towards Can f 2 (22% vs. 0%, p = 0.009) and Equ c 1 (51% vs. 25%, p = 0.03) was more common in severe asthma. IgE levels towards Equ c 1 correlated with asthma control (r = -0.41, p = 0.04). Children with severe allergic asthma had higher sIgE levels to cat, dog and horse. Molecular-based allergy diagnostics revealed a more complex molecular spreading of allergen components in children with the most severe disease. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[CONTEMPORARY MOLECULAR-GENETIC METHODS USED FOR ETIOLOGIC DIAGNOSTICS OF SEPSIS].

PubMed

Gavrilov, S N; Skachkova, T S; Shipulina, O Yu; Savochkina, Yu A; Shipulin, G A; Maleev, V V

2016-01-01

Etiologic diagnostics of sepsis is one of the most difficult problems of contemporary medicine due to a wide variety of sepsis causative agents, many of which are components of normal human microflora. Disadvantages of contemporary "golden standard" of microbiologic diagnostics of sepsis etiology by seeding of blood for sterility are duration of cultivation, limitation in detection of non-cultivable forms of microorganisms, significant effect of preliminary empiric antibiotics therapy on results of the analysis. Methods of molecular diagnostics that are being actively developed and integrated during the last decade are deprived of these disadvantages. Main contemporary methods of molecular-biological diagnostics are examined in the review, actualdata on their diagnostic characteristic are provided. Special attention is given to methods of PCR-diagnostics, including novel Russian developments. Methods of nucleic acid hybridization and proteomic analysis are examined in comparative aspect. Evaluation of application and perspectives of development of methods of molecular diagnostics of sepsis is given.

NANOSTRUCTURED PLANAR WAVEGUIDE DEVICE FOR MOLECULAR IDENTIFICATION OF HAZARDOUS COMPOUNDS IN WATER BY EVANESCENT SURFACE ENHANCED RAMAN SPECTROSCOPY - PHASE I

EPA Science Inventory

Senspex, Inc. proposes to investigate a novel diagnostic tool based upon evanescent field planar waveguide sensing and complementary nanostructured mediated molecular vibration spectroscopy methods for rapid detection and analysis of hazardous biological and chemical targets i...

Minimally Invasive Molecular Staging (MIMS) RT-PCR Breast Cancer Study

DTIC Science & Technology

2007-03-31

per se. 15. SUBJECT TERMS Breast cancer, real-time PCR, molecular diagnostics , micrometastases 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18...a superior molecular marker for detection of non- small cell lung cancer in peripheral blood. Journal of Molecular Diagnostics . 5:23 7-42, 2003. With...Journal of Molecular Diagnostics . 5:237-42, 2003 10. Mikhitarian K, Allen A, Reott S, Hoover L, Allen A, Cole DJ, Gillanders WE, and Mitas M. Enhanced

Implementation of Rapid Molecular Infectious Disease Diagnostics: the Role of Diagnostic and Antimicrobial Stewardship.

PubMed

Messacar, Kevin; Parker, Sarah K; Todd, James K; Dominguez, Samuel R

2017-03-01

New rapid molecular diagnostic technologies for infectious diseases enable expedited accurate microbiological diagnoses. However, diagnostic stewardship and antimicrobial stewardship are necessary to ensure that these technologies conserve, rather than consume, additional health care resources and optimally affect patient care. Diagnostic stewardship is needed to implement appropriate tests for the clinical setting and to direct testing toward appropriate patients. Antimicrobial stewardship is needed to ensure prompt appropriate clinical action to translate faster diagnostic test results in the laboratory into improved outcomes at the bedside. This minireview outlines the roles of diagnostic stewardship and antimicrobial stewardship in the implementation of rapid molecular infectious disease diagnostics. Copyright © 2017 American Society for Microbiology.

Integrating molecular diagnostics into histopathology training: the Belfast model.

PubMed

Flynn, C; James, J; Maxwell, P; McQuaid, S; Ervine, A; Catherwood, M; Loughrey, M B; McGibben, D; Somerville, J; McManus, D T; Gray, M; Herron, B; Salto-Tellez, M

2014-07-01

Molecular medicine is transforming modern clinical practice, from diagnostics to therapeutics. Discoveries in research are being incorporated into the clinical setting with increasing rapidity. This transformation is also deeply changing the way we practise pathology. The great advances in cell and molecular biology which have accelerated our understanding of the pathogenesis of solid tumours have been embraced with variable degrees of enthusiasm by diverse medical professional specialties. While histopathologists have not been prompt to adopt molecular diagnostics to date, the need to incorporate molecular pathology into the training of future histopathologists is imperative. Our goal is to create, within an existing 5-year histopathology training curriculum, the structure for formal substantial teaching of molecular diagnostics. This specialist training has two main goals: (1) to equip future practising histopathologists with basic knowledge of molecular diagnostics and (2) to create the option for those interested in a subspecialty experience in tissue molecular diagnostics to pursue this training. It is our belief that this training will help to maintain in future the role of the pathologist at the centre of patient care as the integrator of clinical, morphological and molecular information. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Diffusion of Molecular Diagnostic Lung Cancer Tests: A Survey of German Oncologists

PubMed Central

Steffen, Julius Alexander

2014-01-01

This study was aimed at examining the diffusion of diagnostic lung cancer tests in Germany. It was motivated by the high potential of detecting and targeting oncogenic drivers. Recognizing that the diffusion of diagnostic tests is a conditio sine qua non for the success of personalized lung cancer therapies, this study analyzed the diffusion of epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) tests in Germany. Qualitative and quantitative research strategies were combined in a mixed-method design. A literature review and subsequent Key Opinion Leader interviews identified a set of qualitative factors driving the diffusion process, which were then translated into an online survey. The survey was conducted among a sample of 961 oncologists (11.34% response rate). The responses were analyzed in a multiple linear regression which identified six statistically significant factors driving the diffusion of molecular diagnostic lung cancer tests: reimbursement, attitude towards R&D, information self-assessment, perceived attitudes of colleagues, age and test-pathway strategies. Besides the important role of adequate reimbursement and relevant guidelines, the results of this study suggest that an increasing usage of test-pathway strategies, especially in an office-based setting, can increase the diffusion of molecular diagnostic lung cancer tests in the future. PMID:25562146

Breakpoint structure of the Anopheles gambiae 2Rb chromosomal inversion.

PubMed

Lobo, Neil F; Sangaré, Djibril M; Regier, Allison A; Reidenbach, Kyanne R; Bretz, David A; Sharakhova, Maria V; Emrich, Scott J; Traore, Sekou F; Costantini, Carlo; Besansky, Nora J; Collins, Frank H

2010-10-25

Alternative arrangements of chromosome 2 inversions in Anopheles gambiae are important sources of population structure, and are associated with adaptation to environmental heterogeneity. The forces responsible for their origin and maintenance are incompletely understood. Molecular characterization of inversion breakpoints provides insight into how they arose, and provides the basis for development of molecular karyotyping methods useful in future studies. Sequence comparison of regions near the cytological breakpoints of 2Rb allowed the molecular delineation of breakpoint boundaries. Comparisons were made between the standard 2R+b arrangement in the An. gambiae PEST reference genome and the inverted 2Rb arrangements in the An. gambiae M and S genome assemblies. Sequence differences between alternative 2Rb arrangements were exploited in the design of a PCR diagnostic assay, which was evaluated against the known chromosomal banding pattern of laboratory colonies and field-collected samples from Mali and Cameroon. The breakpoints of the 7.55 Mb 2Rb inversion are flanked by extensive runs of the same short (72 bp) tandemly organized sequence, which was likely responsible for chromosomal breakage and rearrangement. Application of the molecular diagnostic assay suggested that 2Rb has a single common origin in An. gambiae and its sibling species, Anopheles arabiensis, and also that the standard arrangement (2R+b) may have arisen twice through breakpoint reuse. The molecular diagnostic was reliable when applied to laboratory colonies, but its accuracy was lower in natural populations. The complex repetitive sequence flanking the 2Rb breakpoint region may be prone to structural and sequence-level instability. The 2Rb molecular diagnostic has immediate application in studies based on laboratory colonies, but its usefulness in natural populations awaits development of complementary molecular tools.

The case for DNA methylation based molecular profiling to improve diagnostic accuracy for central nervous system embryonal tumors (not otherwise specified) in adults.

PubMed

Halliday, Gail C; Junckerstorff, Reimar C; Bentel, Jacqueline M; Miles, Andrew; Jones, David T W; Hovestadt, Volker; Capper, David; Endersby, Raelene; Cole, Catherine H; van Hagen, Tom; Gottardo, Nicholas G

2018-01-01

Central nervous system primitive neuro-ectodermal tumors (CNS-PNETs), have recently been re-classified in the most recent 2016 WHO Classification into a standby catch all category, "CNS Embryonal Tumor, not otherwise specified" (CNS embryonal tumor, NOS) based on epigenetic, biologic and histopathologic criteria. CNS embryonal tumors (NOS) are a rare, histologically and molecularly heterogeneous group of tumors that predominantly affect children, and occasionally adults. Diagnosis of this entity continues to be challenging and the ramifications of misdiagnosis of this aggressive class of brain tumors are significant. We report the case of a 45-year-old woman who was diagnosed with a central nervous system embryonal tumor (NOS) based on immunohistochemical analysis of the patient's tumor at diagnosis. However, later genome-wide methylation profiling of the diagnostic tumor undertaken to guide treatment, revealed characteristics most consistent with IDH-mutant astrocytoma. DNA sequencing and immunohistochemistry confirmed the presence of IDH1 and ATRX mutations resulting in a revised diagnosis of high-grade small cell astrocytoma, and the implementation of a less aggressive treatment regime tailored more appropriately to the patient's tumor type. This case highlights the inadequacy of histology alone for the diagnosis of brain tumours and the utility of methylation profiling and integrated genomic analysis for the diagnostic verification of adults with suspected CNS embryonal tumor (NOS), and is consistent with the increasing realization in the field that a combined diagnostic approach based on clinical, histopathological and molecular data is required to more accurately distinguish brain tumor subtypes and inform more effective therapy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Development of a rapid diagnostic assay for the detection of tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification

USDA-ARS?s Scientific Manuscript database

A molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39 °C and target-specific primers and probe were developed for the rapid, sensitive, and specific detection of tomato chlorotic dwarf viroid (TCDVd) in ...

Moving toward rapid and low-cost point-of-care molecular diagnostics with a repurposed 3D printer and RPA.

PubMed

Chan, Kamfai; Wong, Pui-Yan; Parikh, Chaitanya; Wong, Season

2018-03-15

Traditionally, the majority of nucleic acid amplification-based molecular diagnostic tests are done in centralized settings. In recent years, point-of-care tests have been developed for use in low-resource settings away from central laboratories. While most experts agree that point-of-care molecular tests are greatly needed, their availability as cost-effective and easy-to-operate tests remains an unmet goal. In this article, we discuss our efforts to develop a recombinase polymerase amplification reaction-based test that will meet these criteria. First, we describe our efforts in repurposing a low-cost 3D printer as a platform that can carry out medium-throughput, rapid, and high-performing nucleic acid extraction. Next, we address how these purified templates can be rapidly amplified and analyzed using the 3D printer's heated bed or the deconstructed, low-cost thermal cycler we have developed. In both approaches, real-time isothermal amplification and detection of template DNA or RNA can be accomplished using a low-cost portable detector or smartphone camera. Last, we demonstrate the capability of our technologies using foodborne pathogens and the Zika virus. Our low-cost approach does not employ complicated and high-cost components, making it suitable for resource-limited settings. When integrated and commercialized, it will offer simple sample-to-answer molecular diagnostics. Copyright © 2018 Elsevier Inc. All rights reserved.

Can we trust intraoperative culture results in nonunions?

PubMed

Palmer, Michael P; Altman, Daniel T; Altman, Gregory T; Sewecke, Jeffrey J; Ehrlich, Garth D; Hu, Fen Z; Nistico, Laura; Melton-Kreft, Rachel; Gause, Trent M; Costerton, John W

2014-07-01

To identify the presence of bacterial biofilms in nonunions comparing molecular techniques (multiplex polymerase chain reaction and mass spectrometry, fluorescent in situ hybridization) with routine intraoperative cultures. Thirty-four patients with nonunions were scheduled for surgery and enrolled in this ongoing prospective study. Intraoperative specimens were collected from removed implants, surrounding tissue membrane, and local soft tissue followed by standard culture analysis, Ibis's second generation molecular diagnostics (Ibis Biosystems), and bacterial 16S rRNA-based fluorescence in situ hybridization (FISH). Confocal microscopy was used to visualize the tissue specimens reacted with the FISH probes, which were chosen based on the Ibis analysis. Thirty-four patient encounters were analyzed. Eight were diagnosed as infected nonunions by positive intraoperative culture results. Ibis confirmed the presence of bacteria in all 8 samples. Ibis identified bacteria in a total of 30 of 34 encounters, and these data were confirmed by FISH. Twenty-two of 30 Ibis-positive samples were culture-negative. Four samples were negative by all methods of analysis. No samples were positive by culture, but negative by molecular techniques. Our preliminary data indicate that molecular diagnostics are more sensitive for identifying bacteria than cultures in cases of bony nonunion. This is likely because of the inability of cultures to detect biofilms and bacteria previously exposed to antibiotic therapy. Diagnostic Level I. See Instructions for Authors for a complete description of levels of evidence.

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study.

PubMed

Liu, Jie; Kabir, Furqan; Manneh, Jainaba; Lertsethtakarn, Paphavee; Begum, Sharmin; Gratz, Jean; Becker, Steve M; Operario, Darwin J; Taniuchi, Mami; Janaki, Lalitha; Platts-Mills, James A; Haverstick, Doris M; Kabir, Mamun; Sobuz, Shihab U; Nakjarung, Kaewkanya; Sakpaisal, Pimmada; Silapong, Sasikorn; Bodhidatta, Ladaporn; Qureshi, Shahida; Kalam, Adil; Saidi, Queen; Swai, Ndealilia; Mujaga, Buliga; Maro, Athanasia; Kwambana, Brenda; Dione, Michel; Antonio, Martin; Kibiki, Gibson; Mason, Carl J; Haque, Rashidul; Iqbal, Najeeha; Zaidi, Anita K M; Houpt, Eric R

2014-08-01

Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project. Copyright © 2014 Elsevier Ltd. All rights reserved.

Iron deficiency anemia: diagnosis and management.

PubMed

Clark, Susan F

2009-03-01

Iron deficiency anemia (IDA) still remains universally problematic worldwide. The primary focus of this review is to critique articles published over the past 18 months that describe strategies for the diagnosis and management of this prevalent condition. The medical community continues to lack consensus when identifying the optimal approach for the diagnosis and management of IDA. Current diagnostic recommendations revolve around the validity and practicality of current biomarkers such as soluble transferrin-receptor concentrations and others, and cause-based diagnostics that potentially include endoscopy. Management of IDA is based on supplementation combined with effective etiological treatment. Advances in oral and parenteral low-molecular-weight iron preparations has expanded and improved treatment modalities for IDA. Since the introduction of low versus high-molecular-weight intravenous iron administration, there have been fewer serious adverse events associated with parenteral iron preparations. Best practice guidelines for diagnosing and managing IDA should include the design of an algorithm that is inclusive of multiple biomarkers and cause-based diagnostics, which will provide direction in managing IDA, and distinguish between IDA from the anemia of chronic disease.

Progress in the molecular diagnosis of Lyme disease.

PubMed

Ružić-Sabljić, Eva; Cerar, Tjaša

2017-01-01

Current laboratory testing of Lyme borreliosis mostly relies on serological methods with known limitations. Diagnostic modalities enabling direct detection of pathogen at the onset of the clinical signs could overcome some of the limitations. Molecular methods detecting borrelial DNA seem to be the ideal solution, although there are some aspects that need to be considered. Areas covered: This review represent summary and discussion of the published data obtained from literature searches from PubMed and The National Library of Medicine (USA) together with our own experience on molecular diagnosis of Lyme disease. Expert commentary: Molecular methods are promising and currently serve as supporting diagnostic testing in Lyme borreliosis. Since the field of molecular diagnostics is under rapid development, molecular testing could become an important diagnostic modality.

Molecular Diagnostics for Precision Medicine in Colorectal Cancer: Current Status and Future Perspective.

PubMed

Chen, Guoli; Yang, Zhaohai; Eshleman, James R; Netto, George J; Lin, Ming-Tseh

2016-01-01

Precision medicine, a concept that has recently emerged and has been widely discussed, emphasizes tailoring medical care to individuals largely based on information acquired from molecular diagnostic testing. As a vital aspect of precision cancer medicine, targeted therapy has been proven to be efficacious and less toxic for cancer treatment. Colorectal cancer (CRC) is one of the most common cancers and among the leading causes for cancer related deaths in the United States and worldwide. By far, CRC has been one of the most successful examples in the field of precision cancer medicine, applying molecular tests to guide targeted therapy. In this review, we summarize the current guidelines for anti-EGFR therapy, revisit the roles of pathologists in an era of precision cancer medicine, demonstrate the transition from traditional "one test-one drug" assays to multiplex assays, especially by using next-generation sequencing platforms in the clinical diagnostic laboratories, and discuss the future perspectives of tumor heterogeneity associated with anti-EGFR resistance and immune checkpoint blockage therapy in CRC.

Companion diagnostics for the targeted therapy of gastric cancer.

PubMed

Yoo, Changhoon; Park, Young Soo

2015-10-21

Gastric cancer is the fourth most common type of cancer and represents a major cause of cancer-related deaths worldwide. With recent biomedical advances in our understanding of the molecular characteristics of gastric cancer, many genetic alterations have been identified as potential targets for its treatment. Multiple novel agents are currently under development as the demand for active agents that improve the survival of gastric cancer patients constantly increases. Based on lessons from previous trials of targeted agents, it is now widely accepted that the establishment of an optimal diagnostic test to select molecularly defined patients is of equal importance to the development of active agents against targetable genetic alterations. Herein, we highlight the current status and future perspectives of companion diagnostics in the treatment of gastric cancer.

Commercial Molecular Tests for Fungal Diagnosis from a Practical Point of View.

PubMed

Lackner, Michaela; Lass-Flörl, Cornelia

2017-01-01

The increasing interest in molecular diagnostics is a result of tremendously improved knowledge on fungal infections in the past 20 years and the rapid development of new methods, in particular polymerase chain reaction. High expectations have been placed on molecular diagnostics, and the number of laboratories now using the relevant technology is rapidly increasing-resulting in an obvious need for standardization and definition of laboratory organization. In the past 10 years, multiple new molecular tools were marketed for the detection of DNA, antibodies, cell wall components, or other antigens. In contrast to classical culture methods, molecular methods do not detect a viable organisms, but only molecules which indicate its presence; this can be nucleic acids, cell components (antigens), or antibodies (Fig. 1). In this chapter, an overview is provided on commercially available detection tools, their strength and how to use them. A main focus is laid on providing tips and tricks that make daily life easier. We try to focus and mention methodical details which are not highlighted in the manufacturer's instructions of these test kits, but are based on our personal experience in the laboratory. Important to keep in mind is that molecular tools cannot replace culture, microscopy, or a critical view on patients' clinical history, signs, and symptoms, but provide a valuable add on tool. Diagnosis should not be based solely on a molecular test, but molecular tools might deliver an important piece of information that helps matching the diagnostic puzzle to a diagnosis, in particular as few tests are in vitro diagnostic tests (IVD) or only part of the whole test carries the IVD certificate (e.g., DNA extraction is often not included). Please be aware that the authors do not claim to provide a complete overview on all commercially available diagnostic assays being currently marketed for fungal detection, as those are subject to constant change. A main focus is put on commonly used panfungal assays and pathogen-specific assays, including Aspergillus-specific, Candida-specific, Cryptococcus specific, Histoplasma-specific, and Pneumocystis-specific assays. Assays are categorized according to their underlying principle in either antigen-detecting or antibody-detecting or DNA-detecting (Fig. 1). Other non-DNA-detecting nucleic acid methods such as FISH and PNA FISH are not summarized in this chapter and an overview on test performance, common false positives, and the clinical evaluation of commercial tests in studies is provided already in a previous book series by Javier Yugueros Marcos and David H. Pincus (Marcos and Pincus, Methods Mol Biol 968:25-54, 2013).

Genetic testing for patients with renal disease: procedures, pitfalls, and ethical considerations.

PubMed

Korf, B R

1999-07-01

The Human Genome Project is rapidly producing insights into the molecular basis of human genetic disorders. The most immediate clinical benefit is the advent of new diagnostic methods. Molecular diagnostic tools are available for several genetic renal disorders and are in development for many more. Two general approaches to molecular diagnosis are linkage-based testing and direct mutation detection. The former is used when the gene has not been cloned but has been mapped in relation to polymorphic loci. Linkage-based testing is also helpful when a large diversity of mutations makes direct detection difficult. Limitations include the need to study multiple family members, the need for informative polymorphisms, and genetic heterogeneity. Direct mutation detection is limited by genetic heterogeneity and the need to distinguish nonpathogenic allelic variants from pathogenic mutations. Molecular testing raises a number of complex ethical issues, including those associated with prenatal or presymptomatic diagnosis. In addition, there are concerns about informed consent, privacy, genetic discrimination, and technology transfer for newly developed tests. Health professionals need to be aware of the technical and ethical implications of these new methods of testing, as well as the complexities in test interpretation, as molecular approaches are increasingly integrated into medical practice.

Molecular classification of gastric cancer.

PubMed

Röcken, Christoph

2017-03-01

Gastric cancer is among the most common cancers worldwide. Despite declining incidences, the prognosis remains dismal in Western countries and is better in Asian countries with national cancer screening programs. Complete endoscopic or surgical resection of the primary tumor with or without lymphadenectomy offers the only chance of cure in the early stage of the disease. Survival of more locally advanced gastric cancers was improved by the introduction of perioperative, adjuvant and palliative chemotherapy. However, the identification and usage of novel predictive and diagnostic targets is urgently needed. Areas covered: Recent comprehensive molecular profiling of gastric cancer proposed four molecular subtypes, i.e. Epstein-Barr virus-associated, microsatellite instable, chromosomal instable and genomically stable carcinomas. The new molecular classification will spur clinical trials exploring novel targeted therapeutics. This review summarizes recent advancements of the molecular classification, and based on that, putative pitfalls for the development of tissue-based companion diagnostics, i.e. prevalence of actionable targets and therapeutic efficacy, tumor heterogeneity and tumor evolution, impact of ethnicity on gastric cancer biology, and standards of care in the East and West. Expert commentary: The overall low prevalence of actionable targets and tumor heterogeneity are the two main obstacles of precision medicine for gastric cancer.

Point-of-care technologies for molecular diagnostics using a drop of blood.

PubMed

Song, Yujun; Huang, Yu-Yen; Liu, Xuewu; Zhang, Xiaojing; Ferrari, Mauro; Qin, Lidong

2014-03-01

Molecular diagnostics is crucial for prevention, identification, and treatment of disease. Traditional technologies for molecular diagnostics using blood are limited to laboratory use because they rely on sample purification and sophisticated instruments, are labor and time intensive, expensive, and require highly trained operators. This review discusses the frontiers of point-of-care (POC) diagnostic technologies using a drop of blood obtained from a finger prick. These technologies, including emerging biotechnologies, nanotechnologies, and microfluidics, hold the potential for rapid, accurate, and inexpensive disease diagnostics. Copyright © 2014 Elsevier Ltd. All rights reserved.

Field Effect Sensors for Nucleic Acid Detection: Recent Advances and Future Perspectives

PubMed Central

Veigas, Bruno; Fortunato, Elvira; Baptista, Pedro V.

2015-01-01

In the last decade the use of field-effect-based devices has become a basic structural element in a new generation of biosensors that allow label-free DNA analysis. In particular, ion sensitive field effect transistors (FET) are the basis for the development of radical new approaches for the specific detection and characterization of DNA due to FETs’ greater signal-to-noise ratio, fast measurement capabilities, and possibility to be included in portable instrumentation. Reliable molecular characterization of DNA and/or RNA is vital for disease diagnostics and to follow up alterations in gene expression profiles. FET biosensors may become a relevant tool for molecular diagnostics and at point-of-care. The development of these devices and strategies should be carefully designed, as biomolecular recognition and detection events must occur within the Debye length. This limitation is sometimes considered to be fundamental for FET devices and considerable efforts have been made to develop better architectures. Herein we review the use of field effect sensors for nucleic acid detection strategies—from production and functionalization to integration in molecular diagnostics platforms, with special focus on those that have made their way into the diagnostics lab. PMID:25946631

Advancing the education in molecular diagnostics: the IFCC-Initiative "Clinical Molecular Biology Curriculum" (C-CMBC); a ten-year experience.

PubMed

Lianidou, Evi; Ahmad-Nejad, Parviz; Ferreira-Gonzalez, Andrea; Izuhara, Kenji; Cremonesi, Laura; Schroeder, Maria-Eugenia; Richter, Karin; Ferrari, Maurizio; Neumaier, Michael

2014-09-25

Molecular techniques are becoming commonplace in the diagnostic laboratory. Their applications influence all major phases of laboratory medicine including predisposition/genetic risk, primary diagnosis, therapy stratification and prognosis. Readily available laboratory hardware and wetware (i.e. consumables and reagents) foster rapid dissemination to countries that are just establishing molecular testing programs. Appropriate skill levels extending beyond the technical procedure are required for analytical and diagnostic proficiency that is mandatory in molecular genetic testing. An international committee (C-CMBC) of the International Federation for Clinical Chemistry (IFCC) was established to disseminate skills in molecular genetic testing in member countries embarking on the respective techniques. We report the ten-year experience with different teaching and workshop formats for beginners in molecular diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

Lymph Node Metastases Optical Molecular Diagnostic and Radiation Therapy

DTIC Science & Technology

2017-03-01

structures and not molecular functions. The one tool commonly used for metastases imaging is nuclear medicine. Positron emission tomography, PET, is...be visualized at a relevant stage., largely because most imaging is based upon structures and not molecular functions. But there are no tools to...system suitable for imaging signals from in small animals on the standard radiation therapy tools. (3) To evaluate the limits on structural , metabolic

Molecular Diagnostics of Gliomas Using Next Generation Sequencing of a Glioma-Tailored Gene Panel.

PubMed

Zacher, Angela; Kaulich, Kerstin; Stepanow, Stefanie; Wolter, Marietta; Köhrer, Karl; Felsberg, Jörg; Malzkorn, Bastian; Reifenberger, Guido

2017-03-01

Current classification of gliomas is based on histological criteria according to the World Health Organization (WHO) classification of tumors of the central nervous system. Over the past years, characteristic genetic profiles have been identified in various glioma types. These can refine tumor diagnostics and provide important prognostic and predictive information. We report on the establishment and validation of gene panel next generation sequencing (NGS) for the molecular diagnostics of gliomas. We designed a glioma-tailored gene panel covering 660 amplicons derived from 20 genes frequently aberrant in different glioma types. Sensitivity and specificity of glioma gene panel NGS for detection of DNA sequence variants and copy number changes were validated by single gene analyses. NGS-based mutation detection was optimized for application on formalin-fixed paraffin-embedded tissue specimens including small stereotactic biopsy samples. NGS data obtained in a retrospective analysis of 121 gliomas allowed for their molecular classification into distinct biological groups, including (i) isocitrate dehydrogenase gene (IDH) 1 or 2 mutant astrocytic gliomas with frequent α-thalassemia/mental retardation syndrome X-linked (ATRX) and tumor protein p53 (TP53) gene mutations, (ii) IDH mutant oligodendroglial tumors with 1p/19q codeletion, telomerase reverse transcriptase (TERT) promoter mutation and frequent Drosophila homolog of capicua (CIC) gene mutation, as well as (iii) IDH wildtype glioblastomas with frequent TERT promoter mutation, phosphatase and tensin homolog (PTEN) mutation and/or epidermal growth factor receptor (EGFR) amplification. Oligoastrocytic gliomas were genetically assigned to either of these groups. Our findings implicate gene panel NGS as a promising diagnostic technique that may facilitate integrated histological and molecular glioma classification. © 2016 International Society of Neuropathology.

Can we better predict the biologic behavior of incidental IPMN? A comprehensive analysis of molecular diagnostics and biomarkers in intraductal papillary mucinous neoplasms of the pancreas.

PubMed

Tulla, Kiara A; Maker, Ajay V

2018-03-01

Predicting the biologic behavior of intraductal papillary mucinous neoplasm (IPMN) remains challenging. Current guidelines utilize patient symptoms and imaging characteristics to determine appropriate surgical candidates. However, the majority of resected cysts remain low-risk lesions, many of which may be feasible to have under surveillance. We herein characterize the most promising and up-to-date molecular diagnostics in order to identify optimal components of a molecular signature to distinguish levels of IPMN dysplasia. A comprehensive systematic review of pertinent literature, including our own experience, was conducted based on the PRISMA guidelines. Molecular diagnostics in IPMN patient tissue, duodenal secretions, cyst fluid, saliva, and serum were evaluated and organized into the following categories: oncogenes, tumor suppressor genes, glycoproteins, markers of the immune response, proteomics, DNA/RNA mutations, and next-generation sequencing/microRNA. Specific targets in each of these categories, and in aggregate, were identified by their ability to both characterize a cyst as an IPMN and determine the level of cyst dysplasia. Combining molecular signatures with clinical and imaging features in this era of next-generation sequencing and advanced computational analysis will enable enhanced sensitivity and specificity of current models to predict the biologic behavior of IPMN.

GENOSENSE Diagnostics GmbH.

PubMed

Schneeberger, Christian

2004-07-01

GENOSENSE Diagnostics GmbH, a company specialized in preventive genetic diagnostics, has committed itself to applying molecular medical knowledge to realizing the vision of individual, preventive and patient-tailored medicine. GENOSENSE offers a unique line of preventive genomic diagnostic profiles. Each profile focuses on a carefully selected set of polymorphisms associated with particular diseases or physiologic imbalances. GENOSENSE does not only provide the genetic test results, but highly capable medical experts 'translate' the results into a clinical language and assist the customer with established support regarding their medical interpretation. In addition, the company provides academic institutions and pharmaceutical companies with turnkey solutions for research-based projects.

Standing of nucleic acid testing strategies in veterinary diagnosis laboratories to uncover Mycobacterium tuberculosis complex members

PubMed Central

Costa, Pedro; Botelho, Ana; Couto, Isabel; Viveiros, Miguel; Inácio, João

2014-01-01

Nucleic acid testing (NAT) designate any molecular approach used for the detection, identification, and characterization of pathogenic microorganisms, enabling the rapid, specific, and sensitive diagnostic of infectious diseases, such as tuberculosis. These assays have been widely used since the 90s of the last century in human clinical laboratories and, subsequently, also in veterinary diagnostics. Most NAT strategies are based in the polymerase chain reaction (PCR) and its several enhancements and variations. From the conventional PCR, real-time PCR and its combinations, isothermal DNA amplification, to the nanotechnologies, here we review how the NAT assays have been applied to decipher if and which member of the Mycobacterium tuberculosis complex is present in a clinical sample. Recent advances in DNA sequencing also brought new challenges and have made possible to generate rapidly and at a low cost, large amounts of sequence data. This revolution with the high-throughput sequencing (HTS) technologies makes whole genome sequencing (WGS) and metagenomics the trendiest NAT strategies, today. The ranking of NAT techniques in the field of clinical diagnostics is rising, and we provide a SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis with our view of the use of molecular diagnostics for detecting tuberculosis in veterinary laboratories, notwithstanding the gold standard being still the classical culture of the agent. The complementary use of both classical and molecular diagnostics approaches is recommended to speed the diagnostic, enabling a fast decision by competent authorities and rapid tackling of the disease. PMID:25988157

Standing of nucleic acid testing strategies in veterinary diagnosis laboratories to uncover Mycobacterium tuberculosis complex members.

PubMed

Costa, Pedro; Botelho, Ana; Couto, Isabel; Viveiros, Miguel; Inácio, João

2014-01-01

Nucleic acid testing (NAT) designate any molecular approach used for the detection, identification, and characterization of pathogenic microorganisms, enabling the rapid, specific, and sensitive diagnostic of infectious diseases, such as tuberculosis. These assays have been widely used since the 90s of the last century in human clinical laboratories and, subsequently, also in veterinary diagnostics. Most NAT strategies are based in the polymerase chain reaction (PCR) and its several enhancements and variations. From the conventional PCR, real-time PCR and its combinations, isothermal DNA amplification, to the nanotechnologies, here we review how the NAT assays have been applied to decipher if and which member of the Mycobacterium tuberculosis complex is present in a clinical sample. Recent advances in DNA sequencing also brought new challenges and have made possible to generate rapidly and at a low cost, large amounts of sequence data. This revolution with the high-throughput sequencing (HTS) technologies makes whole genome sequencing (WGS) and metagenomics the trendiest NAT strategies, today. The ranking of NAT techniques in the field of clinical diagnostics is rising, and we provide a SWOT (Strengths, Weaknesses, Opportunities, and Threats) analysis with our view of the use of molecular diagnostics for detecting tuberculosis in veterinary laboratories, notwithstanding the gold standard being still the classical culture of the agent. The complementary use of both classical and molecular diagnostics approaches is recommended to speed the diagnostic, enabling a fast decision by competent authorities and rapid tackling of the disease.

The Role of Liquid Based Cytology and Ancillary Techniques in the Peritoneal Washing Analysis: Our Institutional Experience.

PubMed

Rossi, Esther; Bizzarro, Tommaso; Martini, Maurizio; Longatto-Filho, Adhemar; Schmitt, Fernando; Fagotti, Anna; Scambia, Giovanni; Zannoni, Gian Franco

2017-01-01

The cytological analysis of peritoneal effusions serves as a diagnostic and prognostic aid for either primary or metastatic diseases. Among the different cytological preparations, liquid based cytology (LBC) represents a feasible and reliable method ensuring also the application of ancillary techniques (i.e immunocytochemistry-ICC and molecular testing). We recorded 10348 LBC peritoneal effusions between January 2000 and December 2014. They were classified as non-diagnostic (ND), negative for malignancy-NM, atypical-suspicious for malignancy-SM and positive for malignancy-PM. The cytological diagnosis included 218 ND, 9.035 NM, 213 SM and 882 PM. A total of 8048 (7228 NM, 115SM, 705 PM) cases with histological follow-up were included. Our NM included 21 malignant and 7207 benign histological diagnoses. Our 820 SMs+PMs were diagnosed as 107 unknown malignancies (30SM and 77PM), 691 metastatic lesions (81SM and 610PM), 9 lymphomas (2SM and 7PM), 9 mesotheliomas (1SM and 8SM), 4 sarcomas (1SM and 3PM). Primary gynecological cancers contributed with 64% of the cases. We documented 97.4% sensitivity, 99.9% specificity, 98% diagnostic accuracy, 99.7% negative predictive value (NPV) and 99.7% positive predictive value (PPV). Furthermore, the morphological diagnoses were supported by either 173 conclusive ICC results or 50 molecular analyses. Specifically the molecular testing was performed for the EGFR and KRAS mutational analysis based on the previous or contemporary diagnoses of Non Small Cell Lung Cancer (NSCLC) and colon carcinomas. We identified 10 EGFR in NSCCL and 7 KRAS mutations on LBC stored material. Peritoneal cytology is an adjunctive tool in the surgical management of tumors mostly gynecological cancers. LBC maximizes the application of ancillary techniques such as ICC and molecular analysis with feasible diagnostic and predictive yields also in controversial cases.

The Role of Liquid Based Cytology and Ancillary Techniques in the Peritoneal Washing Analysis: Our Institutional Experience

PubMed Central

Rossi, Esther; Bizzarro, Tommaso; Martini, Maurizio; Longatto-Filho, Adhemar; Schmitt, Fernando; Fagotti, Anna; Scambia, Giovanni; Zannoni, Gian Franco

2017-01-01

Background The cytological analysis of peritoneal effusions serves as a diagnostic and prognostic aid for either primary or metastatic diseases. Among the different cytological preparations, liquid based cytology (LBC) represents a feasible and reliable method ensuring also the application of ancillary techniques (i.e immunocytochemistry-ICC and molecular testing). Methods We recorded 10348 LBC peritoneal effusions between January 2000 and December 2014. They were classified as non-diagnostic (ND), negative for malignancy-NM, atypical-suspicious for malignancy-SM and positive for malignancy-PM. Results The cytological diagnosis included 218 ND, 9.035 NM, 213 SM and 882 PM. A total of 8048 (7228 NM, 115SM, 705 PM) cases with histological follow-up were included. Our NM included 21 malignant and 7207 benign histological diagnoses. Our 820 SMs+PMs were diagnosed as 107 unknown malignancies (30SM and 77PM), 691 metastatic lesions (81SM and 610PM), 9 lymphomas (2SM and 7PM), 9 mesotheliomas (1SM and 8SM), 4 sarcomas (1SM and 3PM). Primary gynecological cancers contributed with 64% of the cases. We documented 97.4% sensitivity, 99.9% specificity, 98% diagnostic accuracy, 99.7% negative predictive value (NPV) and 99.7% positive predictive value (PPV). Furthermore, the morphological diagnoses were supported by either 173 conclusive ICC results or 50 molecular analyses. Specifically the molecular testing was performed for the EGFR and KRAS mutational analysis based on the previous or contemporary diagnoses of Non Small Cell Lung Cancer (NSCLC) and colon carcinomas. We identified 10 EGFR in NSCCL and 7 KRAS mutations on LBC stored material. Conclusions Peritoneal cytology is an adjunctive tool in the surgical management of tumors mostly gynecological cancers. LBC maximizes the application of ancillary techniques such as ICC and molecular analysis with feasible diagnostic and predictive yields also in controversial cases. PMID:28099523

Isolation of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays - Application in Aerospace Medicine

DTIC Science & Technology

2004-01-01

of RNA From Peripheral Blood Cells: A Validation Study for Molecular Diagnostics by Microarray and Kinetic RT-PCR Assays  Application in...VALIDATION STUDY FOR MOLECULAR DIAGNOSTICS BY MICROARRAY AND KINETIC RT-PCR ASSAYS  APPLICATION IN AEROSPACE MEDICINE INTRODUCTION Extraction of cellular

The role of molecular diagnostic testing in the management of thyroid nodules.

PubMed

Moore, Maureen D; Panjwani, Suraj; Gray, Katherine D; Finnerty, Brendan M; Zarnegar, Rasa; Fahey, Thomas J

2017-06-01

Fine needle aspiration (FNA) with cytologic examination remains the standard of care for investigation of thyroid nodules. However, as many as 30% of FNA samples are cytologically indeterminate for malignancy, which confounds clinical management. To reduce the burden of repeat diagnostic testing and unnecessary surgery, there has been extensive investigation into molecular markers that can be detected on FNA specimens to more accurately stratify a patient's risk of malignancy. Areas covered: In this review, the authors discuss recent evidence and progress in molecular markers used in the diagnosis of thyroid cancer highlighting somatic gene alterations, molecular technologies and microRNA analysis. Expert commentary: The goal of molecular markers is to improve diagnostic accuracy and aid clinicians in the preoperative management of thyroid lesions. Modalities such as direct mutation analysis, mRNA gene expression profiling, next-generation sequencing, and miRNA expression profiling have been explored to improve the diagnostic accuracy of thyroid nodule FNA. Although no perfect test has been discovered, molecular diagnostic testing has revolutionized the management of thyroid nodules.

Plasmodium vivax molecular diagnostics in community surveys: pitfalls and solutions.

PubMed

Gruenberg, Maria; Moniz, Clara Antunes; Hofmann, Natalie Ellen; Wampfler, Rahel; Koepfli, Cristian; Mueller, Ivo; Monteiro, Wuelton Marcelo; Lacerda, Marcus; de Melo, Gisely Cardoso; Kuehn, Andrea; Siqueira, Andre M; Felger, Ingrid

2018-01-30

A distinctive feature of Plasmodium vivax infections is the overall low parasite density in peripheral blood. Thus, identifying asymptomatic infected individuals in endemic communities requires diagnostic tests with high sensitivity. The detection limits of molecular diagnostic tests are primarily defined by the volume of blood analysed and by the copy number of the amplified molecular marker serving as the template for amplification. By using mitochondrial DNA as the multi-copy template, the detection limit can be improved more than tenfold, compared to standard 18S rRNA targets, thereby allowing detection of lower parasite densities. In a very low transmission area in Brazil, application of a mitochondrial DNA-based assay increased prevalence from 4.9 to 6.5%. The usefulness of molecular tests in malaria epidemiological studies is widely recognized, especially when precise prevalence rates are desired. Of concern, however, is the challenge of demonstrating test accuracy and quality control for samples with very low parasite densities. In this case, chance effects in template distribution around the detection limit constrain reproducibility. Rigorous assessment of false positive and false negative test results is, therefore, required to prevent over- or under-estimation of parasite prevalence in epidemiological studies or when monitoring interventions.

[Application of molecular diagnostic techniques in precision medicine of personalized treatment for colorectal cancer].

PubMed

Fu, Ji; Lin, Guole

2016-01-01

Precision medicine is to customize the treatment options for individual patient based on the personal genome information. Colorectal cancer (CRC) is one of the most common cancer worldwide. Molecular heterogeneity of CRC, which includes the MSI phenotype, hypermutation phenotype, and their relationship with clinical preferences, is believed to be one of the main factors responsible for the considerable variability in treatment response. The development of powerful next-generation sequencing (NGS) technologies allows us to further understand the biological behavior of colorectal cancer, and to analyze the prognosis and chemotherapeutic drug reactions by molecular diagnostic techniques, which can guide the clinical treatment. This paper will introduce the new findings in this field. Meanwhile we integrate the new progress of key pathways including EGFR, RAS, PI3K/AKT and VEGF, and the experience in selective patients through associated molecular diagnostic screening who gain better efficacy after target therapy. The technique for detecting circulating tumor DNA (ctDNA) is introduced here as well, which can identify patients with high risk for recurrence, and demonstrate the risk of chemotherapy resistance. Mechanism of tumor drug resistance may be revealed by dynamic observation of gene alteration during treatment.

Accelerating the Development and Validation of New Value-Based Diagnostics by Leveraging Biobanks.

PubMed

Schneider, Daniel; Riegman, Peter H J; Cronin, Maureen; Negrouk, Anastassia; Moch, Holger; Balling, Rudi; Penault-Llorca, Frederiques; Zatloukal, Kurt; Horgan, Denis

The challenges faced in developing value-based diagnostics has resulted in few of these tests reaching the clinic, leaving many treatment modalities without matching diagnostics to select patients for particular therapies. Many patients receive therapies from which they are unlikely to benefit, resulting in worse outcomes and wasted health care resources. The paucity of value-based diagnostics is a result of the scientific challenges in developing predictive markers, specifically: (1) complex biology, (2) a limited research infrastructure supporting diagnostic development, and (3) the lack of incentives for diagnostic developers to invest the necessary resources. Better access to biospecimens can address some of these challenges. Methodologies developed to evaluate biomarkers from biospecimens archived from patients enrolled in randomized clinical trials offer the greatest opportunity to develop and validate high-value molecular diagnostics. An alternative opportunity is to access high-quality biospecimens collected from large public and private longitudinal observational cohorts such as the UK Biobank, the US Million Veteran Program, the UK 100,000 Genomes Project, or the French E3N cohort. Value-based diagnostics can be developed to work in a range of samples including blood, serum, plasma, urine, and tumour tissue, and better access to these high-quality biospecimens with clinical data can facilitate biomarker research. © 2016 S. Karger AG, Basel.

Multivariate Analysis As a Support for Diagnostic Flowcharts in Allergic Bronchopulmonary Aspergillosis: A Proof-of-Concept Study.

PubMed

Vitte, Joana; Ranque, Stéphane; Carsin, Ania; Gomez, Carine; Romain, Thomas; Cassagne, Carole; Gouitaa, Marion; Baravalle-Einaudi, Mélisande; Bel, Nathalie Stremler-Le; Reynaud-Gaubert, Martine; Dubus, Jean-Christophe; Mège, Jean-Louis; Gaudart, Jean

2017-01-01

Molecular-based allergy diagnosis yields multiple biomarker datasets. The classical diagnostic score for allergic bronchopulmonary aspergillosis (ABPA), a severe disease usually occurring in asthmatic patients and people with cystic fibrosis, comprises succinct immunological criteria formulated in 1977: total IgE, anti- Aspergillus fumigatus ( Af ) IgE, anti- Af "precipitins," and anti- Af IgG. Progress achieved over the last four decades led to multiple IgE and IgG(4) Af biomarkers available with quantitative, standardized, molecular-level reports. These newly available biomarkers have not been included in the current diagnostic criteria, either individually or in algorithms, despite persistent underdiagnosis of ABPA. Large numbers of individual biomarkers may hinder their use in clinical practice. Conversely, multivariate analysis using new tools may bring about a better chance of less diagnostic mistakes. We report here a proof-of-concept work consisting of a three-step multivariate analysis of Af IgE, IgG, and IgG4 biomarkers through a combination of principal component analysis, hierarchical ascendant classification, and classification and regression tree multivariate analysis. The resulting diagnostic algorithms might show the way for novel criteria and improved diagnostic efficiency in Af -sensitized patients at risk for ABPA.

Elucidating the Mechanism of p27 Inactivation by the Bcr-Abl Tyrosine Kinase

DTIC Science & Technology

2005-10-01

Grants 2002- Leukemia Research Fund Membership in Scientific Committees 2000- Molecular diagnostics (German competence network acute...transplantation, including reduced intensity conditioning. Hematological Diagnostics Standard morphology. Supervised molecular diagnostics in the

Benchmarking outcomes in the Neonatal Intensive Care Unit: Cytogenetic and molecular diagnostic rates in a retrospective cohort.

PubMed

Malam, Faheem; Hartley, Taila; Gillespie, Meredith K; Armour, Christine M; Bariciak, Erika; Graham, Gail E; Nikkel, Sarah M; Richer, Julie; Sawyer, Sarah L; Boycott, Kym M; Dyment, David A

2017-05-09

Genetic disease and congenital anomalies continue to be a leading cause of neonate mortality and morbidity. A genetic diagnosis in the neonatal intensive care unit (NICU) can be a challenge given the associated genetic heterogeneity and early stage of a disease. We set out to evaluate the outcomes of Medical Genetics consultation in the NICU in terms of cytogenetic and molecular diagnostic rates and impact on management. We retrospectively reviewed 132 charts from patients admitted to the NICU who received a Medical Genetics diagnostic evaluation over a 2 year period. Of the 132 patients reviewed, 26% (34/132) received a cytogenetic or molecular diagnosis based on the Medical Genetics diagnostic evaluation; only 10% (13/132) received a diagnosis during their admission. The additional 16% (21 patients) received their diagnosis following NICU discharge, but based on a genetic test initiated during hospital-stay. Mean time from NICU admission to confirmed diagnosis was 24 days. For those who received a genetic diagnosis, the information was considered beneficial for clinical management in all, and a direct change to medical management occurred for 12% (4/32). For those non-diagnosed infants seen in out-patient follow-up clinic, diagnoses were made in 8% (3/37). The diagnoses made post-discharge from the NICU comprised a greater number of Mendelian disorders and represent an opportunity to improve genetic care. The adoption of diagnostic tools, such as exome sequencing, used in parallel with traditional approaches will improve rate of diagnoses and will have a significant impact, in particular when the differential diagnosis is broad. © 2017 Wiley Periodicals, Inc.

The role of liquid-based cytology and ancillary techniques in pleural and pericardic effusions: an institutional experience.

PubMed

Rossi, Esther Diana; Bizzarro, Tommaso; Schmitt, Fernando; Longatto-Filho, Adhemar

2015-04-01

Fine-needle aspiration cytology (FNAC) of serous membrane effusions may fulfil a challenging role in the diagnostic analysis of both primary and metastatic disease. From this perspective, liquid-based cytology (LBC) represents a feasible and reliable method for empowering the performance of ancillary techniques (ie, immunocytochemistry and molecular testing) with high diagnostic accuracy. In total, 3171 LBC pleural and pericardic effusions were appraised between January 2000 and December 2013. They were classified as negative for malignancy (NM), suspicious for malignancy (SM), or positive for malignancy (PM). The cytologic diagnoses included 2721 NM effusions (2505 pleural and 216 pericardic), 104 SM effusions (93 pleural and 11 pericardic), and 346 PM effusions (321 pleural and 25 pericardic). The malignant pleural series included 76 unknown malignancies (36 SM and 40 PM effusions), 174 metastatic lesions (85 SM and 89 PM effusions), 14 lymphomas (3 SM and 11 PM effusions), 16 mesotheliomas (5 SM and 11 SM effusions), and 3 myelomas (all SM effusions). The malignant pericardic category included 20 unknown malignancies (5 SM and 15 PM effusions), 15 metastatic lesions (1 SM and 14 PM effusions), and 1 lymphoma (1 PM effusion). There were 411 conclusive immunocytochemical analyses and 47 molecular analyses, and the authors documented 88% sensitivity, 100% specificity, 98% diagnostic accuracy, 98% negative predictive value, and 100% positive predictive value for FNAC. FNAC represents a primary diagnostic tool for effusions and a reliable approach with which to determine the correct follow-up. Furthermore, LBC is useful for ancillary techniques, such as immunocytochemistry and molecular analysis, with feasible diagnostic and predictive utility. © 2015 American Cancer Society.

Disruption of Fibroblast Growth Factor Receptor (FGFR) Signaling as an Approach to Prostate Cancer Therapy

DTIC Science & Technology

2006-04-01

and the abstract was published in the Journal of Molecular Diagnostics . The full length manuscript entitled “Increased expression and activity of...Recurrence. The Journal of Molecular Diagnostics 2004, (6)4: 431. Ozen M, Dixit S and Ittmann M: Molecular profiling of differentially expressed genes...in response to FGFR disruption in prostate cancer. the Journal of Molecular Diagnostics 7 (5): 680, 2005. Full Articles: 1. Ozen M and Ittmann M

Intellectual property considerations for molecular diagnostic development with emphasis on companion diagnostics.

PubMed

Glorikian, Harry; Warburg, Richard Jeremy; Moore, Kelly; Malinowski, Jennifer

2018-02-01

The development of molecular diagnostics is a complex endeavor, with multiple regulatory pathways to consider and numerous approaches to development and commercialization. Companion diagnostics, devices which are "essential for the safe and effective use of a corresponding drug or diagnostic product" (see U.S. Food & Drug Administration, In Vitro Diagnostics - Companion Diagnostics, U.S. Dept. of Health & Human Services(2016), available at https://www.fda.gov/medicaldevices/productsandmedicalprocedures/invitrodiagnostics/ucm407297.htm ) and complementary diagnostics, which are more broadly associated with a class of drug, are becoming increasingly important as integral components of the implementation of precision medicine. Areas covered: The following article will highlight the intellectual property ('IP') considerations pertinent to molecular diagnostics development with special emphasis on companion diagnostics. Expert opinion/commentary Summary: For all molecular diagnostics, intellectual property (IP) concerns are of paramount concern, whether the device will be marketed only in the United States or abroad. Taking steps to protect IP at each stage of product development is critical to optimize profitability of a diagnostic product. Also the legal framework around IP protection of diagnostic technologies has been changing over the previous few years and can be expected to continue to change in the foreseeable near future, thus, a comprehensive IP strategy should take into account the fact that changes in the law can be expected.

2012 HIV Diagnostics Conference: the molecular diagnostics perspective.

PubMed

Branson, Bernard M; Pandori, Mark

2013-04-01

2012 HIV Diagnostic Conference Atlanta, GA, USA, 12-14 December 2012. This report highlights the presentations and discussions from the 2012 National HIV Diagnostic Conference held in Atlanta (GA, USA), on 12-14 December 2012. Reflecting changes in the evolving field of HIV diagnostics, the conference provided a forum for evaluating developments in molecular diagnostics and their role in HIV diagnosis. In 2010, the HIV Diagnostics Conference concluded with the proposal of a new diagnostic algorithm which included nucleic acid testing to resolve discordant screening and supplemental antibody test results. The 2012 meeting, picking up where the 2010 meeting left off, focused on scientific presentations that assessed this new algorithm and the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1 RNA.

Radiation biodosimetry: Applications for spaceflight

NASA Astrophysics Data System (ADS)

Blakely, W. F.; Miller, A. C.; Grace, M. B.; McLeland, C. B.; Luo, L.; Muderhwa, J. M.; Miner, V. L.; Prasanna, P. G. S.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Beckon Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.

dropEst: pipeline for accurate estimation of molecular counts in droplet-based single-cell RNA-seq experiments.

PubMed

Petukhov, Viktor; Guo, Jimin; Baryawno, Ninib; Severe, Nicolas; Scadden, David T; Samsonova, Maria G; Kharchenko, Peter V

2018-06-19

Recent single-cell RNA-seq protocols based on droplet microfluidics use massively multiplexed barcoding to enable simultaneous measurements of transcriptomes for thousands of individual cells. The increasing complexity of such data creates challenges for subsequent computational processing and troubleshooting of these experiments, with few software options currently available. Here, we describe a flexible pipeline for processing droplet-based transcriptome data that implements barcode corrections, classification of cell quality, and diagnostic information about the droplet libraries. We introduce advanced methods for correcting composition bias and sequencing errors affecting cellular and molecular barcodes to provide more accurate estimates of molecular counts in individual cells.

Plasmonic SERS nanochips and nanoprobes for medical diagnostics and bio-energy applications

NASA Astrophysics Data System (ADS)

Ngo, Hoan T.; Wang, Hsin-Neng; Crawford, Bridget M.; Fales, Andrew M.; Vo-Dinh, Tuan

2017-02-01

The development of rapid, easy-to-use, cost-effective, high accuracy, and high sensitive DNA detection methods for molecular diagnostics has been receiving increasing interest. Over the last five years, our laboratory has developed several chip-based DNA detection techniques including the molecular sentinel-on-chip (MSC), the multiplex MSC, and the inverse molecular sentinel-on-chip (iMS-on-Chip). In these techniques, plasmonic surface-enhanced Raman scattering (SERS) Nanowave chips were functionalized with DNA probes for single-step DNA detection. Sensing mechanisms were based on hybridization of target sequences and DNA probes, resulting in a distance change between SERS reporters and the Nanowave chip's gold surface. This distance change resulted in change in SERS intensity, thus indicating the presence and capture of the target sequences. Our techniques were single-step DNA detection techniques. Target sequences were detected by simple delivery of sample solutions onto DNA probe-functionalized Nanowave chips and SERS signals were measured after 1h - 2h incubation. Target sequence labeling or washing to remove unreacted components was not required, making the techniques simple, easy-to-use, and cost effective. The usefulness of the techniques for medical diagnostics was illustrated by the detection of genetic biomarkers for respiratory viral infection and of dengue virus 4 DNA.

Stationary microfluidics: molecular diagnostic assays by moving magnetic beads through non-moving liquids

NASA Astrophysics Data System (ADS)

Becker, Holger; Carstens, Cornelia; Kuhlmeier, Dirk; Sandetskaya, Natalia; Schröter, Nicole; Zilch, Christian; Gärtner, Claudia

2013-03-01

Commonly, microfluidic devices are based on the movement of fluids. For molecular diagnostics assays which often include steps like PCR, this practically always involves a more or less complicated set of external pumps, valves and liquid controls. In the presented paper, we follow a different approach in which the fluid after sample introduction remains stationary and the main bioactive sample molecules are moved through a chain of reaction compartments which contain the different reagents necessary for the assay. The big advantage of this concept is the lack of any external fluid actuation/control. Results on sample carry-over experiments and complete assays will be given.

Bench-to-bedside review: Rapid molecular diagnostics for bloodstream infection - a new frontier?

PubMed Central

2012-01-01

Among critically ill patients, the diagnosis of bloodstream infection poses a major challenge. Current standard bacterial identification based on blood culture platforms is intrinsically time-consuming and slow. The continuous evolvement of molecular techniques has the potential of providing a faster, more sensitive and direct identification of causative pathogens without prior need for cultivation. This may ultimately impact clinical decision-making and antimicrobial treatment. This review summarises the currently available technologies, their strengths and limitations and the obstacles that have to be overcome in order to develop a satisfactory bedside point-of-care diagnostic tool for detection of bloodstream infection. PMID:22647543

Current molecular and emerging nanobiotechnology approaches for the detection of microbial pathogens.

PubMed

Theron, Jacques; Eugene Cloete, Thomas; de Kwaadsteniet, Michele

2010-11-01

Waterborne microbial diseases are escalating worldwide increasing the need for powerful and sensitive diagnostics tools. Molecular methodologies, including immunological and nucleic acid-based methods, have only recently been applied in the water sector. Advances in nanotechnology and nanomaterials have opened the door for the development of new diagnostic tools with increased sensitivity and speed, and reduced cost and labor. Quantum dots, flo dots, gold nanoparticles, magnetic nanoparticles, carbon nanotubes, nanowires, and nanocantilevers, with their unique optical and physical properties, have already been applied in nanodiagnostics. Nanobiotechnology, once remaining technical and practical problems has been addressed, will play an important role in the detection of microbial pathogens.

Molecular diagnostic testing for primary biliary cholangitis.

PubMed

Gatselis, Nikolaos K; Dalekos, George N

2016-09-01

A reliable liver autoimmune serology for the diagnosis of primary biliary cholangitis (PBC) is of particular importance. Recognition of patients at early stages and prompt treatment initiation may alter the outcome, slow progression, delays liver failure, and improves survival. In this review, we summarize and discuss the published data obtained from literature searches from PubMed and The National Library of Medicine (USA) and our own experience on the current and potential molecular based approaches to the diagnosis of PBC. Expert commentary: Standardization of liver diagnostic serology and clinical governance are two major points as antimitochondrial antibodies are the diagnostic hallmark of the disease and PBC-specific antinuclear antibodies could assist in the diagnosis and estimation of prognosis. New biomarkers such as novel autoantibodies, genetic polymorphisms, metabolomic profiling, micro-RNA and epigenetics may assist to the understanding, diagnosis and management of the disease.

Precision medicine based on epigenomics: the paradigm of carcinoma of unknown primary.

PubMed

Moran, Sebastián; Martinez-Cardús, Anna; Boussios, Stergios; Esteller, Manel

2017-11-01

Epigenetic alterations are a common hallmark of human cancer. Single epigenetic markers are starting to be incorporated into clinical practice; however, the translational use of these biomarkers has not been validated at the 'omics' level. The identification of the tissue of origin in patients with cancer of unknown primary (CUP) is an example of how epigenomics can be incorporated in clinical settings, addressing an unmet need in the diagnostic and clinical management of these patients. Despite the great diagnostic advances made in the past decade, the use of traditional diagnostic procedures only enables the tissue of origin to be determined in ∼30% of patients with CUP. Thus, development of molecularly guided diagnostic strategies has emerged to complement traditional procedures, thereby improving the clinical management of patients with CUP. In this Review, we present the latest data on strategies using epigenetics and other molecular biomarkers to guide therapeutic decisions involving patients with CUP, and we highlight areas warranting further research to engage the medical community in this unmet need.

Massively Parallel, Molecular Analysis Platform Developed Using a CMOS Integrated Circuit With Biological Nanopores

PubMed Central

Roever, Stefan

2012-01-01

A massively parallel, low cost molecular analysis platform will dramatically change the nature of protein, molecular and genomics research, DNA sequencing, and ultimately, molecular diagnostics. An integrated circuit (IC) with 264 sensors was fabricated using standard CMOS semiconductor processing technology. Each of these sensors is individually controlled with precision analog circuitry and is capable of single molecule measurements. Under electronic and software control, the IC was used to demonstrate the feasibility of creating and detecting lipid bilayers and biological nanopores using wild type α-hemolysin. The ability to dynamically create bilayers over each of the sensors will greatly accelerate pore development and pore mutation analysis. In addition, the noise performance of the IC was measured to be 30fA(rms). With this noise performance, single base detection of DNA was demonstrated using α-hemolysin. The data shows that a single molecule, electrical detection platform using biological nanopores can be operationalized and can ultimately scale to millions of sensors. Such a massively parallel platform will revolutionize molecular analysis and will completely change the field of molecular diagnostics in the future.

Are the classic diagnostic methods in mycology still state of the art?

PubMed

Wiegand, Cornelia; Bauer, Andrea; Brasch, Jochen; Nenoff, Pietro; Schaller, Martin; Mayser, Peter; Hipler, Uta-Christina; Elsner, Peter

2016-05-01

The diagnostic workup of cutaneous fungal infections is traditionally based on microscopic KOH preparations as well as culturing of the causative organism from sample material. Another possible option is the detection of fungal elements by dermatohistology. If performed correctly, these methods are generally suitable for the diagnosis of mycoses. However, the advent of personalized medicine and the tasks arising therefrom require new procedures marked by simplicity, specificity, and swiftness. The additional use of DNA-based molecular techniques further enhances sensitivity and diagnostic specificity, and reduces the diagnostic interval to 24-48 hours, compared to weeks required for conventional mycological methods. Given the steady evolution in the field of personalized medicine, simple analytical PCR-based systems are conceivable, which allow for instant diagnosis of dermatophytes in the dermatology office (point-of-care tests). © 2016 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.

Companion diagnostics and molecular imaging-enhanced approaches for oncology clinical trials.

PubMed

Van Heertum, Ronald L; Scarimbolo, Robert; Ford, Robert; Berdougo, Eli; O'Neal, Michael

2015-01-01

In the era of personalized medicine, diagnostic approaches are helping pharmaceutical and biotechnology sponsors streamline the clinical trial process. Molecular assays and diagnostic imaging are routinely being used to stratify patients for treatment, monitor disease, and provide reliable early clinical phase assessments. The importance of diagnostic approaches in drug development is highlighted by the rapidly expanding global cancer diagnostics market and the emergent attention of regulatory agencies worldwide, who are beginning to offer more structured platforms and guidance for this area. In this paper, we highlight the key benefits of using companion diagnostics and diagnostic imaging with a focus on oncology clinical trials. Nuclear imaging using widely available radiopharmaceuticals in conjunction with molecular imaging of oncology targets has opened the door to more accurate disease assessment and the modernization of standard criteria for the evaluation, staging, and treatment responses of cancer patients. Furthermore, the introduction and validation of quantitative molecular imaging continues to drive and optimize the field of oncology diagnostics. Given their pivotal role in disease assessment and treatment, the validation and commercialization of diagnostic tools will continue to advance oncology clinical trials, support new oncology drugs, and promote better patient outcomes.

Implementation of Cloud based next generation sequencing data analysis in a clinical laboratory.

PubMed

Onsongo, Getiria; Erdmann, Jesse; Spears, Michael D; Chilton, John; Beckman, Kenneth B; Hauge, Adam; Yohe, Sophia; Schomaker, Matthew; Bower, Matthew; Silverstein, Kevin A T; Thyagarajan, Bharat

2014-05-23

The introduction of next generation sequencing (NGS) has revolutionized molecular diagnostics, though several challenges remain limiting the widespread adoption of NGS testing into clinical practice. One such difficulty includes the development of a robust bioinformatics pipeline that can handle the volume of data generated by high-throughput sequencing in a cost-effective manner. Analysis of sequencing data typically requires a substantial level of computing power that is often cost-prohibitive to most clinical diagnostics laboratories. To address this challenge, our institution has developed a Galaxy-based data analysis pipeline which relies on a web-based, cloud-computing infrastructure to process NGS data and identify genetic variants. It provides additional flexibility, needed to control storage costs, resulting in a pipeline that is cost-effective on a per-sample basis. It does not require the usage of EBS disk to run a sample. We demonstrate the validation and feasibility of implementing this bioinformatics pipeline in a molecular diagnostics laboratory. Four samples were analyzed in duplicate pairs and showed 100% concordance in mutations identified. This pipeline is currently being used in the clinic and all identified pathogenic variants confirmed using Sanger sequencing further validating the software.

NanoString, a novel digital color-coded barcode technology: current and future applications in molecular diagnostics.

PubMed

Tsang, Hin-Fung; Xue, Vivian Weiwen; Koh, Su-Pin; Chiu, Ya-Ming; Ng, Lawrence Po-Wah; Wong, Sze-Chuen Cesar

2017-01-01

Formalin-fixed, paraffin-embedded (FFPE) tissue sample is a gold mine of resources for molecular diagnosis and retrospective clinical studies. Although molecular technologies have expanded the range of mutations identified in FFPE samples, the applications of existing technologies are limited by the low nucleic acids yield and poor extraction quality. As a result, the routine clinical applications of molecular diagnosis using FFPE samples has been associated with many practical challenges. NanoString technologies utilize a novel digital color-coded barcode technology based on direct multiplexed measurement of gene expression and offer high levels of precision and sensitivity. Each color-coded barcode is attached to a single target-specific probe corresponding to a single gene which can be individually counted without amplification. Therefore, NanoString is especially useful for measuring gene expression in degraded clinical specimens. Areas covered: This article describes the applications of NanoString technologies in molecular diagnostics and challenges associated with its applications and the future development. Expert commentary: Although NanoString technology is still in the early stages of clinical use, it is expected that NanoString-based cancer expression panels would play more important roles in the future in classifying cancer patients and in predicting the response to therapy for better personal therapeutic care.

Engineering integrated digital circuits with allosteric ribozymes for scaling up molecular computation and diagnostics.

PubMed

Penchovsky, Robert

2012-10-19

Here we describe molecular implementations of integrated digital circuits, including a three-input AND logic gate, a two-input multiplexer, and 1-to-2 decoder using allosteric ribozymes. Furthermore, we demonstrate a multiplexer-decoder circuit. The ribozymes are designed to seek-and-destroy specific RNAs with a certain length by a fully computerized procedure. The algorithm can accurately predict one base substitution that alters the ribozyme's logic function. The ability to sense the length of RNA molecules enables single ribozymes to be used as platforms for multiple interactions. These ribozymes can work as integrated circuits with the functionality of up to five logic gates. The ribozyme design is universal since the allosteric and substrate domains can be altered to sense different RNAs. In addition, the ribozymes can specifically cleave RNA molecules with triplet-repeat expansions observed in genetic disorders such as oculopharyngeal muscular dystrophy. Therefore, the designer ribozymes can be employed for scaling up computing and diagnostic networks in the fields of molecular computing and diagnostics and RNA synthetic biology.

Molecular Diagnostics for Precision Medicine in Colorectal Cancer: Current Status and Future Perspective

PubMed Central

Chen, Guoli; Yang, Zhaohai; Eshleman, James R.; Netto, George J.

2016-01-01

Precision medicine, a concept that has recently emerged and has been widely discussed, emphasizes tailoring medical care to individuals largely based on information acquired from molecular diagnostic testing. As a vital aspect of precision cancer medicine, targeted therapy has been proven to be efficacious and less toxic for cancer treatment. Colorectal cancer (CRC) is one of the most common cancers and among the leading causes for cancer related deaths in the United States and worldwide. By far, CRC has been one of the most successful examples in the field of precision cancer medicine, applying molecular tests to guide targeted therapy. In this review, we summarize the current guidelines for anti-EGFR therapy, revisit the roles of pathologists in an era of precision cancer medicine, demonstrate the transition from traditional “one test-one drug” assays to multiplex assays, especially by using next-generation sequencing platforms in the clinical diagnostic laboratories, and discuss the future perspectives of tumor heterogeneity associated with anti-EGFR resistance and immune checkpoint blockage therapy in CRC. PMID:27699178

Rapid molecular diagnostics for multi-drug resistant tuberculosis in India.

PubMed

Ramachandran, Rajeswari; Muniyandi, M

2018-03-01

Rapid molecular diagnostic methods help in the detection of TB and Rifampicin resistance. These methods detect TB early, are accurate and play a crucial role in reducing the burden of drug resistant tuberculosis. Areas covered: This review analyses rapid molecular diagnostic tools used in the diagnosis of MDR-TB in India, such as the Line Probe Assay and GeneXpert. We have discussed the burden of MDR-TB and the impact of recent diagnostic tools on case detection and treatment outcomes. This review also discusses the costs involved in establishing these new techniques in India. Expert commentary: Molecular methods have considerable advantages for the programmatic management of drug resistant TB. These include speed, standardization of testing, potentially high throughput and reduced laboratory biosafety requirements. There is a desperate need for India to adopt modern, rapid, molecular tools with point-of-care tests being currently evaluated. New molecular diagnostic tests appear to be cost effective and also help in detecting missing cases. There is enough evidence to support the scaling up of these new tools in India.

Molecular diagnostics of neurodegenerative disorders.

PubMed

Agrawal, Megha; Biswas, Abhijit

2015-01-01

Molecular diagnostics provide a powerful method to detect and diagnose various neurological diseases such as Alzheimer's and Parkinson's disease. The confirmation of such diagnosis allows early detection and subsequent medical counseling that help specific patients to undergo clinically important drug trials. This provides a medical pathway to have better insight of neurogenesis and eventual cure of the neurodegenerative diseases. In this short review, we present recent advances in molecular diagnostics especially biomarkers and imaging spectroscopy for neurological diseases. We describe advances made in Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD), and finally present a perspective on the future directions to provide a framework for further developments and refinements of molecular diagnostics to combat neurodegenerative disorders.

Educational Gaps in Molecular Diagnostics, Genomics, and Personalized Medicine in Dermatopathology Training: A Survey of U.S. Dermatopathology Fellowship Program Directors.

PubMed

Torre, Kristin; Russomanno, Kristen; Ferringer, Tammie; Elston, Dirk; Murphy, Michael J

2018-01-01

Molecular technologies offer clinicians the tools to provide high-quality, cost-effective patient care. We evaluated education focused on molecular diagnostics, genomics, and personalized medicine in dermatopathology fellowship training. A 20-question online survey was emailed to all (n = 53) Accreditation Council for Graduate Medical Education (ACGME)-accredited dermatopathology training programs in the United States. Thirty-one of 53 program directors responded (response rate = 58%). Molecular training is undertaken in 74% of responding dermatopathology fellowships, with levels of instruction varying among dermatology-based and pathology-based programs. Education differed for dermatology- and pathology-trained fellows in approximately one-fifth (19%) of programs. Almost half (48%) of responding program directors believe that fellows are not currently receiving adequate molecular education, although the majority (97%) expect to incorporate additional instruction in the next 2-5 years. Factors influencing the incorporation of relevant education include perceived clinical utility and Accreditation Council for Graduate Medical Education/residency review committee (RRC) requirements. Potential benefits of molecular education include increased medical knowledge, improved patient care, and promotion of effective communication with other healthcare professionals. More than two-thirds (68%) of responding program directors believe that instruction in molecular technologies should be required in dermatopathology fellowship training. Although all responding dermatopathology fellowship program directors agreed that molecular education is important, only a little over half of survey participants believe that their fellows receive adequate instruction. This represents an important educational gap. Discussion among those who oversee fellow education is necessary to best integrate and evaluate teaching of molecular dermatopathology.

Final validation of the ProMisE molecular classifier for endometrial carcinoma in a large population-based case series.

PubMed

Kommoss, S; McConechy, M K; Kommoss, F; Leung, S; Bunz, A; Magrill, J; Britton, H; Kommoss, F; Grevenkamp, F; Karnezis, A; Yang, W; Lum, A; Krämer, B; Taran, F; Staebler, A; Lax, S; Brucker, S Y; Huntsman, D G; Gilks, C B; McAlpine, J N; Talhouk, A

2018-05-01

We have previously developed and confirmed a pragmatic molecular classifier for endometrial cancers; ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Inspired by the Cancer Genome Atlas, ProMisE identifies four prognostically distinct molecular subtypes and can be applied to diagnostic specimens (biopsy/curettings) enabling earlier informed decision-making. We have strictly adhered to the Institute of Medicine (IOM) guidelines for the development of genomic biomarkers, and herein present the final validation step of a locked-down classifier before clinical application. We assessed a retrospective cohort of women from the Tübingen University Women's Hospital treated for endometrial carcinoma between 2003 and 2013. Primary outcomes of overall, disease-specific, and progression-free survival were evaluated for clinical, pathological, and molecular features. Complete clinical and molecular data were evaluable from 452 women. Patient age ranged from 29 to 93 (median 65) years, and 87.8% cases were endometrioid histotype. Grade distribution included 282 (62.4%) G1, 75 (16.6%) G2, and 95 (21.0%) G3 tumors. 276 (61.1%) patients had stage IA disease, with the remaining stage IB [89 (19.7%)], stage II [26 (5.8%)], and stage III/IV [61 (13.5%)]. ProMisE molecular classification yielded 127 (28.1%) MMR-D, 42 (9.3%) POLE, 55 (12.2%) p53abn, and 228 (50.4%) p53wt. ProMisE was a prognostic marker for progression-free (P = 0.001) and disease-specific (P = 0.03) survival even after adjusting for known risk factors. Concordance between diagnostic and surgical specimens was highly favorable; accuracy 0.91, κ 0.88. We have developed, confirmed, and now validated a pragmatic molecular classification tool (ProMisE) that provides consistent categorization of tumors and identifies four distinct prognostic molecular subtypes. ProMisE can be applied to diagnostic samples and thus could be used to inform surgical procedure(s) and/or need for adjuvant therapy. Based on the IOM guidelines this classifier is now ready for clinical evaluation through prospective clinical trials.

Molecular filter-based diagnostics in high speed flows

NASA Technical Reports Server (NTRS)

Elliott, Gregory S.; Samimy, MO; Arnette, Stephen A.

1993-01-01

The use of iodine molecular filters in nonintrusive planar velocimetry methods is examined. Detailed absorption profiles are obtained to highlight the effects that determine the profile shape. It is shown that pressure broadening induced by the presence of a nonabsorbing vapor can be utilized to significantly change the slopes bounding the absorbing region while remaining in the optically-thick regime.

Keeping up with the times: revising the dermatology residency curriculum in the era of molecular diagnostics and personalized medicine.

PubMed

LaChance, Avery; Murphy, Michael J

2014-11-01

The clinical use of molecular diagnostics, genomics, and personalized medicine is increasing and improving rapidly over time. However, medical education incorporating the practical application of these techniques is lagging behind. Although instruction in these areas should be expanded upon and improved at all levels of training, residency provides a concentrated period of time in which to hone in on skills that are practically applicable to a trainee's specialty of choice. Although residencies in some fields, such as pathology, have begun to incorporate practical molecular diagnostics training, this area remains a relative gap in dermatology residency programs. Herein, we advocate for the incorporation of training in molecular diagnostics and personalized medicine into dermatology residency programs and propose a basic curriculum template for how to begin approaching these topics. By incorporating molecular diagnostics into dermatology residency training, dermatologists have the opportunity to lead the way and actively shape the specialty's transition into the era of personalized medicine. © 2014 The International Society of Dermatology.

Synthetic biology platform technologies for antimicrobial applications.

PubMed

Braff, Dana; Shis, David; Collins, James J

2016-10-01

The growing prevalence of antibiotic resistance calls for new approaches in the development of antimicrobial therapeutics. Likewise, improved diagnostic measures are essential in guiding the application of targeted therapies and preventing the evolution of therapeutic resistance. Discovery platforms are also needed to form new treatment strategies and identify novel antimicrobial agents. By applying engineering principles to molecular biology, synthetic biologists have developed platforms that improve upon, supplement, and will perhaps supplant traditional broad-spectrum antibiotics. Efforts in engineering bacteriophages and synthetic probiotics demonstrate targeted antimicrobial approaches that can be fine-tuned using synthetic biology-derived principles. Further, the development of paper-based, cell-free expression systems holds promise in promoting the clinical translation of molecular biology tools for diagnostic purposes. In this review, we highlight emerging synthetic biology platform technologies that are geared toward the generation of new antimicrobial therapies, diagnostics, and discovery channels. Copyright © 2016 Elsevier B.V. All rights reserved.

The value and validation of broad spectrum biosensors for diagnosis and biodefense

PubMed Central

Metzgar, David; Sampath, Rangarajan; Rounds, Megan A; Ecker, David J

2013-01-01

Broad spectrum biosensors capable of identifying diverse organisms are transitioning from the realm of research into the clinic. These technologies simultaneously capture signals from a wide variety of biological entities using universal processes. Specific organisms are then identified through bioinformatic signature-matching processes. This is in contrast to currently accepted molecular diagnostic technologies, which utilize unique reagents and processes to detect each organism of interest. This paradigm shift greatly increases the breadth of molecular diagnostic tools with little increase in biochemical complexity, enabling simultaneous diagnostic, epidemiologic, and biothreat surveillance capabilities at the point of care. This, in turn, offers the promise of increased biosecurity and better antimicrobial stewardship. Efficient realization of these potential gains will require novel regulatory paradigms reflective of the generalized, information-based nature of these assays, allowing extension of empirical data obtained from readily available organisms to support broader reporting of rare, difficult to culture, or extremely hazardous organisms. PMID:24128433

Exosomal microRNA Biomarkers: Emerging Frontiers in Colorectal and Other Human Cancers

PubMed Central

Goel, Ajay; Tovar-Camargo, Oscar A; Toden, Shusuke

2016-01-01

Diagnostic strategies, particularly non-invasive blood-based screening approaches, are gaining increased attention for the early detection and attenuation of mortality associated with colorectal cancer (CRC). However, the majority of current screening approaches are inadequate at replacing the conventional CRC diagnostic procedures. Yet, due to technological advances and a better understanding of molecular events underlying human cancer, a new category of biomarkers are on the horizon. Recent evidence indicates that cells release a distinct class of small vesicles called ‘exosomes’, which contain nucleic acids and proteins that reflect and typify host-cell molecular architecture. Intriguingly, exosomes released from cancer cells have a distinct genetic and epigenetic makeup, which allows them to undertake their tumorigenic function. From a clinical standpoint, these unique cancer-specific fingerprints present in exosomes appear to be detectable in a small amount of blood, making them very attractive substrates for developing cancer biomarkers, particularly noninvasive diagnostic approaches. PMID:26892862

Juvenile retinoschisis: a model for molecular diagnostic testing of X-linked ophthalmic disease.

PubMed

Sieving, P A; Yashar, B M; Ayyagari, R

1999-01-01

X-linked juvenile retinoschisis (RS) provides a starting point to define clinical paradigms and understand the limitations of diagnostic molecular testing. The RS phenotype is specific, but the broad severity range is clinically confusing. Molecular diagnostic testing obviates unnecessary examinations for boys at-risk and identifies carrier females who otherwise show no clinical signs. The XLRS1 gene has 6 exons of 26-196 base-pair size. Each exon is amplified by a single polymerase chain reaction and then sequenced, starting with exons 4 through 6, which contain mutation "hot spots." The 6 XLRS1 exons are sequenced serially. If alterations are found, they are compared with mutations in our > 120 XLRS families and with the > 300 mutations reported worldwide. Point mutations, small deletions, or rearrangements are identified in nearly 90% of males with a clinical diagnosis of RS. XLRS1 has very few sequence polymorphisms. Carrier-state testing produces 1 of 3 results: (1) positive, in which the woman has the same mutation as an affected male relative or known in other RS families; (2) negative, in which she lacks the mutation of her affected male relative; and (3) uninformative, in which no known mutation is identified or no information exists about the familial mutation. Molecular RS screening is an effective diagnostic tool that complements the clinician's skills for early detection of at-risk males. Useful outcomes of carrier testing depend on several factors: (1) a male relative with a clear clinical diagnosis; (2) a well-defined inheritance pattern; (3) high disease penetrance; (4) size and organization of the gene; and (5) the types of disease-associated mutations. Ethical questions include molecular diagnostic testing of young at-risk females before the age of consent, the impact of this information on the emotional health of the patient and family, and issues of employability and insurance coverage.

Juvenile retinoschisis: a model for molecular diagnostic testing of X-linked ophthalmic disease.

PubMed Central

Sieving, P A; Yashar, B M; Ayyagari, R

1999-01-01

BACKGROUND AND PURPOSE: X-linked juvenile retinoschisis (RS) provides a starting point to define clinical paradigms and understand the limitations of diagnostic molecular testing. The RS phenotype is specific, but the broad severity range is clinically confusing. Molecular diagnostic testing obviates unnecessary examinations for boys at-risk and identifies carrier females who otherwise show no clinical signs. METHODS: The XLRS1 gene has 6 exons of 26-196 base-pair size. Each exon is amplified by a single polymerase chain reaction and then sequenced, starting with exons 4 through 6, which contain mutation "hot spots." RESULTS: The 6 XLRS1 exons are sequenced serially. If alterations are found, they are compared with mutations in our > 120 XLRS families and with the > 300 mutations reported worldwide. Point mutations, small deletions, or rearrangements are identified in nearly 90% of males with a clinical diagnosis of RS. XLRS1 has very few sequence polymorphisms. Carrier-state testing produces 1 of 3 results: (1) positive, in which the woman has the same mutation as an affected male relative or known in other RS families; (2) negative, in which she lacks the mutation of her affected male relative; and (3) uninformative, in which no known mutation is identified or no information exists about the familial mutation. CONCLUSIONS: Molecular RS screening is an effective diagnostic tool that complements the clinician's skills for early detection of at-risk males. Useful outcomes of carrier testing depend on several factors: (1) a male relative with a clear clinical diagnosis; (2) a well-defined inheritance pattern; (3) high disease penetrance; (4) size and organization of the gene; and (5) the types of disease-associated mutations. Ethical questions include molecular diagnostic testing of young at-risk females before the age of consent, the impact of this information on the emotional health of the patient and family, and issues of employability and insurance coverage. Images FIGURE 2A FIGURE 2B PMID:10703138

Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

PubMed

Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

2017-01-01

The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning opportunities and workflow improvements in the molecular laboratory.

Leveling the Playing Field: Bringing Development of Biomarkers and Molecular Diagnostics up to the Standards for Drug Development

PubMed Central

Poste, George; Carbone, David P.; Parkinson, David R.; Verweij, Jaap; Hewitt, Stephen; Jessup, J. Milburn

2012-01-01

Molecular diagnostics are increasingly important in clinical research to stratify or identify molecularly profiled patient cohorts for targeted therapies, to modify the dose of a therapeutic, or to assess early response to therapy or monitor patients. Molecular diagnostics can also be used to identify pharmocogenetic risk of adverse drug reactions. The articles of this CCR Focus section on Molecular Diagnosis describe the development and use of markers for medical decision-making in the cancer patient. They define the sources of preanalytic variability to minimize as well as the regulatory and financial challenges in diagnostic development and integration into clinical practice. They also outline an NCI program to assist diagnostic development. Molecular diagnostic clinical tests require rigor in their development and clinical validation with sufficient sensitivity, specificity and validity that is comparable to that used for development of therapeutics. These diagnostics must be offered at a realistic cost that reflects both their clinical value and the costs associated with their development. When genome sequencing technologies move into the clinic, they must be integrated with and traceable to current technology because they may identify more efficient and accurate approaches to drug development. In addition, regulators may define progressive drug approval for companion diagnostics that requires further evidence regarding efficacy and safety before full approval. A way to accomplish this is to emphasize Phase IV post-marketing hypothesis driven clinical trials with biological characterization that permits accurate definition of the association of low prevalence gene alterations with toxicity or response in large cohorts. PMID:22422403

Leveling the playing field: bringing development of biomarkers and molecular diagnostics up to the standards for drug development.

PubMed

Poste, George; Carbone, David P; Parkinson, David R; Verweij, Jaap; Hewitt, Stephen M; Jessup, J Milburn

2012-03-15

Molecular diagnostics are becoming increasingly important in clinical research to stratify or identify molecularly profiled patient cohorts for targeted therapies, to modify the dose of a therapeutic, and to assess early response to therapy or monitor patients. Molecular diagnostics can also be used to identify the pharmacogenetic risk of adverse drug reactions. The articles in this CCR Focus section on molecular diagnosis describe the development and use of markers to guide medical decisions regarding cancer patients. They define sources of preanalytic variability that need to be minimized, as well as the regulatory and financial challenges involved in developing diagnostics and integrating them into clinical practice. They also outline a National Cancer Institute program to assist diagnostic development. Molecular diagnostic clinical tests require rigor in their development and clinical validation, with sensitivity, specificity, and validity comparable to those required for the development of therapeutics. These diagnostics must be offered at a realistic cost that reflects both their clinical value and the costs associated with their development. When genome-sequencing technologies move into the clinic, they must be integrated with and traceable to current technology because they may identify more efficient and accurate approaches to drug development. In addition, regulators may define progressive drug approval for companion diagnostics that requires further evidence regarding efficacy and safety before full approval can be achieved. One way to accomplish this is to emphasize phase IV postmarketing, hypothesis-driven clinical trials with biological characterization that would permit an accurate definition of the association of low-prevalence gene alterations with toxicity or response in large cohorts.

[Microbiological point of care tests].

PubMed

Book, Malte; Lehmann, Lutz Eric; Zhang, Xianghong; Stüber, Frank

2010-11-01

It is well known that the early initiation of a specific antiinfective therapy is crucial to reduce the mortality in severe infection. Procedures culturing pathogens are the diagnostic gold standard in such diseases. However, these methods yield results earliest between 24 to 48 hours. Therefore, severe infections such as sepsis need to be treated with an empirical antimicrobial therapy, which is ineffective in an unknown fraction of these patients. Today's microbiological point of care tests are pathogen specific and therefore not appropriate for an infection with a variety of possible pathogens. Molecular nucleic acid diagnostics such as polymerase chain reaction (PCR) allow the identification of pathogens and resistances. These methods are used routinely to speed up the analysis of positive blood cultures. The newest PCR based system allows the identification of the 25 most frequent sepsis pathogens by PCR in parallel without previous culture in less than 6 hours. Thereby, these systems might shorten the time of possibly insufficient antiinfective therapy. However, these extensive tools are not suitable as point of care diagnostics. Miniaturization and automating of the nucleic acid based method is pending, as well as an increase of detectable pathogens and resistance genes by these methods. It is assumed that molecular PCR techniques will have an increasing impact on microbiological diagnostics in the future. © Georg Thieme Verlag Stuttgart · New York.

[New insights in the differential diagnosis of bladder pain syndrome].

PubMed

Schwalenberg, T; Neuhaus, J; Horn, L-C; Alexander, H; Zimmermann, G; Ho Thi, P; Mallock, T; Stolzenburg, J-U

2010-03-01

The diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC) is challenging, since pathogenetic mechanisms and the definition of clinical relevant parameters are still under lively discussion. The criteria recently proposed by the European Society for the Study of Interstitial Cystitis (ESSIC) define a collective of patients based on the cardinal symptom of bladder pain which is heterogeneous, and therefore cannot receive standardised consistent therapy. Thus an extended diagnosis based on molecular markers seems to be indicated to render individual pharmacotherapy possible, and to contribute to elucidation of BPS/IC pathogenesis. For this purpose we feel the vital need for taking a bladder biopsy. The diagnosis of BPS/IC should rely on 3 "columns": (1) clinical diagnostics; (2) histopathology; (3) molecular diagnostics/protein expression. Since a significant contribution of the 3 functional units of the bladder to the pathophysiology is most evident, the examinations should ideally include urothelium, lamina propria, and detrusor musculature. Generation of receptor profiles of the detrusor muscle represents a first attempt to define a diagnostic tool for the individualisation of BPS/IC pharmacotherapy. Other factors, e.g., beta-hCG expression in the urothelium, need further evaluation. Extended BPS/IC diagnostics could be realistically integrated into routine patient care within a clinic/laboratory network. Georg Thieme Verlag Stuttgart New York.

Emerging Rapid Resistance Testing Methods for Clinical Microbiology Laboratories and Their Potential Impact on Patient Management

PubMed Central

Frickmann, Hagen; Zautner, Andreas E.

2014-01-01

Atypical and multidrug resistance, especially ESBL and carbapenemase expressing Enterobacteriaceae, is globally spreading. Therefore, it becomes increasingly difficult to achieve therapeutic success by calculated antibiotic therapy. Consequently, rapid antibiotic resistance testing is essential. Various molecular and mass spectrometry-based approaches have been introduced in diagnostic microbiology to speed up the providing of reliable resistance data. PCR- and sequencing-based approaches are the most expensive but the most frequently applied modes of testing, suitable for the detection of resistance genes even from primary material. Next generation sequencing, based either on assessment of allelic single nucleotide polymorphisms or on the detection of nonubiquitous resistance mechanisms might allow for sequence-based bacterial resistance testing comparable to viral resistance testing on the long term. Fluorescence in situ hybridization (FISH), based on specific binding of fluorescence-labeled oligonucleotide probes, provides a less expensive molecular bridging technique. It is particularly useful for detection of resistance mechanisms based on mutations in ribosomal RNA. Approaches based on MALDI-TOF-MS, alone or in combination with molecular techniques, like PCR/electrospray ionization MS or minisequencing provide the fastest resistance results from pure colonies or even primary samples with a growing number of protocols. This review details the various approaches of rapid resistance testing, their pros and cons, and their potential use for the diagnostic laboratory. PMID:25343142

Proposed minimal diagnostic criteria for myelodysplastic syndromes (MDS) and potential pre-MDS conditions

PubMed Central

Valent, Peter; Orazi, Attilio; Steensma, David P.; Ebert, Benjamin L.; Haase, Detlef; Malcovati, Luca; van de Loosdrecht, Arjan A.; Haferlach, Torsten; Westers, Theresia M.; Wells, Denise A.; Giagounidis, Aristoteles; Loken, Michael; Orfao, Alberto; Lübbert, Michael; Ganser, Arnold; Hofmann, Wolf-Karsten; Ogata, Kiyoyuki; Schanz, Julie; Béné, Marie C.; Hoermann, Gregor; Sperr, Wolfgang R.; Sotlar, Karl; Bettelheim, Peter; Stauder, Reinhard; Pfeilstöcker, Michael; Horny, Hans-Peter; Germing, Ulrich; Greenberg, Peter; Bennett, John M.

2017-01-01

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of myeloid neoplasms characterized by peripheral cytopenia, dysplasia, and a variable clinical course with about 30% risk to transform to secondary acute myeloid leukemia (AML). In the past 15 years, diagnostic evaluations, prognostication, and treatment of MDS have improved substantially. However, with the discovery of molecular markers and advent of novel targeted therapies, new challenges have emerged in the complex field of MDS. For example, MDS-related molecular lesions may be detectable in healthy individuals and increase in prevalence with age. Other patients exhibit persistent cytopenia of unknown etiology without dysplasia. Although these conditions are potential pre-phases of MDS they may also transform into other bone marrow neoplasms. Recently identified molecular, cytogenetic, and flow-based parameters may add in the delineation and prognostication of these conditions. However, no generally accepted integrated classification and no related criteria are as yet available. In an attempt to address this challenge, an international consensus group discussed these issues in a working conference in July 2016. The outcomes of this conference are summarized in the present article which includes criteria and a proposal for the classification of pre-MDS conditions as well as updated minimal diagnostic criteria of MDS. Moreover, we propose diagnostic standards to delineate between ´normal´, pre-MDS, and MDS. These standards and criteria should facilitate diagnostic and prognostic evaluations in clinical studies as well as in clinical practice. PMID:29088721

Managing the genomic revolution in cancer diagnostics.

PubMed

Nguyen, Doreen; Gocke, Christopher D

2017-08-01

Molecular tumor profiling is now a routine part of patient care, revealing targetable genomic alterations and molecularly distinct tumor subtypes with therapeutic and prognostic implications. The widespread adoption of next-generation sequencing technologies has greatly facilitated clinical implementation of genomic data and opened the door for high-throughput multigene-targeted sequencing. Herein, we discuss the variability of cancer genetic profiling currently offered by clinical laboratories, the challenges of applying rapidly evolving medical knowledge to individual patients, and the need for more standardized population-based molecular profiling.

Sporotrichosis: From KOH to Molecular Biology.

PubMed

Arenas, Roberto; Sánchez-Cardenas, Carlos D; Ramirez-Hobak, Lourdes; Ruíz Arriaga, Leon Felipe; Vega Memije, Ma Elisa

2018-05-23

Sporotrichosis is a cosmopolitan, chronic granulomatous mycosis, acquired by traumatic inoculation and caused by Sporothrix schenckii complex . Several methods of diagnostic are available, from KOH to molecular biology. In this review, we describe from the simplest (clinical diagnosis) to the most advanced diagnostic techniques (molecular biology).

Whole Genome Sequencing Increases Molecular Diagnostic Yield Compared with Current Diagnostic Testing for Inherited Retinal Disease.

PubMed

Ellingford, Jamie M; Barton, Stephanie; Bhaskar, Sanjeev; Williams, Simon G; Sergouniotis, Panagiotis I; O'Sullivan, James; Lamb, Janine A; Perveen, Rahat; Hall, Georgina; Newman, William G; Bishop, Paul N; Roberts, Stephen A; Leach, Rick; Tearle, Rick; Bayliss, Stuart; Ramsden, Simon C; Nemeth, Andrea H; Black, Graeme C M

2016-05-01

To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). Case series. A total of 562 patients diagnosed with IRD. We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. Diagnostic yield of genomic testing. Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield. We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Comprehensive Carrier Screening and Molecular Diagnostic Testing for Recessive Childhood Diseases

PubMed Central

Kingsmore, Stephen

2012-01-01

Of 7,028 disorders with suspected Mendelian inheritance, 1,139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~18% of pediatric hospitalizations. Molecular diagnostic testing is currently available for only ~300 recessive disorders. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening and molecular diagnostic testing to most recessive disease genes has hitherto been impractical. Recently, we reported a preconception carrier screen / molecular diagnostic test for 448 recessive childhood diseases. The current status of this test is reviewed here. Currently, this reports analytical validity of the comprehensive carrier test. As the clinical validity and clinical utility in the contexts described is ascertained, this article will be updated. PMID:22872815

Microfluidic-Based Bacteria Isolation from Whole Blood for Diagnostics of Blood Stream Infection.

PubMed

Zelenin, Sergey; Ramachandraiah, Harisha; Faridi, Asim; Russom, Aman

2017-01-01

Bacterial blood stream infection (BSI) potentially leads to life-threatening clinical conditions and medical emergencies such as severe sepsis, septic shock, and multi organ failure syndrome. Blood culturing is currently the gold standard for the identification of microorganisms and, although it has been automated over the decade, the process still requires 24-72 h to complete. This long turnaround time, especially for the identification of antimicrobial resistance, is driving the development of rapid molecular diagnostic methods. Rapid detection of microbial pathogens in blood related to bloodstream infections will allow the clinician to decide on or adjust the antimicrobial therapy potentially reducing the morbidity, mortality, and economic burden associated with BSI. For molecular-based methods, there is a lot to gain from an improved and straightforward method for isolation of bacteria from whole blood for downstream processing.We describe a microfluidic-based sample-preparation approach that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics.

PubMed

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-20

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

NASA Astrophysics Data System (ADS)

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-02-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas.

Molecular diagnostics of periodontitis.

PubMed

Korona-Głowniak, Izabela; Siwiec, Radosław; Berger, Marcin; Malm, Anna; Szymańska, Jolanta

2017-01-28

The microorganisms that form dental plaque are the main cause of periodontitis. Their identification and the understanding of the complex relationships and interactions that involve these microorganisms, environmental factors and the host's health status enable improvement in diagnostics and targeted therapy in patients with periodontitis. To this end, molecular diagnostics techniques (both techniques based on the polymerase chain reaction and those involving nucleic acid analysis via hybridization) come increasingly into use. On the basis of a literature review, the following methods are presented: polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), 16S rRNA-encoding gene sequencing, checkerboard and reverse-capture checkerboard hybridization, microarrays, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), as well as terminal restriction fragment length polymorphism (TRFLP) and next generation sequencing (NGS). The advantages and drawbacks of each method in the examination of periopathogens are indicated. The techniques listed above allow fast detection of even small quantities of pathogen present in diagnostic material and prove particularly useful to detect microorganisms that are difficult or impossible to grow in a laboratory.

External quality assessment studies for laboratory performance of molecular and serological diagnosis of Chikungunya virus infection.

PubMed

Jacobsen, Sonja; Patel, Pranav; Schmidt-Chanasit, Jonas; Leparc-Goffart, Isabelle; Teichmann, Anette; Zeller, Herve; Niedrig, Matthias

2016-03-01

Since the re-emergence of Chikungunya virus (CHIKV) in Reunion in 2005 and the recent outbreak in the Caribbean islands with an expansion to the Americas the CHIK diagnostic became very important. We evaluate the performance of laboratories regarding molecular and serological diagnostic of CHIK worldwide. A panel of 12 samples for molecular and 13 samples for serology were provided to 60 laboratories in 40 countries for evaluating the sensitivity and specificity of molecular and serology testing. The panel for molecular diagnostic testing was analysed by 56 laboratories returning 60 data sets of results whereas the 56 and 60 data sets were returned for IgG and IgM diagnostic from the participating laboratories. Twenty-three from 60 data sets performed optimal, 7 acceptable and 30 sets of results require improvement. From 50 data sets only one laboratory shows an optimal performance for IgM detection, followed by 9 data sets with acceptable and the rest need for improvement. From 46 IgG serology data sets 20 provide an optimal, 2 an acceptable and 24 require improvement performance. The evaluation of some of the diagnostic performances allows linking the quality of results to the in-house methods or commercial assays used. The external quality assurance for CHIK diagnostics provides a good overview on the laboratory performance regarding sensitivity and specificity for the molecular and serology diagnostic required for the quick and reliable analysis of suspected CHIK patients. Nearly half of the laboratories have to improve their diagnostic profile to achieve a better performance. Copyright © 2016 Z. Published by Elsevier B.V. All rights reserved.

CLINICAL APPROACH TO THE DIAGNOSTIC EVALUATION OF HERDITARY AND ACQUIRED NEUROMUSCULAR DISEASES

PubMed Central

McDonald, Craig M.

2012-01-01

SYNOPSIS In the context of a neuromuscular disease diagnostic evaluation, the clinician still must be able to obtain a relevant patient and family history and perform focused general, musculoskeletal, neurologic and functional physical examinations to direct further diagnostic evaluations. Laboratory studies for hereditary neuromuscular diseases include relevant molecular genetic studies. The EMG and nerve conduction studies remain an extension of the physical examination and help to guide further diagnostic studies such as molecular genetic studies, and muscle and nerve biopsies. All diagnostic information needs to be interpreted not in isolation, but within the context of relevant historical information, family history, physical examination findings, and laboratory data, electrophysiologic findings, pathologic findings, and molecular genetic findings if obtained. PMID:22938875

Wilsonosiphonia gen. nov. (Rhodomelaceae, Rhodophyta) based on molecular and morpho-anatomical characters.

PubMed

Bustamante, Danilo E; Won, Boo Yeon; Miller, Kathy Ann; Cho, Tae Oh

2017-04-01

Morphological, anatomical, and molecular sequence data were used to assess the establishment and phylogenetic position of the genus Wilsonosiphonia gen. nov. Phylogenies based on rbcL and concatenated rbcL and cox1 loci support recognition of Wilsonosiphonia gen. nov., sister to Herposiphonia. Diagnostic features for Wilsonosiphonia are rhizoids located at distal ends of pericentral cells and taproot-shaped multicellular tips of rhizoids. Wilsonosiphonia includes three species with diagnostic rbcL and cox1 sequences, Wilsonosiphonia fujiae sp. nov. (the generitype), W. howei comb. nov., and W. indica sp. nov. These three species resemble each other in external morphology, but W. fujiae is distinguished by having two tetrasporangia per segment rather than one, W. indica by having abundant and persistent trichoblasts, and W. howei by having few and deciduous trichoblasts. © 2017 Phycological Society of America.

Molecular Pathogenesis and Diagnostic, Prognostic and Predictive Molecular Markers in Sarcoma.

PubMed

Mariño-Enríquez, Adrián; Bovée, Judith V M G

2016-09-01

Sarcomas are infrequent mesenchymal neoplasms characterized by notable morphological and molecular heterogeneity. Molecular studies in sarcoma provide refinements to morphologic classification, and contribute diagnostic information (frequently), prognostic stratification (rarely) and predict therapeutic response (occasionally). Herein, we summarize the major molecular mechanisms underlying sarcoma pathogenesis and present clinically useful diagnostic, prognostic and predictive molecular markers for sarcoma. Five major molecular alterations are discussed, illustrated with representative sarcoma types, including 1. the presence of chimeric transcription factors, in vascular tumors; 2. abnormal kinase signaling, in gastrointestinal stromal tumor; 3. epigenetic deregulation, in chondrosarcoma, chondroblastoma, and other tumors; 4. deregulated cell survival and proliferation, due to focal copy number alterations, in dedifferentiated liposarcoma; 5. extreme genomic instability, in conventional osteosarcoma as a representative example of sarcomas with highly complex karyotype. Copyright © 2016 Elsevier Inc. All rights reserved.

Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues.

PubMed

Schaad, Norman W; Frederick, Reid D; Shaw, Joe; Schneider, William L; Hickson, Robert; Petrillo, Michael D; Luster, Douglas G

2003-01-01

Awareness of crop biosecurity and phytosanitation has been heightened since 9/11 and the unresolved anthrax releases in October 2001. Crops are highly vulnerable to accidental or deliberate introductions of crop pathogens from outside U.S. borders. Strategic thinking about protection against deliberate or accidental release of a plant pathogen is an urgent priority. Rapid detection will be the key to success. This review summarizes recent progress in the development of rapid real-time PCR protocols and evaluates their effectiveness in a proposed nationwide network of diagnostic laboratories that will facilitate rapid diagnostics and improved communication.

Cost-effectiveness of using a molecular diagnostic test to improve preoperative diagnosis of thyroid cancer.

PubMed

Najafzadeh, Mehdi; Marra, Carlo A; Lynd, Larry D; Wiseman, Sam M

2012-12-01

Fine-needle aspiration biopsy (FNAB) is a safe and inexpensive diagnostic procedure for evaluating thyroid nodules.Up to 25% of the results from an FNAB, however, may not be diagnostic or may be indeterminate, leading to a subsequent diagnostic thyroid surgery. A new molecularly based diagnostic test could potentially reduce indeterminate cytological results and, with high accuracy, provide a definitive diagnosis for cancer in thyroid nodules. The aim of the study was to estimate the cost-effectiveness of utilizing a molecular diagnostic (DX) test as an adjunct to FNAB, compared with NoDX, to improve the preoperative diagnosis of thyroid nodules. We constructed a patient-level simulation model to estimate the clinical and economic outcomes of using a DX test compared with current practice (NoDX) for the diagnosis of thyroid nodules. By using a cost-effectiveness framework, we measured incremental clinical benefits in terms of quality-adjusted life-years and incremental costs over a 10-year time horizon. Assuming 95% sensitivity and specificity of the Dx test when used as an adjunct to FNAB, the utilization of the DX test resulted in a gain of 0.046 quality-adjusted life-years (95% confidence interval 0.019-0.078) and a saving of $1087 (95% confidence interval $691-$1533) in direct costs per patient. If the cost of the Dx test is less than $1087 per test, we expect to save quality-adjusted life-years and reduce costs when it is utilized. Sensitivity of the DX test, compared with specificity, had a larger influence on the overall outcomes. Copyright © 2012 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

77 FR 77032 - Healthcare Trade Mission to Russia, June 3-7, 2013

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-31

... humans. The world market for biotechnology (used for molecular genetics diagnostic technologies) was $13... generation vaccines, antibiotics and bacteriophages) 2. Biomedicine (molecular diagnostics, personalized...

78 FR 32369 - Healthcare Trade Mission to Russia, October 21-25, 2013

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-30

... environment on humans. The world market of biotechnology (used for molecular genetics diagnostic technologies..., phytomedicines, new generation vaccines, antibiotics and bacteriophages). Biomedicine (molecular diagnostics...

SELDI-TOF-based serum proteomic pattern diagnostics for early detection of cancer.

PubMed

Petricoin, Emanuel F; Liotta, Lance A

2004-02-01

Proteomics is more than just generating lists of proteins that increase or decrease in expression as a cause or consequence of pathology. The goal should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. The nature of this information can be a cause, or a consequence, of disease and toxicity-based processes. Serum proteomic pattern diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier. This approach has recently shown tremendous promise in the detection of early-stage cancers. The biomarkers found by SELDI-TOF-based pattern recognition analysis are mostly low molecular weight fragments produced at the specific tumor microenvironment.

DNA-programmed dynamic assembly of quantum dots for molecular computation.

PubMed

He, Xuewen; Li, Zhi; Chen, Muzi; Ma, Nan

2014-12-22

Despite the widespread use of quantum dots (QDs) for biosensing and bioimaging, QD-based bio-interfaceable and reconfigurable molecular computing systems have not yet been realized. DNA-programmed dynamic assembly of multi-color QDs is presented for the construction of a new class of fluorescence resonance energy transfer (FRET)-based QD computing systems. A complete set of seven elementary logic gates (OR, AND, NOR, NAND, INH, XOR, XNOR) are realized using a series of binary and ternary QD complexes operated by strand displacement reactions. The integration of different logic gates into a half-adder circuit for molecular computation is also demonstrated. This strategy is quite versatile and straightforward for logical operations and would pave the way for QD-biocomputing-based intelligent molecular diagnostics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scratching the surface of tomorrow's diagnostics: the Editor-in-Chief's opinion at the 15th year of Expert Review of Molecular Diagnostics.

PubMed

Lorincz, Attila; Raison, Claire

2015-01-01

Interview with Attila Lorincz by Claire Raison (Commissioning Editor) To mark the beginning of the 15th year of Expert Review of Molecular Diagnostics, the journal's Editor-in-Chief shares his expert knowledge on translational diagnostics, his opinion on recent controversies and his predictions for molecular diagnostics in 2015 and beyond. Attila Lorincz received his doctorate from Trinity College, Dublin, Republic of Ireland, and went on to become a research fellow at the University of California, Santa Barbara, CA, USA. During Professor Lorincz's research on human papillomavirus (HPV), he found several important and novel carcinogenic HPV types and pioneered the use of HPV DNA testing for clinical diagnostics. In 1988, Professor Lorincz's team produced the first HPV test to be FDA-approved for patients and in 2003, for general population cervical precancer screening. Now Professor of Molecular Epidemiology at the Centre for Cancer Prevention, Queen Mary University of London, UK, he and his team are furthering translational research into DNA methylation assays for cancer risk prediction.

Screening the low molecular weight fraction of human serum using ATR-IR spectroscopy.

PubMed

Bonnier, Franck; Brachet, Guillaume; Duong, Romain; Sojinrin, Tobiloba; Respaud, Renaud; Aubrey, Nicolas; Baker, Matthew J; Byrne, Hugh J; Chourpa, Igor

2016-10-01

Vibrational spectroscopic techniques can detect small variations in molecular content, linked with disease, showing promise for screening and early diagnosis. Biological fluids, particularly blood serum, are potentially valuable for diagnosis purposes. The so-called Low Molecular Weight Fraction (LMWF) contains the associated peptidome and metabolome and has been identified as potentially the most relevant molecular population for disease-associated biomarker research. Although vibrational spectroscopy can deliver a specific chemical fingerprint of the samples, the High Molecular Weight Fraction (HMWF), composed of the most abundant serum proteins, strongly dominates the response and ultimately makes the detection of minor spectral variations a challenging task. Spectroscopic detection of potential serum biomarkers present at relatively low concentrations can be improved using pre-analytical depletion of the HMWF. In the present study, human serum fractionation by centrifugal filtration was used prior to analysis by Attenuated Total Reflection infrared spectroscopy. Using a model sample based on glycine spiked serum, it is demonstrated that the screening of the LMWF can be applied to quantify blinded concentrations up to 50 times lower. Moreover, the approach is easily transferable to different bodily fluids which would support the development of more efficient and suitable clinical protocols exploring vibrational spectroscopy based ex-vivo diagnostic tools. Revealing serum LMWF for spectral serological diagnostic applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiplexed color-coded probe-based gene expression assessment for clinical molecular diagnostics in formalin-fixed paraffin-embedded human renal allograft tissue.

PubMed

Adam, Benjamin; Afzali, Bahman; Dominy, Katherine M; Chapman, Erin; Gill, Reeda; Hidalgo, Luis G; Roufosse, Candice; Sis, Banu; Mengel, Michael

2016-03-01

Histopathologic diagnoses in transplantation can be improved with molecular testing. Preferably, molecular diagnostics should fit into standard-of-care workflows for transplant biopsies, that is, formalin-fixed paraffin-embedded (FFPE) processing. The NanoString(®) gene expression platform has recently been shown to work with FFPE samples. We aimed to evaluate its methodological robustness and feasibility for gene expression studies in human FFPE renal allograft samples. A literature-derived antibody-mediated rejection (ABMR) 34-gene set, comprised of endothelial, NK cell, and inflammation transcripts, was analyzed in different retrospective biopsy cohorts and showed potential to molecularly discriminate ABMR cases, including FFPE samples. NanoString(®) results were reproducible across a range of RNA input quantities (r = 0.998), with different operators (r = 0.998), and between different reagent lots (r = 0.983). There was moderate correlation between NanoString(®) with FFPE tissue and quantitative reverse transcription polymerase chain reaction (qRT-PCR) with corresponding dedicated fresh-stabilized tissue (r = 0.487). Better overall correlation with histology was observed with NanoString(®) (r = 0.354) than with qRT-PCR (r = 0.146). Our results demonstrate the feasibility of multiplexed gene expression quantification from FFPE renal allograft tissue. This represents a method for prospective and retrospective validation of molecular diagnostics and its adoption in clinical transplantation pathology. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Use of multiplex PCR based molecular diagnostics in diagnosis of suspected CNS infections in tertiary care setting-A retrospective study.

PubMed

Javali, Mahendra; Acharya, Purushottam; Mehta, Aneesh; John, Aju Abraham; Mahale, Rohan; Srinivasa, R

2017-10-01

CNS infections like meningitis and encephalitis pose enormous healthcare challenges due to mortality, sequelae and socioeconomic burden. In tertiary setting, clinical, microbiological, cytological and radiological investigations are not distinctive enough for diagnosing microbial etiology. Molecular diagnostics is filling this gap. We evaluated the clinical impact of a commercially available multiplex molecular diagnostic system - SES for diagnosing suspected CNS infections. This study was conducted in our tertiary level Neurology ICU. 110 patients admitted during Nov-2010 to April-2014 were included. CSF samples of patients clinically suspected of having CNS infections were subjected to routine investigation in our laboratory and SES test at XCyton Diagnostics. We studied the impact of SES in diagnosis of CNS infections and its efficacy in helping therapeutic management. SES showed detection rate of 42.18% and clinical specificity of 100%. It had 10 times higher detection rate than conventional tests. Streptococcus pneumoniae and Mycobacterium tuberculosis were two top bacterial pathogens. VZV was most detected viral pathogen. SES results elicited changes in therapy in both positive and negative cases. We observed superior patient outcomes as measured by GCS scale. 75% and 82.14% of the patients positive and negative on SES respectively, recovered fully. Detecting causative organism and ruling out infectious etiology remain the most critical aspect for management and prognosis of patients with suspected CNS infections. In this study, we observed higher detection rate of pathogens, target specific escalation and evidence based de-escalation of antimicrobials using SES. Institution of appropriate therapy helped reduce unnecessary use of antimicrobials. Copyright © 2017 Elsevier B.V. All rights reserved.

A Simple, Inexpensive Device for Nucleic Acid Amplification without Electricity—Toward Instrument-Free Molecular Diagnostics in Low-Resource Settings

PubMed Central

LaBarre, Paul; Hawkins, Kenneth R.; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Boyle, David; Weigl, Bernhard

2011-01-01

Background Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). Methodology/Principal Findings In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. Conclusions/Significance We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes. PMID:21573065

Optimized acoustic biochip integrated with microfluidics for biomarkers detection in molecular diagnostics.

PubMed

Papadakis, G; Friedt, J M; Eck, M; Rabus, D; Jobst, G; Gizeli, E

2017-09-01

The development of integrated platforms incorporating an acoustic device as the detection element requires addressing simultaneously several challenges of technological and scientific nature. The present work was focused on the design of a microfluidic module, which, combined with a dual or array type Love wave acoustic chip could be applied to biomedical applications and molecular diagnostics. Based on a systematic study we optimized the mechanics of the flow cell attachment and the sealing material so that fluidic interfacing/encapsulation would impose minimal losses to the acoustic wave. We have also investigated combinations of operating frequencies with waveguide materials and thicknesses for maximum sensitivity during the detection of protein and DNA biomarkers. Within our investigations neutravidin was used as a model protein biomarker and unpurified PCR amplified Salmonella DNA as the model genetic target. Our results clearly indicate the need for experimental verification of the optimum engineering and analytical parameters, in order to develop commercially viable systems for integrated analysis. The good reproducibility of the signal together with the ability of the array biochip to detect multiple samples hold promise for the future use of the integrated system in a Lab-on-a-Chip platform for application to molecular diagnostics.

Addressing Loss of Efficiency Due to Misclassification Error in Enriched Clinical Trials for the Evaluation of Targeted Therapies Based on the Cox Proportional Hazards Model.

PubMed

Tsai, Chen-An; Lee, Kuan-Ting; Liu, Jen-Pei

2016-01-01

A key feature of precision medicine is that it takes individual variability at the genetic or molecular level into account in determining the best treatment for patients diagnosed with diseases detected by recently developed novel biotechnologies. The enrichment design is an efficient design that enrolls only the patients testing positive for specific molecular targets and randomly assigns them for the targeted treatment or the concurrent control. However there is no diagnostic device with perfect accuracy and precision for detecting molecular targets. In particular, the positive predictive value (PPV) can be quite low for rare diseases with low prevalence. Under the enrichment design, some patients testing positive for specific molecular targets may not have the molecular targets. The efficacy of the targeted therapy may be underestimated in the patients that actually do have the molecular targets. To address the loss of efficiency due to misclassification error, we apply the discrete mixture modeling for time-to-event data proposed by Eng and Hanlon [8] to develop an inferential procedure, based on the Cox proportional hazard model, for treatment effects of the targeted treatment effect for the true-positive patients with the molecular targets. Our proposed procedure incorporates both inaccuracy of diagnostic devices and uncertainty of estimated accuracy measures. We employed the expectation-maximization algorithm in conjunction with the bootstrap technique for estimation of the hazard ratio and its estimated variance. We report the results of simulation studies which empirically investigated the performance of the proposed method. Our proposed method is illustrated by a numerical example.

Molecular Diagnostics Update for the Emerging (If Not Already Widespread) Sexually Transmitted Infection Agent Mycoplasma genitalium: Just About Ready for Prime Time.

PubMed

Munson, Erik

2017-10-01

Mycoplasma genitalium is an important and emerging agent of sexually transmitted infection in females and males, carrying the potential for postinfection genital tract sequelae. Past efforts to identify this organism on a routine basis, which were problematic due to the fastidious nature of the bacterium and its antigenic intricacies, have recently become supplemented by molecular diagnostics. A number of these assays are available commercially. This minireview describes the format and performance indices of a number of M. genitalium DNA- and RNA-based amplification assays; many of these assays have contributed to an improved clinical and epidemiologic understanding of this organism. Copyright © 2017 American Society for Microbiology.

Isoenzymes.

ERIC Educational Resources Information Center

Daugherty, N. A.

1979-01-01

The uses of isoenzyme assays are reviewed as they relate to solutions of practical problems. Emphasizes their use of diagnostic tools in medicine, specifically in cardiac profiling. Suggests that principles of isoenzyme assays readily demonstrate molecular structure, acid-base equilibria, kinetics, and electrochemistry to general chemistry…

Detection of mRNA by reverse-transcription PCR as an indicator of viability in Phytophthora ramorum

Treesearch

A. Chimento; S.O. Cacciola; M. Garbelotto

2011-01-01

In the last few decades, the use of molecular tools has greatly improved the efficiency of plant disease diagnosis. However, one of the major setbacks of most molecular diagnostic approaches is their inability to differentiate between dead and viable pathogens. We propose a new strategy for the detection of plant pathogens, based on the use of mRNA as a viability...

Lung tumor diagnosis and subtype discovery by gene expression profiling.

PubMed

Wang, Lu-yong; Tu, Zhuowen

2006-01-01

The optimal treatment of patients with complex diseases, such as cancers, depends on the accurate diagnosis by using a combination of clinical and histopathological data. In many scenarios, it becomes tremendously difficult because of the limitations in clinical presentation and histopathology. To accurate diagnose complex diseases, the molecular classification based on gene or protein expression profiles are indispensable for modern medicine. Moreover, many heterogeneous diseases consist of various potential subtypes in molecular basis and differ remarkably in their response to therapies. It is critical to accurate predict subgroup on disease gene expression profiles. More fundamental knowledge of the molecular basis and classification of disease could aid in the prediction of patient outcome, the informed selection of therapies, and identification of novel molecular targets for therapy. In this paper, we propose a new disease diagnostic method, probabilistic boosting tree (PB tree) method, on gene expression profiles of lung tumors. It enables accurate disease classification and subtype discovery in disease. It automatically constructs a tree in which each node combines a number of weak classifiers into a strong classifier. Also, subtype discovery is naturally embedded in the learning process. Our algorithm achieves excellent diagnostic performance, and meanwhile it is capable of detecting the disease subtype based on gene expression profile.

Diffuse Gliomas for Nonneuropathologists: The New Integrated Molecular Diagnostics.

PubMed

Lee, Sunhee C

2018-05-18

Diffuse gliomas comprise the bulk of "brain cancer" in adults. The recent update to the 4th edition of the World Health Organization's classification of tumors of the central nervous system reflects an unprecedented change in the landscape of the diagnosis and management of diffuse gliomas that will affect all those involved in the management and care of patients. Of the recently discovered gene alterations, mutations in the Krebs cycle enzymes isocitrate dehydrogenases (IDHs) 1 and 2 have fundamentally changed the way the gliomas are understood and classified. Incorporating information on a few genetic parameters (IDH, ATRX and/or p53, and chromosome 1p19q codeletion), a relatively straightforward diagnostic algorithm has been generated with robust and reproducible results that correlate with patients' survival far better than relying on conventional histology alone. Evidence also supports the conclusion that the vast majority of diffuse gliomas without IDH mutations (IDH-wild-type astrocytomas) behave like IDH-wild-type glioblastomas ("molecular GBM"). Together, these changes reflect a big shift in the practice of diagnostic neuropathology in which tumor risk stratification aligns better with molecular information than histology/grading. The purpose of this review is to provide the readers with a brief synopsis of the changes in the 2016 World Health Organization update with an emphasis on diffuse gliomas and to summarize key gene abnormalities on which these classifications are based. Practical points involved in day-to-day diagnostic workup are also discussed, along with a comparison of the various diagnostic tests, including immunohistochemistry, with an emphasis on targeted next-generation sequencing panel technology as a future universal approach.

Regulation Effects by Programmed Molecules for Transcription-Based Diagnostic Automata towards Therapeutic Use

NASA Astrophysics Data System (ADS)

Hirabayashi, Miki; Ohashi, Hirotada; Kubo, Tai

We have presented experimental analysis on the controllability of our transcription-based diagnostic biomolecular automata by programmed molecules. Focusing on the noninvasive transcriptome diagnosis by salivary mRNAs, we already proposed the novel concept of diagnostic device using DNA computation. This system consists of the main computational element which has a stem shaped promoter region and a pseudo-loop shaped read-only memory region for transcription regulation through the conformation change caused by the recognition of disease-related biomarkers. We utilize the transcription of malachite green aptamer sequence triggered by the target recognition for observation of detection. This algorithm makes it possible to release RNA-aptamer drugs multiply, different from the digestion-based systems by the restriction enzyme which was proposed previously, for the in-vivo use, however, the controllability of aptamer release is not enough at the previous stage. In this paper, we verified the regulation effect on aptamer transcription by programmed molecules in basic conditions towards the developm! ent of therapeutic automata. These results would bring us one step closer to the realization of new intelligent diagnostic and therapeutic automata based on molecular circuits.

Ten years of R&D and full automation in molecular diagnosis.

PubMed

Greub, Gilbert; Sahli, Roland; Brouillet, René; Jaton, Katia

2016-01-01

A 10-year experience of our automated molecular diagnostic platform that carries out 91 different real-time PCR is described. Progresses and future perspectives in molecular diagnostic microbiology are reviewed: why automation is important; how our platform was implemented; how homemade PCRs were developed; the advantages/disadvantages of homemade PCRs, including the critical aspects of troubleshooting and the need to further reduce the turnaround time for specific samples, at least for defined clinical settings such as emergencies. The future of molecular diagnosis depends on automation, and in a novel perspective, it is time now to fully acknowledge the true contribution of molecular diagnostic and to reconsider the indication for PCR, by also using these tests as first-line assays.

Miniaturized multiplex label-free electronic chip for rapid nucleic acid analysis based on carbon nanotube nanoelectrode arrays

NASA Technical Reports Server (NTRS)

Koehne, Jessica E.; Chen, Hua; Cassell, Alan M.; Ye, Qi; Han, Jie; Meyyappan, Meyya; Li, Jun

2004-01-01

BACKGROUND: Reducing cost and time is the major concern in clinical diagnostics, particularly in molecular diagnostics. Miniaturization technologies have been recognized as promising solutions to provide low-cost microchips for diagnostics. With the recent advancement in nanotechnologies, it is possible to further improve detection sensitivity and simplify sample preparation by incorporating nanoscale elements in diagnostics devices. A fusion of micro- and nanotechnologies with biology has great potential for the development of low-cost disposable chips for rapid molecular analysis that can be carried out with simple handheld devices. APPROACH: Vertically aligned multiwalled carbon nanotubes (MWNTs) are fabricated on predeposited microelectrode pads and encapsulated in SiO2 dielectrics with only the very end exposed at the surface to form an inlaid nanoelectrode array (NEA). The NEA is used to collect the electrochemical signal associated with the target molecules binding to the probe molecules, which are covalently attached to the end of the MWNTs. CONTENT: A 3 x 3 microelectrode array is presented to demonstrate the miniaturization and multiplexing capability. A randomly distributed MWNT NEA is fabricated on each microelectrode pad. Selective functionalization of the MWNT end with a specific oligonucleotide probe and passivation of the SiO2 surface with ethylene glycol moieties are discussed. Ru(bpy)2+ -mediator-amplified guanine oxidation is used to directly measure the electrochemical signal associated with target molecules. SUMMARY: The discussed MWNT NEAs have ultrahigh sensitivity in direct electrochemical detection of guanine bases in the nucleic acid target. Fewer than approximately 1000 target nucleic acid molecules can be measured with a single microelectrode pad of approximately 20 x 20 microm2, which approaches the detection limit of laser scanners in fluorescence-based DNA microarray techniques. MWNT NEAs can be easily integrated with microelectronic circuitry and microfluidics for development of a fully automated system for rapid molecular analysis with minimum cost.

Molecular diagnostics in medical microbiology: yesterday, today and tomorrow.

PubMed

van Belkum, Alex

2003-10-01

Clinical microbiology is clearly on the move, and various new diagnostic technologies have been introduced into laboratory practice over the past few decades. However, Henri D Isenberg recently stated that molecular biology techniques promised to revolutionise the diagnosis of infectious disease, but that, to date, this promise is still in its infancy. Molecular diagnostics have now surpassed these early stages and have definitely reached puberty. Currently, a second generation of automated molecular approaches is already within the microbiologists' reach. Quantitative amplification tests in combination with genomics, transcriptomics, proteomics and related methodologies will pave the way to further enhancement of innovative microbial detection and identification.

Diagnosis of Neisseria gonorrhoeae Using Molecular Beacon

PubMed Central

Patel, Achchhe Lal; Sonkar, Subash Chandra; Kumari, Indu; Saluja, Daman

2015-01-01

Neisseria gonorrhoeae is an important sexually transmitted diseases (STD) causing pathogen worldwide. Due to absence of an affordable diagnostic assay, routine screening of gonococcal infection becomes impossible in developing countries where infection rates are maximum. Treatment is given on the basis of symptoms alone which leads to spread of infection. Thus, development of a rapid, sensitive, specific, and PCR based visual diagnostic assay suitable for developing countries, required for better disease management, is aimed at in present study. Endocervical swabs were collected from patients visiting gynecology department of various hospitals in Delhi. In-house PCR based assay was developed and modified to visual assay using molecular beacon for end-point detection. It was evaluated against Roche AMPLICOR NG kit and rmp gene. Specificity of beacon was confirmed by competition experiments. Diagnostic test was 98.21% specific and 99.59% sensitive whereas negative and positive predicted value were 99.40% and 98.78%, respectively. We also observed that twice the concentration (2X) of premix was stable at 4°C for 4 months and dry swab samples gave concordant results with that of wet swabs. These features make the test best suitable for routine diagnosis of genital infections in developing countries. PMID:25802857

Recognition of the genus Thaumatophyllum Schott − formerly Philodendron subg. Meconostigma (Araceae) − based on molecular and morphological evidence

PubMed Central

Sakuragui, Cassia Mônica; Calazans, Luana Silva Braucks; de Oliveira, Leticia Loss; de Morais, Érica Barroso; Benko-Iseppon, Ana Maria; Vasconcelos, Santelmo; Schrago, Carlos Eduardo Guerra; Mayo, Simon Joseph

2018-01-01

Abstract Philodendron subgenus Meconostigma has been a well-circumscribed group since 1829. Members of this group are easily distinguished by diagnostic morphological characters as well as by a distinct ecology and geographical distribution. Based on molecular, morphological and cytological evidence, we propose the recognition of P. subg. Meconostigma as a distinct genus, Thaumatophyllum Schott. We also present the necessary new combinations, an emended key and some nomenclatural and taxonomic corrections regarding 21 names of Thaumatophyllum. PMID:29750071

Toward theragnostics.

PubMed

Pene, Frédéric; Courtine, Emilie; Cariou, Alain; Mira, Jean-Paul

2009-01-01

Theragnostics is a treatment strategy that combines therapeutics with diagnostics. It associates both a diagnostic test that identifies patients most likely to be helped or harmed by a new medication, and targeted drug therapy based on the test results. Bioinformatics, genomics, proteomics, and functional genomics are molecular biology tools essential for the progress of molecular theragnostics. These tools generate the genetic and protein information required for the development of diagnostic assays. Theragnostics includes a wide range of subjects, including personalized medicine, pharmacogenomics, and molecular imaging to develop efficient new targeted therapies with adequate benefit/risk to patients and a better molecular understanding of how to optimize drug selection. Furthermore, theragnostics aims to monitor the response to the treatment, to increase drug efficacy and safety. In addition, theragnostics could eliminate the unnecessary treatment of patients for whom therapy is not appropriate, resulting in significant drug cost savings for the healthcare system. However, the introduction of theragnostic tests into routine health care requires both a demonstration of cost-effectiveness and the availability of appropriate accessible testing systems. This review reports validation studies in oncology and infectious diseases that have demonstrated the benefits of such approach in well-defined subpopulations of patients, moving the field from the drug development process toward clinical practice and routine application. Theragnostics may change the usual business model of pharmaceutical companies from the classic blockbuster model toward targeted therapies.

Modeling Complex Workflow in Molecular Diagnostics

PubMed Central

Gomah, Mohamed E.; Turley, James P.; Lu, Huimin; Jones, Dan

2010-01-01

One of the hurdles to achieving personalized medicine has been implementing the laboratory processes for performing and reporting complex molecular tests. The rapidly changing test rosters and complex analysis platforms in molecular diagnostics have meant that many clinical laboratories still use labor-intensive manual processing and testing without the level of automation seen in high-volume chemistry and hematology testing. We provide here a discussion of design requirements and the results of implementation of a suite of lab management tools that incorporate the many elements required for use of molecular diagnostics in personalized medicine, particularly in cancer. These applications provide the functionality required for sample accessioning and tracking, material generation, and testing that are particular to the evolving needs of individualized molecular diagnostics. On implementation, the applications described here resulted in improvements in the turn-around time for reporting of more complex molecular test sets, and significant changes in the workflow. Therefore, careful mapping of workflow can permit design of software applications that simplify even the complex demands of specialized molecular testing. By incorporating design features for order review, software tools can permit a more personalized approach to sample handling and test selection without compromising efficiency. PMID:20007844

[Diagnostic algorithm in chronic myeloproliferative diseases (CMPD)].

PubMed

Haferlach, Torsten; Bacher, Ulrike; Kern, Wolfgang; Schnittger, Susanne; Haferlach, Claudia

2007-09-15

The Philadelphia-negative chronic myeloproliferative diseases (CMPD) are very complex and heterogeneous disorders. They are represented by polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD/unclassifiable (CMPD-U), chronic neutrophilic leukemia (CNL), and chronic eosinophilic leukemia/hypereosinophilic syndrome (CEL/HES) according to the WHO classification. Before, diagnostics were mainly focused on clinical and morphological aspects, but in recent years cytogenetics and fluorescence in situ hybridization (FISH) found entrance in routine schedules as chromosomal abnormalities are relevant for prognosis and classification. Recently, there is rapid progress in the field of molecular characterization: the JAK2V617F mutation which shows a high incidence in PV, CIMF, and ET already plays a central role and will probably soon be included in follow-up procedures. Due to the detection of mutations in exon 12 of the JAK2 gene or mutations in the MPL gene the variety of activating mutations in the CMPD is still increasing. In CEL/HES the detection of the FIP1L1-PDGFRA fusion gene and overexpression of PDGFRA and PDGFRB led to targeted therapy with tyrosine kinase inhibitors. Thus, diagnostics in the CMPD transform toward a multimodal diagnostic concept based on a combination of methods - cyto-/histomorphology, cytogenetics, and individual molecular methods which can be included in a diagnostic algorithm.

Cancer diagnostics: The journey from histomorphology to molecular profiling.

PubMed

Ahmed, Atif A; Abedalthagafi, Malak

2016-09-06

Although histomorphology has made significant advances into the understanding of cancer etiology, classification and pathogenesis, it is sometimes complicated by morphologic ambiguities, and other shortcomings that necessitate the development of ancillary tests to complement its diagnostic value. A new approach to cancer patient management consists of targeting specific molecules or gene mutations in the cancer genome by inhibitory therapy. Molecular diagnostic tests and genomic profiling methods are increasingly being developed to identify tumor targeted molecular profile that is the basis of targeted therapy. Novel targeted therapy has revolutionized the treatment of gastrointestinal stromal tumor, renal cell carcinoma and other cancers that were previously difficult to treat with standard chemotherapy. In this review, we discuss the role of histomorphology in cancer diagnosis and management and the rising role of molecular profiling in targeted therapy. Molecular profiling in certain diagnostic and therapeutic difficulties may provide a practical and useful complement to histomorphology and opens new avenues for targeted therapy and alternative methods of cancer patient management.

Democratizing molecular diagnostics for the developing world.

PubMed

Abou Tayoun, Ahmad N; Burchard, Paul R; Malik, Imran; Scherer, Axel; Tsongalis, Gregory J

2014-01-01

Infectious diseases that are largely treatable continue to pose a tremendous burden on the developing world despite the availability of highly potent drugs. The high mortality and morbidity rates of these diseases are largely due to a lack of affordable diagnostics that are accessible to resource-limited areas and that can deliver high-quality results. In fact, modified molecular diagnostics for infectious diseases were rated as the top biotechnology to improve health in developing countries. In this review, we describe the characteristics of accessible molecular diagnostic tools and discuss the challenges associated with implementing such tools at low infrastructure sites. We highlight our experience as part of the "Grand Challenge" project supported by the Gates Foundation for addressing global health inequities and describe issues and solutions associated with developing adequate technologies or molecular assays needed for broad access in the developing world. We believe that sharing this knowledge will facilitate the development of new molecular technologies that are extremely valuable for improving global health.

Point-of-care oral-based diagnostics

PubMed Central

Hart, RW; Mauk, MG; Liu, C; Qiu, X; Thompson, JA; Chen, D; Malamud, D; Abrams, WR; Bau, HH

2014-01-01

Many of the target molecules that reside in blood are also present in oral fluids, albeit at lower concentrations. Oral fluids are, however, relatively easy and safe to collect without the need for specialized equipment and training. Thus, oral fluids provide convenient samples for medical diagnostics. Recent advances in lab-on-a-chip technologies have made minute, fully integrated diagnostic systems practical for an assortment of point-of-care tests. Such systems can perform either immunoassays or molecular diagnostics outside centralized laboratories within time periods ranging from minutes to an hour. The article briefly reviews recent advances in devices for point-of-care testing with a focus on work that has been carried out by the authors as part of a NIH program. PMID:21521419

Gene and genetic diagnostic method patent claims: a comparison under current European and US patent law

PubMed Central

Huys, Isabelle; Van Overwalle, Geertrui; Matthijs, Gert

2011-01-01

The paper focuses on the fundamental debate that is going on in Europe and the United States about whether genes and genetic diagnostic methods are to be regarded as inventions or subject matter eligible for patent protection, or whether they are discoveries or principles of nature and thus excluded from patentability. The study further explores some possible scenarios of American influences on European patent applications with respect to genetic diagnostic methods. Our analysis points out that patent eligibility for genes and genetic diagnostic methods, as discussed in the United States in the Association of Molecular Pathology versus US Patent and Trademark Office decision, is based on a different reasoning compared with the European Patent Convention. PMID:21654725

Gene and genetic diagnostic method patent claims: a comparison under current European and US patent law.

PubMed

Huys, Isabelle; Van Overwalle, Geertrui; Matthijs, Gert

2011-10-01

The paper focuses on the fundamental debate that is going on in Europe and the United States about whether genes and genetic diagnostic methods are to be regarded as inventions or subject matter eligible for patent protection, or whether they are discoveries or principles of nature and thus excluded from patentability. The study further explores some possible scenarios of American influences on European patent applications with respect to genetic diagnostic methods. Our analysis points out that patent eligibility for genes and genetic diagnostic methods, as discussed in the United States in the Association of Molecular Pathology versus US Patent and Trademark Office decision, is based on a different reasoning compared with the European Patent Convention.

Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

PubMed

Schwalbe, E C; Hicks, D; Rafiee, G; Bashton, M; Gohlke, H; Enshaei, A; Potluri, S; Matthiesen, J; Mather, M; Taleongpong, P; Chaston, R; Silmon, A; Curtis, A; Lindsey, J C; Crosier, S; Smith, A J; Goschzik, T; Doz, F; Rutkowski, S; Lannering, B; Pietsch, T; Bailey, S; Williamson, D; Clifford, S C

2017-10-18

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

Current status and future perspectives on molecular and serological methods in diagnostic mycology.

PubMed

Lau, Anna; Chen, Sharon; Sleiman, Sue; Sorrell, Tania

2009-11-01

Invasive fungal infections are an important cause of infectious morbidity. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve patient outcomes. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Since laboratories are increasingly reliant on DNA sequencing for fungal identification, measures to improve sequence interpretation should support validation of reference isolates and quality control in public gene repositories. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex xMAP) and/or commercial PCR assays warrant further evaluation for routine diagnostic use. Notwithstanding the advantages of molecular tests, serological assays remain clinically useful for patient management. The serum Aspergillus galactomannan test has been incorporated into diagnostic algorithms of invasive aspergillosis. Both the galactomannan and the serum beta-D-glucan test have value for diagnosing infection and monitoring therapeutic response.

Serum markers for prostate cancer: a rational approach to the literature.

PubMed

Steuber, Thomas; O'Brien, Matthew Frank; Lilja, Hans

2008-07-01

Due to its universal applicability for early detection and prediction of cancer stage and disease recurrence, widespread implementation of serum-based prostate-specific antigen (PSA) measurements has a significant influence on current treatment strategies for men with prostate cancer (PCa). However, over-detection and the resultant over-treatment of indolent cancers have been strongly implicated to occur. Using current recommended guidelines, the PSA test suffers from both limited sensitivity and specificity to enable efficacious population-based cancer detection. Therefore, novel biomarkers are much needed to complement PSA by enhancing its diagnostic and prognostic performance. The present literature on serum markers for PCa was reviewed. PSA derivatives, molecular PSA isoforms, and novel molecular targets in blood were summarized and weighted against their potential to improve decision-making of men with PCa. Current evidence suggests that no single analyte is likely to achieve the desired level of diagnostic and prognostic accuracy for PCa. However, the combination of biomarkers with clinical and demographic data, for example, using established standard nomograms, has produced progress toward the goal of both optimal screening and risk assessment. Furthermore, potential candidate molecular markers for PCa can be derived from high-throughput technologies. Current studies demonstrate that understanding dynamic PSA changes over time may offer diagnostic and prognostic information. Bridging the gap between basic science and clinical practice represents the main goal in the near future to enable physicians to tailor risk-adjusted screening and treatment strategies for current patients with PCa.

The Evolution of Advanced Molecular Diagnostics for the Detection and Characterization of Mycoplasma pneumoniae.

PubMed

Diaz, Maureen H; Winchell, Jonas M

2016-01-01

Over the past decade there have been significant advancements in the methods used for detecting and characterizing Mycoplasma pneumoniae, a common cause of respiratory illness and community-acquired pneumonia worldwide. The repertoire of available molecular diagnostics has greatly expanded from nucleic acid amplification techniques (NAATs) that encompass a variety of chemistries used for detection, to more sophisticated characterizing methods such as multi-locus variable-number tandem-repeat analysis (MLVA), Multi-locus sequence typing (MLST), matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), single nucleotide polymorphism typing, and numerous macrolide susceptibility profiling methods, among others. These many molecular-based approaches have been developed and employed to continually increase the level of discrimination and characterization in order to better understand the epidemiology and biology of M. pneumoniae. This review will summarize recent molecular techniques and procedures and lend perspective to how each has enhanced the current understanding of this organism and will emphasize how Next Generation Sequencing may serve as a resource for researchers to gain a more comprehensive understanding of the genomic complexities of this insidious pathogen.

Sequencing-based diagnostics for pediatric genetic diseases: progress and potential

PubMed Central

Tayoun, Ahmad Abou; Krock, Bryan; Spinner, Nancy B.

2016-01-01

Introduction The last two decades have witnessed revolutionary changes in clinical diagnostics, fueled by the Human Genome Project and advances in high throughput, Next Generation Sequencing (NGS). We review the current state of sequencing-based pediatric diagnostics, associated challenges, and future prospects. Areas Covered We present an overview of genetic disease in children, review the technical aspects of Next Generation Sequencing and the strategies to make molecular diagnoses for children with genetic disease. We discuss the challenges of genomic sequencing including incomplete current knowledge of variants, lack of data about certain genomic regions, mosaicism, and the presence of regions with high homology. Expert Commentary NGS has been a transformative technology and the gap between the research and clinical communities has never been so narrow. Therapeutic interventions are emerging based on genomic findings and the applications of NGS are progressing to prenatal genetics, epigenomics and transcriptomics. PMID:27388938

Molecular Pathology and Personalized Medicine: The Dawn of a New Era in Companion Diagnostics-Practical Considerations about Companion Diagnostics for Non-Small-Cell-Lung-Cancer.

PubMed

Plönes, Till; Engel-Riedel, Walburga; Stoelben, Erich; Limmroth, Christina; Schildgen, Oliver; Schildgen, Verena

2016-01-15

Companion diagnostics (CDx) have become a major tool in molecular pathology and assist in therapy decisions in an increasing number of various cancers. Particularly, the developments in lung cancer have been most impressing in the last decade and consequently lung cancer mutation testing and molecular profiling has become a major business of diagnostic laboratories. However, it has become difficult to decide which biomarkers are currently relevant for therapy decisions, as many of the new biomarkers are not yet approved as therapy targets, remain in the status of clinical studies, or still have not left the experimental phase. The current review is focussed on those markers that do have current therapy implications, practical implications arising from the respective companion diagnostics, and thus is focused on daily practice.

Molecular diagnostics of inflammatory disease: New tools and perspectives.

PubMed

Garzorz-Stark, Natalie; Lauffer, Felix

2017-08-01

This essay reviews current approaches to establish novel molecular diagnostic tools for inflammatory skin diseases. Moreover, it highlights the importance of stratifying patients according to molecular signatures and revising current outdated disease classification systems to eventually reach the goal of personalized medicine. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular pathogenesis and clinical management of Fanconi anemia

PubMed Central

Kee, Younghoon; D’Andrea, Alan D.

2012-01-01

Fanconi anemia (FA) is a rare genetic disorder associated with a high frequency of hematological abnormalities and congenital anomalies. Based on multilateral efforts from basic scientists and clinicians, significant advances in our knowledge of FA have been made in recent years. Here we review the clinical features, the diagnostic criteria, and the current and future therapies of FA and describe the current understanding of the molecular basis of the disease. PMID:23114602

Multispectral optical tweezers for molecular diagnostics of single biological cells

NASA Astrophysics Data System (ADS)

Butler, Corey; Fardad, Shima; Sincore, Alex; Vangheluwe, Marie; Baudelet, Matthieu; Richardson, Martin

2012-03-01

Optical trapping of single biological cells has become an established technique for controlling and studying fundamental behavior of single cells with their environment without having "many-body" interference. The development of such an instrument for optical diagnostics (including Raman and fluorescence for molecular diagnostics) via laser spectroscopy with either the "trapping" beam or secondary beams is still in progress. This paper shows the development of modular multi-spectral imaging optical tweezers combining Raman and Fluorescence diagnostics of biological cells.

Smart Cup: A Minimally-Instrumented, Smartphone-Based Point-of-Care Molecular Diagnostic Device.

PubMed

Liao, Shih-Chuan; Peng, Jing; Mauk, Michael G; Awasthi, Sita; Song, Jinzhao; Friedman, Harvey; Bau, Haim H; Liu, Changchun

2016-06-28

Nucleic acid amplification-based diagnostics offer rapid, sensitive, and specific means for detecting and monitoring the progression of infectious diseases. However, this method typically requires extensive sample preparation, expensive instruments, and trained personnel. All of which hinder its use in resource-limited settings, where many infectious diseases are endemic. Here, we report on a simple, inexpensive, minimally-instrumented, smart cup platform for rapid, quantitative molecular diagnostics of pathogens at the point of care. Our smart cup takes advantage of water-triggered, exothermic chemical reaction to supply heat for the nucleic acid-based, isothermal amplification. The amplification temperature is regulated with a phase-change material (PCM). The PCM maintains the amplification reactor at a constant temperature, typically, 60-65°C, when ambient temperatures range from 12 to 35°C. To eliminate the need for an optical detector and minimize cost, we use the smartphone's flashlight to excite the fluorescent dye and the phone camera to record real-time fluorescence emission during the amplification process. The smartphone can concurrently monitor multiple amplification reactors and analyze the recorded data. Our smart cup's utility was demonstrated by amplifying and quantifying herpes simplex virus type 2 (HSV-2) with LAMP assay in our custom-made microfluidic diagnostic chip. We have consistently detected as few as 100 copies of HSV-2 viral DNA per sample. Our system does not require any lab facilities and is suitable for use at home, in the field, and in the clinic, as well as in resource-poor settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent.

Solar thermal polymerase chain reaction for smartphone-assisted molecular diagnostics

PubMed Central

Jiang, Li; Mancuso, Matthew; Lu, Zhengda; Akar, Gunkut; Cesarman, Ethel; Erickson, David

2014-01-01

Nucleic acid-based diagnostic techniques such as polymerase chain reaction (PCR) are used extensively in medical diagnostics due to their high sensitivity, specificity and quantification capability. In settings with limited infrastructure and unreliable electricity, however, access to such devices is often limited due to the highly specialized and energy-intensive nature of the thermal cycling process required for nucleic acid amplification. Here we integrate solar heating with microfluidics to eliminate thermal cycling power requirements as well as create a simple device infrastructure for PCR. Tests are completed in less than 30 min, and power consumption is reduced to 80 mW, enabling a standard 5.5 Wh iPhone battery to provide 70 h of power to this system. Additionally, we demonstrate a complete sample-to-answer diagnostic strategy by analyzing human skin biopsies infected with Kaposi's Sarcoma herpesvirus (KSHV/HHV-8) through the combination of solar thermal PCR, HotSHOT DNA extraction and smartphone-based fluorescence detection. We believe that exploiting the ubiquity of solar thermal energy as demonstrated here could facilitate broad availability of nucleic acid-based diagnostics in resource-limited areas. PMID:24553130

Information engineering for molecular diagnostics.

PubMed Central

Sorace, J. M.; Ritondo, M.; Canfield, K.

1994-01-01

Clinical laboratories are beginning to apply the recent advances in molecular biology to the testing of patient samples. The emerging field of Molecular Diagnostics will require a new Molecular Diagnostics Laboratory Information System which handles the data types, samples and test methods found in this field. The system must be very flexible in regards to supporting ad-hoc queries. The requirements which are shaping the developments in this field are reviewed and a data model developed. Several queries which demonstrate the data models ability to support the information needs of this area have been developed and run. These results demonstrate the ability of the purposed data model to meet the current and projected needs of this rapidly expanding field. PMID:7949937

Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays

PubMed Central

Agosto-Arroyo, Emmanuel; Coshatt, Gina M.; Winokur, Thomas S.; Harada, Shuko; Park, Seung L.

2017-01-01

Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning opportunities and workflow improvements in the molecular laboratory. PMID:28480121

Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches.

PubMed

Khodakov, Dmitriy; Wang, Chunyan; Zhang, David Yu

2016-10-01

Nucleic acid sequence variations have been implicated in many diseases, and reliable detection and quantitation of DNA/RNA biomarkers can inform effective therapeutic action, enabling precision medicine. Nucleic acid analysis technologies being translated into the clinic can broadly be classified into hybridization, PCR, and sequencing, as well as their combinations. Here we review the molecular mechanisms of popular commercial assays, and their progress in translation into in vitro diagnostics. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Disruption of Fibroblast Growth Factor Receptor (FGFR) Signaling as an Approach to Prostate Cancer Therapy

DTIC Science & Technology

2005-04-01

presented in tenth Annual Meeting of Association of Molecular Pathology and the abstract was published in the Journal of Molecular Diagnostics . The full...CDC25C Phosphatase Activity in Prostate Cancer: Correlation to Biochemical Recurrence. Journal of Molecular Diagnostics 2004, (6)4: 431. A manuscript in...receptor signaling. Clinical Cancer Research (in press). Acceptance letter for the above manuscript An abstract published in the Journal of Molecular

78 FR 3906 - Prospective Grant of a Co-Exclusive License: Adenovirus-Based Controls and Calibrators for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-17

... contemplated license should be directed to: Uri Reichman, Ph.D., M.B.A, Office of Technology Transfer, National... calibrators and controls for molecular diagnostics (e.g. real time PCR tests). The prospective co-exclusive...

Progress in molecular-based management of differentiated thyroid cancer

PubMed Central

Xing, Mingzhao; Haugen, Bryan R; Schlumberger, Martin

2014-01-01

Substantial developments have occurred in the past 5–10 years in clinical translational research of thyroid cancer. Diagnostic molecular markers, such as RET-PTC, RAS, and BRAFV600E mutations; galectin 3; and a new gene expression classifier, are outstanding examples that have improved diagnosis of thyroid nodules. BRAF mutation is a prognostic genetic marker that has improved risk stratification and hence tailored management of patients with thyroid cancer, including those with conventionally low risks. Novel molecular-targeted treatments hold great promise for radioiodine-refractory and surgically inoperable thyroid cancers as shown in clinical trials; such treatments are likely to become a component of the standard treatment regimen for patients with thyroid cancer in the near future. These novel molecular-based management strategies for thyroid nodules and thyroid cancer are the most exciting developments in this unprecedented era of molecular thyroid-cancer medicine. PMID:23668556

Additional burden of asymptomatic and sub-patent malaria infections during low transmission season in forested tribal villages in Chhattisgarh, India.

PubMed

Chourasia, Mehul Kumar; Raghavendra, Kamaraju; Bhatt, Rajendra M; Swain, Dipak Kumar; Meshram, Hemraj M; Meshram, Jayant K; Suman, Shrity; Dubey, Vinita; Singh, Gyanendra; Prasad, Kona Madhavinadha; Kleinschmidt, Immo

2017-08-08

The burden of sub-patent malaria is difficult to recognize in low endemic areas due to limitation of diagnostic tools, and techniques. Polymerase chain reaction (PCR), a molecular based technique, is one of the key methods for detection of low parasite density infections. The study objective was to assess the additional burden of asymptomatic and sub-patent malaria infection among tribal populations inhabiting three endemic villages in Keshkal sub-district, Chhattisgarh, India. A cross-sectional survey was conducted in March-June 2016, during the low transmission season, to measure and compare prevalence of malaria infection using three diagnostics: rapid diagnostic test, microscopy and nested-PCR. Out of 437 individuals enrolled in the study, 103 (23.6%) were malaria positive by PCR and/or microscopy of whom 89.3% were Plasmodium falciparum cases, 77.7% were afebrile and 35.9% had sub-patent infections. A substantial number of asymptomatic and sub-patent malaria infections were identified in the survey. Hence, strategies for identifying and reducing the hidden burden of asymptomatic and sub-patent infections should focus on forest rural tribal areas using more sensitive molecular diagnostic methods to curtail malaria transmission.

Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae.

PubMed

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Clancy, Eoin; Barry, Thomas

2015-10-01

Haemophilus influenzae is recognised as an important human pathogen associated with invasive infections, including bloodstream infection and meningitis. Currently used molecular-based diagnostic assays lack specificity in correctly detecting and identifying H. influenzae. As such, there is a need to develop novel diagnostic assays for the specific identification of H. influenzae. Whole genome comparative analysis was performed to identify putative diagnostic targets, which are unique in nucleotide sequence to H. influenzae. From this analysis, we identified 2H. influenzae putative diagnostic targets, phoB and pstA, for use in real-time PCR diagnostic assays. Real-time PCR diagnostic assays using these targets were designed and optimised to specifically detect and identify all 55H. influenzae strains tested. These novel rapid assays can be applied to the specific detection and identification of H. influenzae for use in epidemiological studies and could also enable improved monitoring of invasive disease caused by these bacteria. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular property diagnostic suite (MPDS): Development of disease-specific open source web portals for drug discovery.

PubMed

Nagamani, S; Gaur, A S; Tanneeru, K; Muneeswaran, G; Madugula, S S; Consortium, Mpds; Druzhilovskiy, D; Poroikov, V V; Sastry, G N

2017-11-01

Molecular property diagnostic suite (MPDS) is a Galaxy-based open source drug discovery and development platform. MPDS web portals are designed for several diseases, such as tuberculosis, diabetes mellitus, and other metabolic disorders, specifically aimed to evaluate and estimate the drug-likeness of a given molecule. MPDS consists of three modules, namely data libraries, data processing, and data analysis tools which are configured and interconnected to assist drug discovery for specific diseases. The data library module encompasses vast information on chemical space, wherein the MPDS compound library comprises 110.31 million unique molecules generated from public domain databases. Every molecule is assigned with a unique ID and card, which provides complete information for the molecule. Some of the modules in the MPDS are specific to the diseases, while others are non-specific. Importantly, a suitably altered protocol can be effectively generated for another disease-specific MPDS web portal by modifying some of the modules. Thus, the MPDS suite of web portals shows great promise to emerge as disease-specific portals of great value, integrating chemoinformatics, bioinformatics, molecular modelling, and structure- and analogue-based drug discovery approaches.

Near instrument-free, simple molecular device for rapid detection of herpes simplex viruses.

PubMed

Lemieux, Bertrand; Li, Ying; Kong, Huimin; Tang, Yi-Wei

2012-06-01

The first near instrument-free, inexpensive and simple molecular diagnostic device (IsoAmp HSV, BioHelix Corp., MA, USA) recently received US FDA clearance for use in the detection of herpes simplex viruses (HSV) in genital and oral lesion specimens. The IsoAmp HSV assay uses isothermal helicase-dependent amplification in combination with a disposable, hermetically-sealed, vertical-flow strip identification. The IsoAmp HSV assay has a total test-to-result time of less than 1.5 h by omitting the time-consuming nucleic acid extraction. The diagnostic sensitivity and specificity are comparable to PCR and are superior to culture-based methods. The near instrument-free, rapid and simple characteristics of the IsoAmp HSV assay make it potentially suitable for point-of-care testing.

DHB Task Force Review of DoD Biodefense Infrastructure and Biological Research Portfolio

DTIC Science & Technology

2009-04-29

Center and Biological Defense and Research Directorate (BORD) biodefense research portfolio includes vaccines, molecular diagnostics , genomics, and...human monoclonal antibodies, and innate immune agonists); molecular diagnostics (including recombinant reagents, assay development, reagent production

Diagnostic Accuracy of Molecular Amplification Tests for Human African Trypanosomiasis—Systematic Review

PubMed Central

Boer, Kimberly R.; Dyserinck, Heleen C.; Büscher, Philippe; Schallig, Henk D. H. F.; Leeflang, Mariska M. G.

2012-01-01

Background A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Methodology Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. Results 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Conclusion Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately. PMID:22253934

Diagnostic accuracy of molecular amplification tests for human African trypanosomiasis--systematic review.

PubMed

Mugasa, Claire M; Adams, Emily R; Boer, Kimberly R; Dyserinck, Heleen C; Büscher, Philippe; Schallig, Henk D H F; Leeflang, Mariska M G

2012-01-01

A range of molecular amplification techniques have been developed for the diagnosis of Human African Trypanosomiasis (HAT); however, careful evaluation of these tests must precede implementation to ensure their high clinical accuracy. Here, we investigated the diagnostic accuracy of molecular amplification tests for HAT, the quality of articles and reasons for variation in accuracy. Data from studies assessing diagnostic molecular amplification tests were extracted and pooled to calculate accuracy. Articles were included if they reported sensitivity and specificity or data whereby values could be calculated. Study quality was assessed using QUADAS and selected studies were analysed using the bivariate random effects model. 16 articles evaluating molecular amplification tests fulfilled the inclusion criteria: PCR (n = 12), NASBA (n = 2), LAMP (n = 1) and a study comparing PCR and NASBA (n = 1). Fourteen articles, including 19 different studies were included in the meta-analysis. Summary sensitivity for PCR on blood was 99.0% (95% CI 92.8 to 99.9) and the specificity was 97.7% (95% CI 93.0 to 99.3). Differences in study design and readout method did not significantly change estimates although use of satellite DNA as a target significantly lowers specificity. Sensitivity and specificity of PCR on CSF for staging varied from 87.6% to 100%, and 55.6% to 82.9% respectively. Here, PCR seems to have sufficient accuracy to replace microscopy where facilities allow, although this conclusion is based on multiple reference standards and a patient population that was not always representative. Future studies should, therefore, include patients for which PCR may become the test of choice and consider well designed diagnostic accuracy studies to provide extra evidence on the value of PCR in practice. Another use of PCR for control of disease could be to screen samples collected from rural areas and test in reference laboratories, to spot epidemics quickly and direct resources appropriately.

Statistical inference on censored data for targeted clinical trials under enrichment design.

PubMed

Chen, Chen-Fang; Lin, Jr-Rung; Liu, Jen-Pei

2013-01-01

For the traditional clinical trials, inclusion and exclusion criteria are usually based on some clinical endpoints; the genetic or genomic variability of the trial participants are not totally utilized in the criteria. After completion of the human genome project, the disease targets at the molecular level can be identified and can be utilized for the treatment of diseases. However, the accuracy of diagnostic devices for identification of such molecular targets is usually not perfect. Some of the patients enrolled in targeted clinical trials with a positive result for the molecular target might not have the specific molecular targets. As a result, the treatment effect may be underestimated in the patient population truly with the molecular target. To resolve this issue, under the exponential distribution, we develop inferential procedures for the treatment effects of the targeted drug based on the censored endpoints in the patients truly with the molecular targets. Under an enrichment design, we propose using the expectation-maximization algorithm in conjunction with the bootstrap technique to incorporate the inaccuracy of the diagnostic device for detection of the molecular targets on the inference of the treatment effects. A simulation study was conducted to empirically investigate the performance of the proposed methods. Simulation results demonstrate that under the exponential distribution, the proposed estimator is nearly unbiased with adequate precision, and the confidence interval can provide adequate coverage probability. In addition, the proposed testing procedure can adequately control the size with sufficient power. On the other hand, when the proportional hazard assumption is violated, additional simulation studies show that the type I error rate is not controlled at the nominal level and is an increasing function of the positive predictive value. A numerical example illustrates the proposed procedures. Copyright © 2013 John Wiley & Sons, Ltd.

Integrating molecular markers into the World Health Organization classification of CNS tumors: a survey of the neuro-oncology community.

PubMed

Aldape, Kenneth; Nejad, Romina; Louis, David N; Zadeh, Gelareh

2017-03-01

Molecular markers provide important biological and clinical information related to the classification of brain tumors, and the integration of relevant molecular parameters into brain tumor classification systems has been a widely discussed topic in neuro-oncology over the past decade. With recent advances in the development of clinically relevant molecular signatures and the 2016 World Health Organization (WHO) update, the views of the neuro-oncology community on such changes would be informative for implementing this process. A survey with 8 questions regarding molecular markers in tumor classification was sent to an email list of Society for Neuro-Oncology members and attendees of prior meetings (n=5065). There were 403 respondents. Analysis was performed using whole group response, based on self-reported subspecialty. The survey results show overall strong support for incorporating molecular knowledge into the classification and clinical management of brain tumors. Across all 7 subspecialty groups, ≥70% of respondents agreed to this integration. Interestingly, some variability is seen among subspecialties, notably with lowest support from neuropathologists, which may reflect their roles in implementing such diagnostic technologies. Based on a survey provided to the neuro-oncology community, we report strong support for the integration of molecular markers into the WHO classification of brain tumors, as well as for using an integrated "layered" diagnostic format. While membership from each specialty showed support, there was variation by specialty in enthusiasm regarding proposed changes. The initial results of this survey influenced the deliberations underlying the 2016 WHO classification of tumors of the central nervous system. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.

A systematic review of diagnostic criteria for psoriasis in adults and children: evidence from studies with a primary aim to develop or validate diagnostic criteria.

PubMed

Burden-Teh, E; Phillips, R C; Thomas, K S; Ratib, S; Grindlay, D; Murphy, R

2017-11-06

The diagnosis of psoriasis in adults and children is made clinically, for both patient management and the selection of participants in research. Diagnostic criteria provide a structure for clinical assessment, which in turn helps standardize patient recruitment into clinical trials and case definitions in observational studies. The aim of this systematic review was to identify and critically appraise the published studies to date that had a primary research aim to develop or validate diagnostic criteria for psoriasis. A search of Ovid MEDLINE and Ovid Embase was conducted in October 2016. The primary objective was to record the sensitivity and specificity of diagnostic criteria for psoriasis. Secondary objectives included diagnostic recommendations, applicability to children and study characteristics. Diagnostic accuracy studies were critically appraised for risk of bias using the QUADAS-2 tool. Twenty-three studies met the inclusion criteria. None detailed clinical examination-based diagnostic criteria. The included criteria varied from genetic and molecular diagnostic models to skin imaging, histopathology, and questionnaire-based, computer-aided and traditional Chinese medicine criteria. High sensitivity and specificity (> 90%) were reported in many studies. However, the study authors often did not specify how the criteria would be used clinically or in research. This review identified studies with varying risk of bias, and due to each study developing separate criteria meta-analysis was not possible. Clinical examination-based diagnostic criteria are currently lacking for psoriasis. Future research could follow an international collaborative approach and employ study designs allowing high-quality diagnostic accuracy testing. Existing and newly developed criteria require validation. © 2017 British Association of Dermatologists.

Diagnostic Tests to Support Late-Stage Control Programs for Schistosomiasis and Soil-Transmitted Helminthiases.

PubMed

Hawkins, Kenneth R; Cantera, Jason L; Storey, Helen L; Leader, Brandon T; de Los Santos, Tala

2016-12-01

Global efforts to address schistosomiasis and soil-transmitted helminthiases (STH) include deworming programs for school-aged children that are made possible by large-scale drug donations. Decisions on these mass drug administration (MDA) programs currently rely on microscopic examination of clinical specimens to determine the presence of parasite eggs. However, microscopy-based methods are not sensitive to the low-intensity infections that characterize populations that have undergone MDA. Thus, there has been increasing recognition within the schistosomiasis and STH communities of the need for improved diagnostic tools to support late-stage control program decisions, such as when to stop or reduce MDA. Failure to adequately address the need for new diagnostics could jeopardize achievement of the 2020 London Declaration goals. In this report, we assess diagnostic needs and landscape potential solutions and determine appropriate strategies to improve diagnostic testing to support control and elimination programs. Based upon literature reviews and previous input from experts in the schistosomiasis and STH communities, we prioritized two diagnostic use cases for further exploration: to inform MDA-stopping decisions and post-MDA surveillance. To this end, PATH has refined target product profiles (TPPs) for schistosomiasis and STH diagnostics that are applicable to these use cases. We evaluated the limitations of current diagnostic methods with regards to these use cases and identified candidate biomarkers and diagnostics with potential application as new tools. Based on this analysis, there is a need to develop antigen-detecting rapid diagnostic tests (RDTs) with simplified, field-deployable sample preparation for schistosomiasis. Additionally, there is a need for diagnostic tests that are more sensitive than the current methods for STH, which may include either a field-deployable molecular test or a simple, low-cost, rapid antigen-detecting test.

GENE-07. MOLECULAR NEUROPATHOLOGY 2.0 - INCREASING DIAGNOSTIC ACCURACY IN PEDIATRIC NEUROONCOLOGY

PubMed Central

Sturm, Dominik; Jones, David T.W.; Capper, David; Sahm, Felix; von Deimling, Andreas; Rutkoswki, Stefan; Warmuth-Metz, Monika; Bison, Brigitte; Gessi, Marco; Pietsch, Torsten; Pfister, Stefan M.

2017-01-01

Abstract The classification of central nervous system (CNS) tumors into clinically and biologically distinct entities and subgroups is challenging. Children and adolescents can be affected by >100 histological variants with very variable outcomes, some of which are exceedingly rare. The current WHO classification has introduced a number of novel molecular markers to aid routine neuropathological diagnostics, and DNA methylation profiling is emerging as a powerful tool to distinguish CNS tumor classes. The Molecular Neuropathology 2.0 study aims to integrate genome wide (epi-)genetic diagnostics with reference neuropathological assessment for all newly-diagnosed pediatric brain tumors in Germany. To date, >350 patients have been enrolled. A molecular diagnosis is established by epigenetic tumor classification through DNA methylation profiling and targeted panel sequencing of >130 genes to detect diagnostically and/or therapeutically useful DNA mutations, structural alterations, and fusion events. Results are aligned with the reference neuropathological diagnosis, and discrepant findings are discussed in a multi-disciplinary tumor board including reference neuroradiological evaluation. Ten FFPE sections as input material are sufficient to establish a molecular diagnosis in >95% of tumors. Alignment with reference pathology results in four broad categories: a) concordant classification (~77%), b) discrepant classification resolvable by tumor board discussion and/or additional data (~5%), c) discrepant classification without currently available options to resolve (~8%), and d) cases currently unclassifiable by molecular diagnostics (~10%). Discrepancies are enriched in certain histopathological entities, such as histological high grade gliomas with a molecularly low grade profile. Gene panel sequencing reveals predisposing germline events in ~10% of patients. Genome wide (epi-)genetic analyses add a valuable layer of information to routine neuropathological diagnostics. Our study provides insight into CNS tumors with divergent histopathological and molecular classification, opening new avenues for research discoveries and facilitating optimization of clinical management for affected patients in the future.

Optimising molecular diagnostic capacity for effective control of tuberculosis in high-burden settings.

PubMed

Sabiiti, W; Mtafya, B; Kuchaka, D; Azam, K; Viegas, S; Mdolo, A; Farmer, E C W; Khonga, M; Evangelopoulos, D; Honeyborne, I; Rachow, A; Heinrich, N; Ntinginya, N E; Bhatt, N; Davies, G R; Jani, I V; McHugh, T D; Kibiki, G; Hoelscher, M; Gillespie, S H

2016-08-01

The World Health Organization's 2035 vision is to reduce tuberculosis (TB) associated mortality by 95%. While low-burden, well-equipped industrialised economies can expect to see this goal achieved, it is challenging in the low- and middle-income countries that bear the highest burden of TB. Inadequate diagnosis leads to inappropriate treatment and poor clinical outcomes. The roll-out of the Xpert(®) MTB/RIF assay has demonstrated that molecular diagnostics can produce rapid diagnosis and treatment initiation. Strong molecular services are still limited to regional or national centres. The delay in implementation is due partly to resources, and partly to the suggestion that such techniques are too challenging for widespread implementation. We have successfully implemented a molecular tool for rapid monitoring of patient treatment response to anti-tuberculosis treatment in three high TB burden countries in Africa. We discuss here the challenges facing TB diagnosis and treatment monitoring, and draw from our experience in establishing molecular treatment monitoring platforms to provide practical insights into successful optimisation of molecular diagnostic capacity in resource-constrained, high TB burden settings. We recommend a holistic health system-wide approach for molecular diagnostic capacity development, addressing human resource training, institutional capacity development, streamlined procurement systems, and engagement with the public, policy makers and implementers of TB control programmes.

VLPs of HCV local isolates for HCV immunoassay diagnostic approach in Indonesia

NASA Astrophysics Data System (ADS)

Prasetyo, Afiono Agung

2017-01-01

Hepatitis C Virus (HCV) infection is a major global disease which often leads to morbidity and mortality. Low survival is related to the lack of adequate diagnostic because HCV infection is frequently asymptomatic and there are no specific diagnostic tests due to the fast transformation of the virus. Here, we investigated the VLPs (virus-like particles) of HCV local isolate as an immunoassay diagnostic approach to detect HCV infection, especially in Indonesia. The core, E1, and E2 of HCV local isolate genes were cloned and molecular analyzed, either as single or in recombinant-VLP form, to determine the molecular and chemical characteristics of each VLPs related to their potential use as an immunoassay detection method for HCV infection. The results indicated the molecular and chemical character of each VLPs are comparable. Conclusion: VLPs of HCV has the potential as an immunoassay diagnostic approach to detect HCV infection.

Studying the Impact of Spaceflight Environment on Immune Functions Using New Molecular Diagnostics System

NASA Astrophysics Data System (ADS)

Cohen, Luchino

Immune functions are altered during space flights. Latent virus reactivation, reduction in the number of immune cells, decreased cell activation and increased sensitivity of astronauts to infections following their return on Earth demonstrate that the immune system is less efficient during space flight. The causes of this immune deficiency are not fully understood and this dysfunction during long-term missions could result in the appearance of opportunistic infections or a decrease in the immuno-surveillance mechanisms that eradicate cancer cells. Therefore, the immune functions of astronauts will have to be monitored continuously during long-term missions in space, using miniature and semi-automated diagnostic systems. The objectives of this project are to study the causes of space-related immunodeficiency, to develop countermeasures to maintain an optimal immune function and to improve our capacity to detect infectious diseases during space missions through the monitoring of astronauts' immune system. In order to achieve these objectives, an Immune Function Diagnostic System (IFDS) will be designed to perform a set of immunological assays on board spacecrafts or on planet-bound bases. Through flow cytometric assays and molecular biology analyses, this diagnostic system could improve medical surveillance of astronauts and could be used to test countermeasures aimed at preventing immune deficiency during space missions. The capacity of the instrument to assess cellular fluorescence and to quantify the presence of soluble molecules in biological samples would support advanced molecular studies in space life sciences. Finally, such diagnostic system could also be used on Earth in remote areas or in mobile hospitals following natural disasters to fight against infectious diseases and other pathologies.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection.

PubMed

Lau, Han Yih; Botella, Jose R

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail.

Advanced DNA-Based Point-of-Care Diagnostic Methods for Plant Diseases Detection

PubMed Central

Lau, Han Yih; Botella, Jose R.

2017-01-01

Diagnostic technologies for the detection of plant pathogens with point-of-care capability and high multiplexing ability are an essential tool in the fight to reduce the large agricultural production losses caused by plant diseases. The main desirable characteristics for such diagnostic assays are high specificity, sensitivity, reproducibility, quickness, cost efficiency and high-throughput multiplex detection capability. This article describes and discusses various DNA-based point-of care diagnostic methods for applications in plant disease detection. Polymerase chain reaction (PCR) is the most common DNA amplification technology used for detecting various plant and animal pathogens. However, subsequent to PCR based assays, several types of nucleic acid amplification technologies have been developed to achieve higher sensitivity, rapid detection as well as suitable for field applications such as loop-mediated isothermal amplification, helicase-dependent amplification, rolling circle amplification, recombinase polymerase amplification, and molecular inversion probe. The principle behind these technologies has been thoroughly discussed in several review papers; herein we emphasize the application of these technologies to detect plant pathogens by outlining the advantages and disadvantages of each technology in detail. PMID:29375588

Raptor: A Portable Biosensor Upgraded for Reliability and Sensitivity

DTIC Science & Technology

2003-07-01

Research Center for Molecular Diagnostics and Therapy. The F. tularensis antigen (killed vaccine strain), affinity-purified rabbit anti-B...Russian Research Center of Molecular Diagnostics and Therapies, for their generous donations materials. The views expressed here are those of the

Opportunities and challenges associated with clinical diagnostic genome sequencing: a report of the Association for Molecular Pathology.

PubMed

Schrijver, Iris; Aziz, Nazneen; Farkas, Daniel H; Furtado, Manohar; Gonzalez, Andrea Ferreira; Greiner, Timothy C; Grody, Wayne W; Hambuch, Tina; Kalman, Lisa; Kant, Jeffrey A; Klein, Roger D; Leonard, Debra G B; Lubin, Ira M; Mao, Rong; Nagan, Narasimhan; Pratt, Victoria M; Sobel, Mark E; Voelkerding, Karl V; Gibson, Jane S

2012-11-01

This report of the Whole Genome Analysis group of the Association for Molecular Pathology illuminates the opportunities and challenges associated with clinical diagnostic genome sequencing. With the reality of clinical application of next-generation sequencing, technical aspects of molecular testing can be accomplished at greater speed and with higher volume, while much information is obtained. Although this testing is a next logical step for molecular pathology laboratories, the potential impact on the diagnostic process and clinical correlations is extraordinary and clinical interpretation will be challenging. We review the rapidly evolving technologies; provide application examples; discuss aspects of clinical utility, ethics, and consent; and address the analytic, postanalytic, and professional implications. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

[Diagnostic molecular pathology of lymphatic and myeloid neoplasms].

PubMed

Klapper, W; Kreipe, H

2015-03-01

Molecular pathology has been an integral part of the diagnostics of tumors of the hematopoietic system substantially longer than for solid neoplasms. In contrast to solid tumors, the primary objective of molecular pathology in hematopoietic neoplasms is not the prediction of drug efficacy but the diagnosis itself by excluding reactive proliferation and by using molecular features for tumor classification. In the case of malignant lymphomas, the most commonly applied molecular tests are those for gene rearrangements for immunoglobulin heavy chains and T-cell receptors. However, this article puts the focus on new and diagnostically relevant assays in hematopathology. Among these are mutations of MYD88 codon 265 in lymphoplasmacytic lymphomas, B-raf V600E in hairy cell leukemia and Stat3 exon 21 in indolent T-cell lymphomas. In myeloproliferative neoplasms, MPL W515, calreticulin exon 9 and the BCR-ABL and JAK2 V617F junctions are the most frequently analyzed differentiation series. In myelodysplastic and myeloproliferative neoplasms, SRSF2, SETBP1 and CSF3R mutations provide important differential diagnostic information. Genes mutated in myelodysplastic syndromes (MDS) are particularly diverse but their analysis significantly improves the differential diagnostics between reactive conditions and MDS. The most frequent changes in MDS include mutations of TET2 and various genes encoding splicing factors.

Molecular filter based planar Doppler velocimetry

NASA Astrophysics Data System (ADS)

Elliott, Gregory S.; Beutner, Thomas J.

1999-11-01

Molecular filter based diagnostics are continuing to gain popularity as a research tool for investigations in areas of aerodynamics, fluid mechanics, and combustion. This class of diagnostics has gone by many terms including Filtered Rayleigh Scattering, Doppler Global Velocimetry, and Planar Doppler Velocimetry. The majority of this article reviews recent advances in Planar Doppler Velocimetry in measuring up to three velocity components over a planar region in a flowfield. The history of the development of these techniques is given with a description of typical systems, components, and levels of uncertainty in the measurement. Current trends indicate that uncertainties on the order of 1 m/s are possible with these techniques. A comprehensive review is also given on the application of Planar Doppler Velocimetry to laboratory flows, supersonic flows, and large scale subsonic wind tunnels. The article concludes with a description of future trends, which may simplify the technique, followed by a description of techniques which allow multi-property measurements (i.e. velocity, density, temperature, and pressure) simultaneously.

Diagnostic of students' misconceptions using the Biological Concepts Instrument (BCI): A method for conducting an educational needs assessment

PubMed Central

Champagne Queloz, Annie; Klymkowsky, Michael W.; Stern, Elsbeth; Hafen, Ernst; Köhler, Katja

2017-01-01

Concept inventories, constructed based on an analysis of students’ thinking and their explanations of scientific situations, serve as diagnostics for identifying misconceptions and logical inconsistencies and provide data that can help direct curricular reforms. In the current project, we distributed the Biological Concepts Instrument (BCI) to 17-18-year-old students attending the highest track of the Swiss school system (Gymnasium). Students’ performances on many questions related to evolution, genetics, molecular properties and functions were diverse. Important common misunderstandings were identified in the areas of evolutionary processes, molecular properties and an appreciation of stochastic processes in biological systems. Our observations provide further evidence that the BCI is efficient in identifying specific areas where targeted instruction is required. Based on these observations we have initiated changes at several levels to reconsider how biological systems are presented to university biology studies with the goal of improving student’s foundational understanding. PMID:28493960

Verification of performance specifications for a US Food and Drug Administration-approved molecular microbiology test: Clostridium difficile cytotoxin B using the Becton, Dickinson and Company GeneOhm Cdiff assay.

PubMed

Schlaberg, Robert; Mitchell, Michael J; Taggart, Edward W; She, Rosemary C

2012-01-01

US Food and Drug Administration (FDA)-approved diagnostic tests based on molecular genetic technologies are becoming available for an increasing number of microbial pathogens. Advances in technology and lower costs have moved molecular diagnostic tests formerly performed for research purposes only into much wider use in clinical microbiology laboratories. To provide an example of laboratory studies performed to verify the performance of an FDA-approved assay for the detection of Clostridium difficile cytotoxin B compared with the manufacturer's performance standards. We describe the process and protocols used by a laboratory for verification of an FDA-approved assay, assess data from the verification studies, and implement the assay after verification. Performance data from the verification studies conducted by the laboratory were consistent with the manufacturer's performance standards and the assay was implemented into the laboratory's test menu. Verification studies are required for FDA-approved diagnostic assays prior to use in patient care. Laboratories should develop a standardized approach to verification studies that can be adapted and applied to different types of assays. We describe the verification of an FDA-approved real-time polymerase chain reaction assay for the detection of a toxin gene in a bacterial pathogen.

Current and proposed molecular diagnostics in a genitourinary service line laboratory at a tertiary clinical institution.

PubMed

Udager, Aaron M; Alva, Ajjai; Mehra, Rohit

2014-01-01

The idea that detailed knowledge of molecular oncogenesis will drive diagnostic, prognostic, and therapeutic clinical decision making in an increasingly multidisciplinary practice of oncologic care has been anticipated for many years. With the recent rapid advancement in our understanding of the molecular underpinnings of genitourinary malignancies, this concept is now starting to take shape in the fields of prostate, kidney, bladder, testicular, and penile cancer. Such breakthroughs necessitate the development of robust clinical-grade assays that can be quickly made available for patients to facilitate diagnosis in challenging cases, risk-stratify patients for subsequent clinical management, select the appropriate targeted therapy from among increasingly diverse and numerous options, and enroll patients in advanced clinical trials. This rapid translation of basic and clinical cancer research requires a streamlined, multidisciplinary approach to clinical assay development, termed here the molecular diagnostics service line laboratory. In this review, we summarize the current state and explore the future of molecular diagnostics in genitourinary oncology to conceptualize a genitourinary service line laboratory at a tertiary clinical institution.

Precision medicine for cancer with next-generation functional diagnostics.

PubMed

Friedman, Adam A; Letai, Anthony; Fisher, David E; Flaherty, Keith T

2015-12-01

Precision medicine is about matching the right drugs to the right patients. Although this approach is technology agnostic, in cancer there is a tendency to make precision medicine synonymous with genomics. However, genome-based cancer therapeutic matching is limited by incomplete biological understanding of the relationship between phenotype and cancer genotype. This limitation can be addressed by functional testing of live patient tumour cells exposed to potential therapies. Recently, several 'next-generation' functional diagnostic technologies have been reported, including novel methods for tumour manipulation, molecularly precise assays of tumour responses and device-based in situ approaches; these address the limitations of the older generation of chemosensitivity tests. The promise of these new technologies suggests a future diagnostic strategy that integrates functional testing with next-generation sequencing and immunoprofiling to precisely match combination therapies to individual cancer patients.

RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics

PubMed Central

Best, Myron G.; Sol, Nik; Kooi, Irsan; Tannous, Jihane; Westerman, Bart A.; Rustenburg, François; Schellen, Pepijn; Verschueren, Heleen; Post, Edward; Koster, Jan; Ylstra, Bauke; Ameziane, Najim; Dorsman, Josephine; Smit, Egbert F.; Verheul, Henk M.; Noske, David P.; Reijneveld, Jaap C.; Nilsson, R. Jonas A.; Tannous, Bakhos A.; Wesseling, Pieter; Wurdinger, Thomas

2015-01-01

Summary Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”. PMID:26525104

Multifunctional Nanomaterials Utilizing Hybridization Chain Reaction for Molecular Diagnostics and Bioanalytical Applications

NASA Astrophysics Data System (ADS)

Rana, Md. Muhit

DNA nanotechnology has shown great promise in molecular diagnostic, bioanalytical and biomedical applications. The great challenge of detecting target analytes, biomarkers and small molecules, in molecular diagnostics is low yield sensitivity. To address this challenge, different nanomaterials have been used for a long time and to date there is no such cost-effective bioanalytical technique which can detect these target biomarkers (DNA, RNA, circulating DNA/miRNA) or environmental heavy metal ions (Hg2+ and Ag+) in a cost-effective and efficient manner. Herein, we initially discuss two possible bioanalytical detection methods- a) colorimetric and b) fluorometric assays which are very popular nowadays due to their distinctive spectroscopic properties. Finally, we report the promising colorimetric assay using a novel DNA based amplification strategy know as hybridization chain reaction (HCR) for potential application in the visual detection of low copies of biomarkers (miRNAs as little as 20 femtomole in an RNA pool and cell extracts in seven different combinations and Ebola virus DNA as low as 400 attomoles in liquid biopsy mimics in sixteen different combinations), environmental and biological heavy metal ions (mercury and silver concentrations as low as 10 pM in water, soil and urine samples) and also successfully applied to a molecular logic gate operation to distinguish OR and AND logic gates. No results showed any false-positive or false-negative information. On the other hand, we also discuss the future possibilities of HCR amplification technology, which is very promising for fluorometric bioanalysis. The HCR based nanoprobe technology has numerous remarkable advantages over other methods. It is re-programmable, simple, inexpensive, easy to assemble and operate and can be performed with visual and spectroscopic read-outs upon recognition of the target analytes. This rapid, specific and sensitive approach for biomarkers and heavy metal ion detection generates an on-site signal while eliminating the use of sophisticated high-maintenance instrumentation. We demonstrate that this state-of-the-art technology and methodology can potentially serve as an alternative approach to detect novel disease biomarkers, small molecules and inorganic compounds. This approach can be combined with the current existing methods for real-time point-of-care molecular diagnostics and is significant for preclinical or clinical studies.

Twenty-four cases of imported zika virus infections diagnosed by molecular methods.

PubMed

Alejo-Cancho, Izaskun; Torner, Nuria; Oliveira, Inés; Martínez, Ana; Muñoz, José; Jane, Mireia; Gascón, Joaquim; Requena-Méndez, Ana; Vilella, Anna; Marcos, M Ángeles; Pinazo, María Jesús; Gonzalo, Verónica; Rodriguez, Natalia; Martínez, Miguel J

2016-10-01

Zika virus is an emerging flavivirus widely spreading through Latin America. Molecular diagnosis of the infection can be performed using serum, urine and saliva samples, although a well-defined diagnostic algorithm is not yet established. We describe a series of 24 cases of imported zika virus infection into Catalonia (northeastern Spain). Based on our findings, testing of paired serum and urine samples is recommended. Copyright © 2016 Elsevier Inc. All rights reserved.

Liposome Formulation of Fullerene-Based Molecular Diagnostic and Therapeutic Agents

PubMed Central

Zhou, Zhiguo

2013-01-01

Fullerene medicine is a new but rapidly growing research subject. Fullerene has a number of desired structural, physical and chemical properties to be adapted for biological use including antioxidants, anti-aging, anti-inflammation, photodynamic therapy, drug delivery, and magnetic resonance imaging contrast agents. Chemical functionalization of fullerenes has led to several interesting compounds with very promising preclinical efficacy, pharmacokinetic and safety data. However, there is no clinical evaluation or human use except in fullerene-based cosmetic products for human skincare. This article summarizes recent advances in liposome formulation of fullerenes for the use in therapeutics and molecular imaging. PMID:24300561

[Hepatocellular tumours in noncirrhotic liver tissue].

PubMed

Goltz, D; Fischer, H-P

2015-11-01

In recent years, the spectrum of tissue-based diagnostics of hepatocellular tumours has changed due to novel molecular pathological findings. Innovative radiographics filter out small lesions and ambiguous tumours for bioptical sampling. The spectrum of these tumours includes hepatocellular carcinoma, hepatocellular adenomas, focal nodular hyperplasia and macroregenerative nodules. Primarily, morphological analysis should identify the dignity of a lesion. After exclusion of HCC and reactive liver cell nodules, hepatocellular adenomas should be further subclassified based on immunohistochemical/molecular pathological criteria according to the WHO classification of liver tumours. This procedure provides significant additional information regarding the prognosis and therapeutic implications of hepatocellular adenomas.

Delimiting species of Protaphorura (Collembola: Onychiuridae): integrative evidence based on morphology, DNA sequences and geography.

PubMed

Sun, Xin; Zhang, Feng; Ding, Yinhuan; Davies, Thomas W; Li, Yu; Wu, Donghui

2017-08-15

Species delimitation remains a significant challenge when the diagnostic morphological characters are limited. Integrative taxonomy was applied to the genus Protaphorura (Collembola: Onychiuridae), which is one of most difficult soil animals to distinguish taxonomically. Three delimitation approaches (morphology, molecular markers and geography) were applied providing rigorous species validation criteria with an acceptably low error rate. Multiple molecular approaches, including distance- and evolutionary model-based methods, were used to determine species boundaries based on 144 standard barcode sequences. Twenty-two molecular putative species were consistently recovered across molecular and geographical analyses. Geographic criteria were was proved to be an efficient delimitation method for onychiurids. Further morphological examination, based on the combination of the number of pseudocelli, parapseudocelli and ventral mesothoracic chaetae, confirmed 18 taxa of 22 molecular units, with six of them described as new species. These characters were found to be of high taxonomical value. This study highlights the potential benefits of integrative taxonomy, particularly simultaneous use of molecular/geographical tools, as a powerful way of ascertaining the true diversity of the Onychiuridae. Our study also highlights that discovering new morphological characters remains central to achieving a full understanding of collembolan taxonomy.

ROLE OF MOLECULAR MARKERS IN THYROID NODULE MANAGEMENT: THEN AND NOW.

PubMed

Nikiforov, Yuri E

2017-08-01

To describe the evolution and clinical utility of molecular testing for thyroid nodules and cancer achieved over the last 2 decades. Scientific reports on thyroid cancer genetics and molecular diagnostics in thyroid nodules. Over the last 2 decades, our understanding of the genetic mechanisms of thyroid cancer has dramatically expanded, such that most thyroid cancers now have known gene driver events. This knowledge provides the basis for establishing and further improving molecular tests for thyroid nodules and cancer and for the introduction of new entities such as noninvasive follicular thyroid neoplasm with papillary-like nuclear features. The progress with molecular tests for thyroid nodules started in the 1990s from demonstrating feasibility of detecting various molecular alterations in fine-needle aspiration (FNA) material collected from thyroid nodules. It was followed by the introduction of the first single-gene mutational markers, such as BRAF, and a small mutational panel into clinical practice in the mid 2000s. Currently, several more advanced molecular tests are available for clinical use. They are based on multiple molecular markers and have increasing impact on the clinical management of patients with thyroid nodules. The evolution of molecular tests for thyroid nodules followed the discovery of various diagnostic and prognostic molecular markers of thyroid cancer that can be applied to thyroid FNA samples to inform more individualized management of these patients. FNA = fine-needle aspiration miRNA = micro RNA NGS = next-generation sequencing NIFTP = noninvasive follicular thyroid neoplasm with papillary-like nuclear features NPV = negative predictive value PPV = positive predictive value PTC = papillary thyroid carcinoma RAI = radioactive iodine.

Single-cell proteomics: potential implications for cancer diagnostics.

PubMed

Gavasso, Sonia; Gullaksen, Stein-Erik; Skavland, Jørn; Gjertsen, Bjørn T

2016-01-01

Single-cell proteomics in cancer is evolving and promises to provide more accurate diagnoses based on detailed molecular features of cells within tumors. This review focuses on technologies that allow for collection of complex data from single cells, but also highlights methods that are adaptable to routine cancer diagnostics. Current diagnostics rely on histopathological analysis, complemented by mutational detection and clinical imaging. Though crucial, the information gained is often not directly transferable to defined therapeutic strategies, and predicting therapy response in a patient is difficult. In cancer, cellular states revealed through perturbed intracellular signaling pathways can identify functional mutations recurrent in cancer subsets. Single-cell proteomics remains to be validated in clinical trials where serial samples before and during treatment can reveal excessive clonal evolution and therapy failure; its use in clinical trials is anticipated to ignite a diagnostic revolution that will better align diagnostics with the current biological understanding of cancer.

Molecular diagnostic experience of whole-exome sequencing in adult patients.

PubMed

Posey, Jennifer E; Rosenfeld, Jill A; James, Regis A; Bainbridge, Matthew; Niu, Zhiyv; Wang, Xia; Dhar, Shweta; Wiszniewski, Wojciech; Akdemir, Zeynep H C; Gambin, Tomasz; Xia, Fan; Person, Richard E; Walkiewicz, Magdalena; Shaw, Chad A; Sutton, V Reid; Beaudet, Arthur L; Muzny, Donna; Eng, Christine M; Yang, Yaping; Gibbs, Richard A; Lupski, James R; Boerwinkle, Eric; Plon, Sharon E

2016-07-01

Whole-exome sequencing (WES) is increasingly used as a diagnostic tool in medicine, but prior reports focus on predominantly pediatric cohorts with neurologic or developmental disorders. We describe the diagnostic yield and characteristics of WES in adults. We performed a retrospective analysis of consecutive WES reports for adults from a diagnostic laboratory. Phenotype composition was determined using Human Phenotype Ontology terms. Molecular diagnoses were reported for 17.5% (85/486) of adults, which is lower than that for a primarily pediatric population (25.2%; P = 0.0003); the diagnostic rate was higher (23.9%) for those 18-30 years of age compared to patients older than 30 years (10.4%; P = 0.0001). Dual Mendelian diagnoses contributed to 7% of diagnoses, revealing blended phenotypes. Diagnoses were more frequent among individuals with abnormalities of the nervous system, skeletal system, head/neck, and growth. Diagnostic rate was independent of family history information, and de novo mutations contributed to 61.4% of autosomal dominant diagnoses. Early WES experience in adults demonstrates molecular diagnoses in a substantial proportion of patients, informing clinical management, recurrence risk, and recommendations for relatives. A positive family history was not predictive, consistent with molecular diagnoses often revealed by de novo events, informing the Mendelian basis of genetic disease in adults.Genet Med 18 7, 678-685.

Isothermal multiple displacement amplification: a methodical approach enhancing molecular routine diagnostics of microcarcinomas and small biopsies.

PubMed

Mairinger, Fabian D; Walter, Robert Fh; Vollbrecht, Claudia; Hager, Thomas; Worm, Karl; Ting, Saskia; Wohlschläger, Jeremias; Zarogoulidis, Paul; Zarogoulidis, Konstantinos; Schmid, Kurt W

2014-01-01

Isothermal multiple displacement amplification (IMDA) can be a powerful tool in molecular routine diagnostics for homogeneous and sequence-independent whole-genome amplification of notably small tumor samples, eg, microcarcinomas and biopsies containing a small amount of tumor. Currently, this method is not well established in pathology laboratories. We designed a study to confirm the feasibility and convenience of this method for routine diagnostics with formalin-fixed, paraffin-embedded samples prepared by laser-capture microdissection. A total of 250 μg DNA (concentration 5 μg/μL) was generated by amplification over a period of 8 hours with a material input of approximately 25 cells, approximately equivalent to 175 pg of genomic DNA. In the generated DNA, a representation of all chromosomes could be shown and the presence of elected genes relevant for diagnosis in clinical samples could be proven. Mutational analysis of clinical samples could be performed without any difficulty and showed concordance with earlier diagnostic findings. We established the feasibility and convenience of IMDA for routine diagnostics. We also showed that small amounts of DNA, which were not analyzable with current molecular methods, could be sufficient for a wide field of applications in molecular routine diagnostics when they are preamplified with IMDA.

Molecular Diagnostic Experience of Whole-Exome Sequencing in Adult Patients

PubMed Central

Posey, Jennifer E.; Rosenfeld, Jill A.; James, Regis A.; Bainbridge, Matthew; Niu, Zhiyv; Wang, Xia; Dhar, Shweta; Wiszniewski, Wojciech; Akdemir, Zeynep H.C.; Gambin, Tomasz; Xia, Fan; Person, Richard E.; Walkiewicz, Magdalena; Shaw, Chad A.; Sutton, V. Reid; Beaudet, Arthur L.; Muzny, Donna; Eng, Christine M.; Yang, Yaping; Gibbs, Richard A.; Lupski, James R.; Boerwinkle, Eric; Plon, Sharon E.

2015-01-01

Purpose Whole exome sequencing (WES) is increasingly used as a diagnostic tool in medicine, but prior reports focus on predominantly pediatric cohorts with neurologic or developmental disorders. We describe the diagnostic yield and characteristics of whole exome sequencing in adults. Methods We performed a retrospective analysis of consecutive WES reports for adults from a diagnostic laboratory. Phenotype composition was determined using Human Phenotype Ontology terms. Results Molecular diagnoses were reported for 17.5% (85/486) of adults, lower than a primarily pediatric population (25.2%; p=0.0003); the diagnostic rate was higher (23.9%) in those 18–30 years of age compared to patients over 30 years (10.4%; p=0.0001). Dual Mendelian diagnoses contributed to 7% of diagnoses, revealing blended phenotypes. Diagnoses were more frequent among individuals with abnormalities of the nervous system, skeletal system, head/neck, and growth. Diagnostic rate was independent of family history information, and de novo mutations contributed to 61.4% of autosomal dominant diagnoses. Conclusion Early WES experience in adults demonstrates molecular diagnoses in a substantial proportion of patients, informing clinical management, recurrence risk and recommendations for relatives. A positive family history was not predictive, consistent with molecular diagnoses often revealed by de novo events, informing the Mendelian basis of genetic disease in adults. PMID:26633545

Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer.

PubMed

Oberauner-Wappis, Lisa; Loibner, Martina; Viertler, Christian; Groelz, Daniel; Wyrich, Ralf; Zatloukal, Kurt

2016-04-01

Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue-based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin-free, non-cross-linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene-fixed and paraffin-embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24-h formalin postfixation step of slides from PAXgene-fixed and paraffin-embedded tissues allowed us to use the assays approved for formalin-fixed and paraffin-embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24-h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene-fixed and paraffin-embedded tissue to be used for further molecular testing. © 2016 The Authors. International Journal of Experimental Pathology published by John Wiley & Sons Ltd on behalf of Company of the International Journal of Experimental Pathology (CIJEP).

Recent technological advancements in tuberculosis diagnostics - A review.

PubMed

Gupta, Shagun; Kakkar, Vipan

2018-09-15

Early diagnosis and on-time effective treatment are indispensable for Tuberculosis (TB) control - a life threatening infectious communicable disease. The conventional techniques for diagnosing TB normally take two to three weeks. This delay in diagnosis and further increase in detection complexity due to the emerging risks of XDR-TB (Extensively drug Resistant-TB) and MDR-TB (Multidrug Resistant-TB) are evoking interest of researchers in the field of developing rapid TB detection techniques such as biosensing and other point-of-care (POC) techniques. Biosensing technologies along with the collaboration with nanotechnology have enormous potential to boost the MTB detection and for overall management in clinical diagnosis. A diverse range of portable, sensitive and rapid biosensors based on different signal transducer principles and with different biomarkers detection capabilities have been developed for TB detection in the early stages. Further, a lot of progress has been achieved over the years in developing various point-of-care diagnostic tools including non-molecular methods and molecular techniques. The objective of this study is to present a succinct review of the available TB detection techniques that are either in use or under development. The focus of this review is on the current developments occurred in nano-biosensing technologies. A synopsis of ameliorations in different non-molecular diagnostic tools and progress in the field of molecular techniques along with the role of emerging Lab-on-Chip technology for diagnosing and mitigating the TB consequences have also been presented. Copyright © 2018 Elsevier B.V. All rights reserved.

Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy.

PubMed

Helbig, Katherine L; Farwell Hagman, Kelly D; Shinde, Deepali N; Mroske, Cameron; Powis, Zöe; Li, Shuwei; Tang, Sha; Helbig, Ingo

2016-09-01

To assess the yield of diagnostic exome sequencing (DES) and to characterize the molecular findings in characterized and novel disease genes in patients with epilepsy. In an unselected sample of 1,131 patients referred for DES, overall results were compared between patients with and without epilepsy. DES results were examined based on age of onset and epilepsy diagnosis. Positive/likely positive results were identified in 112/293 (38.2%) epilepsy patients compared with 210/732 (28.7%) patients without epilepsy (P = 0.004). The diagnostic yield in characterized disease genes among patients with epilepsy was 33.4% (105/314). KCNQ2, MECP2, FOXG1, IQSEC2, KMT2A, and STXBP1 were most commonly affected by de novo alterations. Patients with epileptic encephalopathies had the highest rate of positive findings (43.4%). A likely positive novel genetic etiology was proposed in 14/200 (7%) patients with epilepsy; this frequency was highest in patients with epileptic encephalopathies (17%). Three genes (COQ4, DNM1, and PURA) were initially reported as likely positive novel disease genes and were subsequently corroborated in independent peer-reviewed publications. DES with analysis and interpretation of both characterized and novel genetic etiologies is a useful diagnostic tool in epilepsy, particularly in severe early-onset epilepsy. The reporting on novel genetic etiologies may further increase the diagnostic yield.Genet Med 18 9, 898-905.

Applied Genomics: Data Mining Reveals Species-Specific Malaria Diagnostic Targets More Sensitive than 18S rRNA▿†‡

PubMed Central

Demas, Allison; Oberstaller, Jenna; DeBarry, Jeremy; Lucchi, Naomi W.; Srinivasamoorthy, Ganesh; Sumari, Deborah; Kabanywanyi, Abdunoor M.; Villegas, Leopoldo; Escalante, Ananias A.; Kachur, S. Patrick; Barnwell, John W.; Peterson, David S.; Udhayakumar, Venkatachalam; Kissinger, Jessica C.

2011-01-01

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ∼100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms. PMID:21525225

Chasing down the triple-negative myeloproliferative neoplasms: Implications for molecular diagnostics.

PubMed

Langabeer, Stephen E

2016-01-01

The majority of patients with classical myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia, and primary myelofibrosis harbor distinct disease-driving mutations within the JAK2 , CALR , or MPL genes. The term triple-negative has been recently applied to those MPN without evidence of these consistent mutations, prompting whole or targeted exome sequencing approaches to determine the driver mutational status of this subgroup. These strategies have identified numerous novel mutations that occur in alternative exons of both JAK2 and MPL , the majority of which result in functional activation. Current molecular diagnostic approaches may possess insufficient coverage to detect these alternative mutations, prompting further consideration of targeted exon sequencing into routine diagnostic practice. How to incorporate these illuminating findings into the expanding molecular diagnostic algorithm for MPN requires continual attention.

Molecular diagnostics in the neoplasms of the pancreas, liver, gall bladder, and extrahepatic biliary tract.

PubMed

Weindel, Michael; Zulfiqar, Muhammad; Bhalla, Amarpreet; Shidham, Vinod B

2013-12-01

Pancreatic neoplasms, including ductal adenocarcinoma, intraductal papillary mucinous neoplasm, solid pseudopapillary neoplasm, pancreatic endocrine neoplasms, acinar cell carcinoma, and ampullary carcinoma, are associated with different genetic abnormalities. Liver neoplasms, including hepatic adenomas, hepatocellular carcinomas, and cholangiocarcinomas, are associated with identifiable risk factors and genetic changes. Gall bladder adenomas and adenocarcinomas arise from distinct molecular pathways. The molecular abnormalities seen in these tumors are not used routinely in the molecular diagnostic laboratory. Copyright © 2013 Elsevier Inc. All rights reserved.

On-chip purification and detection of hepatitis C virus RNA from human plasma.

PubMed

Vaghi, V; Potrich, C; Pasquardini, L; Lunelli, L; Vanzetti, L; Ebranati, E; Lai, A; Zehender, G; Mombello, D; Cocuzza, M; Pirri, C F; Pederzolli, C

2016-01-01

Hepatitis C virus (HCV) is one of the main causes of chronic liver disease worldwide. The diagnosis and monitoring of HCV infection is a crucial need in the clinical management. The conventional diagnostic technologies are challenged when trying to address molecular diagnostics, especially because they require a complex and time-consuming sample preparation phase. Here, a new concept based on surface functionalization was applied to viral RNA purification: first of all polydimethylsiloxane (PDMS) flat surfaces were modified to hold RNA adsorption. After a careful chemical and morphological analysis of the modified surfaces, the functionalization protocols giving the best RNA adsorbing surfaces were applied to PDMS microdevices. The functionalized microdevices were then used for RNA purification from HCV infected human plasma samples. RNA purification and RT were successfully performed in the same microdevice chamber, saving time of analysis, reagents, and labor. The PCR protocol for HCV cDNA amplification was also implemented in the microdevice, demonstrating that the entire process of HCV analysis, from plasma to molecular readout, could be performed on-chip. Not only HCV but also other microdevice-based viral RNA detection could therefore result in a successful Point-of-Care (POC) diagnostics for resource-limited settings. Copyright © 2015 Elsevier B.V. All rights reserved.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing.

PubMed

Yu, Stephanie C Y; Chan, K C Allen; Zheng, Yama W L; Jiang, Peiyong; Liao, Gary J W; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y; Go, Attie T J I; van Vugt, John M G; Minekawa, Ryoko; Oudejans, Cees B M; Nicolaides, Kypros H; Chiu, Rossa W K; Lo, Y M Dennis

2014-06-10

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring.

Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing

PubMed Central

Yu, Stephanie C. Y.; Chan, K. C. Allen; Zheng, Yama W. L.; Jiang, Peiyong; Liao, Gary J. W.; Sun, Hao; Akolekar, Ranjit; Leung, Tak Y.; Go, Attie T. J. I.; van Vugt, John M. G.; Minekawa, Ryoko; Oudejans, Cees B. M.; Nicolaides, Kypros H.; Chiu, Rossa W. K.; Lo, Y. M. Dennis

2014-01-01

Noninvasive prenatal testing using fetal DNA in maternal plasma is an actively researched area. The current generation of tests using massively parallel sequencing is based on counting plasma DNA sequences originating from different genomic regions. In this study, we explored a different approach that is based on the use of DNA fragment size as a diagnostic parameter. This approach is dependent on the fact that circulating fetal DNA molecules are generally shorter than the corresponding maternal DNA molecules. First, we performed plasma DNA size analysis using paired-end massively parallel sequencing and microchip-based capillary electrophoresis. We demonstrated that the fetal DNA fraction in maternal plasma could be deduced from the overall size distribution of maternal plasma DNA. The fetal DNA fraction is a critical parameter affecting the accuracy of noninvasive prenatal testing using maternal plasma DNA. Second, we showed that fetal chromosomal aneuploidy could be detected by observing an aberrant proportion of short fragments from an aneuploid chromosome in the paired-end sequencing data. Using this approach, we detected fetal trisomy 21 and trisomy 18 with 100% sensitivity (T21: 36/36; T18: 27/27) and 100% specificity (non-T21: 88/88; non-T18: 97/97). For trisomy 13, the sensitivity and specificity were 95.2% (20/21) and 99% (102/103), respectively. For monosomy X, the sensitivity and specificity were both 100% (10/10 and 8/8). Thus, this study establishes the principle of size-based molecular diagnostics using plasma DNA. This approach has potential applications beyond noninvasive prenatal testing to areas such as oncology and transplantation monitoring. PMID:24843150

A methodological overview on molecular preimplantation genetic diagnosis and screening: a genomic future?

PubMed

Vendrell, Xavier; Bautista-Llácer, Rosa

2012-12-01

The genetic diagnosis and screening of preimplantation embryos generated by assisted reproduction technology has been consolidated in the prenatal care framework. The rapid evolution of DNA technologies is tending to molecular approaches. Our intention is to present a detailed methodological view, showing different diagnostic strategies based on molecular techniques that are currently applied in preimplantation genetic diagnosis. The amount of DNA from one single, or a few cells, obtained by embryo biopsy is a limiting factor for the molecular analysis. In this sense, genetic laboratories have developed molecular protocols considering this restrictive condition. Nevertheless, the development of whole-genome amplification methods has allowed preimplantation genetic diagnosis for two or more indications simultaneously, like the selection of histocompatible embryos plus detection of monogenic diseases or aneuploidies. Moreover, molecular techniques have permitted preimplantation genetic screening to progress, by implementing microarray-based comparative genome hybridization. Finally, a future view of the embryo-genetics field based on molecular advances is proposed. The normalization, cost-effectiveness analysis, and new technological tools are the next topics for preimplantation genetic diagnosis and screening. Concomitantly, these additions to assisted reproduction technologies could have a positive effect on the schedules of preimplantation studies.

Molecular diagnostics for hereditary hearing loss in children.

PubMed

Sommen, Manou; Wuyts, Wim; Van Camp, Guy

2017-08-01

Hearing loss (HL) is the most common birth defect in industrialized countries with far-reaching social, psychological and cognitive implications. It is an extremely heterogeneous disease, complicating molecular testing. The introduction of next-generation sequencing (NGS) has resulted in great progress in diagnostics allowing to study all known HL genes in a single assay. The diagnostic yield is currently still limited, but has the potential to increase substantially. Areas covered: In this review the utility of NGS and the problems for comprehensive molecular testing for HL are evaluated and discussed. Expert commentary: Different publications have proven the appropriateness of NGS for molecular testing of heterogeneous diseases such as HL. However, several problems still exist, such as pseudogenic background of some genes and problematic copy number variant analysis on targeted NGS data. Another main challenge for the future will be the establishment of population specific mutation-spectra to achieve accurate personalized comprehensive molecular testing for HL.

Molecular Pathology: Predictive, Prognostic, and Diagnostic Markers in Uterine Tumors.

PubMed

Ritterhouse, Lauren L; Howitt, Brooke E

2016-09-01

This article focuses on the diagnostic, prognostic, and predictive molecular biomarkers in uterine malignancies, in the context of morphologic diagnoses. The histologic classification of endometrial carcinomas is reviewed first, followed by the description and molecular classification of endometrial epithelial malignancies in the context of histologic classification. Taken together, the molecular and histologic classifications help clinicians to approach troublesome areas encountered in clinical practice and evaluate the utility of molecular alterations in the diagnosis and subclassification of endometrial carcinomas. Putative prognostic markers are reviewed. The use of molecular alterations and surrogate immunohistochemistry as prognostic and predictive markers is also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular diagnostics for Chagas disease: up to date and novel methodologies.

PubMed

Alonso-Padilla, Julio; Gallego, Montserrat; Schijman, Alejandro G; Gascon, Joaquim

2017-07-01

Chagas disease is caused by the parasite Trypanosoma cruzi. It affects 7 million people, mainly in Latin America. Diagnosis is usually made serologically, but at some clinical scenarios serology cannot be used. Then, molecular detection is required for early detection of congenital transmission, treatment response follow up, and diagnosis of immune-suppression reactivation. However, present tests are technically demanding and require well-equipped laboratories which make them unfeasible in low-resources endemic regions. Areas covered: Available molecular tools for detection of T. cruzi DNA, paying particular attention to quantitative PCR protocols, and to the latest developments of user-friendly molecular diagnostic methodologies. Expert commentary: In the absence of appropriate biomarkers, molecular diagnosis is the only option for the assessment of treatment response. Besides, it is very useful for the early detection of acute infections, like congenital cases. Since current Chagas disease molecular tests are restricted to referential labs, research efforts must focus in the implementation of easy-to-use diagnostic tools in order to overcome the access to diagnosis gap.

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

PubMed Central

Khan, A S; Moe, C L; Glass, R I; Monroe, S S; Estes, M K; Chapman, L E; Jiang, X; Humphrey, C; Pon, E; Iskander, J K

1994-01-01

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis. Images PMID:8150941

Molecular Diagnostics of Fusion and Laboratory Plasmas

NASA Astrophysics Data System (ADS)

Fantz, U.

2005-05-01

The presence of molecules in the cold scrape-off layer of fusion experiments and industrial plasmas requires an understanding of the molecular dynamics in these low temperature plasmas. Suitable diagnostic methods can provide an insight in molecular processes in the plasma volume as well as for plasma surface interactions. A very simple but powerful technique is the molecular emission spectroscopy. Spectra are obtained easily, whereas interpretation might be very complex and relies on the availability of atomic and molecular data. Examples are given for hydrogen plasmas and plasmas with hydrocarbons which both are of importance in industrial applications as well as in fusion experiments.

Molecular Approaches to Thyroid Cancer Diagnosis

PubMed Central

Hsiao, Susan J.; Nikiforov, Yuri E.

2014-01-01

Thyroid nodules are common, and the accurate diagnosis of cancer or benign disease is important for the effective clinical management of these patients. Molecular markers are a helpful diagnostic tool, particularly for cytologically indeterminate thyroid nodules. In the past few years, significant progress has been made in developing molecular markers for clinical use in fine needle aspiration (FNA) specimens, including gene mutation panels and gene expression classifiers. With the availability of next generation sequencing technology, gene mutation panels can be expanded to interrogate multiple genes simultaneously and to provide yet more accurate diagnostic information. In addition, recently several new molecular markers in thyroid cancer have been identified that offer diagnostic, prognostic, and therapeutic information that could potentially be of value in guiding individualized management of patients with thyroid nodules. PMID:24829266

Intestinal cestodes.

PubMed

Webb, Camille; Cabada, Miguel M

2017-10-01

Cestodes infections in humans are among the most prevalent parasitosis worldwide. Although tapeworm infection is often asymptomatic, they can be associated with a range of symptoms. The landscape of cestode infections is changing with rapid diagnosis techniques and advanced molecular diagnosis aiding in identification of species specific epidemiology. Traditional descriptions of species by location have been challenged with molecular diagnostic techniques, which show variation in distribution of species, thought to be because of globalization and importation of disease. Epidemiology, molecular diagnostic techniques. Infection by tapeworms is often asymptomatic or accompanied by mild symptoms though can occasionally cause severe disease and contribute to anemia and malnutrition. Tapeworm infection is most prevalent in resource-poor countries but the distribution is worldwide. Epidemiology of infection is changing because of molecular diagnostics, which allow more accurate tracking of species.

Disseminated histoplasmosis in a domestic cat imported from the USA to Austria

PubMed Central

Klang, Andrea; Loncaric, Igor; Spergser, Joachim; Eigelsreiter, Sabine; Weissenböck, Herbert

2013-01-01

We present a case of disseminated histoplasmosis in a domestic cat imported from the USA to Austria. Histopathological examination revealed a systemic mycosis with most severe involvement of the lungs suggestive of Histoplasma (H.) capsulatum-infection. Molecular confirmation was based on polymerase chain reaction (PCR) and sequence analysis of a fungal culture from liver samples. This is the first case of feline histoplasmosis proven by molecular diagnostic technique in Europe and reported in Austria, etc. PMID:24432230

A new molecular diagnostic tool for quantitatively detecting and genotyping “Candidatus Liberibacter species”

USDA-ARS?s Scientific Manuscript database

A new molecular diagnostic method was developed for quantitative detection of “Candidatus Liberibacter” species associated with citrus Huanglongbing (“Ca. Liberibacter asiaticus”, “Ca. Liberibacter africanus” and “Ca. Liberibacter americanus”) and potato zebra chip disorder (“Ca. Liberibacter solana...

Chordoma of the Head and Neck: A Review.

PubMed

Wasserman, Jason K; Gravel, Denis; Purgina, Bibianna

2018-06-01

Chordoma is a rare malignant bone tumor that can arise anywhere along the central neural axis and many involve head and neck sites, most commonly the skull base. The relative rarity of these tumors, combined with the complex anatomy of the head and neck, pose diagnostic challenges to pathologists. This article describes the pertinent clinical, pathologic, and molecular features of chordomas and describes how these features can be used to aid in formulating a differential diagnosis. Emphasis is placed on key diagnostic pitfalls and the importance of incorporating immunohistochemical information into the diagnosis.

Molecular Pathology: A Requirement for Precision Medicine in Cancer.

PubMed

Dietel, Manfred

2016-01-01

The increasing importance of targeting drugs and check-point inhibitors in the treatment of several tumor entities (breast, colon, lung, malignant melanoma, lymphoma, etc.) and the necessity of a companion diagnostic (HER2, (pan)RAS, EGFR, ALK, BRAF, ROS1, MET, PD-L1, etc.) is leading to new challenges for surgical pathology. Since almost all the biomarkers to be specifically detected are tissue based, a precise and reliable diagnostic is absolutely crucial. To meet this challenge surgical pathology has adapted a number of molecular methods (semi-quantitative immunohistochemistry, fluorescence in situ hybridization, PCR and its multiple variants, (pyro/Sanger) sequencing, next generation sequencing (amplicon, whole exome, whole genome), DNA arrays, methylation analyses, etc.) to be applicable for formalin-fixed paraffin-embedded tissue. Reading a patient's tissue as 'deeply' as possible and obtaining information on the morphological, genetic, proteomic and epigenetic background are the tasks of pathologists and molecular biologists and provide the clinicians with information relevant for precision medicine. Intensified cooperation between clinicians and pathologists will provide the basis of improved clinical drug selection and guide development of new cancer gene therapies and molecularly targeted drugs by research units and the pharmaceutical industry. © 2016 S. Karger GmbH, Freiburg.

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

PubMed Central

Rodríguez-Lázaro, David; D'Agostino, Martin; Pla, Maria; Cook, Nigel

2004-01-01

An important analytical control in molecular amplification-based methods is an internal amplification control (IAC), which should be included in each reaction mixture. An IAC is a nontarget nucleic acid sequence which is coamplified simultaneously with the target sequence. With negative results for the target nucleic acid, the absence of an IAC signal indicates that amplification has failed. A general strategy for the construction of an IAC for inclusion in molecular beacon-based real-time nucleic acid sequence-based amplification (NASBA) assays is presented. Construction proceeds in two phases. In the first phase, a double-stranded DNA molecule that contains nontarget sequences flanked by target sequences complementary to the NASBA primers is produced. At the 5′ end of this DNA molecule is a T7 RNA polymerase binding sequence. In the second phase of construction, RNA transcripts are produced from the DNA by T7 RNA polymerase. This RNA is the IAC; it is amplified by the target NASBA primers and is detected by a molecular beacon probe complementary to the internal nontarget sequences. As a practical example, an IAC for use in an assay for the detection of Mycobacterium avium subsp. paratuberculosis is described, its incorporation and optimization within the assay are detailed, and its application to spiked and natural clinical samples is shown to illustrate the correct interpretation of the diagnostic results. PMID:15583319

Basis for molecular diagnostics and immunotherapy for esophageal cancer.

PubMed

Abdo, Joe; Agrawal, Devendra K; Mittal, Sumeet K

2017-01-01

Esophageal cancer (EC) is an extremely aggressive neoplasm, diagnosed in about 17,000 Americans every year with a mortality rate of more than 80% within five years and a median overall survival of just 13 months. For decades, the go-to regimen for esophageal cancer patients has been the use of taxane and platinum-based chemotherapy regimens, which has yielded the field's most dire survival statistics. Areas covered: Combination immunotherapy and a more robust molecular diagnostic platform for esophageal tumors could improve patient management strategies and potentially extend lives beyond the current survival figures. Analyzing a panel of biomarkers including those affiliated with taxane and platinum resistance (ERCC1 and TUBB3) as well as immunotherapy effectiveness (PD-L1) would provide oncologists more information on how to optimize first-line therapy for EC. Expert commentary: Of the 12 FDA-approved therapies in EC, zero target the genome. A majority of the approved drugs either target or are effected by proteomic expression. Therefore, a broader understanding of diagnostic biomarkers could give more clarity and direction in treating esophageal cancer in concert with a greater use of immunotherapy.

A national study of breast and colorectal cancer patients' decision-making for novel personalized medicine genomic diagnostics.

PubMed

Issa, Amalia M; Tufail, Waqas; Atehortua, Nelson; McKeever, John

2013-05-01

Molecular diagnostics are increasingly being used to help guide decision-making for personalized medical treatment of breast and colorectal cancer patients. The main aim of this study was to better understand and determine breast and colorectal cancer patients' decision-making strategies and the trade-offs they make in deciding about characteristics of molecular genomic diagnostics for breast and colorectal cancer. We surveyed a nationally representative sample of 300 breast and colorectal cancer patients using a previously developed web-administered instrument. Eligibility criteria included patients aged 18 years and older with either breast or colorectal cancer. We explored several attributes and attribute levels of molecular genomic diagnostics in 20 scenarios. Our analysis revealed that both breast and colorectal cancer patients weighted the capability of molecular genomic diagnostics to determine the probability of treatment efficacy as being of greater importance than information provided to detect adverse events. The probability of either false-positive or -negative results was ranked highly as a potential barrier by both breast and colorectal patients. However, 78.6% of breast cancer patients ranked the possibility of a 'false-negative test result leading to undertreatment' higher than the 'chance of a false positive, which may lead to overtreatment' (68%). This finding contrasted with the views of colorectal cancer patients who ranked the chance of a false positive as being of greater concern than a false negative (72.8 vs 63%). Overall, cancer patients exhibited a high willingness to accept and pay for genomic diagnostic tests, especially among breast cancer patients. Cancer patients seek a test accuracy rate of 90% or higher. Breast and colorectal cancer patients' decisions about genomic diagnostics are influenced more by the probability of being cured than by avoiding potential severe adverse events. This study provides insights into the relative weight that breast and colorectal cancer patients place on various aspects of molecular genomic diagnostics, and the trade-offs they are willing to make among attributes of such tests.

Molecular Diagnostic for Boll Weevil (Coleoptera: Curculionidae) Based on Amplification of Three Species-specific Microsatellites

USDA-ARS?s Scientific Manuscript database

The boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae), is a serious pest of cultivated cotton in the Americas, and reinfestation of zones from which they have been eradicated is of perpetual concern. Extensive arrays of pheromone traps monitor for reintroductions, but occasionally...

Protease biosensors based on peptide-nanocellulose conjugates: from molecular design to dressing interface

USDA-ARS?s Scientific Manuscript database

The development of point of care diagnostic protease sensors applied to wound healing has received increased interest for chronic wound treatment and as an interface with chronic wound dressings. Biosensor technology has grown exponentially in recent years. Here we focus on nanocelluosic biosensor t...

Next generation of optical diagnostics for bladder cancer using probe-based confocal laser endomicroscopy

NASA Astrophysics Data System (ADS)

Liu, Jen-Jane; Chang, Timothy C.; Pan, Ying; Hsiao, Shelly T.; Mach, Kathleen E.; Jensen, Kristin C.; Liao, Joseph C.

2012-02-01

Real-time imaging with confocal laser endomicroscopy (CLE) probes that fit in standard endoscopes has emerged as a clinically feasible technology for optical biopsy of bladder cancer. Confocal images of normal, inflammatory, and neoplastic urothelium obtained with intravesical fluorescein can be differentiated by morphologic characteristics. We compiled a confocal atlas of the urinary tract using these diagnostic criteria to be used in a prospective diagnostic accuracy study. Patients scheduled to undergo transurethral resection of bladder tumor underwent white light cystoscopy (WLC), followed by CLE, and histologic confirmation of resected tissue. Areas that appeared normal by WLC were imaged and biopsied as controls. We imaged and prospectively analyzed 135 areas in 57 patients. We show that CLE improves the diagnostic accuracy of WLC for diagnosing benign tissue, low and high grade cancer. Interobserver studies showed a moderate level of agreement by urologists and nonclinical researchers. Despite morphologic differences between inflammation and cancer, real-time differentiation can still be challenging. Identification of bladder cancer-specific contrast agents could provide molecular specificity to CLE. By using fluorescently-labeled antibodies or peptides that bind to proteins expressed in bladder cancer, we have identified putative molecular contrast agents for targeted imaging with CLE. We describe one candidate agent - anti-CD47 - that was instilled into bladder specimens. The tumor and normal urothelium were imaged with CLE, with increased fluorescent signal demonstrated in areas of tumor compared to normal areas. Thus, cancer-specificity can be achieved using molecular contrast agents ex vivo in conjunction with CLE.

Targeted Next Generation Sequencing with ThyroSeq v2.1 for Indeterminate Thyroid Nodules in Clinical Practice.

PubMed

Witt, Robert L

2016-12-01

To determine if patients elect molecular testing over diagnostic surgery or repeat fine needle aspiration for indeterminate thyroid nodules. Can ThyroSeq v2.1 molecular testing reduce diagnostic thyroid surgery and rule out cancer? Retrospective review Setting: Single institution, single-practice surgeon. Fifteen month retrospective review of indeterminate thyroid nodules that went on to ThyroSeq v2.1 testing. 286 patients met American Thyroid Association guideline criteria for surgeon- performed, ultrasound-guided fine needle aspiration for a thyroid nodule with on-site cytopathology. The indeterminate (Bethesda III or IV) fine needle aspiration cytology rate was 9.1 percent. Prevalence of malignancy in indeterminate nodules was 19 percent. 26/26 (100 percent) patients with indeterminate thyroid nodules elected molecular testing. 16 patients had no mutation, 9 had one or more mutations, and I had no result. 16 of 25 (64 percent) patients with no mutation elected not to undergo diagnostic surgery for indeterminate thyroid nodules. Patients demonstrated a strong preference for molecular testing instead of diagnostic thyroid surgery for indeterminate thyroid nodules. All patients in this series, 25/25 (100 percent) with indeterminate thyroid nodules elected molecular testing instead of repeat biopsy or diagnostic thyroid surgery. 16 of 25 (64 percent) patients tested had no mutation. All 16/16 (100 percent) patients with no mutation on ThyroSeq "rule out" testing elected active surveillance rather than surgery or biopsy, reducing diagnostic surgery. The risk of malignancy among mutation negative patients was not definitively established. There are a number of factors currently that may mute the power of "rule in" testing.

Conventional and molecular diagnostic strategies for prosthetic joint infections.

PubMed

Esteban, Jaime; Sorlí, Luisa; Alentorn-Geli, Eduard; Puig, Lluís; Horcajada, Juan P

2014-01-01

An accurate diagnosis of prosthetic joint infection (PJI) is the mainstay for an optimized clinical management. This review analyzes different diagnostic strategies of PJI, with special emphasis on molecular diagnostic tools and their current and future applications. Until now, the culture of periprosthetic tissues has been considered the gold standard for the diagnosis of PJI. However, sonication of the implant increases the sensitivity of those cultures and is being increasingly adopted by many centers. Molecular diagnostic methods compared with intraoperative tissue culture, especially if combined with sonication, have a higher sensitivity, a faster turnaround time and are not influenced by previous antimicrobial therapy. However, they still lack a system for detection of antimicrobial susceptibility, which is crucial for an optimized and less toxic therapy of PJI. More studies are needed to assess the clinical value of these methods and their cost-effectiveness.

Novel molecular diagnostic tools for malaria elimination: a review of options from the point of view of high-throughput and applicability in resource limited settings.

PubMed

Britton, Sumudu; Cheng, Qin; McCarthy, James S

2016-02-16

As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral flow rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination.

Recent Advances in Cardiac Computed Tomography: Dual Energy, Spectral and Molecular CT Imaging

PubMed Central

Danad, Ibrahim; Fayad, Zahi A.; Willemink, Martin J.; Min, James K.

2015-01-01

Computed tomography (CT) evolved into a powerful diagnostic tool and it is impossible to imagine current clinical practice without CT imaging. Due to its widespread availability, ease of clinical application, superb sensitivity for detection of CAD, and non-invasive nature, CT has become a valuable tool within the armamentarium of the cardiologist. In the last few years, numerous technological advances in CT have occurred—including dual energy CT (DECT), spectral CT and CT-based molecular imaging. By harnessing the advances in technology, cardiac CT has advanced beyond the mere evaluation of coronary stenosis to an imaging modality tool that permits accurate plaque characterization, assessment of myocardial perfusion and even probing of molecular processes that are involved in coronary atherosclerosis. Novel innovations in CT contrast agents and pre-clinical spectral CT devices have paved the way for CT-based molecular imaging. PMID:26068288

How have advances in our understanding of the molecular genetics of paediatric leukaemia led to improved targeted therapies for these diseases?

PubMed

Szychot, Elwira; Brodkiewicz, Andrzej; Peregud-Pogorzelski, Jarosław

2014-01-01

The term "leukaemia" refers to a large and heterogenous group of diseases, with treatment response and outcome dependent on the specific type of malignancy. New molecular methods allow us to specifically evaluate the type of disorder, and provide treatment of necessary intensity. The aim of this review is to provide insight into the progress in leukaemia treatment that had been possible due to advances in molecular genetics over the last few decades. Those new sophisticated diagnostic methods have allowed us not only to predict patients' prognosis but also to provide a specific therapy depending on the molecular and genetic characteristics of patients. Our review is based on 25 articles regarding novel diagnostic and therapeutic methods as well as prognostic factors, released between 1992 and 2011. Those articles focus mostly on molecular and cytogenetic testing allowing revolutionary methods of patient classification and individual therapy for this highly heterogeneous group of disorders. Implementation of molecular genetic testing to evaluate the type of leukaemia allowed paediatric oncologists and haematologists to adjust the intensity of treatment, improve outcome, minimize toxicity of therapies and considerably lower the risk of side effects. In the last few decades there has been a great improvement in survival among children suffering from haematopoietic malignancies. Progress made in molecular genetics allowed the creation of new treatment protocols that are designed to maintain a high cure rate for children with leukaemia while reducing toxicity.

RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics.

PubMed

Best, Myron G; Sol, Nik; Kooi, Irsan; Tannous, Jihane; Westerman, Bart A; Rustenburg, François; Schellen, Pepijn; Verschueren, Heleen; Post, Edward; Koster, Jan; Ylstra, Bauke; Ameziane, Najim; Dorsman, Josephine; Smit, Egbert F; Verheul, Henk M; Noske, David P; Reijneveld, Jaap C; Nilsson, R Jonas A; Tannous, Bakhos A; Wesseling, Pieter; Wurdinger, Thomas

2015-11-09

Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based "liquid biopsies". Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Infectious Disease Management through Point-of-Care Personalized Medicine Molecular Diagnostic Technologies

PubMed Central

Bissonnette, Luc; Bergeron, Michel G.

2012-01-01

Infectious disease management essentially consists in identifying the microbial cause(s) of an infection, initiating if necessary antimicrobial therapy against microbes, and controlling host reactions to infection. In clinical microbiology, the turnaround time of the diagnostic cycle (>24 hours) often leads to unnecessary suffering and deaths; approaches to relieve this burden include rapid diagnostic procedures and more efficient transmission or interpretation of molecular microbiology results. Although rapid nucleic acid-based diagnostic testing has demonstrated that it can impact on the transmission of hospital-acquired infections, we believe that such life-saving procedures should be performed closer to the patient, in dedicated 24/7 laboratories of healthcare institutions, or ideally at point of care. While personalized medicine generally aims at interrogating the genomic information of a patient, drug metabolism polymorphisms, for example, to guide drug choice and dosage, personalized medicine concepts are applicable in infectious diseases for the (rapid) identification of a disease-causing microbe and determination of its antimicrobial resistance profile, to guide an appropriate antimicrobial treatment for the proper management of the patient. The implementation of point-of-care testing for infectious diseases will require acceptance by medical authorities, new technological and communication platforms, as well as reimbursement practices such that time- and life-saving procedures become available to the largest number of patients. PMID:25562799

Lyssaviruses: special emphasis on rabies virus and other members of the lyssavirus genus.

PubMed

Harkess, Graeme; Fooks, Anthony R

2011-01-01

Rabies is routinely diagnosed based on the clinical description and history of exposure in a rabies-endemic country. A negative diagnostic test for rabies virus or a related lyssavirus does not exclude the clinical diagnosis. Diagnostic tests are never optimal and are entirely dependent on the nature and quality of the sample supplied. Often, only a sample from a single time point is investigated reducing the overall sensitivity of any diagnosis. With the advent of molecular biology, tests have been developed that are rapid, robust, and sensitive in support of the rapid detection and strain identification of rabies virus from clinical specimens. These molecular tests complement conventional tests in rabies diagnosis, particularly for human cases, for which an early laboratory diagnosis is critical and may decrease the number of unnecessary contacts with the patient, reduce the requirement for invasive and costly interventions, and enable the appropriate medical treatment regimen to be administered for the patient. The barrier to success is in transferring the technology for the latest techniques in rabies diagnosis to rabies-endemic countries. These barriers are not insurmountable and in liaison with international organisations, especially OIE, FAO, and WHO, these diagnostic tests will be validated for rabies diagnosis and surveillance, and implemented in modern and well-equipped diagnostic laboratories throughout the world.

Diagnosis of inflammatory bowel disease: Potential role of molecular biometrics.

PubMed

M'Koma, Amosy E

2014-11-27

Accurate diagnosis of predominantly colonic inflammatory bowel disease (IBD) is not possible in 30% of patients. For decades, scientists have worked to find a solution to improve diagnostic accuracy for IBD, encompassing Crohn's colitis and ulcerative colitis. Evaluating protein patterns in surgical pathology colectomy specimens of colonic mucosal and submucosal compartments, individually, has potential for diagnostic medicine by identifying integrally independent, phenotype-specific cellular and molecular characteristics. Mass spectrometry (MS) and imaging (I) MS are analytical technologies that directly measure molecular species in clinical specimens, contributing to the in-depth understanding of biological molecules. The biometric-system complexity and functional diversity is well suited to proteomic and diagnostic studies. The direct analysis of cells and tissues by Matrix-Assisted-Laser Desorption/Ionization (MALDI) MS/IMS has relevant medical diagnostic potential. MALDI-MS/IMS detection generates molecular signatures obtained from specific cell types within tissue sections. Herein discussed is a perspective on the use of MALDI-MS/IMS and bioinformatics technologies for detection of molecular-biometric patterns and identification of differentiating proteins. I also discuss a perspective on the global challenge of transferring technologies to clinical laboratories dealing with IBD issues. The significance of serologic-immunometric advances is also discussed.

Diagnosis of inflammatory bowel disease: Potential role of molecular biometrics

PubMed Central

M’Koma, Amosy E

2014-01-01

Accurate diagnosis of predominantly colonic inflammatory bowel disease (IBD) is not possible in 30% of patients. For decades, scientists have worked to find a solution to improve diagnostic accuracy for IBD, encompassing Crohn’s colitis and ulcerative colitis. Evaluating protein patterns in surgical pathology colectomy specimens of colonic mucosal and submucosal compartments, individually, has potential for diagnostic medicine by identifying integrally independent, phenotype-specific cellular and molecular characteristics. Mass spectrometry (MS) and imaging (I) MS are analytical technologies that directly measure molecular species in clinical specimens, contributing to the in-depth understanding of biological molecules. The biometric-system complexity and functional diversity is well suited to proteomic and diagnostic studies. The direct analysis of cells and tissues by Matrix-Assisted-Laser Desorption/Ionization (MALDI) MS/IMS has relevant medical diagnostic potential. MALDI-MS/IMS detection generates molecular signatures obtained from specific cell types within tissue sections. Herein discussed is a perspective on the use of MALDI-MS/IMS and bioinformatics technologies for detection of molecular-biometric patterns and identification of differentiating proteins. I also discuss a perspective on the global challenge of transferring technologies to clinical laboratories dealing with IBD issues. The significance of serologic-immunometric advances is also discussed. PMID:25429322

Using next-generation sequencing to develop molecular diagnostics for Pseudoperonospora cubensis, the cucurbit downy mildew pathogen

USDA-ARS?s Scientific Manuscript database

Advances in Next Generation Sequencing (NGS) allow for rapid development of genomics resources needed to generate molecular diagnostics assays for infectious agents. NGS approaches are particularly helpful for organisms that cannot be cultured, such as the downy mildew pathogens, a group of biotrop...

Biosensor Detection of Neuropathy Target Esterase in Whole Blood as a Biomarker of Exposure to Neuropathic Organophosphorus Compounds

DTIC Science & Technology

2002-10-15

Chernogolovka, Moscow Region, Russia Arkady V. Eremenko Research Center for Molecular Diagnostics and Therapy, Moscow, Russia Ilya N. Kurochkin...Faculty of Chemistry, M. V. Lomonosov Moscow State University, and Research Center for Molecular Diagnostics and Therapy, Moscow, Russia Vladimir V

78 FR 21128 - Molecular Diagnostic Instruments With Combined Functions; Draft Guidance for Industry and Food...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-09

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2013-D-0258] Molecular Diagnostic Instruments With Combined Functions; Draft Guidance for Industry and Food and Drug Administration Staff; Availability AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food...

Rayleigh Scattering Diagnostic for Simultaneous Measurements of Dynamic Density and Velocity

NASA Technical Reports Server (NTRS)

Seasholtz, Richard G.; Panda, J.

2000-01-01

A flow diagnostic technique based on the molecular Rayleigh scattering of laser light is used to obtain dynamic density and velocity data in turbulent flows. The technique is based on analyzing the Rayleigh scattered light with a Fabry-Perot interferometer and recording information about the interference pattern with a multiple anode photomultiplier tube (PMT). An artificial neural network is used to process the signals from the PMT to recover the velocity time history, which is then used to calculate the velocity power spectrum. The technique is illustrated using simulated data. The results of an experiment to measure the velocity power spectrum in a low speed (100 rn/sec) flow are also presented.

An update on clinical applications of magnetic nanoparticles for increasing the resolution of magnetic resonance imaging.

PubMed

Zeinali Sehrig, Fatemeh; Majidi, Sima; Asvadi, Sahar; Hsanzadeh, Arash; Rasta, Seyed Hossein; Emamverdy, Masumeh; Akbarzadeh, Jamshid; Jahangiri, Sahar; Farahkhiz, Shahrzad; Akbarzadeh, Abolfazl

2016-11-01

Today, technologies based on magnetic nanoparticles (MNPs) are regularly applied to biological systems with diagnostic or therapeutic aims. Nanoparticles made of the elements iron (Fe), gadolinium (Gd) or manganese (Mn) are generally used in many diagnostic applications performed under magnetic resonance imaging (MRI). Similar to molecular-based contrast agents, nanoparticles can be used to increase the resolution of imaging while offering well biocompatibility, poisonousness and biodistribution. Application of MNPs enhanced MRI sensitivity due to the accumulation of iron in the liver caused by discriminating action of the hepatobiliary system. The aim of this study is about the use, properties and advantages of MNPs in MRI.

A diagnostic biochip for the comprehensive analysis of MLL translocations in acute leukemia.

PubMed

Maroc, N; Morel, A; Beillard, E; De La Chapelle, A L; Fund, X; Mozziconacci, M-J; Dupont, M; Cayuela, J-M; Gabert, J; Koki, A; Fert, V; Hermitte, F

2004-09-01

Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10(-1), and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.

Rapid, Affordable and Portable Medium-Throughput Molecular Device for Zika Virus

PubMed Central

Chan, Kamfai; Weaver, Scott C.; Wong, Pui-Yan; Lie, Sherly; Wang, Eryu; Guerbois, Mathilde; Vayugundla, Siva Praneeth; Wong, Season

2016-01-01

Zika virus (ZIKV) has gained global attention as an etiologic agent of fetal microcephaly and Guillain-Barré syndrome. Existing immuno-based rapid tests often fail to distinguish between Zika and related flaviviruses that are common in affected regions of Central and South Americas and the Caribbean. The US CDC and qualified state health department laboratories can perform the reverse transcription polymerase chain reaction (RT-PCR) ZIKV test using highly sophisticated instruments with long turnaround times. The preliminary results of a portable and low-cost molecular diagnostics system for ZIKV infection are reported here. In less than 15 minutes, this low-cost platform can automatically perform high quality RNA extraction from up to 12 ZIKV-spiked urine samples simultaneously. It can also perform reverse transcription recombinase polymerase amplification reaction (RT-RPA) in ≤15 minutes. The fluorescent signal produced from probe-based RT-RPA or RT-PCR assays can be monitored using LEDs and a smartphone camera. In addition, the RT-RPA and RT-PCR assays do not cross-react with dengue and chikungunya viral RNA. This low-cost system lacks complicated, sensitive and high cost components, making it suitable for resource-limited settings. It has the potential to offer simple sample-to-answer molecular diagnostics and can inform healthcare workers of patients’ diagnosis promptly. PMID:27934884

Personalized Cancer Medicine: Molecular Diagnostics, Predictive biomarkers, and Drug Resistance

PubMed Central

Gonzalez de Castro, D; Clarke, P A; Al-Lazikani, B; Workman, P

2013-01-01

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution. PMID:23361103

Molecular Networking As a Drug Discovery, Drug Metabolism, and Precision Medicine Strategy.

PubMed

Quinn, Robert A; Nothias, Louis-Felix; Vining, Oliver; Meehan, Michael; Esquenazi, Eduardo; Dorrestein, Pieter C

2017-02-01

Molecular networking is a tandem mass spectrometry (MS/MS) data organizational approach that has been recently introduced in the drug discovery, metabolomics, and medical fields. The chemistry of molecules dictates how they will be fragmented by MS/MS in the gas phase and, therefore, two related molecules are likely to display similar fragment ion spectra. Molecular networking organizes the MS/MS data as a relational spectral network thereby mapping the chemistry that was detected in an MS/MS-based metabolomics experiment. Although the wider utility of molecular networking is just beginning to be recognized, in this review we highlight the principles behind molecular networking and its use for the discovery of therapeutic leads, monitoring drug metabolism, clinical diagnostics, and emerging applications in precision medicine. Copyright © 2016. Published by Elsevier Ltd.

Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics.

PubMed

Ngo, Hoan T; Gandra, Naveen; Fales, Andrew M; Taylor, Steve M; Vo-Dinh, Tuan

2016-07-15

One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method's simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications. Copyright © 2016. Published by Elsevier B.V.

Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen Candida parapsilosis

PubMed Central

Nosek, Jozef; Tomáška, L'ubomír; Ryčovská, Adriana; Fukuhara, Hiroshi

2002-01-01

Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeast pathogen Candida parapsilosis. The strategy for identification of C. parapsilosis strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay is complemented by immunodetection of a protein component of mitochondrial telomeres. The results demonstrate that mitochondrial telomeres represent specific molecular markers with potential applications in yeast diagnostics and taxonomy. PMID:11923346

Molecular Methods for the Detection of Mycoplasma and Ureaplasma Infections in Humans

PubMed Central

Waites, Ken B.; Xiao, Li; Paralanov, Vanya; Viscardi, Rose M.; Glass, John I.

2012-01-01

Mycoplasma and Ureaplasma species are well-known human pathogens responsible for a broad array of inflammatory conditions involving the respiratory and urogenital tracts of neonates, children, and adults. Greater attention is being given to these organisms in diagnostic microbiology, largely as a result of improved methods for their laboratory detection, made possible by powerful molecular-based techniques that can be used for primary detection in clinical specimens. For slow-growing species, such as Mycoplasma pneumoniae and Mycoplasma genitalium, molecular-based detection is the only practical means for rapid microbiological diagnosis. Most molecular-based methods used for detection and characterization of conventional bacteria have been applied to these organisms. A complete genome sequence is available for one or more strains of all of the important human pathogens in the Mycoplasma and Ureaplasma genera. Information gained from genome analyses and improvements in efficiency of DNA sequencing are expected to significantly advance the field of molecular detection and genotyping during the next few years. This review provides a summary and critical review of methods suitable for detection and characterization of mycoplasmas and ureaplasmas of humans, with emphasis on molecular genotypic techniques. PMID:22819362

Surveyor assay to diagnose persistent Müllerian duct syndrome in Miniature Schnauzers.

PubMed

Kim, Young June; Kwon, Hyuk Jin; Byun, Hyuk Soo; Yeom, Donguk; Choi, Jea-Hong; Kim, Joong-Hyun; Shim, Hosup

2017-12-31

Persistent Müllerian duct syndrome (PMDS) is a pseudohermaphroditism in males characterized by the presence of Müllerian duct derivatives. As PMDS dogs often lack clinical symptoms, a molecular diagnosis is essential to identify the syndrome in these animals. In this study, a new molecular method using DNA mismatch-specific Surveyor nuclease was developed. The Surveyor nuclease assay identified the AMHR2 mutation that produced PMDS in a Miniature Schnauzer as accurately as that obtained by using the conventional method based on restriction digestion. As an alternative to the current molecular diagnostic method, the new method may result in increased accuracy when detecting PMDS.

Surveyor assay to diagnose persistent Müllerian duct syndrome in Miniature Schnauzers

PubMed Central

Kim, Young June; Kwon, Hyuk Jin; Byun, Hyuk Soo; Yeom, Donguk; Choi, Jea-Hong; Kim, Joong-Hyun

2017-01-01

Persistent Müllerian duct syndrome (PMDS) is a pseudohermaphroditism in males characterized by the presence of Müllerian duct derivatives. As PMDS dogs often lack clinical symptoms, a molecular diagnosis is essential to identify the syndrome in these animals. In this study, a new molecular method using DNA mismatch-specific Surveyor nuclease was developed. The Surveyor nuclease assay identified the AMHR2 mutation that produced PMDS in a Miniature Schnauzer as accurately as that obtained by using the conventional method based on restriction digestion. As an alternative to the current molecular diagnostic method, the new method may result in increased accuracy when detecting PMDS. PMID:27515263

Parasitological and molecular diagnostic of a clinical Babesia caballi outbreak in Southern Romania.

PubMed

Ionita, Mariana; Nicorescu, Isabela Madalina; Pfister, Kurt; Mitrea, Ioan Liviu

2018-05-15

Equine piroplasmosis (EP) is a tick-borne disease of equids caused by Babesia caballi and/or Theileria equi, which is endemic in many tropical and temperate areas of the world. However, clinical outbreaks of EP in Romania during the last decades have not been reported Therefore, the aim of this paper is (i) to describe a clinical B. caballi outbreak in horses on several farms in Southern Romania using a diagnostic and therapeutic approach and (ii) the molecular diagnostic of EP in an endemic area of Romania. In the first case, a 10-month-old stallion male was presented with lethargy, anorexia, fever (40.9 °C), pale mucosal/mucous/membranes and a marked anemia. In the subsequent weeks, three horses from other farms located in the same area, displayed similar clinical signs. B. caballi was diagnosed in all the horses based on Giemsa-stained blood smears and the diagnosis was further confirmed by polymerase chain reaction (PCR), using a single-round and multiplex PCR and sequencing. All four horses were treated with imidocarb dipropionate, at a dose rate of 2.2 mg/kg body weight (two injections at 48 h apart), and all horses clinically recovered within 24-48 h, post-treatment. This report presents the first molecularly characterized B. caballi outbreak in Romania in clinically affected horses, confirmed by DNA sequencing.

Greater Real-Life Diagnostic Efficacy of Allergen Molecule-Based Diagnosis for Prescription of Immunotherapy in an Area with Multiple Pollen Exposure

PubMed Central

Saltabayeva, Ulbosin; Garib, Victoria; Morenko, Marina; Rosenson, Rafail; Ispayeva, Zhanat; Gatauova, Madina; Zulus, Loreta; Karaulov, Alexander; Gastager, Felix; Valenta, Rudolf

2017-01-01

Background Allergen molecule-based diagnosis has been suggested to facilitate the identification of disease-causing allergen sources and the prescription of allergen-specific immunotherapy (AIT). The aim of the current study was to compare allergen molecule-based IgE serology with allergen extract-based skin testing for the identification of the disease-causing allergen sources. The study was conducted in an area where patients are exposed to pollen from multiple sources (trees, grasses, and weeds) at the same time to compare the diagnostic efficiency of the 2 forms of diagnosis. Methods Patients from Astana, Kazakhstan, who suffered from pollen-induced allergy (n = 95) were subjected to skin prick testing (SPT) with a local panel of tree pollen, grass pollen, and weed pollen allergen extracts and IgE antibodies specific for marker allergen molecules (nArt v 1, nArt v 3, rAmb a 1, rPhl p 1, rPhl p 5, rBet v 1) were measured by ImmunoCAP. Direct and indirect costs for diagnosis based on SPT and marker allergen-based IgE serology as well as direct costs for immunotherapy depending on SPT and serological test results were calculated. Results The costs for SPT-based diagnosis per patient were lower than the costs for allergen molecule-based IgE serology. However, allergen molecule-based serology was more precise in detecting the disease-causing allergen sources. A lower number of immunotherapy treatments (n = 119) was needed according to molecular diagnosis as compared to extract-based diagnosis (n = 275), which considerably reduced the total costs for diagnosis and for a 3-year treatment from EUR 1,112.30 to 521.77 per patient. Conclusions The results from this real-life study show that SPT is less expensive than allergen molecule-based diagnostic testing, but molecular diagnosis allowed more precise prescription of immunotherapy which substantially reduced treatment costs and combined costs for diagnosis and treatment. PMID:28654920

Molecular Tools for Diagnosis of Visceral Leishmaniasis: Systematic Review and Meta-Analysis of Diagnostic Test Accuracy

PubMed Central

de Ruiter, C. M.; van der Veer, C.; Leeflang, M. M. G.; Deborggraeve, S.; Lucas, C.

2014-01-01

Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies. PMID:24829226

Multiple biomarkers in molecular oncology. I. Molecular diagnostics applications in cervical cancer detection.

PubMed

Malinowski, Douglas P

2007-03-01

The screening for cervical carcinoma and its malignant precursors (cervical neoplasia) currently employs morphology-based detection methods (Papanicolaou [Pap] smear) in addition to the detection of high-risk human papillomavirus. The combination of the Pap smear with human papillomavirus testing has achieved significant improvements in sensitivity for the detection of cervical disease. Diagnosis of cervical neoplasia is dependent upon histology assessment of cervical biopsy specimens. Attempts to improve the specificity of cervical disease screening have focused on the investigation of molecular biomarkers for adjunctive use in combination with the Pap smear. Active research into the genomic and proteomic alterations that occur during human papillomavirus-induced neoplastic transformation have begun to characterize some of the basic mechanisms inherent to the disease process of cervical cancer development. This research continues to demonstrate the complexity of multiple genomic and proteomic alterations that accumulate during the tumorigenesis process. Despite this diversity, basic patterns of uncontrolled signal transduction, cell cycle deregulation, activation of DNA replication and altered extracellular matrix interactions are beginning to emerge as common features inherent to cervical cancer development. Some of these gene or protein expression alterations have been investigated as potential biomarkers for screening and diagnostics applications. The contribution of multiple gene alterations in the development of cervical cancer suggests that the application of multiple biomarker panels has the potential to develop clinically useful molecular diagnostics. In this review, the application of biomarkers for the improvement of sensitivity and specificity of the detection of cervical neoplasia within cytology specimens will be discussed.

Treatment algorithm in 2014 for advanced non-small cell lung cancer: therapy selection by tumour histology and molecular biology.

PubMed

Manegold, Christian

2014-09-01

The availability of antineoplastic monoclonal antibodies, small molecules and newer cytotoxics such as pemetrexed, the EGFR-tyrosine kinase inhibitors erlotinib, gefitinib, afatinib as well as the anti-angiogenic bevacizumab and the ALK-inhibitor crizotinib has recently changes the treatment algorithm of advanced non-small cell lung cancer. Decision making in 2014 is characterized by customizing therapy, by selecting a specific therapeutic regimen based on the histotype and the genotype of the tumour. This refers to first-line induction therapy and maintenance therapy as well, but also to subsequent lines of therapy since anti-neoplastic drugs and regimens used upfront clinically influence the selection of agents/regimes considered for second-/third-line treatment. Consequently, therapy customization through tumour histology and molecular markers has significantly influenced the work of pathologists around the globe and the process of obtaining an extended therapeutically relevant tumour diagnosis. Not only histological sub-typing became standard but molecular information is also considered of increasing importance for treatment selection. Routine molecular testing in certified laboratories must be established, and the diagnostic process should ideally be performed under the guidance of evidence based recommendation. The process of investigating and implementing medical targeting in lung cancer therefore, requires advanced diagnostic techniques and expertise and because of its large dimension is costly and influenced by the limitation of financial and clinical resources. Copyright © 2014. Published by Elsevier Urban & Partner Sp. z o.o.

Nanostructured sensors for biomedical applications--a current perspective.

PubMed

Krishnamoorthy, Sivashankar

2015-08-01

Nanostructured sensors have unique capabilities that can be tailored to advantage in advancing the diagnosis, monitoring and cure of several diseases and health conditions. This report aims at providing a current perspective on, (a) the emerging clinical needs that defines the challenges to be addressed by nanostructured sensors, with specific emphasis on early stage diagnosis, drug-diagnostic combinations, and predictive models to design therapy, (b) the emerging industry trends in in vitro diagnostics, mobile health care, high-throughput molecular and cell-based diagnostic platforms, and (c) recent instances of nanostructured biosensors, including promising sensing concepts that can be enhanced using nanostructures that carry high promise towards catering to the emerging clinical needs, as well as the market/industry trends. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biomarkers for personalized oncology: recent advances and future challenges.

PubMed

Kalia, Madhu

2015-03-01

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells and oncology is a branch of medicine that deals with tumors. The last decade has seen significant advances in the development of biomarkers in oncology that play a critical role in understanding molecular and cellular mechanisms which drive tumor initiation, maintenance and progression. Clinical molecular diagnostics and biomarker discoveries in oncology are advancing rapidly as we begin to understand the complex mechanisms that transform a normal cell into an abnormal one. These discoveries have fueled the development of novel drug targets and new treatment strategies. The standard of care for patients with advanced-stage cancers has shifted away from an empirical treatment strategy based on the clinical-pathological profile to one where a biomarker driven treatment algorithm based on the molecular profile of the tumor is used. Recent advances in multiplex genotyping technologies and high-throughput genomic profiling by next-generation sequencing make possible the rapid and comprehensive analysis of the cancer genome of individual patients even from very little tumor biopsy material. Predictive (diagnostic) biomarkers are helpful in matching targeted therapies with patients and in preventing toxicity of standard (systemic) therapies. Prognostic biomarkers identify somatic germ line mutations, changes in DNA methylation, elevated levels of microRNA (miRNA) and circulating tumor cells (CTC) in blood. Predictive biomarkers using molecular diagnostics are currently in use in clinical practice of personalized oncotherapy for the treatment of five diseases: chronic myeloid leukemia, colon, breast, lung cancer and melanoma and these biomarkers are being used successfully to evaluate benefits that can be achieved through targeted therapy. Examples of these molecularly targeted biomarker therapies are: tyrosine kinase inhibitors in chronic myeloid leukemia and gastrointestinal tumors; anaplastic lymphoma kinase (ALK) inhibitors in lung cancer with EML4-ALk fusion; HER2/neu blockage in HER2/neu-positive breast cancer; and epidermal growth factor receptors (EGFR) inhibition in EGFR-mutated lung cancer. This review presents the current state of our knowledge of biomarkers in five selected cancers: chronic myeloid leukemia, colorectal cancer, breast cancer, non-small cell lung cancer and melanoma. Copyright © 2015 Elsevier Inc. All rights reserved.

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

PubMed Central

Wilkinson, Samuel L.; John, Shibu; Walsh, Roddy; Novotny, Tomas; Valaskova, Iveta; Gupta, Manu; Game, Laurence; Barton, Paul J R.; Cook, Stuart A.; Ware, James S.

2013-01-01

Background Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations. Methodology/Principal Findings We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS. Conclusions/Significance MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive. PMID:23861798

Low-Molecular-Weight Plasma Proteome Analysis Using Top-Down Mass Spectrometry.

PubMed

Cheon, Dong Huey; Yang, Eun Gyeong; Lee, Cheolju; Lee, Ji Eun

2017-01-01

While human plasma has a wealth of diagnostic information regarding the state of the human body in heath and disease, low molecular weight (LMW) proteome (<30 kDa) has been shown to contain a rich source of diagnostic biomarkers. Here we describe a protocol for top-down proteomic analysis to identify and characterize the LMW proteoforms present in four types of human plasma samples without immunoaffinity depletion and with depletion of the top two, six, and seven high-abundance proteins. Each type of plasma sample was first fractionated based on molecular weight using gel-eluted liquid fraction entrapment electrophoresis (GELFrEE). Then, the GELFrEE fractions containing up to 30 kDa were subjected to nanocapillary-LC-MS/MS, and the high-resolution MS and MS/MS data were processed using ProSightPC software. As a result, a total of 442 LMW proteins and cleaved products, including those with posttranslational modifications (PTMs) and single amino acid variations (SAAVs), were identified with a threshold E-value of 1 × 10 -4 from the four types of plasma samples.

Rett syndrome: revised diagnostic criteria and nomenclature.

PubMed

Neul, Jeffrey L; Kaufmann, Walter E; Glaze, Daniel G; Christodoulou, John; Clarke, Angus J; Bahi-Buisson, Nadia; Leonard, Helen; Bailey, Mark E S; Schanen, N Carolyn; Zappella, Michele; Renieri, Alessandra; Huppke, Peter; Percy, Alan K

2010-12-01

Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in Methyl-CpG-binding protein 2 (MECP2). Despite distinct clinical features, the accumulation of clinical and molecular information in recent years has generated considerable confusion regarding the diagnosis of RTT. The purpose of this work was to revise and clarify 2002 consensus criteria for the diagnosis of RTT in anticipation of treatment trials. RettSearch members, representing the majority of the international clinical RTT specialists, participated in an iterative process to come to a consensus on a revised and simplified clinical diagnostic criteria for RTT. The clinical criteria required for the diagnosis of classic and atypical RTT were clarified and simplified. Guidelines for the diagnosis and molecular evaluation of specific variant forms of RTT were developed. These revised criteria provide clarity regarding the key features required for the diagnosis of RTT and reinforce the concept that RTT is a clinical diagnosis based on distinct clinical criteria, independent of molecular findings. We recommend that these criteria and guidelines be utilized in any proposed clinical research.

Implementation and new insights in molecular diagnostics for HIV infection.

PubMed

Tsang, Hin-Fung; Chan, Lawrence Wing-Chi; Tong, Jennifer Chiu-Hung; Wong, Heong-Ting; Lai, Christopher Koon-Chi; Au, Thomas Chi-Chuen; Chan, Amanda Kit-Ching; Ng, Lawrence Po-Wah; Cho, William Chi-Shing; Wong, Sze-Chuen Cesar

2018-05-01

Acquired immunodeficiency syndrome (AIDS) is a kind of acquired disease that breaks down the immune system. Human immunodeficiency virus (HIV) is the causative agent of AIDS. By the end of 2016, there were 36.7 million people living with HIV worldwide. Early diagnosis can alert infected individuals to risk behaviors in order to control HIV transmission. Infected individuals are also benefited from proper treatment and management upon early diagnosis. Thanks to the public awareness of the disease, the annual increase of new HIV infections has been slowly declining over the past decades. The advent of molecular diagnostics has allowed early detection and better management of HIV infected patients. Areas covered: In this review, the authors summarized and discussed the current and future technologies in molecular diagnosis as well as the biomarkers developed for HIV infection. Expert Commentary: A simple and rapid detection of viral load is important for patients and doctors to monitor HIV progression and antiretroviral treatment efficiency. In the near future, it is expected that new technologies such as digital PCR and CRISPR-based technology will play more important role in HIV detection and patient management.

Rayleigh Scattering Diagnostic for Measurement of Velocity and Density Fluctuation Spectra

NASA Technical Reports Server (NTRS)

Seasholtz, Richard G.; Panda, Jayanta; Elam, Kristie A.

2002-01-01

A new molecular Rayleigh scattering based flow diagnostic is used for the first time to measure the power spectrum of gas density and radial velocity component in the plumes of high speed jets. The technique is based on analyzing the Rayleigh scattered light with a Fabry-Perot interferometer used in the static, imaging mode. The PC based data acquisition system is capable of simultaneous sampling of velocity and density at rates to 100 kHz and data record lengths to 10 million. Velocity and density power spectra and velocity-density cross spectra are presented for a subsonic jet, an underexpanded screeching jet, and for Mach 1.4 and Mach 1.8 supersonic jets. Software and hardware interfaces were developed to allow computer control of all aspects of the experiment and data acquisition.

[Allergy and autoimmunity: Molecular diagnostics, therapy, and presumable pathogenesis].

PubMed

Arefieva, A S; Smoldovskaya, O V; Tikhonov, A A; Rubina, A Yu

2017-01-01

Allergic and autoimmune diseases represent immunopathological reactions of an organism to antigens. Despite that the allergy is a result of exaggerated immune response to foreign antigens (allergens) and autoimmune diseases are characterized by the pathological response to internal antigens (autoantigens), the underlying mechanisms of these diseases are probably common. Thus, both types of diseases represent variations in the hypersensitivity reaction. A large percentage of both the adult and pediatric population is in need of early diagnostics of these pathologies of the immune system. Considering the diversity of antibodies produced in allergic and autoimmune disease and the difficulties accompanying clinical diagnosing, molecular diagnostics of these pathological processes should be carried out in several stages, including screening and confirmatory studies. In this review, we summarize the available data on the molecular diagnostics and therapy of allergic and autoimmune diseases and discuss the basic similarities and differences in the mechanisms of their development.

Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative.

PubMed

Duval, Kristin; Aubin, Rémy A; Elliott, James; Gorn-Hondermann, Ivan; Birnboim, H Chaim; Jonker, Derek; Fourney, Ron M; Frégeau, Chantal J

2010-02-01

Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.

Pitfalls Associated With the Use of Molecular Diagnostic Panels in the Diagnosis of Cryptococcal Meningitis.

PubMed

O'Halloran, Jane A; Franklin, Alexander; Lainhart, William; Burnham, Carey-Ann; Powderly, William; Dubberke, Erik

2017-01-01

We report the case of a kidney transplantation patient on chronic immunosuppressive therapy presenting with subacute meningitis. The final diagnosis of cryptococcal meningitis was delayed due to 2 false-negative cryptococcal results on a molecular diagnostic panel. Caution with such platforms in suspected cryptococcal meningitis is needed.

External Quality Assessment for Zika Virus Molecular Diagnostic Testing, Brazil.

PubMed

Fischer, Carlo; Pedroso, Celia; Mendrone, Alfredo; Bispo de Filippis, Ana Maria; Vallinoto, Antonio Carlos Rosário; Ribeiro, Bergmann Morais; Durigon, Edison Luiz; Marques, Ernesto T A; Campos, Gubio S; Viana, Isabelle F T; Levi, José Eduardo; Scarpelli, Luciano Cesar; Nogueira, Mauricio Lacerda; Bastos, Michele de Souza; Souza, Nathalia C Santiago; Khouri, Ricardo; Lira, Sanny; Komninakis, Shirley Vasconcelos; Baronti, Cécile; Charrel, Rémi N; Kümmerer, Beate M; Drosten, Christian; Brites, Carlos; de Lamballerie, Xavier; Niedrig, Matthias; Netto, Eduardo Martins; Drexler, Jan Felix

2018-05-01

We conducted an external quality assessment of Zika virus molecular diagnostic tests in Brazil using a new Zika virus standard. Of 15 laboratories, 73% showed limited sensitivity and specificity. Viral load estimates varied significantly. Continuous quality assurance is needed to adequately estimate risk for Zika virus-associated disease and determine patient care.

Molecular markers and new diagnostic methods to differentiate malignant from benign mesothelial pleural proliferations: a literature review.

PubMed

Bruno, Rossella; Alì, Greta; Fontanini, Gabriella

2018-01-01

Malignant pleural mesothelioma (MPM) is an aggressive tumor associated with asbestos exposure. Histopathological analysis of pleural tissues is the gold standard for diagnosis; however, it can be difficult to differentiate malignant from benign pleural lesions. The purpose of this review is to describe the most important biomarkers and new diagnostic tools suggested for this differential diagnosis. There are many studies concerning the separation between MPM and benign pleural proliferations from both pleural tissues or effusions; most of them are based on the evaluation of one or few biomarkers by immunohistochemistry (IHC) or enzyme-linked immunosorbent assays (ELISAs), whereas others focused on the identification of MPM signatures given by microRNA (miRNA) or gene expression profiles as well as on the combination of molecular data and classification algorithms. None of the reported biomarkers showed adequate diagnostic accuracy, except for p16 [evaluated by fluorescent in situ hybridization (FISH)] and BAP1 (evaluated by IHC), both biomarkers are recommended by the International Mesothelioma Interest Group guidelines for histological and cytological diagnosis. BAP1 and p16 showed a specificity of 100% in discerning malignant from benign lesions because they are exclusively unexpressed or deleted in MPM. However, their sensitivity, even when used together, is not completely sufficient, and absence of their alterations cannot confirm the benign nature of the lesion. Recently, the availability of new techniques and increasing knowledge regarding MPM genetics led to the definition of some molecular panels, including genes or miRNAs specifically deregulated in MPM, that are extremely valuable for differential diagnosis. Moreover, the development of classification algorithms is facilitating the application of molecular data for clinical practice. Data regarding new diagnostic tools and MPM signatures are absolutely promising; however, before their application in clinical practice, a prospective validation is necessary, as these approaches could surely improve the differential diagnosis between malignant and benign pleural lesions.

Carbon Nanotube Biosensors for Space Molecule Detection and Clinical Molecular Diagnostics

NASA Technical Reports Server (NTRS)

Han, Jie

2001-01-01

Both space molecule detection and clinical molecule diagnostics need to develop ultra sensitive biosensors for detection of less than attomole molecules such as amino acids for DNA. However all the electrode sensor systems including those fabricated from the existing carbon nanotubes, have a background level of nA (nanoAmp). This has limited DNA or other molecule detection to nA level or molecules whose concentration is, much higher than attomole level. A program has been created by NASA and NCI (National Cancer Institute) to exploit the possibility of carbon nanotube based biosensors to solve this problem for both's interest. In this talk, I will present our effort on the evaluation and novel design of carbon nanotubes as electrode biosensors with strategies to minimize background currents while maximizing signal intensity.The fabrication of nanotube electrode arrays, immobilization of molecular probes on nanotube electrodes and in vitro biosensor testing will also be discussed.

Fluorescence-Raman Dual Modal Endoscopic System for Multiplexed Molecular Diagnostics

NASA Astrophysics Data System (ADS)

Jeong, Sinyoung; Kim, Yong-Il; Kang, Homan; Kim, Gunsung; Cha, Myeong Geun; Chang, Hyejin; Jung, Kyung Oh; Kim, Young-Hwa; Jun, Bong-Hyun; Hwang, Do Won; Lee, Yun-Sang; Youn, Hyewon; Lee, Yoon-Sik; Kang, Keon Wook; Lee, Dong Soo; Jeong, Dae Hong

2015-03-01

Optical endoscopic imaging, which was recently equipped with bioluminescence, fluorescence, and Raman scattering, allows minimally invasive real-time detection of pathologies on the surface of hollow organs. To characterize pathologic lesions in a multiplexed way, we developed a dual modal fluorescence-Raman endomicroscopic system (FRES), which used fluorescence and surface-enhanced Raman scattering nanoprobes (F-SERS dots). Real-time, in vivo, and multiple target detection of a specific cancer was successful, based on the fast imaging capability of fluorescence signals and the multiplex capability of simultaneously detected SERS signals using an optical fiber bundle for intraoperative endoscopic system. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) on the breast cancer xenografts in a mouse orthotopic model were successfully detected in a multiplexed way, illustrating the potential of FRES as a molecular diagnostic instrument that enables real-time tumor characterization of receptors during routine endoscopic procedures.

The application of genome-wide 5-hydroxymethylcytosine studies in cancer research.

PubMed

Thomson, John P; Meehan, Richard R

2017-01-01

Early detection and characterization of molecular events associated with tumorgenesis remain high priorities. Genome-wide epigenetic assays are promising diagnostic tools, as aberrant epigenetic events are frequent and often cancer specific. The deposition and analysis of multiple patient-derived cancer epigenomic profiles contributes to our appreciation of the underlying biology; aiding the detection of novel identifiers for cancer subtypes. Modifying enzymes and co-factors regulating these epigenetic marks are frequently mutated in cancers, and as epigenetic modifications themselves are reversible, this makes their study very attractive with respect to pharmaceutical intervention. Here we focus on the novel modified base, 5-hydoxymethylcytosine, and discuss how genome-wide 5-hydoxymethylcytosine profiling expedites our molecular understanding of cancer, serves as a lineage tracer, classifies the mode of action of potentially carcinogenic agents and clarifies the roles of potential novel cancer drug targets; thus assisting the development of new diagnostic/prognostic tools.

Development of a miRNA-based diagnostic assay for pancreatic ductal adenocarcinoma.

PubMed

Szafranska-Schwarzbach, Anna E; Adai, Alex T; Lee, Linda S; Conwell, Darwin L; Andruss, Bernard F

2011-04-01

Diagnosis of pancreatic cancer remains a clinical challenge. Both chronic pancreatitis and pancreatic cancer may present with similar symptoms and similar imaging features, often leading to incorrect interpretation. Thus, the use of an objective molecular test that can discriminate between chronic pancreatitis and pancreatic cancer will be a valuable asset in obtaining a definitive diagnosis of pancreatic cancer. Following Clinical Laboratory Improvement Amendments and College of American Pathologists guidelines, Asuragen Clinical Services Laboratory has developed and validated a laboratory-developed test, miRInform(®) Pancreas, to aid in the identification of pancreatic ductal adenocarcinoma. This molecular diagnostic tool uses reverse-transcription quantitative PCR to measure the expression difference between two miRNAs, miR-196a and miR-217, in fixed tissue specimens. This article describes the test validation process as well as determination of performance parameters of miRInform Pancreas.

[Liquid biopsy analysis using cell-free DNA (cfDNA): Opportunities and limitations].

PubMed

Dahl, E; Kloten, V

2015-11-01

Molecular biological analysis of nucleic acids in blood or other bodily fluids (i.e. liquid biopsy analyses) may supplement the pathologists' diagnostic armamentarium in a reasonable way-particularly in cancer precision medicine. Within the field of oncology, liquid biopsy can potentially be used to monitor tumor burden in the blood and to early detect emerging resistance in the course of targeted cancer therapies. An already approved application of liquid biopsy is the detection of epidermal growth factor receptor (EGFR) driver mutations in blood samples of lung cancer patients in those cases where no tissue biopsy is available. However, there is still currently considerable insecurity associated with blood-based DNA analytic methods that must be solved before liquid biopsy can be implemented for broader routine application in the diagnosis of cancer. In this article, the current state of development of liquid biopsy in molecular diagnostics from a pathology point of view is presented.

Bone Sarcoma Pathology: Diagnostic Approach for Optimal Therapy.

PubMed

Rosenberg, Andrew E

2017-01-01

The pathologic interpretation of malignant bone tumors is one of the more challenging areas in surgical pathology. This is based on the reality that primary bone sarcomas are uncommon, demonstrate significant morphologic heterogeneity, and have a broad spectrum of biology. Accordingly, it is difficult for pathologists to acquire the necessary experience to confidently and accurately diagnose bone sarcomas. The task is further complicated by the fact that it requires the integration of clinical and radiologic information into the diagnostic process. Lastly, molecular aberrations in sarcomas are being newly discovered and their identification is often critical to make specific diagnoses. The pathologist's role in guiding optimal treatment in biopsy specimens is to make an accurate diagnosis and provide the grade and molecular aberrations when appropriate. The pathology report of resected tumors must confirm this information and assess the surgical resection margins and the percentage of necrosis if the sarcoma has been treated with neoadjuvant systemic therapy.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

PubMed

Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers.

PubMed

Zhou, Lei; Wang, Rui; Yao, Chi; Li, Xiaomin; Wang, Chengli; Zhang, Xiaoyan; Xu, Congjian; Zeng, Aijun; Zhao, Dongyuan; Zhang, Fan

2015-04-24

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.

Fully 3D printed integrated reactor array for point-of-care molecular diagnostics.

PubMed

Kadimisetty, Karteek; Song, Jinzhao; Doto, Aoife M; Hwang, Young; Peng, Jing; Mauk, Michael G; Bushman, Frederic D; Gross, Robert; Jarvis, Joseph N; Liu, Changchun

2018-06-30

Molecular diagnostics that involve nucleic acid amplification tests (NAATs) are crucial for prevention and treatment of infectious diseases. In this study, we developed a simple, inexpensive, disposable, fully 3D printed microfluidic reactor array that is capable of carrying out extraction, concentration and isothermal amplification of nucleic acids in variety of body fluids. The method allows rapid molecular diagnostic tests for infectious diseases at point of care. A simple leak-proof polymerization strategy was developed to integrate flow-through nucleic acid isolation membranes into microfluidic devices, yielding a multifunctional diagnostic platform. Static coating technology was adopted to improve the biocompatibility of our 3D printed device. We demonstrated the suitability of our device for both end-point colorimetric qualitative detection and real-time fluorescence quantitative detection. We applied our diagnostic device to detection of Plasmodium falciparum in plasma samples and Neisseria meningitides in cerebrospinal fluid (CSF) samples by loop-mediated, isothermal amplification (LAMP) within 50 min. The detection limits were 100 fg for P. falciparum and 50 colony-forming unit (CFU) for N. meningitidis per reaction, which are comparable to that of benchtop instruments. This rapid and inexpensive 3D printed device has great potential for point-of-care molecular diagnosis of infectious disease in resource-limited settings. Copyright © 2018 Elsevier B.V. All rights reserved.

Morphological and molecular diagnostics of Phytoseiulus persimilis and Phytoseiulus macropilis (Acari: Phytoseiidae).

PubMed

Okassa, Mireille; Tixier, Marie-Stéphane; Kreiter, Serge

2010-11-01

This study focuses on the diagnostics of two natural enemy species, belonging to the genus Phytoseiulus in the family Phytoseiidae (sub-family Amblyseiinae): P. macropilis and P. persimilis. These two species are of primary importance in biological control all over the world. However, they are morphologically very similar and specific diagnostics is difficult. This study utilizes mitochondrial molecular markers (12S rRNA and Cytb mtDNA) to differentiate these two species. Morphological analyses showed significant differences between P. persimilis and P. macropilis for 17 morphological characters of the 32 considered. However, despite these significant differences, the ranges of all characters overlap. Only the serration of the macroseta on the basitarsus (StIV) allows the differentiation between P. persimilis and P. macropilis. Despite these small morphological differences, molecular results, for both mitochondrial DNA fragments considered (rRNA and Cytb mtDNA), showed a clear delineation between the specimens of P. macropilis and P. persimilis. This study emphasizes (i) that only one morphological character (serration of the seta StIV) clearly separates these two species, and (ii) the usefulness of an automatical molecular and simple diagnostic tool for accurate differentiation of the two species and ensure the morphological diagnostics. Further studies are proposed, including more DNA sequences especially for P. macropilis.

Supramolecular Nanoparticles for Molecular Diagnostics and Therapeutics

NASA Astrophysics Data System (ADS)

Chen, Kuan-Ju

Over the past decades, significant efforts have been devoted to explore the use of various nanoparticle-based systems in the field of nanomedicine, including molecular imaging and therapy. Supramolecular synthetic approaches have attracted lots of attention due to their flexibility, convenience, and modularity for producing nanoparticles. In this dissertation, the developmental story of our size-controllable supramolecular nanoparticles (SNPs) will be discussed, as well as their use in specific biomedical applications. To achieve the self-assembly of SNPs, the well-characterized molecular recognition system (i.e., cyclodextrin/adamantane recognition) was employed. The resulting SNPs, which were assembled from three molecular building blocks, possess incredible stability in various physiological conditions, reversible size-controllability and dynamic disassembly that were exploited for various in vitro and in vivo applications. An advantage of using the supramolecular approach is that it enables the convenient incorporation of functional ligands onto SNP surface that confers functionality ( e.g., targeting, cell penetration) to SNPs. We utilized SNPs for molecular imaging such as magnetic resonance imaging (MRI) and positron emission tomography (PET) by introducing reporter systems (i.e., radio-isotopes, MR contrast agents, and fluorophores) into SNPs. On the other hand, the incorporation of various payloads, including drugs, genes and proteins, into SNPs showed improved delivery performance and enhanced therapeutic efficacy for these therapeutic agents. Leveraging the powers of (i) a combinatorial synthetic approach based on supramolecular assembly and (ii) a digital microreactor, a rapid developmental pathway was developed that is capable of screening SNP candidates for the ideal structural and functional properties that deliver optimal performance. Moreover, SNP-based theranostic delivery systems that combine reporter systems and therapeutic payloads into a single SNP for both diagnosis and therapy were generated. The results show that this type of theranostic SNPs may have a great contribution in the optimization of therapeutic efficacy for individual patients in clinical translation in the near future. It is anticipated that our supramolecular synthetic approach could be adopted to assemble various SNP-based delivery agents for molecular diagnostics and therapeutics that pave the way toward personalized medicine.

Quality in the molecular microbiology laboratory.

PubMed

Wallace, Paul S; MacKay, William G

2013-01-01

In the clinical microbiology laboratory advances in nucleic acid detection, quantification, and sequence analysis have led to considerable improvements in the diagnosis, management, and monitoring of infectious diseases. Molecular diagnostic methods are routinely used to make clinical decisions based on when and how to treat a patient as well as monitor the effectiveness of a therapeutic regime and identify any potential drug resistant strains that may impact on the long term patient treatment program. Therefore, confidence in the reliability of the result provided by the laboratory service to the clinician is essential for patient treatment. Hence, suitable quality assurance and quality control measures are important to ensure that the laboratory methods and service meet the necessary regulatory requirements both at the national and international level. In essence, the modern clinical microbiology laboratory ensures the appropriateness of its services through a quality management system that monitors all aspects of the laboratory service pre- and post-analytical-from patient sample receipt to reporting of results, from checking and upholding staff competency within the laboratory to identifying areas for quality improvements within the service offered. For most European based clinical microbiology laboratories this means following the common International Standard Organization (ISO9001) framework and ISO15189 which sets out the quality management requirements for the medical laboratory (BS EN ISO 15189 (2003) Medical laboratories-particular requirements for quality and competence. British Standards Institute, Bristol, UK). In the United States clinical laboratories performing human diagnostic tests are regulated by the Centers for Medicare and Medicaid Services (CMS) following the requirements within the Clinical Laboratory Improvement Amendments document 1988 (CLIA-88). This chapter focuses on the key quality assurance and quality control requirements within the modern microbiology laboratory providing molecular diagnostics.

A novel method for room temperature distribution and conservation of RNA and DNA reference materials for guaranteeing performance of molecular diagnostics in onco-hematology: A GBMHM study.

PubMed

Cayuela, Jean-Michel; Mauté, Carole; Fabre, Anne-Lise; Nibourel, Olivier; Dulucq, Stéphanie; Delabesse, Eric; Villarèse, Patrick; Hayette, Sandrine; Mozziconacci, Marie-Joelle; Macintyre, Elizabeth

2015-10-01

Performance of methods used for molecular diagnostics must be closely controlled by regular analysis of internal quality controls. However, conditioning, shipping and long lasting storage of nucleic acid controls remain problematic. Therefore, we evaluated the minicapsule-based innovative process developed by Imagene (Evry, France) for implementing DNA and RNA controls designed for clonality assessment of lymphoproliferations and BCR-ABL1 mRNA quantification, respectively. DNA samples were extracted from 12 cell lines selected for giving specific amplifications with most BIOMED-2 PCR tubes. RNA samples were extracted from 8 cell line mixtures expressing various BCR-ABL1 transcript levels. DNA and RNA were encapsulated by Imagene and shipped at room temperature to participating laboratories. Biologists were asked to report quality data of recovered nucleic acids as well as PCR results. Encapsulated nucleic acids samples were easily and efficiently recovered from minicapsules. The expected rearrangements at immunoglobulin, T-cell receptor and BCL2 loci were detected in DNA samples by all laboratories. Quality of RNA was consistent between laboratories and met the criteria requested for quantification of BCR-ABL1 transcripts. Expression levels measured by the 5 laboratories were within ±2 fold interval from the corresponding pre-encapsulation reference value. Moreover aging studies of encapsulated RNA simulating up to 100 years storage at room temperature show no bias in quantitative outcome. Therefore, Imagene minicapsules are suitable for storage and distribution at room temperature of genetic material designed for proficiency control of molecular diagnostic methods based on end point or real-time quantitative PCR. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.

PubMed

Clancy, Eoin; Coughlan, Helena; Higgins, Owen; Boo, Teck Wee; Cormican, Martin; Barrett, Louise; Smith, Terry J; Reddington, Kate; Barry, Thomas

2016-08-01

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a High Throughput Assay for Rapid and Accurate 10-Plex Detection of Citrus Pathogens

USDA-ARS?s Scientific Manuscript database

The need to reliably detect and identify multiple plant pathogens simultaneously, especially in woody perennial hosts, has led to development of new molecular diagnostic approaches. In this study, a Luminex-based system was developed that provided a robust and sensitive test for simultaneous detect...

Childhood Central Nervous System Germ Cell Tumors Treatment (PDQ®)—Health Professional Version

Cancer.gov

CNS germ cell tumors can be diagnosed and classified based on histology, tumor markers, or a combination of both. Get detailed information about newly diagnosed and recurrent childhood CNS germ cell tumors including molecular features and clinical features, diagnostic and staging evaluation, and treatment in this summary for clinicians.

Efficacy of the FilmArray blood culture identification panel for direct molecular diagnosis of infectious diseases from samples other than blood.

PubMed

Micó, Miquel; Navarro, Ferran; de Miniac, Daniela; González, Yésica; Brell, Albert; López, Cristina; Sánchez-Reus, Ferran; Mirelis, Beatriz; Coll, Pere

2015-12-01

Molecular-based techniques reduce the delay in diagnosing infectious diseases and therefore contribute to better patient outcomes. We assessed the FilmArray blood culture identification (BCID) panel (Biofire Diagnostics/bioMérieux) directly on clinical specimens other than blood: cerebrospinal, joint, pleural and ascitic fluids, bronchoscopy samples and abscesses. We compared the results from 88 samples obtained by culture-based techniques. The percentage of agreement between the two methods was 75 % with a Cohen κ value of 0.51. Global sensitivity and specificity using the FilmArray BCID panel were 71 and 97 %, respectively. Sensitivity was poorer in samples with a low bacterial load, such as ascitic and pleural fluids (25 %), whereas the sensitivity for abscess samples was high (89 %). These findings suggest that the FilmArray BCID panel could be useful to perform microbiological diagnosis directly from samples other than positive blood cultures, as it offers acceptable sensitivity and moderate agreement with conventional microbiological methods. Nevertheless, cost-benefit studies should be performed before introducing this method into algorithms for microbiological diagnostics.

Strategies for Characterization of Low-Abundant Intact or Truncated Low-Molecular-Weight Proteins From Human Plasma.

PubMed

Cai, Tanxi; Yang, Fuquan

2017-01-01

Low-molecular-weight region (LMW, MW≤30kDa) of human serum/plasma proteins, including small intact proteins, truncated fragments of larger proteins, along with some other small components, has been associated with the ongoing physiological and pathological events, and thereby represent a treasure trove of diagnostic molecules. Great progress in the mining of novel biomarkers from this diagnostic treasure trove for disease diagnosis and health monitoring has been achieved based on serum samples from healthy individuals and patients and powerful new approaches in biochemistry and systems biology. However, cumulative evidence indicates that many potential LMW protein biomarkers might still have escaped from detection due to their low abundance, the dynamic complexity of serum/plasma, and the limited efficiency of characterization approaches. Here, we provide an overview of the current state of knowledge with respect to strategies for the characterization of low-abundant LMW proteins (small intact or truncated proteins) from human serum/plasma, involving prefractionation or enrichment methods to reduce dynamic range and mass spectrometry-based characterization of low-abundant LMW proteins. © 2017 Elsevier Inc. All rights reserved.

[Epidemiologic diagnostic of nosocomial suppurative-septic infections of Pseudomonas etiology based on intraspecies typing of causative agent].

PubMed

Fel'dblium, I V; Zakharova, Iu A; Nikolaeva, A M; Fedotova, O S

2013-01-01

Scientific justification of optimization of epidemiologic diagnostic of suppurative-septic infection (SSI) caused by Pseudomonas aeruginosa based on comparability of antibiotic sensitivity and beta-lactamase production. Intraspecies typing of 37 P. aeruginosa strains isolated during microbiological monitoring of 106 patients and 131 objects of clinical environment of surgical and obstetrician hospitals by using a complex ofphenotypic and molecular-biological methods including determination of sensitivity to antibiotics by serial dilutions method and PCR-diagnostics with determination of TEM, SHV, CTX, OXA, MBL, VIM genes was performed. P. aeruginosa strains combined into groups by isolation location during studies turned out to be heterogeneous by sensitivity to antibiotics and beta-lactamase production that allowed to form subgroups of strains by focality attribute. Isolates recovered from different SSI foci had significant differences in minimal inhibitory concentration (MIC) reaching 1024 times. MIC parameter within subgroups did not exceed 8 - 16 consequent dilutions. Use of a complex of phenotypic and molecular-biologic methods of causative agent typing including determination of sensitivity to antibiotics by serial dilutions method and evaluation of beta-lactamase production allowed to establish a mechanism of development of SSI epidemic process caused by P. aeruginosa, detect origins and reservoirs of infection in hospital, modes and factors of transmission and reach maximum justification of epidemiologic control and prophylaxis measures of localization of foci of nosocomial infections of pseudomonas etiology.

Identification of Five Novel Variants in Chinese Oculocutaneous Albinism by Targeted Next-Generation Sequencing.

PubMed

Qiu, Biyuan; Ma, Tao; Peng, Chunyan; Zheng, Xiaoqin; Yang, Jiyun

2018-04-01

The diagnosis of oculocutaneous albinism (OCA) is established using clinical signs and symptoms. OCA is, however, a highly genetically heterogeneous disease with mutations identified in at least nineteen unique genes, many of which produce overlapping phenotypic traits. Thus, differentiating genetic OCA subtypes for diagnoses and genetic counseling is challenging, based on clinical presentation alone, and would benefit from a comprehensive molecular diagnostic. To develop and validate a more comprehensive, targeted, next-generation-sequencing-based diagnostic for the identification of OCA-causing variants. The genomic DNA samples from 28 OCA probands were analyzed by targeted next-generation sequencing (NGS), and the candidate variants were confirmed through Sanger sequencing. We observed mutations in the TYR, OCA2, and SLC45A2 genes in 25/28 (89%) patients with OCA. We identified 38 pathogenic variants among these three genes, including 5 novel variants: c.1970G>T (p.Gly657Val), c.1669A>C (p.Thr557Pro), c.2339-2A>C, and c.1349C>G (p.Thr450Arg) in OCA2; c.459_470delTTTTGCTGCCGA (p.Ala155_Phe158del) in SLC45A2. Our findings expand the mutational spectrum of OCA in the Chinese population, and the assay we developed should be broadly useful as a molecular diagnostic, and as an aid for genetic counseling for OCA patients.

Demise of Polymerase Chain Reaction/Electrospray Ionization-Mass Spectrometry as an Infectious Diseases Diagnostic Tool.

PubMed

Özenci, Volkan; Patel, Robin; Ullberg, Måns; Strålin, Kristoffer

2018-01-18

Although there are several US Food and Drug Administration (FDA)-approved/cleared molecular microbiology diagnostics for direct analysis of patient samples, all are single target or panel-based tests. There is no FDA-approved/cleared diagnostic for broad microbial detection. Polymerase chain reaction (PCR)/electrospray ionization-mass spectrometry (PCR/ESI-MS), commercialized as the IRIDICA system (Abbott) and formerly PLEX-ID, had been under development for over a decade and had become CE-marked and commercially available in Europe in 2014. Capable of detecting a large number of microorganisms, it was under review at the FDA when, in April 2017, Abbott discontinued it. This turn of events represents not only the loss of a potential diagnostic tool for infectious diseases but may be a harbinger of similar situations with other emerging and expensive microbial diagnostics, especially genomic tests. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Wavenumber selection based analysis in Raman spectroscopy improves skin cancer diagnostic specificity at high sensitivity levels (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zhao, Jianhua; Zeng, Haishan; Kalia, Sunil; Lui, Harvey

2017-02-01

Background: Raman spectroscopy is a non-invasive optical technique which can measure molecular vibrational modes within tissue. A large-scale clinical study (n = 518) has demonstrated that real-time Raman spectroscopy could distinguish malignant from benign skin lesions with good diagnostic accuracy; this was validated by a follow-up independent study (n = 127). Objective: Most of the previous diagnostic algorithms have typically been based on analyzing the full band of the Raman spectra, either in the fingerprint or high wavenumber regions. Our objective in this presentation is to explore wavenumber selection based analysis in Raman spectroscopy for skin cancer diagnosis. Methods: A wavenumber selection algorithm was implemented using variably-sized wavenumber windows, which were determined by the correlation coefficient between wavenumbers. Wavenumber windows were chosen based on accumulated frequency from leave-one-out cross-validated stepwise regression or least and shrinkage selection operator (LASSO). The diagnostic algorithms were then generated from the selected wavenumber windows using multivariate statistical analyses, including principal component and general discriminant analysis (PC-GDA) and partial least squares (PLS). A total cohort of 645 confirmed lesions from 573 patients encompassing skin cancers, precancers and benign skin lesions were included. Lesion measurements were divided into training cohort (n = 518) and testing cohort (n = 127) according to the measurement time. Result: The area under the receiver operating characteristic curve (ROC) improved from 0.861-0.891 to 0.891-0.911 and the diagnostic specificity for sensitivity levels of 0.99-0.90 increased respectively from 0.17-0.65 to 0.20-0.75 by selecting specific wavenumber windows for analysis. Conclusion: Wavenumber selection based analysis in Raman spectroscopy improves skin cancer diagnostic specificity at high sensitivity levels.

CT-guided transthoracic core needle biopsy for small pulmonary lesions: diagnostic performance and adequacy for molecular testing.

PubMed

Tian, Panwen; Wang, Ye; Li, Lei; Zhou, Yongzhao; Luo, Wenxin; Li, Weimin

2017-02-01

Computed tomography (CT)-guided transthoracic needle biopsy is a well-established, minimally invasive diagnostic tool for pulmonary lesions. Few large studies have been conducted on the diagnostic performance and adequacy for molecular testing of transthoracic core needle biopsy (TCNB) for small pulmonary lesions. This study included CT-guided TCNB with 18-gauge cutting needles in 560 consecutive patients with small (≤3 cm) pulmonary lesions from January 2012 to January 2015. There were 323 males and 237 females, aged 51.8±12.7 years. The size of the pulmonary lesions was 1.8±0.6 cm. The sensitivity, specificity, accuracy and complications of the biopsies were investigated. The risk factors of diagnostic failure were assessed using univariate and multivariate analyses. The sample's adequacy for molecular testing of non-small cell lung cancer (NSCLC) was analyzed. The overall sensitivity, specificity, and accuracy for diagnosis of malignancy were 92.0% (311/338), 98.6% (219/222), and 94.6% (530/560), respectively. The incidence of bleeding complications was 22.9% (128/560), and the incidence of pneumothorax was 10.4% (58/560). Logistic multivariate regression analysis showed that the independent risk factors for diagnostic failure were a lesion size ≤1 cm [odds ratio (OR), 3.95; P=0.007], lower lobe lesions (OR, 2.83; P=0.001), and pneumothorax (OR, 1.98; P=0.004). Genetic analysis was successfully performed on 95.45% (168/176) of specimens diagnosed as NSCLC. At least 96.8% of samples with two or more passes from a lesion were sufficient for molecular testing. The diagnostic yield of small pulmonary lesions by CT-guided TCNB is high, and the procedure is relatively safe. A lesion size ≤1 cm, lower lobe lesions, and pneumothorax are independent risk factors for biopsy diagnostic failure. TCNB specimens could provide adequate tissues for molecular testing.

Design of a Tablet Computer App for Facilitation of a Molecular Blood Culture Test in Clinical Microbiology and Preliminary Usability Evaluation.

PubMed

Samson, Lasse L; Pape-Haugaard, Louise; Meltzer, Michelle C; Fuchs, Martin; Schønheyder, Henrik C; Hejlesen, Ole

2016-03-18

User mobility is an important aspect of the development of clinical information systems for health care professionals. Mobile phones and tablet computers have obtained widespread use by health care professionals, offering an opportunity for supporting the access to patient information through specialized applications (apps) while supporting the mobility of the users. The use of apps for mobile phones and tablet computers may support workflow of complex tasks, for example, molecular-based diagnostic tests in clinical microbiology. Multiplex Blood Culture Test (MuxBCT) is a molecular-based diagnostic test used for rapid identification of pathogens in positive blood cultures. To facilitate the workflow of the MuxBCT, a specialized tablet computer app was developed as an accessory to the diagnostic test. The app aims to reduce the complexity of the test by step-by-step guidance of microscopy and to assist users in reaching an exact bacterial or fungal diagnosis based on blood specimen observations and controls. Additionally, the app allows for entry of test results, and communication thereof to the laboratory information system (LIS). The objective of the study was to describe the design considerations of the MuxBCT app and the results of a preliminary usability evaluation. The MuxBCT tablet app was developed and set up for use in a clinical microbiology laboratory. A near-live simulation study was conducted in the clinical microbiology laboratory to evaluate the usability of the MuxBCT app. The study was designed to achieve a high degree of realism as participants carried out a scenario representing the context of use for the MuxBCT app. As the MuxBCT was under development, the scenario involved the use of molecular blood culture tests similar to the MuxBCT for identification of microorganisms from positive blood culture samples. The study participants were observed, and their interactions with the app were recorded. After the study, the participants were debriefed to clarify observations. Four medical laboratory technicians, for example, representative of end users of the app, participated in the clinical simulation study. Using the MuxBCT app, the study participants successfully identified and reported all microorganisms from the positive blood cultures examined. Three of the four participants reported that they found the app useful, while one study participant reported that she would prefer to make notes on paper and later enter them into the LIS. The preliminary usability evaluation results indicate that use of the MuxBCT tablet app can facilitate the workflow of the MuxBCT diagnostic test.

[Assessment of laboratory diagnostic network in the implementation of the Program for Viral Hepatitis Prevention and Control in São Paulo State, Brazil, 1997-2012].

PubMed

Marques, Cristiano Corrêa de Azevedo; Carvalheiro, José da Rocha

2017-01-01

to assess the performance of the diagnostic network in the implementation process of the Program for Viral Hepatitis Prevention and Control in São Paulo State, Brazil, from 1997 to 2012. evaluation study based on documentary research and structured interviews, combined with a historical series analysis of indicators developed to assess the implementation process of the program, using data from the Department of the Brazilian National Health System. from 1997 to 2012, the serology, biopsy and molecular biology diagnostic networks showed an increase in the coefficients of coverage of 7.4, 7.3, and 62.0 times, respectively, with an increase in cases detection and treatment access. despite the effective implementation of the diagnostic network, there is a need to review the search strategy for new cases, and access to liver biopsy, still insufficient to the program demand.

Radiation biodosimetry: applications for spaceflight

NASA Astrophysics Data System (ADS)

Blakely, W.; Miller, A.; Grace, M.; Prasanna, P.; Muderhwa, J.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a dose assessment software application, a portable blood cell counter, and molecular biodosimetry using miniaturized equipment. The Biodosimetry Assessment Tool (BAT) software application calculates radiation dose based on a patient's physical signs and symptoms and blood analysis, annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. The dry reagent centrifuge-based blood cell counter (QBC Autoread Plus, Beckon Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins), once dose-dependent targets are identified, optimized, and validated, will make use of miniaturized diagnostic equipment for nucleic acid sequence and antigen-based biosensor detection technologies. These radiation assessment diagnostic technologies can have dual use for other medical related applications. [The Armed Forces Radiobiology Research Institute, under work unit AFRRI-01-3, and the Defense Threat Reduction Agency, under contract GG4661, supported this research.

Clinical Relevance of Prognostic and Predictive Molecular Markers in Gliomas.

PubMed

Siegal, Tali

2016-01-01

Sorting and grading of glial tumors by the WHO classification provide clinicians with guidance as to the predicted course of the disease and choice of treatment. Nonetheless, histologically identical tumors may have very different outcome and response to treatment. Molecular markers that carry both diagnostic and prognostic information add useful tools to traditional classification by redefining tumor subtypes within each WHO category. Therefore, molecular markers have become an integral part of tumor assessment in modern neuro-oncology and biomarker status now guides clinical decisions in some subtypes of gliomas. The routine assessment of IDH status improves histological diagnostic accuracy by differentiating diffuse glioma from reactive gliosis. It carries a favorable prognostic implication for all glial tumors and it is predictive for chemotherapeutic response in anaplastic oligodendrogliomas with codeletion of 1p/19q chromosomes. Glial tumors that contain chromosomal codeletion of 1p/19q are defined as tumors of oligodendroglial lineage and have favorable prognosis. MGMT promoter methylation is a favorable prognostic marker in astrocytic high-grade gliomas and it is predictive for chemotherapeutic response in anaplastic gliomas with wild-type IDH1/2 and in glioblastoma of the elderly. The clinical implication of other molecular markers of gliomas like mutations of EGFR and ATRX genes and BRAF fusion or point mutation is highlighted. The potential of molecular biomarker-based classification to guide future therapeutic approach is discussed and accentuated.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe.

PubMed

Lee, Hyeran; Akers, Walter J; Cheney, Philip P; Edwards, W Barry; Liang, Kexian; Culver, Joseph P; Achilefu, Samuel

2009-01-01

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with (64)Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k(cat) and K(M) of 0.55+/-0.01 s(-1) and 1.12+/-0.06 microM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled (64)Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe

NASA Astrophysics Data System (ADS)

Lee, Hyeran; Akers, Walter J.; Cheney, Philip P.; Edwards, W. Barry; Liang, Kexian; Culver, Joseph P.; Achilefu, Samuel

2009-07-01

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with 64Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters kcat and KM of 0.55+/-0.01 s-1 and 1.12+/-0.06 μM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled 64Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Molecular Diagnosis and Biomarker Identification on SELDI proteomics data by ADTBoost method.

PubMed

Wang, Lu-Yong; Chakraborty, Amit; Comaniciu, Dorin

2005-01-01

Clinical proteomics is an emerging field that will have great impact on molecular diagnosis, identification of disease biomarkers, drug discovery and clinical trials in the post-genomic era. Protein profiling in tissues and fluids in disease and pathological control and other proteomics techniques will play an important role in molecular diagnosis with therapeutics and personalized healthcare. We introduced a new robust diagnostic method based on ADTboost algorithm, a novel algorithm in proteomics data analysis to improve classification accuracy. It generates classification rules, which are often smaller and easier to interpret. This method often gives most discriminative features, which can be utilized as biomarkers for diagnostic purpose. Also, it has a nice feature of providing a measure of prediction confidence. We carried out this method in amyotrophic lateral sclerosis (ALS) disease data acquired by surface enhanced laser-desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF MS) experiments. Our method is shown to have outstanding prediction capacity through the cross-validation, ROC analysis results and comparative study. Our molecular diagnosis method provides an efficient way to distinguish ALS disease from neurological controls. The results are expressed in a simple and straightforward alternating decision tree format or conditional format. We identified most discriminative peaks in proteomic data, which can be utilized as biomarkers for diagnosis. It will have broad application in molecular diagnosis through proteomics data analysis and personalized medicine in this post-genomic era.

Clinical relevance of molecular diagnostics in gastrointestinal (GI) cancer: European Society of Digestive Oncology (ESDO) expert discussion and recommendations from the 17th European Society for Medical Oncology (ESMO)/World Congress on Gastrointestinal Cancer, Barcelona.

PubMed

Baraniskin, Alexander; Van Laethem, Jean-Luc; Wyrwicz, Lucjan; Guller, Ulrich; Wasan, Harpreet S; Matysiak-Budnik, Tamara; Gruenberger, Thomas; Ducreux, Michel; Carneiro, Fatima; Van Cutsem, Eric; Seufferlein, Thomas; Schmiegel, Wolff

2017-11-01

In the epoch of precision medicine and personalised oncology, which aims to deliver the right treatment to the right patient, molecular genetic biomarkers are a topic of growing interest. The aim of this expert discussion and position paper is to review the current status of various molecular tests for gastrointestinal (GI) cancers and especially considering their significance for the clinical routine use. Opinion leaders and experts from diverse nationalities selected on scientific merit were asked to answer to a prepared set of questions about the current status of molecular diagnostics in different GI cancers. All answers were then discussed during a plenary session and reported here in providing a well-balanced reflection of both clinical expertise and updated evidence-based medicine. Preselected molecular genetic biomarkers that are described and disputed in the current medical literature in different GI cancers were debated, and recommendations for clinical routine practice were made whenever possible. Furthermore, the preanalytical variations were commented and proposals for quality controls of biospecimens were made. The current article summarises the recommendations of the expert committee regarding prognostic and predictive molecular genetic biomarkers in different entities of GI cancers. The briefly and comprehensively formulated guidelines should assist clinicians in the process of decision making in daily clinical practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

Implication of Gastric Cancer Molecular Genetic Markers in Surgical Practice.

PubMed

Nemtsova, Marina V; Strelnikov, Vladimir V; Tanas, Alexander S; Bykov, Igor I; Zaletaev, Dmitry V; Rudenko, Viktoria V; Glukhov, Alexander I; Kchorobrich, Tatiana V; Li, Yi; Tarasov, Vadim V; Barreto, George E; Aliev, Gjumrakch

2017-10-01

We have investigated aberrant methylation of genes CDH1, RASSF1A, MLH1, N33, DAPK, expression of genes hTERT, MMP7, MMP9, BIRC5 (survivin), PTGS2, and activity of telomerase of 106 gastric tumor samples obtained intra-operatively and 53 gastric tumor samples from the same group of patients obtained endoscopically before surgery. Biopsy specimens obtained from 50 patients with chronic calculous cholecystitis were used as a control group. Together with tissue samples obtained from different sites remote to tumors, a total of 727 samples have been studied. The selected parameters comprise a system of molecular markers that can be used in both diagnostics of gastric cancer and in dynamic monitoring of patients after surgery. Special attention was paid to the use of molecular markers for the diagnostics of malignant process in the material obtained endoscopically since the efficacy of morphological diagnostics in biopsies is compromised by intratumoral heterogeneity, which may prevent reliable identification of tumor cells in the sampling. Our data indicated that certain molecular genetic events provided more sensitive yet specific markers of the tumor. We demonstrated that molecular profiles detected in preoperative biopsies were confirmed by the material obtained intra-operatively. The use of endoscopic material facilitates gastric tumors pre-operative diagnostics, improving early detection of gastric cancer and potential effective treatment strategies.

Molecular engineering of antibodies for therapeutic and diagnostic purposes

PubMed Central

Ducancel, Frédéric; Muller, Bruno H.

2012-01-01

During the past ten years, monoclonal antibodies (mAbs) have taken center stage in the field of targeted therapy and diagnosis. This increased interest in mAbs is due to their binding accuracy (affinity and specificity) together with the original molecular and structural rules that govern interactions with their cognate antigen. In addition, the effector properties of antibodies constitute a second major advantage associated with their clinical use. The development of molecular and structural engineering and more recently of in vitro evolution of antibodies has opened up new perspectives in the de novo design of antibodies more adapted to clinical and diagnostic use. Thus, efforts are regularly made by researchers to improve or modulate antibody recognition properties, to adapt their pharmacokinetics, engineer their stability, and control their immunogenicity. This review presents the latest molecular engineering results on mAbs with therapeutic and diagnostic applications. PMID:22684311

Rapid Detection of Pathogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

David Perlin

2005-08-14

Pathogen identification is a crucial first defense against bioterrorism. A major emphasis of our national biodefense strategy is to establish fast, accurate and sensitive assays for diagnosis of infectious diseases agents. Such assays will ensure early and appropriate treatment of infected patients. Rapid diagnostics can also support infection control measures, which monitor and limit the spread of infectious diseases agents. Many select agents are highly transmissible in the early stages of disease, and it is critical to identify infected patients and limit the risk to the remainder of the population and to stem potential panic in the general population. Nucleicmore » acid-based molecular approaches for identification overcome many of the deficiencies associated with conventional culture methods by exploiting both large- and small-scale genomic differences between organisms. PCR-based amplification of highly conserved ribosomal RNA (rRNA) genes, intergenic sequences, and specific toxin genes is currently the most reliable approach for bacterial, fungal and many viral pathogenic agents. When combined with fluorescence-based oligonucleotide detection systems, this approach provides real-time, quantitative, high fidelity analysis capable of single nucleotide allelic discrimination (4). These probe systems offer rapid turn around time (<2 h) and are suitable for high throughput, automated multiplex operations that are critical for clinical diagnostic laboratories. In this pilot program, we have used molecular beacon technology invented at the Public health Research Institute to develop a new generation of molecular probes to rapidly detect important agents of infectious diseases. We have also developed protocols to rapidly extract nucleic acids from a variety of clinical specimen including and blood and tissue to for detection in the molecular assays. This work represented a cooperative research development program between the Kramer-Tyagi/Perlin labs on probe development and the Perlin lab in sample preparation and testing in animal models.« less

The laboratory diagnosis of bacterial vaginosis

PubMed Central

Money, Deborah

2005-01-01

Bacterial vaginosis (BV) is an extremely common health problem for women. In addition to the troublesome symptoms often associated with a disruption in the balance of vaginal flora, BV is associated with adverse gynecological and pregnancy outcomes. Although not technically a sexually transmitted infection, BV is a sexually associated condition. Diagnostic tests include real-time clinical/microbiological diagnosis, and the current gold standard, the standardized evaluation of morphotypes on Gram stain analysis. The inappropriate use of vaginal culture can be misleading. Future developments into molecular-based diagnostics will be important to further understand this complex endogenous flora disruption. PMID:18159532

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

PubMed

Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro G; Gállego, Montserrat; Muñoz, Carmen

2018-01-01

Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Nanomedicine: nanoparticles, molecular biosensors, and targeted gene/drug delivery for combined single-cell diagnostics and therapeutics

NASA Astrophysics Data System (ADS)

Prow, Tarl W.; Salazar, Jose H.; Rose, William A.; Smith, Jacob N.; Reece, Lisa; Fontenot, Andrea A.; Wang, Nan A.; Lloyd, R. Stephen; Leary, James F.

2004-07-01

Next generation nanomedicine technologies are being developed to provide for continuous and linked molecular diagnostics and therapeutics. Research is being performed to develop "sentinel nanoparticles" which will seek out diseased (e.g. cancerous) cells, enter those living cells, and either perform repairs or induce those cells to die through apoptosis. These nanoparticles are envisioned as multifunctional "smart drug delivery systems". The nanosystems are being developed as multilayered nanoparticles (nanocrystals, nanocapsules) containing cell targeting molecules, intracellular re-targeting molecules, molecular biosensor molecules, and drugs/enzymes/gene therapy. These "nanomedicine systems" are being constructed to be autonomous, much like present-day vaccines, but will have sophisticated targeting, sensing, and feedback control systems-much more sophisticated than conventional antibody-based therapies. The fundamental concept of nanomedicine is to not to just kill all aberrant cells by surgery, radiation therapy, or chemotherapy. Rather it is to fix cells, when appropriate, one cell-at-a-time, to preserve and re-build organ systems. When cells should not be fixed, such as in cases where an improperly repaired cell might give rise to cancer cells, the nanomedical therapy would be to induce apoptosis in those cells to eliminate them without the damagin bystander effects of the inflammatory immune response system reacting to necrotic cells or those which have died from trauma or injury. The ultimate aim of nanomedicine is to combine diagnostics and therapeutics into "real-time medicine", using where possible in-vivo cytometry techniques for diagnostics and therapeutics. A number of individual components of these multi-component nanoparticles are already working in in-vitro and ex-vivo cell and tissue systems. Work has begun on construction of integrated nanomedical systems.

The Ebola Spatial Care Path™: Accelerating point-of-care diagnosis, decision making, and community resilience in outbreaks.

PubMed

Kost, Gerald J; Ferguson, William J; Hoe, Jackie; Truong, Anh-Thu; Banpavichit, Arirat; Kongpila, Surin

2015-01-01

To present a vision where point-of-care testing (POCT) accelerates an Ebola Spatial Care Path™ (SCP) and future molecular diagnostics enable facilitated-access self-testing (FAST POC); to design an alternate care facility (ACF) for the SCP; to innovate an Ebola diagnostic center (DC); and to propel rapid POCT to the frontline to create resilience that stops future outbreaks. PubMed, literature, and web searches. Centers for Disease Control and Prevention (CDC), Food and Drug Administration (FDA), Medicine Without Frontiers, and World Health Organization (WHO) document analyses. Investigations in China, the Philippines, Thailand, and the United States. Review of SE Asia, US, and West Africa isolation-treatment centers. Innovation of a SCP, ACF, and DC suitable for American and other communities. The authors designed an ACF and DC to integrate SCP principles for urgent Ebola care. FDA emergency use authorizations for Ebola molecular diagnostics were discovered, but no portable, handheld, or self-contained molecular POC instruments are yet available, although feasible. The WHO initiated design criteria and an acceptance protocol for testing. Financial investment in POCT will downsize Ebola outbreaks. POCT is facilitating global health. Now, global health problems are elevating POCT to new levels of importance for accelerating diagnosis and evidence-based decision making during disease outbreaks. Authorities concur that rapid diagnosis has potential to stop disease spread. With embedded POCT, strategic SCPs planned by communities fulfill CDC recommendations. POC devices should consolidate multiplex test clusters supporting patients with Ebola in isolation. The ultimate future solution is FAST POC. New technologies offer minimally significant risks. Diagnostic centers in ACFs and transportable formats also will optimize Ebola SCPs.

Magnetically engineered smart thin films: toward lab-on-chip ultra-sensitive molecular imaging.

PubMed

Hassan, Muhammad A; Saqib, Mudassara; Shaikh, Haseeb; Ahmad, Nasir M; Elaissari, Abdelhamid

2013-03-01

Magnetically responsive engineered smart thin films of nanoferrites as contrast agent are employed to develop surface based magnetic resonance imaging to acquire simple yet fast molecular imaging. The work presented here can be of significant potential for future lab-on-chip point-of-care diagnostics from the whole blood pool on almost any substrates to reduce or even prevent clinical studies involve a living organism to enhance the non-invasive imaging to advance the '3Rs' of work in animals-replacement, refinement and reduction.

Molecular phylogenetic analysis of Enterobius vermicularis and development of an 18S ribosomal DNA-targeted diagnostic PCR.

PubMed

Zelck, Ulrike E; Bialek, Ralf; Weiss, Michael

2011-04-01

We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed.

ACVP-02: Plasma SIV/SHIV RNA Viral Load Measurements through the AIDS and Cancer Virus Program Quantitative Molecular Diagnostics Core | Frederick National Laboratory for Cancer Research

Cancer.gov

The SIV plasma viral load assay performed by the Quantitative Molecular Diagnostics Core (QMDC) utilizes reagents specifically designed to detect and accurately quantify the full range of SIV/SHIV viral variants and clones in common usage in the rese

Recommendations for accreditation of laboratories in molecular biology of hematologic malignancies.

PubMed

Flandrin-Gresta, Pascale; Cornillet, Pascale; Hayette, Sandrine; Gachard, Nathalie; Tondeur, Sylvie; Mauté, Carole; Cayuela, Jean-Michel

2015-01-01

Over recent years, the development of molecular biology techniques has improved the hematological diseases diagnostic and follow-up. Consequently, these techniques are largely used in the biological screening of these diseases; therefore the Hemato-oncology molecular diagnostics laboratories must be actively involved in the accreditation process according the ISO 15189 standard. The French group of molecular biologists (GBMHM) provides requirements for the implementation of quality assurance for the medical molecular laboratories. This guideline states the recommendations for the pre-analytical, analytical (methods validation procedures, quality controls, reagents), and post-analytical conditions. In addition, herein we state a strategy for the internal quality control management. These recommendations will be regularly updated.

Quantum Dot Platform for Single-Cell Molecular Profiling

NASA Astrophysics Data System (ADS)

Zrazhevskiy, Pavel S.

In-depth understanding of the nature of cell physiology and ability to diagnose and control the progression of pathological processes heavily rely on untangling the complexity of intracellular molecular mechanisms and pathways. Therefore, comprehensive molecular profiling of individual cells within the context of their natural tissue or cell culture microenvironment is essential. In principle, this goal can be achieved by tagging each molecular target with a unique reporter probe and detecting its localization with high sensitivity at sub-cellular resolution, primarily via microscopy-based imaging. Yet, neither widely used conventional methods nor more advanced nanoparticle-based techniques have been able to address this task up to date. High multiplexing potential of fluorescent probes is heavily restrained by the inability to uniquely match probes with corresponding molecular targets. This issue is especially relevant for quantum dot probes---while simultaneous spectral imaging of up to 10 different probes is possible, only few can be used concurrently for staining with existing methods. To fully utilize multiplexing potential of quantum dots, it is necessary to design a new staining platform featuring unique assignment of each target to a corresponding quantum dot probe. This dissertation presents two complementary versatile approaches towards achieving comprehensive single-cell molecular profiling and describes engineering of quantum dot probes specifically tailored for each staining method. Analysis of expanded molecular profiles is achieved through augmenting parallel multiplexing capacity with performing several staining cycles on the same specimen in sequential manner. In contrast to other methods utilizing quantum dots or other nanoparticles, which often involve sophisticated probe synthesis, the platform technology presented here takes advantage of simple covalent bioconjugation and non-covalent self-assembly mechanisms for straightforward probe preparation and specimen labeling, requiring no advanced technical skills and being directly applicable for a wide range of molecular profiling studies. Utilization of quantum dot platform for single-cell molecular profiling promises to greatly benefit both biomedical research and clinical diagnostics by providing a tool for addressing phenotypic heterogeneity within large cell populations, opening access to studying low-abundance events often masked or completely erased by batch processing, and elucidating biomarker signatures of diseases critical for accurate diagnostics and targeted therapy.

Quantum Dots for Molecular Diagnostics of Tumors

PubMed Central

Zdobnova, T.A.; Lebedenko, E.N.; Deyev, S.М.

2011-01-01

Semiconductor quantum dots (QDs) are a new class of fluorophores with unique physical and chemical properties, which allow to appreciably expand the possibilities for the current methods of fluorescent imaging and optical diagnostics. Here we discuss the prospects of QD application for molecular diagnostics of tumors ranging from cancer-specific marker detection on microplates to non-invasive tumor imagingin vivo. We also point out the essential problems that require resolution in order to clinically promote QD, and we indicate innovative approaches to oncology which are implementable using QD. PMID:22649672

Identification of species based on DNA barcode using k-mer feature vector and Random forest classifier.

PubMed

Meher, Prabina Kumar; Sahu, Tanmaya Kumar; Rao, A R

2016-11-05

DNA barcoding is a molecular diagnostic method that allows automated and accurate identification of species based on a short and standardized fragment of DNA. To this end, an attempt has been made in this study to develop a computational approach for identifying the species by comparing its barcode with the barcode sequence of known species present in the reference library. Each barcode sequence was first mapped onto a numeric feature vector based on k-mer frequencies and then Random forest methodology was employed on the transformed dataset for species identification. The proposed approach outperformed similarity-based, tree-based, diagnostic-based approaches and found comparable with existing supervised learning based approaches in terms of species identification success rate, while compared using real and simulated datasets. Based on the proposed approach, an online web interface SPIDBAR has also been developed and made freely available at http://cabgrid.res.in:8080/spidbar/ for species identification by the taxonomists. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of applicability of triplet-state emission of molecular hydrogen for spectral diagnostics of a DC discharge

NASA Astrophysics Data System (ADS)

Shakhatov, V. A.; Lebedev, Yu. A.

2017-10-01

The applicability of emission of the N 3Λσ triplet states of molecular hydrogen for spectral diagnostics of the positive column of a dc glow discharge in hydrogen at translational gas temperatures of 360-600 K, specific absorbed powers of 0.8-4.25 W/cm, gas pressures of p = 0.3-15.0 Torr, reduced fields of E/N = 30-130 Td, and electron densities of n e = 4.0 × 109-6.5 × 1010 cm-3 is analyzed by using an advanced level-based semi-empirical collisional-radiative model. It is found that secondary processes make the main contribution to the population and decay of the N 3Λσ = a 3Σ+ g , c 3Π u , g 3Σ+ g , h 3Σ+ g , and i 3Π g triplet states. The dipole-allowed transitions e 3Σ+ g → a 3Σ+ g , f 3Σ+ g → a 3Σ+ g , g 3Σ+ g and k 3Π u → a 3Σ+ g can be used for spectral diagnostics of a dc discharge within a simplified coronal model.

[New etiological concepts in uveitis].

PubMed

Bodaghi, B

2005-05-01

Uveitis remains an important cause of visual impairment, particularly in young patients. Idiopathic forms of intraocular inflammation should no longer be regarded as a presumed clinical entity, and the ophthalmologist must reconsider the specific etiology of primary uveitis when the clinical examination does not yield a definitive diagnosis or when the course of the disease on corticosteroids remains atypical. Laboratory tests based on serum analysis have limited value and should not be considered as diagnostic proof in different clinical presentations. The diagnostic management of infectious uveitis has been greatly improved by the use of molecular techniques applied to ocular fluids and tissues. Polymerase chain reaction (PCR) technology is a powerful tool that should be proposed in atypical cases of uveitis or retinitis of unclear but potentially infectious origin. This strategy is a major step before using unconventional and new immunomodulatory agents such as anti-TNF-alpha molecules. Under strict experimental conditions including adequate testing to rule out a possible contamination, PCR and its variants have changed our practical approach to intraocular inflammatory disorders and have provided new details for the understanding of infectious uveitis. The concept of pathogen-induced intraocular inflammation can be revisited in the light of molecular data obtained after anterior chamber paracentesis or diagnostic vitrectomy.

Basis for molecular diagnostics and immunotherapy for esophageal cancer

PubMed Central

Abdo, Joe; Agrawal, Devendra K.; Mittal, Sumeet K.

2017-01-01

Introduction Esophageal cancer is an extremely aggressive neoplasm, diagnosed in about 17,000 Americans every year with a mortality rate of more than 80% within five years and a median overall survival of just 13 months. For decades, the go-to regimen for esophageal cancer patients has been the use of taxane and platinum-based chemotherapy regimens, which has yielded the field’s most dire survival statistics. Areas covered Combination immunotherapy and a more robust molecular diagnostic platform for esophageal tumors could improve patient management strategies and potentially extend lives beyond the current survival figures. Analyzing a panel of biomarkers including those affiliated with taxane and platinum resistance (ERCC1 and TUBB3) as well as immunotherapy effectiveness (PD-L1) would provide oncologists more information on how to optimize first-line therapy for esophageal cancer. Expert commentary Of the 12 FDA-approved therapies in esophageal cancer, zero target the genome. A majority of the approved drugs either target or are effected by proteomic expression. Therefore, a broader understanding of diagnostic biomarkers could give more clarity and direction in treating esophageal cancer in concert with a greater use of immunotherapy. PMID:27838937

Treatment Options to Manage Wound Biofilm

PubMed Central

Jones, Curtis E.; Kennedy, John P.

2012-01-01

Background Bioburden is an accepted barrier to chronic wound healing. Defining the significance, phenotype, clinical classification, and treatment guidelines has been historically lacking of evidence and based on paradigms that do not represent the scientific or clinical reality. The Problem Chronic wound bioburden is typically abundant, polymicrobial, and extremely diverse. These microbes naturally adopt biofilm phenotypes, which are quite often viable but not culturable, thereby going undetected. The failures of culture-based detection have led to abandonment of routine bioburden evaluation and aggressive treatment or, worse, to assume bioburden is not a significant barrier. Predictably, treatment regimens to address biofilm phenotypes lagged behind our diagnostic tools and understanding. Basic/Clinical Science Advances Microbial DNA-based diagnostic tools and treatment regimens have emerged, which provide and leverage objective information, resulting in a dramatic impact on outcomes. Relevance to Clinical Care Modern medicine demands decisions based on objective evidence. The diagnostic and treatment protocols reviewed herein empower clinicians to practice modern medicine with regard to bioburden, with DNA level certainty. Conclusion Bioburden is a significant barrier to healing for all chronic wounds. Molecular diagnostics provide the first objective means of assessing wound bioburden. The accuracy and comprehensive data from such diagnostic methodologies provide clinicians with the ability to employ patient-specific treatment options, targeted to each patient's microbial wound census. Based on current outcomes data, the most effective therapeutic options are topical (TPL) antibiofilm agents (ABF) combined with TPL antibiotics (ABX). In specific patients, systemic ABX and selective biocides are also appropriate, but not exclusive of ABF combined with TPL ABX. PMID:24527291

Personalized Medicine Based on Theranostic Radioiodine Molecular Imaging for Differentiated Thyroid Cancer.

PubMed

Ahn, Byeong-Cheol

2016-01-01

Molecular imaging based personalized therapy has been a fascinating concept for individualized therapeutic strategy, which is able to attain the highest efficacy and reduce adverse effects in certain patients. Theranostics, which integrates diagnostic testing to detect molecular targets for particular therapeutic modalities, is one of the key technologies that contribute to the success of personalized medicine. Although the term "theranostics" was used after the second millennium, its basic principle was applied more than 70 years ago in the field of thyroidology with radioiodine molecular imaging. Differentiated thyroid cancer, which arises from follicular cells in the thyroid, is the most common endocrine malignancy, and theranostic radioiodine has been successfully applied to diagnose and treat differentiated thyroid cancer, the applications of which were included in the guidelines published by various thyroid or nuclear medicine societies. Through better pathophysiologic understanding of thyroid cancer and advancements in nuclear technologies, theranostic radioiodine contributes more to modern tailored personalized management by providing high therapeutic effect and by avoiding significant adverse effects in differentiated thyroid cancer. This review details the inception of theranostic radioiodine and recent radioiodine applications for differentiated thyroid cancer management as a prototype of personalized medicine based on molecular imaging.

Advanced Mass Spectrometry Technologies for the Study of Microbial Pathogenesis

PubMed Central

Moore, Jessica L.; Caprioli, Richard M.; Skaar, Eric P.

2014-01-01

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been successfully applied to the field of microbial pathogenesis with promising results, principally in diagnostic microbiology to rapidly identify bacteria based on the molecular profiles of small cell populations. Direct profiling of molecules from serum and tissue samples by MALDI MS providesa means to study the pathogen-host interaction and to discover potential markers of infection. Systematic molecular profiling across tissue sections represents a new imaging modality, enabling regiospecific molecular measurements to be made in situ, in both two- and three-dimensional analyses. Herein, we briefly summarize work that employs MALDI MS to study the pathogenesis of microbial infection. PMID:24997399

Differences in the molecular biology of adenocarcinoma of the esophagus, gastric cardia, and upper gastric third.

PubMed

Lehmann, Kuno; Schneider, Paul M

2010-01-01

Adenocarcinoma of the distal esophagus, gastric cardia, and upper gastric third are grouped in type I-III by the Siewert classification. This classification is based on the endoscopic localisation of the tumor center, and is the most important diagnostic tool to group these tumors. On a molecular level, there is currently no marker that would allow to differentiate the three different types. Furthermore, the Siewert classification was not uniformly used in the recent literature, making interpretation and generalization of these results difficult. However, several potential targets have been identified that may help to separate these tumors by molecular markers, and are summarized in this chapter.

Molecular Detection of Chilo infuscatellus

PubMed Central

Wang, Weizhong; Wang, Rong; Zheng, Haotian; Gao, Sanji

2017-01-01

Abstract The Chilo infuscatellus (Snellen; Lepidoptera: Pyralidae) is the main pest of sugarcane in China. The shortage of easily distinguishable morphological characters especially in early stage make it challenging to diagnosed and promptly take steps for pest management. In the present study, we described a PCR method for the molecular identification of based on barcode region of COI sequences between C. infuscatellus and four other sugarcane borer species. A 285bp fragment was successfully amplified from all life stages and different geographical populations. Sensitivity tests revealed that diagnostic bands were generated as low as the DNA template concentration of 5 ng/µl. Our work demonstrated a rapid and accurate way for the molecular diagnosis of C. infuscatellus. PMID:29117377

Deep Sequencing of Urinary RNAs for Bladder Cancer Molecular Diagnostics.

PubMed

Sin, Mandy L Y; Mach, Kathleen E; Sinha, Rahul; Wu, Fan; Trivedi, Dharati R; Altobelli, Emanuela; Jensen, Kristin C; Sahoo, Debashis; Lu, Ying; Liao, Joseph C

2017-07-15

Purpose: The majority of bladder cancer patients present with localized disease and are managed by transurethral resection. However, the high rate of recurrence necessitates lifetime cystoscopic surveillance. Developing a sensitive and specific urine-based test would significantly improve bladder cancer screening, detection, and surveillance. Experimental Design: RNA-seq was used for biomarker discovery to directly assess the gene expression profile of exfoliated urothelial cells in urine derived from bladder cancer patients ( n = 13) and controls ( n = 10). Eight bladder cancer specific and 3 reference genes identified by RNA-seq were quantitated by qPCR in a training cohort of 102 urine samples. A diagnostic model based on the training cohort was constructed using multiple logistic regression. The model was further validated in an independent cohort of 101 urines. Results: A total of 418 genes were found to be differentially expressed between bladder cancer and controls. Validation of a subset of these genes was used to construct an equation for computing a probability of bladder cancer score (P BC ) based on expression of three markers ( ROBO1, WNT5A , and CDC42BPB ). Setting P BC = 0.45 as the cutoff for a positive test, urine testing using the three-marker panel had overall 88% sensitivity and 92% specificity in the training cohort. The accuracy of the three-marker panel in the independent validation cohort yielded an AUC of 0.87 and overall 83% sensitivity and 89% specificity. Conclusions: Urine-based molecular diagnostics using this three-marker signature could provide a valuable adjunct to cystoscopy and may lead to a reduction of unnecessary procedures for bladder cancer diagnosis. Clin Cancer Res; 23(14); 3700-10. ©2017 AACR . ©2017 American Association for Cancer Research.

Establishment of a Molecular Serotyping Scheme and a Multiplexed Luminex-Based Array for Enterobacter aerogenes

PubMed Central

Guo, Xi; Wang, Min; Wang, Lu; Wang, Yao; Chen, Tingting; Wu, Pan; Chen, Min; Liu, Bin; Feng, Lu

2018-01-01

Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens. Polysaccharide gene clusters (PSgcs) are typically responsible for the diversity of bacterial surface polysaccharides. Through whole-genome sequencing and analysis, eight putative PSgc types were identified in 23 Enterobacter aerogenes strains from several geographic areas, allowing us to present the first molecular serotyping system for E. aerogenes. A conventional antigenic scheme was also established and correlated well with the molecular serotyping system that was based on PSgc genetic variation, indicating that PSgc-based molecular typing and immunological serology provide equally valid results. Further, a multiplex Luminex-based array was developed, and a double-blind test was conducted with 97 clinical specimens from Shanghai, China, to validate our array. The results of these analyses indicated that strains containing PSgc4 and PSgc7 comprised the predominant groups. We then examined 86 publicly available E. aerogenes strain genomes and identified an additional seven novel PSgc types, with PSgc10 being the most abundant type. In total, our study identified 15 PSgc types in E. aerogenes, providing the basis for a molecular serotyping scheme. From these results, differing epidemic patterns were identified between strains that were predominant in different regions. Our study highlights the feasibility and reliability of a serotyping system based on PSgc diversity, and for the first time, presents a molecular serotyping system, as well as an antigenic scheme for E. aerogenes, providing the basis for molecular diagnostics and epidemiological surveillance of this important emerging pathogen. PMID:29616012

Establishment of a Molecular Serotyping Scheme and a Multiplexed Luminex-Based Array for Enterobacter aerogenes.

PubMed

Guo, Xi; Wang, Min; Wang, Lu; Wang, Yao; Chen, Tingting; Wu, Pan; Chen, Min; Liu, Bin; Feng, Lu

2018-01-01

Serotyping based on surface polysaccharide antigens is important for the clinical detection and epidemiological surveillance of pathogens. Polysaccharide gene clusters (PSgcs) are typically responsible for the diversity of bacterial surface polysaccharides. Through whole-genome sequencing and analysis, eight putative PSgc types were identified in 23 Enterobacter aerogenes strains from several geographic areas, allowing us to present the first molecular serotyping system for E. aerogenes . A conventional antigenic scheme was also established and correlated well with the molecular serotyping system that was based on PSgc genetic variation, indicating that PSgc-based molecular typing and immunological serology provide equally valid results. Further, a multiplex Luminex-based array was developed, and a double-blind test was conducted with 97 clinical specimens from Shanghai, China, to validate our array. The results of these analyses indicated that strains containing PSgc4 and PSgc7 comprised the predominant groups. We then examined 86 publicly available E. aerogenes strain genomes and identified an additional seven novel PSgc types, with PSgc10 being the most abundant type. In total, our study identified 15 PSgc types in E. aerogenes , providing the basis for a molecular serotyping scheme. From these results, differing epidemic patterns were identified between strains that were predominant in different regions. Our study highlights the feasibility and reliability of a serotyping system based on PSgc diversity, and for the first time, presents a molecular serotyping system, as well as an antigenic scheme for E. aerogenes , providing the basis for molecular diagnostics and epidemiological surveillance of this important emerging pathogen.

UV-vis, IR and 1H NMR spectroscopic studies of some mono- and bis-azo-compounds based on 2,7-dihydroxynaphthalene and aniline derivatives

NASA Astrophysics Data System (ADS)

Issa, Raafat M.; Fayed, Tarek A.; Awad, Mohammed K.; El-Kony, Sanaa M.

2005-12-01

The absorption spectra of mono- and bis-azo-derivatives obtained by coupling the diazonium salts of aromatic amines and 2,7-dihydroxynaphthalene have been studied in six organic solvents. The different absorption bands have been assigned and the effect of solvents on the charge transfer band is also discussed. The diagnostic IR spectral bands and 1H NMR signals are assigned and discussed in relation to molecular structure. Also, semi-empirical molecular orbital calculations using the atom superposition and electron delocalization molecular orbital (ASED-MO) theory have been performed to investigate the molecular and electronic structures of these compounds. According to these calculations, an intramolecular hydrogen bonding is essential for stabilization of such molecules.

Molecular Diagnostics in Autosomal Dominant Polycystic Kidney Disease: Utility and Limitations

PubMed Central

Zhao, Xiao; Paterson, Andrew D.; Zahirieh, Alireza; He, Ning; Wang, Kairong; Pei, York

2008-01-01

Background and objectives: Gene-based mutation screening is now available and has the potential to provide diagnostic confirmation or exclusion of autosomal dominant polycystic kidney disease. This study illustrates its utility and limitations in the clinical setting. Design, setting, participants, & measurements: Using a molecular diagnostic service, genomic DNA of one affected individual from each study family was screened for pathologic PKD1 and PKD2 mutations. Bidirectional sequencing was performed to identify sequence variants in all exons and splice junctions of both genes and to confirm the specific mutations in other family members. In two multiplex families, microsatellite markers were genotyped at both PDK1 and PKD2 loci, and pair-wise and multipoint linkage analysis was performed. Results: Three of five probands studied were referred for assessment of renal cystic disease without a family history of autosomal dominant polycystic kidney disease, and two others were younger at-risk members of families with autosomal dominant polycystic kidney disease being evaluated as living-related kidney donors. Gene-based mutation screening identified pathogenic mutations that provided confirmation or exclusion of disease in three probands, but in the other two, only unclassified variants were identified. In one proband in which mutation screening was indeterminate, DNA linkage studies provided strong evidence for disease exclusion. Conclusions: Gene-based mutation screening or DNA linkage analysis should be considered in individuals in whom the diagnosis of autosomal dominant polycystic kidney disease is uncertain because of a lack of family history or equivocal imaging results and in younger at-risk individuals who are being evaluated as living-related kidney donors. PMID:18077784

A Rapid and Low-Cost PCR Thermal Cycler for Infectious Disease Diagnostics

PubMed Central

Chan, Kamfai; Wong, Pui-Yan; Yu, Peter; Hardick, Justin; Wong, Kah-Yat; Wilson, Scott A.; Wu, Tiffany; Hui, Zoe; Gaydos, Charlotte; Wong, Season S.

2016-01-01

The ability to make rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a great step forward in global health. Many molecular diagnostic assays are developed based on using thermal cyclers to carry out polymerase chain reaction (PCR) and reverse-transcription PCR for DNA and RNA amplification and detection, respectively. Unfortunately, most commercial thermal cyclers are expensive and need continuous electrical power supply, so they are not suitable for uses in low-resource settings. We have previously reported a low-cost and simple approach to amplify DNA using vacuum insulated stainless steel thermoses food cans, which we have named it thermos thermal cycler or TTC. Here, we describe the use of an improved set up to enable the detection of viral RNA targets by reverse-transcription PCR (RT-PCR), thus expanding the TTC’s ability to identify highly infectious, RNA virus-based diseases in low resource settings. The TTC was successful in demonstrating high-speed and sensitive detection of DNA or RNA targets of sexually transmitted diseases, HIV/AIDS, Ebola hemorrhagic fever, and dengue fever. Our innovative TTC costs less than $200 to build and has a capacity of at least eight tubes. In terms of speed, the TTC’s performance exceeded that of commercial thermal cyclers tested. When coupled with low-cost endpoint detection technologies such as nucleic acid lateral-flow assay or a cell-phone-based fluorescence detector, the TTC will increase the availability of on-site molecular diagnostics in low-resource settings. PMID:26872358

The Evolving Classification of Pulmonary Hypertension.

PubMed

Foshat, Michelle; Boroumand, Nahal

2017-05-01

- An explosion of information on pulmonary hypertension has occurred during the past few decades. The perception of this disease has shifted from purely clinical to incorporate new knowledge of the underlying pathology. This transfer has occurred in light of advancements in pathophysiology, histology, and molecular medical diagnostics. - To update readers about the evolving understanding of the etiology and pathogenesis of pulmonary hypertension and to demonstrate how pathology has shaped the current classification. - Information presented at the 5 World Symposia on pulmonary hypertension held since 1973, with the last meeting occurring in 2013, was used in this review. - Pulmonary hypertension represents a heterogeneous group of disorders that are differentiated based on differences in clinical, hemodynamic, and histopathologic features. Early concepts of pulmonary hypertension were largely influenced by pharmacotherapy, hemodynamic function, and clinical presentation of the disease. The initial nomenclature for pulmonary hypertension segregated the clinical classifications from pathologic subtypes. Major restructuring of this disease classification occurred between the first and second symposia, which was the first to unite clinical and pathologic information in the categorization scheme. Additional changes were introduced in subsequent meetings, particularly between the third and fourth World Symposia meetings, when additional pathophysiologic information was gained. Discoveries in molecular diagnostics significantly progressed the understanding of idiopathic pulmonary arterial hypertension. Continued advancements in imaging modalities, mechanistic pathogenicity, and molecular biomarkers will enable physicians to define pulmonary hypertension phenotypes based on the pathobiology and allow for treatment customization.

Development of a multiplex assay for genus and species-specific detection of Phytophthora based on differences in mitochondrial gene order

USDA-ARS?s Scientific Manuscript database

The availability of a molecular diagnostic assay for Phytophthora that is specific, sensitive, has both genus and species specific detection capabilities multiplexed and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory ef...

From diagnostics to metagenomics: Applications of DNA-based tools in forest pathology

Treesearch

Amy L. Ross-Davis; Mee-Sook Kim; Jane E. Stewart; John W. Hanna; John D. Shaw; Ned B. Klopfenstein

2013-01-01

Advances in molecular technology provide an accessible set of tools to 1) help forest pathologists detect, identify, and monitor forest pathogens, 2) examine the evolutionary relationships and global distributions of forest pathogens and their hosts, 3) assess the diversity and structure of host and pathogen populations, and 4) evaluate the structure and function of...

Campylobacter ureolyticus

PubMed Central

O'Donovan, Dylan; Corcoran, Gerard D; Lucey, Brigid; Sleator, Roy D

2014-01-01

Herein, we provide a brief overview of the emerging bacterial pathogen Campylobacter ureolyticus. We describe the identification of the pathogen by molecular as opposed to classical culture based diagnostics and discuss candidate reservoirs of infection. We also review the available genomic data, outlining some of the major virulence factors, and discuss how these mechanisms likely contribute to pathogenesis of the organism. PMID:24717836

Molecular Phylogenetic Analysis of Enterobius vermicularis and Development of an 18S Ribosomal DNA-Targeted Diagnostic PCR▿

PubMed Central

Zelck, Ulrike E.; Bialek, Ralf; Weiß, Michael

2011-01-01

We genetically characterized pinworms obtained from 37 children from different regions of Germany and established new species-specific molecular diagnostic tools. No ribosomal DNA diversity was found; the phylogenetic position of Enterobius vermicularis within the Oxyurida order and its close relationship to the Ascaridida and Spirurida orders was confirmed. PMID:21248085

Subatomic turf wars. Molecular imaging holds the promise of personalized medicine, while pitting radiologists against oncologists, others over diagnostic role.

PubMed

Becker, Cinda

2004-11-29

Molecular imaging is expanding radiologists' territory and holds the promise of personalizing treatment. But a turf war is looming over who has the diagnostic role-radiologists, oncologists or others. "We're talking about a whole new way of thinking in terms of diagnosing and treating diseases," David Rollo says.

Emerging Pathogens: Challenges and Successes of Molecular Diagnostics

PubMed Central

Dong, Jianli; Olano, Juan P.; McBride, Jere W.; Walker, David H.

2008-01-01

More than 50 emerging and reemerging pathogens have been identified during the last 40 years. Until 1992 when the Institute of Medicine issued a report that defined emerging infectious diseases, medicine had been complacent about such infectious diseases despite the alarm bells of infections with human immunodeficiency virus. Molecular tools have proven useful in discovering and characterizing emerging viruses and bacteria such as Sin Nombre virus (hantaviral pulmonary syndrome), hepatitis C virus, Bartonella henselae (cat scratch disease, bacillary angiomatosis), and Anaplasma phagocytophilum (human granulocytotropic anaplasmosis). The feasibility of applying molecular diagnostics to dangerous, fastidious, and uncultivated agents for which conventional tests do not yield timely diagnoses has achieved proof of concept for many agents, but widespread use of cost-effective, validated commercial assays has yet to occur. This review presents representative emerging viral respiratory infections, hemorrhagic fevers, and hepatitides, as well as bacterial and parasitic zoonotic, gastrointestinal, and pulmonary infections. Agent characteristics, epidemiology, clinical manifestations, and diagnostic methods are tabulated for another 22 emerging viruses and five emerging bacteria. The ongoing challenge to the field of molecular diagnostics is to apply contemporary knowledge to facilitate agent diagnosis as well as to further discoveries of novel pathogens. PMID:18403608

Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy.

PubMed

de Ruiter, C M; van der Veer, C; Leeflang, M M G; Deborggraeve, S; Lucas, C; Adams, E R

2014-09-01

Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Role of molecular testing in the multidisciplinary diagnostic approach of ichthyosis.

PubMed

Diociaiuti, Andrea; El Hachem, May; Pisaneschi, Elisa; Giancristoforo, Simona; Genovese, Silvia; Sirleto, Pietro; Boldrini, Renata; Angioni, Adriano

2016-01-13

The term ichthyosis describes a generalized disorder of cornification characterized by scaling and/or hyperkeratosis of different skin regions. Mutations in a broad group of genes related to keratinocyte differentiation and epidermal barrier function have been demonstrated to play a causative role in disease development. Ichthyosis may be classified in syndromic or non-syndromic forms based on the occurrence or absence of extracutaneous signs. In this setting, the diagnosis of ichthyosis is an integrated multistep process requiring a multidisciplinary approach in order to formulate the appropriate diagnostic hypothesis and to address the genetic testing. Due to the complex features of the different ichthyoses and the high number of genes involved we have investigated a group of 64 patients, affected by syndromic and non-syndromic diseases, using Next Generation Sequencing as a new tool for the molecular diagnosis. Using this innovative molecular approach we were able to find pathogenic mutations in 53 out of 64 patients resulting in 82.8 % total detection rate. An interesting result from the analysis of the data is the high rate of novel sequence variations found compared to known mutations and the relevant rate of homozygous mutations. The possibility to analyze a large number of genes associated with various diseases allows to study cases with phenotypes not well-determined, giving the opportunity to make new genotype-phenotype correlation. In some cases there were discrepancies between clinical features and histology or electron microscopy and only molecular analysis allowed to definitively resolve the diagnostic dilemma. The genetic diagnosis of ichthyosis leads to a more accurate and effective genetic counseling, allowing correct evaluation of the risk of recurrence, particularly in families with consanguineous background.

A cDNA microarray gene expression data classifier for clinical diagnostics based on graph theory.

PubMed

Benso, Alfredo; Di Carlo, Stefano; Politano, Gianfranco

2011-01-01

Despite great advances in discovering cancer molecular profiles, the proper application of microarray technology to routine clinical diagnostics is still a challenge. Current practices in the classification of microarrays' data show two main limitations: the reliability of the training data sets used to build the classifiers, and the classifiers' performances, especially when the sample to be classified does not belong to any of the available classes. In this case, state-of-the-art algorithms usually produce a high rate of false positives that, in real diagnostic applications, are unacceptable. To address this problem, this paper presents a new cDNA microarray data classification algorithm based on graph theory and is able to overcome most of the limitations of known classification methodologies. The classifier works by analyzing gene expression data organized in an innovative data structure based on graphs, where vertices correspond to genes and edges to gene expression relationships. To demonstrate the novelty of the proposed approach, the authors present an experimental performance comparison between the proposed classifier and several state-of-the-art classification algorithms.

Company Profile: Selventa, Inc.

PubMed

Fryburg, David A; Latino, Louis J; Tagliamonte, John; Kenney, Renee D; Song, Diane H; Levine, Arnold J; de Graaf, David

2012-08-01

Selventa, Inc. (MA, USA) is a biomarker discovery company that enables personalized healthcare. Originally founded as Genstruct, Inc., Selventa has undergone significant evolution from a technology-based service provider to an active partner in the development of diagnostic tests, functioning as a molecular dashboard of disease activity using a unique platform. As part of that evolution, approximately 2 years ago the company was rebranded as Selventa to reflect its new identity and mission. The contributions to biomedical research by Selventa are based on in silico, reverse-engineering methods to determine biological causality. That is, given a set of in vitro or in vivo biological observations, which biological mechanisms can explain the measured results? Facilitated by a large and carefully curated knowledge base, these in silico methods generated new insights into the mechanisms driving a disease. As Selventa's methods would enable biomarker discovery and be directly applicable to generating novel diagnostics, the scientists at Selventa have focused on the development of predictive biomarkers of response in autoimmune and oncologic diseases. Selventa is presently building a portfolio of independent, as well as partnered, biomarker projects with the intention to create diagnostic tests that predict response to therapy.

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

PubMed

Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

2016-04-15

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Molecular diagnostics for human leptospirosis.

PubMed

Waggoner, Jesse J; Pinsky, Benjamin A

2016-10-01

The definitive diagnosis of leptospirosis, which results from infection with spirochetes of the genus Leptospira, currently relies on the use of culture, serological testing (microscopic agglutination testing), and molecular detection. The purpose of this review is to describe new molecular diagnostics for Leptospira and discuss advancements in the use of available methods. Efforts have been focused on improving the clinical sensitivity of Leptospira detection using molecular methods. In this review, we describe a reoptimized pathogenic species-specific real-time PCR (targeting lipL32) that has demonstrated improved sensitivity, findings by two groups that real-time reverse-transcription PCR assays targeting the 16S rrs gene can improve detection, and two new loop-mediated amplification techniques. Quantitation of leptospiremia, detection in different specimen types, and the complementary roles played by molecular detection and microscopic agglutination testing will be discussed. Finally, a protocol for Leptospira strain subtyping using variable number tandem repeat targets and high-resolution melting will be described. Molecular diagnostics have an established role for the diagnosis of leptospirosis and provide an actionable diagnosis in the acute setting. The use of real-time reverse-transcription PCR for testing serum/plasma and cerebrospinal fluid, when available, may improve the detection of Leptospira without decreasing clinical specificity.

[Molecular biological detection of dermatophytes in clinical samples when onychomycosis or tinea pedis is suspected. A prospective study comparing conventional dermatomycological diagnostics and polymerase chain reaction].

PubMed

Winter, I; Uhrlaß, S; Krüger, C; Herrmann, J; Bezold, G; Winter, A; Barth, S; Simon, J C; Gräser, Y; Nenoff, P

2013-04-01

The prevalence of onychomycosis is rising worldwide. Before starting antifungal treatment, an exact mycological diagnosis should be obtained. The current laboratory diagnosis of dermatomycoses is based on the detection of the causative agent by microscopy and culture. These conventional diagnostic methods for fungal infections often are not the best solution because they are time-consuming, cultures are false-negative and direct examination identifies non-vital structures which cannot be used for speciation. A total of 218 patients presenting in a surgical practice over 3 months with clinical signs of tinea pedis and/or onychomycosis were involved in the prospective study. All patients had predisposing factors for tinea pedis and tinea unguium, such as vascular insufficiency, diabetes mellitus, and leg ulcers. Nail specimens and skin scrapings were investigated for fungi using Blancophor® preparation, and cultured. In addition to conventional diagnostics, PCR (polymerase chain reaction) for detection of dermatophyte DNA was employed. This PCR-Elisa assay is based on the use of specific primers which target the topoisomerase II gene. This allows the highly specific molecular identification of Trichophyton (T.) rubrum, T. interdigitale, and Epidermophyton floccosum directly in clinical samples. 23.9 % of patients were culture-positive for dermatophytes (either T. rubrum, or T. interdigitale). With PCR, dermatophyte DNA either of T. rubrum or T. interdigitale could be detected in nail samples and skin scrapings from at least 29.9 % of all patients. Epidermophyton floccosum was not found in this study, neither by cultivation nor by PCR. The diagnostic sensitivity of the PCR-Elisa assay was calculated as 79.0% ; the diagnostic specificity as 85.5 %. PCR-Elisa evaluation makes possible a rapid, specific and sensitive diagnosis of dermatophytosis of the nails and skin within 24 (maximal 48) hours with identification of the involved species.

[Molecular diagnostics of ALK-positive lung cancer].

PubMed

Tímár, József; Lotz, Gábor; Rásó, Erzsébet; Moldvay, Judit

2017-09-20

ALK translocation is the 3rd most frequent genetic aberration in lung adenocarcinoma, and several inhibitors are now clinically available in first and second line settings. Accordingly, molecular diagnostics of ALK-positive lung cancer is very important and can be done with the rational combination of several methods. All international recommendations suggest that, except for cytological samples, screening technology for ALK-positive tumors is immunohistochemistry using a validated test. It is highly recommended that in case of ALK protein positive samples gene translocation must be confirmed by fluorescent in situ hybridization (FISH). In case of cytological samples FISH technique must be used as ALK diagnostics. In equivocal cases the genetic alteration of ALK can be confirmed by alternative molecular techniques such as next generation sequencing or RNAbased PCR methods. Upon administration of ALK inhibitors, acquired resistance is frequent which is mostly due to ALK amplification and/or mutation. It is evident that the diagnostics of these secondary ALK gene alterations must be done from recurrent tumors or circulating nucleic acids.

The cobas® 6800/8800 System: a new era of automation in molecular diagnostics.

PubMed

Cobb, Bryan; Simon, Christian O; Stramer, Susan L; Body, Barbara; Mitchell, P Shawn; Reisch, Natasa; Stevens, Wendy; Carmona, Sergio; Katz, Louis; Will, Stephen; Liesenfeld, Oliver

2017-02-01

Molecular diagnostics is a key component of laboratory medicine. Here, the authors review key triggers of ever-increasing automation in nucleic acid amplification testing (NAAT) with a focus on specific automated Polymerase Chain Reaction (PCR) testing and platforms such as the recently launched cobas® 6800 and cobas® 8800 Systems. The benefits of such automation for different stakeholders including patients, clinicians, laboratory personnel, hospital administrators, payers, and manufacturers are described. Areas Covered: The authors describe how molecular diagnostics has achieved total laboratory automation over time, rivaling clinical chemistry to significantly improve testing efficiency. Finally, the authors discuss how advances in automation decrease the development time for new tests enabling clinicians to more readily provide test results. Expert Commentary: The advancements described enable complete diagnostic solutions whereby specific test results can be combined with relevant patient data sets to allow healthcare providers to deliver comprehensive clinical recommendations in multiple fields ranging from infectious disease to outbreak management and blood safety solutions.

Using comparative genomics to develop a molecular diagnostic for the identification of an emerging pest Drosophila suzukii.

PubMed

Murphy, K A; Unruh, T R; Zhou, L M; Zalom, F G; Shearer, P W; Beers, E H; Walton, V M; Miller, B; Chiu, J C

2015-06-01

Drosophila suzukii (Spotted Wing Drosophila) has recently become a serious invasive pest of fruit crops in the USA, Canada, and Europe, leading to substantial economic losses. D. suzukii is a direct pest, ovipositing directly into ripe or ripening fruits; in contrast, other Drosophilids utilize decaying or blemished fruits and are nuisance pests at worst. Immature stages of D. suzukii are difficult to differentiate from other Drosophilids, posing problems for research and for meeting quarantine restrictions designed to prevent the spread of this pest in fruit exports. Here we used a combined phylogenetic and bioinformatic approach to discover genetic markers suitable for a species diagnostic protocol of this agricultural pest. We describe a molecular diagnostic for rapid identification of single D. suzukii larva using multiplex polymerase chain reaction. Our molecular diagnostic was validated using nine different species of Drosophila for specificity and 19 populations of D. suzukii from different geographical regions to ensure utility within species.

Values of molecular markers in the differential diagnosis of thyroid abnormalities.

PubMed

Tennakoon, T M P B; Rushdhi, M; Ranasinghe, A D C U; Dassanayake, R S

2017-06-01

Thyroid cancer (TC), follicular adenoma (FA) and Hashimoto's thyroiditis (HT) are three of the most frequently reported abnormalities that affect the thyroid gland. A frequent co-occurrence along with similar histopathological features is observed between TC and FA as well as between TC and HT. The conventional diagnostic methods such as histochemical analysis present complications in differential diagnosis when these abnormalities occur simultaneously. Hence, the authors recognize novel methods based on screening genetic defects of thyroid abnormalities as viable diagnostic and prognostic methods that could complement the conventional methods. We have extensively reviewed the existing literature on TC, FA and HT and also on three genes, namely braf, nras and ret/ptc, that could be used to differentially diagnose the three abnormalities. Emphasis was also given to the screening methods available to detect the said molecular markers. It can be conferred from the analysis of the available data that the utilization of braf, nras and ret/ptc as markers for the therapeutic evaluation of FA and HT is debatable. However, molecular screening for braf, nras and ret/ptc mutations proves to be a conclusive method that could be employed to differentially diagnose TC from HT and FA in the instance of a suspected co-occurrence. Thyroid cancer patients can be highly benefited from the screening for the said genetic markers, especially the braf gene due to its diagnostic value as well as due to the availability of personalized medicine targeted specifically for braf mutants.

A NASBA on microgel-tethered molecular-beacon microarray for real-time microbial molecular diagnostics.

PubMed

Ma, Y; Dai, X; Hong, T; Munk, G B; Libera, M

2016-12-19

Despite their many advantages and successes, molecular beacon (MB) hybridization probes have not been extensively used in microarray formats because of the complicating probe-substrate interactions that increase the background intensity. We have previously shown that tethering to surface-patterned microgels is an effective means for localizing MB probes to specific surface locations in a microarray format while simultaneously maintaining them in as water-like an environment as possible and minimizing probe-surface interactions. Here we extend this approach to include both real-time detection together with integrated NASBA amplification. We fabricate small (∼250 μm × 250 μm) simplex, duplex, and five-plex assays with microarray spots of controllable size (∼20 μm diameter), position, and shape to detect bacteria and fungi in a bloodstream-infection model. The targets, primers, and microgel-tethered probes can be combined in a single isothermal reaction chamber with no post-amplification labelling. We extract total RNA from clinical blood samples and differentiate between Gram-positive and Gram-negative bloodstream infection in a duplex assay to detect RNA- amplicons. The sensitivity based on our current protocols in a simplex assay to detect specific ribosomal RNA sequences within total RNA extracted from S. aureus and E. coli cultures corresponds to tens of bacteria per ml. We furthermore show that the platform can detect RNA- amplicons from synthetic target DNA with 1 fM sensitivity in sample volumes that contain about 12 000 DNA molecules. These experiments demonstrate an alternative approach that can enable rapid and real-time microarray-based molecular diagnostics.

Molecular imaging assessment of periodontitis lesions in an experimental mouse model.

PubMed

Ideguchi, Hidetaka; Yamashiro, Keisuke; Yamamoto, Tadashi; Shimoe, Masayuki; Hongo, Shoichi; Kochi, Shinsuke; Yoshihara-Hirata, Chiaki; Aoyagi, Hiroaki; Kawamura, Mari; Takashiba, Shogo

2018-06-06

We aimed to evaluate molecular imaging as a novel diagnostic tool for mice periodontitis model induced by ligature and Porphyromonas gingivalis (Pg) inoculation. Twelve female mice were assigned to the following groups: no treatment as control group (n = 4); periodontitis group induced by ligature and Pg as Pg group (n = 4); and Pg group treated with glycyrrhizinic acid (GA) as Pg + GA group (n = 4). All mice were administered a myeloperoxidase (MPO) activity-specific luminescent probe and observed using a charge-coupled device camera on day 14. Image analysis on all mice was conducted using software to determine the signal intensity of inflammation. Additionally, histological and radiographic evaluation for periodontal inflammation and bone resorption at the site of periodontitis, and quantitative enzyme-linked immunosorbent assay (ELISA) were conducted on three mice for each group. Each experiment was performed three times. Levels of serum IgG antibody against P. gingivalis were significantly higher in the Pg than in the Pg + GA group. Histological analyses indicated that the number of osteoclasts and neutrophils were significantly lower in the Pg + GA than in the Pg group. Micro-CT image analysis indicated no difference in bone resorption between the Pg and Pg + GA groups. The signal intensity of MPO activity was detected on the complete craniofacial image; moreover, strong signal intensity was localized specifically at the periodontitis site in the ex vivo palate, with group-wise differences. Molecular imaging analysis based on MPO activity showed high sensitivity of detection of periodontal inflammation in mice. Molecular imaging analysis based on MPO activity has potential as a diagnostic tool for periodontitis.

Development of a curriculum in molecular diagnostics, genomics and personalized medicine for dermatology trainees.

PubMed

Murphy, Michael J; Shahriari, Neda; Payette, Michael; Mnayer, Laila; Elaba, Zendee

2016-10-01

Results of molecular studies are redefining the diagnosis and management of a wide range of skin disorders. Dermatology training programs maintain a relative gap in relevant teaching. To develop a curriculum in molecular diagnostics, genomics and personalized medicine for dermatology trainees at our institution. The aim is to provide trainees with a specialty-appropriate, working knowledge in clinical molecular dermatology. The Departments of Dermatology and Pathology and Laboratory Medicine collaborated on the design and implementation of educational objectives and teaching modalities for the new curriculum. A multidisciplinary curriculum was developed. It comprises: (i) assigned reading from the medical literature and reference textbook; (ii) review of teaching sets; (iii) two 1 hour lectures; (iv) trainee presentations; (v) 1-week rotation in a clinical molecular pathology and cytogenetics laboratory; and (vi) assessments and feedback. Residents who participated in the curriculum to date have found the experience to be of value. Our curriculum provides a framework for other dermatology residency programs to develop their own specific approach to molecular diagnostics education. Such training will provide a foundation for lifelong learning as molecular testing evolves and becomes integral to the practice of dermatology. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Recent advances in photodynamic diagnosis of gastric cancer using 5-aminolevulinic acid.

PubMed

Koizumi, Noriaki; Harada, Yoshinori; Minamikawa, Takeo; Tanaka, Hideo; Otsuji, Eigo; Takamatsu, Tetsuro

2016-01-21

Photodynamic diagnosis based on 5-aminolevulinic acid-induced protoporphyrin IX has been clinically applied in many fields based upon its evidenced efficacy and adequate safety. In order to establish a personalized medicine approach for treating gastric cancer patients, rapid intraoperative detection of malignant lesions has become important. Feasibility of photodynamic diagnosis using 5-aminolevulinic acid for gastric cancer patients has been investigated, especially for the detection of peritoneal dissemination and lymph node metastasis. This method enables intraoperative real-time fluorescence detection of peritoneal dissemination, exhibiting higher sensitivity than white light observation without histopathological examination. The method also enables detection of metastatic foci within excised lymph nodes, exhibiting a diagnostic accuracy comparable to that of a current molecular diagnostics technique. Although several complicating issues still need to be resolved, such as the effect of tissue autofluorescence and the insufficient depth penetration of excitation light, this simple and rapid method has the potential to become a useful diagnostic tool for gastric cancer, as well as urinary bladder cancer and glioma.

How recent advances in molecular tests could impact the diagnosis of pneumonia.

PubMed

Murdoch, David R

2016-01-01

Molecular diagnostic tests have been the single major development in pneumonia diagnostics over recent years. Nucleic acid detection tests (NATs) have greatly improved the ability to detect respiratory viruses and bacterial pathogens that do not normally colonize the respiratory tract. In contrast, NATs do not yet have an established role for diagnosing pneumonia caused by bacteria that commonly colonize the nasopharynx due to difficulties discriminating between pathogens and coincidental carriage strains. New approaches are needed to distinguish infection from colonization, such as through use of quantitative methods and identification of discriminating cut-off levels. The recent realization that the lung microbiome exists has provided new insights into the pathogenesis of pneumonia involving the interaction between multiple microorganisms. New developments in molecular diagnostics must account for this new paradigm.

Invasive candidiasis: future directions in non-culture based diagnosis.

PubMed

Posch, Wilfried; Heimdörfer, David; Wilflingseder, Doris; Lass-Flörl, Cornelia

2017-09-01

Delayed initial antifungal therapy is associated with high mortality rates caused by invasive candida infections, since accurate detection of the opportunistic pathogenic yeast and its identification display a diagnostic challenge. diagnosis of candida infections relies on time-consuming methods such as blood cultures, serologic and histopathologic examination. to allow for fast detection and characterization of invasive candidiasis, there is a need to improve diagnostic tools. trends in diagnostics switch to non-culture-based methods, which allow specified diagnosis within significantly shorter periods of time in order to provide early and appropriate antifungal treatment. Areas covered: within this review comprise novel pathogen- and host-related testing methods, e.g. multiplex-PCR analyses, T2 magnetic resonance, fungus-specific DNA microarrays, microRNA characterization or analyses of IL-17 as biomarker for early detection of invasive candidiasis. Expert commentary: Early recognition and diagnosis of fungal infections is a key issue for improved patient management. As shown in this review, a broad range of novel molecular based tests for the detection and identification of Candida species is available. However, several assays are in-house assays and lack standardization, clinical validation as well as data on sensitivity and specificity. This underscores the need for the development of faster and more accurate diagnostic tests.

Engineered Aptamers to Probe Molecular Interactions on the Cell Surface

PubMed Central

Batool, Sana; Bhandari, Sanam; George, Shanell; Okeoma, Precious; Van, Nabeela; Zümrüt, Hazan E.; Mallikaratchy, Prabodhika

2017-01-01

Significant progress has been made in understanding the nature of molecular interactions on the cell membrane. To decipher such interactions, molecular scaffolds can be engineered as a tool to modulate these events as they occur on the cell membrane. To guarantee reliability, scaffolds that function as modulators of cell membrane events must be coupled to a targeting moiety with superior chemical versatility. In this regard, nucleic acid aptamers are a suitable class of targeting moieties. Aptamers are inherently chemical in nature, allowing extensive site-specific chemical modification to engineer sensing molecules. Aptamers can be easily selected using a simple laboratory-based in vitro evolution method enabling the design and development of aptamer-based functional molecular scaffolds against wide range of cell surface molecules. This article reviews the application of aptamers as monitors and modulators of molecular interactions on the mammalian cell surface with the aim of increasing our understanding of cell-surface receptor response to external stimuli. The information gained from these types of studies could eventually prove useful in engineering improved medical diagnostics and therapeutics. PMID:28850067

Significance and integration of molecular diagnostics in the framework of veterinary practice.

PubMed

Aranaz, Alicia

2015-01-01

The field of molecular diagnostics in veterinary practice is rapidly evolving. An array of molecular techniques of different complexity is available to facilitate the fast and specific diagnosis of animal diseases. The choice for the adequate technique is dependent on the mission and attributions of the laboratory and requires both a knowledge of the molecular biology basis and of its limitations. The ability to quickly detect pathogens and their characteristics would allow for precise decision-making and target measures such as prophylaxis, appropriate therapy, and biosafety plans to control disease outbreaks. In practice, taking benefit of the huge amount of data that can be obtained using molecular techniques highlights the need of collaboration between veterinarians in the laboratory and practitioners.

Molecular Biomarkers for the Evaluation of Colorectal Cancer: Guideline From the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology.

PubMed

Sepulveda, Antonia R; Hamilton, Stanley R; Allegra, Carmen J; Grody, Wayne; Cushman-Vokoun, Allison M; Funkhouser, William K; Kopetz, Scott E; Lieu, Christopher; Lindor, Noralane M; Minsky, Bruce D; Monzon, Federico A; Sargent, Daniel J; Singh, Veena M; Willis, Joseph; Clark, Jennifer; Colasacco, Carol; Rumble, R Bryan; Temple-Smolkin, Robyn; Ventura, Christina B; Nowak, Jan A

2017-03-01

To develop evidence-based guideline recommendations through a systematic review of the literature to establish standard molecular biomarker testing of colorectal cancer (CRC) tissues to guide epidermal growth factor receptor (EGFR) therapies and conventional chemotherapy regimens. The American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology convened an expert panel to develop an evidence-based guideline to establish standard molecular biomarker testing and guide therapies for patients with CRC. A comprehensive literature search that included more than 4,000 articles was conducted. Twenty-one guideline statements were established. Evidence supports mutational testing for EGFR signaling pathway genes, since they provide clinically actionable information as negative predictors of benefit to anti-EGFR monoclonal antibody therapies for targeted therapy of CRC. Mutations in several of the biomarkers have clear prognostic value. Laboratory approaches to operationalize CRC molecular testing are presented. Key Words: Molecular diagnostics; Gastrointestinal; Histology; Genetics; Oncology. Copyright © 2017 American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, American Society for Clinical Oncology, and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Syndromic Panel-Based Testing in Clinical Microbiology.

PubMed

Ramanan, Poornima; Bryson, Alexandra L; Binnicker, Matthew J; Pritt, Bobbi S; Patel, Robin

2018-01-01

The recent development of commercial panel-based molecular diagnostics for the rapid detection of pathogens in positive blood culture bottles, respiratory specimens, stool, and cerebrospinal fluid has resulted in a paradigm shift in clinical microbiology and clinical practice. This review focuses on U.S. Food and Drug Administration (FDA)-approved/cleared multiplex molecular panels with more than five targets designed to assist in the diagnosis of bloodstream, respiratory tract, gastrointestinal, or central nervous system infections. While these panel-based assays have the clear advantages of a rapid turnaround time and the detection of a large number of microorganisms and promise to improve health care, they present certain challenges, including cost and the definition of ideal test utilization strategies (i.e., optimal ordering) and test interpretation. Copyright © 2017 American Society for Microbiology.

Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

PubMed

Goller, K V; Dill, V; Madi, M; Martin, P; Van der Stede, Y; Vandenberge, V; Haas, B; Van Borm, S; Koenen, F; Kasanga, C J; Ndusilo, N; Beer, M; Liu, L; Mioulet, V; Armson, B; King, D P; Fowler, V L

2018-04-01

Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab ® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world. © 2017 The Authors. Transboundary and Emerging Diseases Published by Blackwell Verlag GmbH.

Towards a molecular taxonomic key of the Aurantioideae subfamily using chloroplastic SNP diagnostic markers of the main clades genotyped by competitive allele-specific PCR.

PubMed

Oueslati, Amel; Ollitrault, Frederique; Baraket, Ghada; Salhi-Hannachi, Amel; Navarro, Luis; Ollitrault, Patrick

2016-08-18

Chloroplast DNA is a primary source of molecular variations for phylogenetic analysis of photosynthetic eukaryotes. However, the sequencing and analysis of multiple chloroplastic regions is difficult to apply to large collections or large samples of natural populations. The objective of our work was to demonstrate that a molecular taxonomic key based on easy, scalable and low-cost genotyping method should be developed from a set of Single Nucleotide Polymorphisms (SNPs) diagnostic of well-established clades. It was applied to the Aurantioideae subfamily, the largest group of the Rutaceae family that includes the cultivated citrus species. The publicly available nucleotide sequences of eight plastid genomic regions were compared for 79 accessions of the Aurantioideae subfamily to search for SNPs revealing taxonomic differentiation at the inter-tribe, inter-subtribe, inter-genus and interspecific levels. Diagnostic SNPs (DSNPs) were found for 46 of the 54 clade levels analysed. Forty DSNPs were selected to develop KASPar markers and their taxonomic value was tested by genotyping 108 accessions of the Aurantioideae subfamily. Twenty-seven markers diagnostic of 24 clades were validated and they displayed a very high rate of transferability in the Aurantioideae subfamily (only 1.2 % of missing data on average). The UPGMA from the validated markers produced a cladistic organisation that was highly coherent with the previous phylogenetic analysis based on the sequence data of the eight plasmid regions. In particular, the monophyletic origin of the "true citrus" genera plus Oxanthera was validated. However, some clarification remains necessary regarding the organisation of the other wild species of the Citreae tribe. We validated the concept that with well-established clades, DSNPs can be selected and efficiently transformed into competitive allele-specific PCR markers (KASPar method) allowing cost-effective highly efficient cladistic analysis in large collections at subfamily level. The robustness of this genotyping method is an additional decisive advantage for network collaborative research. The availability of WGS data for the main "true citrus" species should soon make it possible to develop a set of DSNP markers allowing very fine resolution of this very important horticultural group.

Evidence of clinical utility: an unmet need in molecular diagnostics for patients with cancer.

PubMed

Parkinson, David R; McCormack, Robert T; Keating, Susan M; Gutman, Steven I; Hamilton, Stanley R; Mansfield, Elizabeth A; Piper, Margaret A; Deverka, Patricia; Frueh, Felix W; Jessup, J Milburn; McShane, Lisa M; Tunis, Sean R; Sigman, Caroline C; Kelloff, Gary J

2014-03-15

This article defines and describes best practices for the academic and business community to generate evidence of clinical utility for cancer molecular diagnostic assays. Beyond analytical and clinical validation, successful demonstration of clinical utility involves developing sufficient evidence to demonstrate that a diagnostic test results in an improvement in patient outcomes. This discussion is complementary to theoretical frameworks described in previously published guidance and literature reports by the U.S. Food and Drug Administration, Centers for Disease Control and Prevention, Institute of Medicine, and Center for Medical Technology Policy, among others. These reports are comprehensive and specifically clarify appropriate clinical use, adoption, and payer reimbursement for assay manufacturers, as well as Clinical Laboratory Improvement Amendments-certified laboratories, including those that develop assays (laboratory developed tests). Practical criteria and steps for establishing clinical utility are crucial to subsequent decisions for reimbursement without which high-performing molecular diagnostics will have limited availability to patients with cancer and fail to translate scientific advances into high-quality and cost-effective cancer care. See all articles in this CCR Focus section, "The Precision Medicine Conundrum: Approaches to Companion Diagnostic Co-development." ©2014 AACR.

PSMA-Based Radioligand Therapy for Metastatic Castration-Resistant Prostate Cancer: The Bad Berka Experience Since 2013.

PubMed

Kulkarni, Harshad R; Singh, Aviral; Schuchardt, Christiane; Niepsch, Karin; Sayeg, Manal; Leshch, Yevgeniy; Wester, Hans-Juergen; Baum, Richard P

2016-10-01

A potential milestone in personalized nuclear medicine is theranostics of metastatic castration-resistant prostate cancer (mCRPC) based on molecular imaging using PET/CT with 68 Ga-labeled prostate-specific membrane antigen (PSMA) ligands and molecular radiotherapy using PSMA-targeted radioligand therapy (PRLT) with 177 Lu-PSMA ligands. 68 Ga-PSMA PET/CT enables accurate detection of mCRPC lesions with high diagnostic sensitivity and specificity and provides quantitative and reproducible data that can be used to select patients for PRLT and therapeutic monitoring. Our comprehensive experience over the last 3 years using different radioligands indicates that PRLT is highly effective for the treatment of mCRPC, even in advanced cases, and potentially lends a significant benefit to overall and progression-free survival. Additionally, significant improvement in clinical symptoms and excellent palliation of pain can be achieved. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Towards the automated identification of Chrysomya blow flies from wing images.

PubMed

Macleod, N; Hall, M J R; Wardhana, A H

2018-04-15

The Old World screwworm fly (OWSF), Chrysomya bezziana (Diptera: Calliphoridae), is an important agent of traumatic myiasis and, as such, a major human and animal health problem. In the implementation of OWSF control operations, it is important to determine the geographical origins of such disease-causing species in order to establish whether they derive from endemic or invading populations. Gross morphological and molecular studies have demonstrated the existence of two distinct lineages of this species, one African and the other Asian. Wing morphometry is known to be of substantial assistance in identifying the geographical origin of individuals because it provides diagnostic markers that complement molecular diagnostics. However, placement of the landmarks used in traditional geometric morphometric analysis can be time-consuming and subject to error caused by operator subjectivity. Here we report results of an image-based approach to geometric morphometric analysis for delivering wing-based identifications. Our results indicate that this approach can produce identifications that are practically indistinguishable from more traditional landmark-based results. In addition, we demonstrate that the direct analysis of digital wing images can be used to discriminate between three Chrysomya species of veterinary and forensic importance and between C. bezziana genders. © 2018 The Trustees of the Natural History Museum, London. Medical and Veterinary Entomology © 2018 Royal Entomological Society.

Analysis of the Human Prostate-Specific Proteome Defined by Transcriptomics and Antibody-Based Profiling Identifies TMEM79 and ACOXL as Two Putative, Diagnostic Markers in Prostate Cancer

PubMed Central

O'Hurley, Gillian; Busch, Christer; Fagerberg, Linn; Hallström, Björn M.; Stadler, Charlotte; Tolf, Anna; Lundberg, Emma; Schwenk, Jochen M.; Jirström, Karin; Bjartell, Anders; Gallagher, William M.; Uhlén, Mathias; Pontén, Fredrik

2015-01-01

To better understand prostate function and disease, it is important to define and explore the molecular constituents that signify the prostate gland. The aim of this study was to define the prostate specific transcriptome and proteome, in comparison to 26 other human tissues. Deep sequencing of mRNA (RNA-seq) and immunohistochemistry-based protein profiling were combined to identify prostate specific gene expression patterns and to explore tissue biomarkers for potential clinical use in prostate cancer diagnostics. We identified 203 genes with elevated expression in the prostate, 22 of which showed more than five-fold higher expression levels compared to all other tissue types. In addition to previously well-known proteins we identified two poorly characterized proteins, TMEM79 and ACOXL, with potential to differentiate between benign and cancerous prostatic glands in tissue biopsies. In conclusion, we have applied a genome-wide analysis to identify the prostate specific proteome using transcriptomics and antibody-based protein profiling to identify genes with elevated expression in the prostate. Our data provides a starting point for further functional studies to explore the molecular repertoire of normal and diseased prostate including potential prostate cancer markers such as TMEM79 and ACOXL. PMID:26237329

Investigative pathology: leading the post-genomic revolution.

PubMed

Berman, David M; Bosenberg, Marcus W; Orwant, Robin L; Thurberg, Beth L; Draetta, Gulio F; Fletcher, Christopher D M; Loda, Massimo

2012-01-01

The completion of the Human Genome Project and the development of genome-based technologies over the past decade have set the stage for a new era of personalized medicine. By all rights, molecularly trained investigative pathologists should be leading this revolution. Singularly well suited for this work, molecular pathologists have the rare ability to wed genomic tools with unique diagnostic skills and tissue-based pathology techniques for integrated diagnosis of human disease. However, the number of pathologists with expertise in genome-based research has remained relatively low due to outdated training methods and a reluctance among some traditional pathologists to embrace new technologies. Moreover, because budding pathologists may not appreciate the vast selection of jobs available to them, they often end up choosing jobs that focus almost entirely on routine diagnosis rather than new frontiers in molecular pathology. This review calls for changes aimed at rectifying these troubling trends to ensure that pathology continues to guide patient care in a post-genomic era.

Early detection: the impact of genomics.

PubMed

van Lanschot, M C J; Bosch, L J W; de Wit, M; Carvalho, B; Meijer, G A

2017-08-01

The field of genomics has shifted our view on disease development by providing insights in the molecular and functional processes encoded in the genome. In the case of cancer, many alterations in the DNA accumulate that enable tumor growth or even metastatic dissemination. Identification of molecular signatures that define different stages of progression towards cancer can enable early tumor detection. In this review, the impact of genomics will be addressed using early detection of colorectal cancer (CRC) as an example. Increased understanding of the adenoma-to-carcinoma progression has led to the discovery of several diagnostic biomarkers. This combined with technical advancements, has facilitated the development of molecular tests for non-invasive early CRC detection in stool and blood samples. Even though several tests have already made it to clinical practice, sensitivity and specificity for the detection of precancerous lesions still need improvement. Besides the diagnostic qualities, also the accuracy of the intermediate endpoint is an important issue on how the effectiveness of a novel test is perceived. Here, progression biomarkers may provide a more precise measure than the currently used morphologically based features. Similar developments in biomarker use for early detection have taken place in other cancer types.

Rett Syndrome: Revised Diagnostic Criteria and Nomenclature

PubMed Central

Neul, Jeffrey L.; Kaufmann, Walter E.; Glaze, Daniel G.; Christodoulou, John; Clarke, Angus J.; Bahi-Buisson, Nadia; Leonard, Helen; Bailey, Mark E. S.; Schanen, N. Carolyn; Zappella, Michele; Renieri, Alessandra; Huppke, Peter; Percy, Alan K.

2010-01-01

Objective Rett syndrome (RTT) is a severe neurodevelopmental disease that affects approximately 1 in 10,000 live female births and is often caused by mutations in Methyl-CpG-binding protein 2 (MECP2). Despite distinct clinical features, the accumulation of clinical and molecular information in recent years has generated considerable confusion regarding the diagnosis of RTT. The purpose of this work was revise and clarify 2002 consensus criteria for the diagnosis of RTT in anticipation of treatment trials. Method RettSearch members, representing the majority of the international clinical RTT specialists, participated in an iterative process to come to a consensus on a revised and simplified clinical diagnostic criteria for RTT. Results The clinical criteria required for the diagnosis of classic and atypical RTT were clarified and simplified. Guidelines for the diagnosis and molecular evaluation of specific variant forms of RTT were developed. Interpretation These revised criteria provide clarity regarding the key features required for the diagnosis of RTT and reinforce the concept that RTT is a clinical diagnosis based on distinct clinical criteria, independent of molecular findings. We recommend that these criteria and guidelines be utilized in any proposed clinical research. PMID:21154482

Radiolabeled Nanoparticles for Multimodality Tumor Imaging

PubMed Central

Xing, Yan; Zhao, Jinhua; Conti, Peter S.; Chen, Kai

2014-01-01

Each imaging modality has its own unique strengths. Multimodality imaging, taking advantages of strengths from two or more imaging modalities, can provide overall structural, functional, and molecular information, offering the prospect of improved diagnostic and therapeutic monitoring abilities. The devices of molecular imaging with multimodality and multifunction are of great value for cancer diagnosis and treatment, and greatly accelerate the development of radionuclide-based multimodal molecular imaging. Radiolabeled nanoparticles bearing intrinsic properties have gained great interest in multimodality tumor imaging over the past decade. Significant breakthrough has been made toward the development of various radiolabeled nanoparticles, which can be used as novel cancer diagnostic tools in multimodality imaging systems. It is expected that quantitative multimodality imaging with multifunctional radiolabeled nanoparticles will afford accurate and precise assessment of biological signatures in cancer in a real-time manner and thus, pave the path towards personalized cancer medicine. This review addresses advantages and challenges in developing multimodality imaging probes by using different types of nanoparticles, and summarizes the recent advances in the applications of radiolabeled nanoparticles for multimodal imaging of tumor. The key issues involved in the translation of radiolabeled nanoparticles to the clinic are also discussed. PMID:24505237

Bio-Mimetic Sensors Based on Molecularly Imprinted Membranes

PubMed Central

Algieri, Catia; Drioli, Enrico; Guzzo, Laura; Donato, Laura

2014-01-01

An important challenge for scientific research is the production of artificial systems able to mimic the recognition mechanisms occurring at the molecular level in living systems. A valid contribution in this direction resulted from the development of molecular imprinting. By means of this technology, selective molecular recognition sites are introduced in a polymer, thus conferring it bio-mimetic properties. The potential applications of these systems include affinity separations, medical diagnostics, drug delivery, catalysis, etc. Recently, bio-sensing systems using molecularly imprinted membranes, a special form of imprinted polymers, have received the attention of scientists in various fields. In these systems imprinted membranes are used as bio-mimetic recognition elements which are integrated with a transducer component. The direct and rapid determination of an interaction between the recognition element and the target analyte (template) was an encouraging factor for the development of such systems as alternatives to traditional bio-assay methods. Due to their high stability, sensitivity and specificity, bio-mimetic sensors-based membranes are used for environmental, food, and clinical uses. This review deals with the development of molecularly imprinted polymers and their different preparation methods. Referring to the last decades, the application of these membranes as bio-mimetic sensor devices will be also reported. PMID:25196110

Endorsing good quality assurance practices in molecular pathology: risks and recommendations for diagnostic laboratories and external quality assessment providers.

PubMed

Tembuyser, Lien; Dequeker, Elisabeth M C

2016-01-01

Quality assurance is an indispensable element in a molecular diagnostic laboratory. The ultimate goal is to warrant patient safety. Several risks that can compromise high quality procedures are at stake, from sample collection to the test performed by the laboratory, the reporting of test results to clinicians, and the organization of effective external quality assessment schemes. Quality assurance should therefore be safeguarded at each level and should imply a holistic multidisciplinary approach. This review aims to provide an overview of good quality assurance practices and discusses certain risks and recommendations to promote and improve quality assurance for both diagnostic laboratories and for external quality assessment providers. The number of molecular targets is continuously rising, and new technologies are evolving. As this poses challenges for clinical implementation and increases the demand for external quality assessment, the formation of an international association for improving quality assurance in molecular pathology is called for.

Nanoparticles for Imaging: Top or Flop?

PubMed Central

Mertens, Marianne E.; Grimm, Jan; Lammers, Twan

2014-01-01

Nanoparticles are frequently suggested as diagnostic agents. However, except for iron oxide nanoparticles, diagnostic nanoparticles have been barely incorporated into clinical use so far. This is predominantly due to difficulties in achieving acceptable pharmacokinetic properties and reproducible particle uniformity as well as to concerns about toxicity, biodegradation, and elimination. Reasonable indications for the clinical utilization of nanoparticles should consider their biologic behavior. For example, many nanoparticles are taken up by macrophages and accumulate in macrophage-rich tissues. Thus, they can be used to provide contrast in liver, spleen, lymph nodes, and inflammatory lesions (eg, atherosclerotic plaques). Furthermore, cells can be efficiently labeled with nanoparticles, enabling the localization of implanted (stem) cells and tissue-engineered grafts as well as in vivo migration studies of cells. The potential of using nanoparticles for molecular imaging is compromised because their pharmacokinetic properties are difficult to control. Ideal targets for nanoparticles are localized on the endothelial luminal surface, whereas targeted nanoparticle delivery to extravascular structures is often limited and difficult to separate from an underlying enhanced permeability and retention (EPR) effect. The majority of clinically used nanoparticle-based drug delivery systems are based on the EPR effect, and, for their more personalized use, imaging markers can be incorporated to monitor biodistribution, target site accumulation, drug release, and treatment efficacy. In conclusion, although nanoparticles are not always the right choice for molecular imaging (because smaller or larger molecules might provide more specific information), there are other diagnostic and theranostic applications for which nanoparticles hold substantial clinical potential. PMID:25247562

The potential of liquid-based cytology in lymph node cytological evaluation: the role of morphology and the aid of ancillary techniques.

PubMed

Rossi, E D; Martini, M; Straccia, P; Bizzarro, T; Fadda, G; Larocca, L M

2016-02-01

Our aim was to evaluate the feasibility and diagnostic accuracy of liquid-based cytology (LBC) on lymph node fine needle aspiration (FNA). FNA may fulfil a challenging role in the evaluation of the majority of primary (benign and malignant) diagnoses as well as metastatic lymph node lesions. Although the morphological features may be quite easily recognized, cytological samples with a scant cellular component may raise some issues. We appraised 263 cytological lymph nodes from different body regions analysed between January and December 2013, including 137 male and 126 female patients, and processed with LBC. The cytological diagnoses included 160 benign and 103 malignant lesions. We reported 35 benign and 73 malignant lesions from 108 with surgical follow-up. The latter malignant series included 68 metastatic lesions, four suspicious for malignancy and one inadequate sample. The cytological diagnoses were supported by 62 conclusive immunocytochemical and 28 molecular analyses. Of the 108 cases, we documented 35 true negatives, 72 true positives, one false negative and no false positives, resulting in 98.6% sensitivity, 100% specificity, 99% diagnostic accuracy, 97.2% negative predictive value and 100% positive predictive value. FNA represents the first diagnostic tool in lymph node management and a reliable approach in order to avoid an excision biopsy. Furthermore, LBC is a feasible method for ancillary tests for which methanol-fixed samples are suitable, such as immunocytochemistry and molecular analysis. © 2014 John Wiley & Sons Ltd.

Recent Developments in Synthetic Carbohydrate-Based Diagnostics, Vaccines, and Therapeutics.

PubMed

Fernández-Tejada, Alberto; Cañada, F Javier; Jiménez-Barbero, Jesús

2015-07-20

Glycans are everywhere in biological systems, being involved in many cellular events with important implications for medical purposes. Building upon a detailed understanding of the functional roles of carbohydrates in molecular recognition processes and disease states, glycans are increasingly being considered as key players in pharmacological research. On the basis of the important progress recently made in glycochemistry, glycobiology, and glycomedicine, we provide a complete overview of successful applications and future perspectives of carbohydrates in the biopharmaceutical and medical fields. This review highlights the development of carbohydrate-based diagnostics, exemplified by glycan imaging techniques and microarray platforms, synthetic oligosaccharide vaccines against infectious diseases (e.g., HIV) and cancer, and finally carbohydrate-derived therapeutics, including glycomimetic drugs and glycoproteins. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phytophthora database 2.0: update and future direction.

PubMed

Park, Bongsoo; Martin, Frank; Geiser, David M; Kim, Hye-Seon; Mansfield, Michele A; Nikolaeva, Ekaterina; Park, Sook-Young; Coffey, Michael D; Russo, Joseph; Kim, Seong H; Balci, Yilmaz; Abad, Gloria; Burgess, Treena; Grünwald, Niklaus J; Cheong, Kyeongchae; Choi, Jaeyoung; Lee, Yong-Hwan; Kang, Seogchan

2013-12-01

The online community resource Phytophthora database (PD) was developed to support accurate and rapid identification of Phytophthora and to help characterize and catalog the diversity and evolutionary relationships within the genus. Since its release in 2008, the sequence database has grown to cover 1 to 12 loci for ≈2,600 isolates (representing 138 described and provisional species). Sequences of multiple mitochondrial loci were added to complement nuclear loci-based phylogenetic analyses and diagnostic tool development. Key characteristics of most newly described and provisional species have been summarized. Other additions to improve the PD functionality include: (i) geographic information system tools that enable users to visualize the geographic origins of chosen isolates on a global-scale map, (ii) a tool for comparing genetic similarity between isolates via microsatellite markers to support population genetic studies, (iii) a comprehensive review of molecular diagnostics tools and relevant references, (iv) sequence alignments used to develop polymerase chain reaction-based diagnostics tools to support their utilization and new diagnostic tool development, and (v) an online community forum for sharing and preserving experience and knowledge accumulated in the global Phytophthora community. Here we present how these improvements can support users and discuss the PD's future direction.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

PubMed

Hirsch, B; Endris, V; Lassmann, S; Weichert, W; Pfarr, N; Schirmacher, P; Kovaleva, V; Werner, M; Bonzheim, I; Fend, F; Sperveslage, J; Kaulich, K; Zacher, A; Reifenberger, G; Köhrer, K; Stepanow, S; Lerke, S; Mayr, T; Aust, D E; Baretton, G; Weidner, S; Jung, A; Kirchner, T; Hansmann, M L; Burbat, L; von der Wall, E; Dietel, M; Hummel, M

2018-04-01

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Broad spectrum microarray for fingerprint-based bacterial species identification

PubMed Central

2010-01-01

Background Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. Results A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. Conclusions These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups. PMID:20163710

A fluorogenic molecular nanoprobe with an engineered internal environment for sensitive and selective detection of biological hydrogen sulfide.

PubMed

Kim, Myung; Seo, Young Hun; Kim, Youngsun; Heo, Jeongyun; Jang, Woo-Dong; Sim, Sang Jun; Kim, Sehoon

2017-02-14

A nanoreactor approach based on the amphiphilic assembly of various molecules offers a chance to finely engineer the internal reaction medium to enable highly selective and sensitive detection of H 2 S in biological media, being useful for microscopic imaging of cellular processes and in vitro diagnostics with blood samples.

Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order

Treesearch

G. J. Bilodeau; F. N. Martin; M. D. Coffey; C. L. Blomquist

2014-01-01

A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed...

Low dose radiation effects on the brain - from mechanisms and behavioral outcomes to mitigation strategies.

PubMed

Kovalchuk, Anna; Kolb, Bryan

2017-07-03

Based on the most recent estimates by the Canadian Cancer Society, 2 in 5 Canadians will develop cancer in their lifetimes. More than half of all cancer patients receive some type of radiation therapy, and all patients undergo radiation-based diagnostics. While radiation is one of the most important diagnostic and treatments modalities, high-dose cranial radiation therapy causes numerous central nervous system side-effects, including declines in cognitive function, memory, and attention. While the mechanisms of these effects have been studies, they still need to be further elucidated. On the other hand, the effects of low dose radiation as well as indirect radiation bystander effects on the brain remain elusive. We pioneered analysis of the molecular and cellular effects of low dose direct, bystander and scatter radiation on the brain. Using a rat model, we showed that low dose radiation exposures cause molecular and cellular changes in the brain and impacts animal behavior. Here we reflect upon our recent findings and current state of knowledge in the field, and suggest novel radiation effect biomarkers and means of prevention. We propose strategies and interventions to prevent and mitigate radiation effects on the brain.

From molecular biology to nanotechnology and nanomedicine.

PubMed

Bogunia-Kubik, Katarzyna; Sugisaka, Masanori

2002-01-01

Great progress in the development of molecular biology techniques has been seen since the discovery of the structure of deoxyribonucleic acid (DNA) and the implementation of a polymerase chain reaction (PCR) method. This started a new era of research on the structure of nucleic acids molecules, the development of new analytical tools, and DNA-based analyses. The latter included not only diagnostic procedures but also, for example, DNA-based computational approaches. On the other hand, people have started to be more interested in mimicking real life, and modeling the structures and organisms that already exist in nature for the further evaluation and insight into their behavior and evolution. These factors, among others, have led to the description of artificial organelles or cells, and the construction of nanoscale devices. These nanomachines and nanoobjects might soon find a practical implementation, especially in the field of medical research and diagnostics. The paper presents some examples, illustrating the progress in multidisciplinary research in the nanoscale area. It is focused especially on immunogenetics-related aspects and the wide usage of DNA molecules in various fields of science. In addition, some proposals for nanoparticles and nanoscale tools and their applications in medicine are reviewed and discussed.

Greater Real-Life Diagnostic Efficacy of Allergen Molecule-Based Diagnosis for Prescription of Immunotherapy in an Area with Multiple Pollen Exposure.

PubMed

Saltabayeva, Ulbosin; Garib, Victoria; Morenko, Marina; Rosenson, Rafail; Ispayeva, Zhanat; Gatauova, Madina; Zulus, Loreta; Karaulov, Alexander; Gastager, Felix; Valenta, Rudolf

2017-01-01

Allergen molecule-based diagnosis has been suggested to facilitate the identification of disease-causing allergen sources and the prescription of allergen-specific immunotherapy (AIT). The aim of the current study was to compare allergen molecule-based IgE serology with allergen extract-based skin testing for the identification of the disease-causing allergen sources. The study was conducted in an area where patients are exposed to pollen from multiple sources (trees, grasses, and weeds) at the same time to compare the diagnostic efficiency of the 2 forms of diagnosis. Patients from Astana, Kazakhstan, who suffered from pollen-induced allergy (n = 95) were subjected to skin prick testing (SPT) with a local panel of tree pollen, grass pollen, and weed pollen allergen extracts and IgE antibodies specific for marker allergen molecules (nArt v 1, nArt v 3, rAmb a 1, rPhl p 1, rPhl p 5, rBet v 1) were measured by ImmunoCAP. Direct and indirect costs for diagnosis based on SPT and marker allergen-based IgE serology as well as direct costs for immunotherapy depending on SPT and serological test results were calculated. The costs for SPT-based diagnosis per patient were lower than the costs for allergen molecule-based IgE serology. However, allergen molecule-based serology was more precise in detecting the disease-causing allergen sources. A lower number of immunotherapy treatments (n = 119) was needed according to molecular diagnosis as compared to extract-based diagnosis (n = 275), which considerably reduced the total costs for diagnosis and for a 3-year treatment from EUR 1,112.30 to 521.77 per patient. The results from this real-life study show that SPT is less expensive than allergen molecule-based diagnostic testing, but molecular diagnosis allowed more precise prescription of immunotherapy which substantially reduced treatment costs and combined costs for diagnosis and treatment. © 2017 The Author(s) Published by S. Karger AG, Basel.

Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018.

PubMed

Bhalla, Amarpreet; Zulfiqar, Muhammad; Bluth, Martin H

2018-06-01

The molecular pathogenesis and classification of colorectal carcinoma are based on the traditional adenomaecarcinoma sequence, serrated polyp pathway, and microsatellite instability (MSI). The genetic basis for hereditary nonpolyposis colorectal cancer is the detection of mutations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes. Genetic testing for Lynch syndrome includes MSI testing, methylator phenotype testing, BRAF mutation testing, and molecular testing for germline mutations in MMR genes. Molecular makers with predictive and prognostic implications include quantitative multigene reverse transcriptase polymerase chain reaction assay and KRAS and BRAF mutation analysis. Mismatch repair-deficient tumors have higher rates of programmed death-ligand 1 expression. Cell-free DNA analysis in fluids are proving beneficial for diagnosis and prognosis in these disease states towards effective patient management. Copyright © 2018 Elsevier Inc. All rights reserved.

Oxidant/Antioxidant Imbalance in Alzheimer's Disease: Therapeutic and Diagnostic Prospects

PubMed Central

2018-01-01

Alzheimer's disease (AD) is the most common cause of dementia and a great socioeconomic burden in the aging society. Compelling evidence demonstrates that molecular change characteristics for AD, such as oxidative stress and amyloid β (Aβ) oligomerization, precede by decades the onset of clinical dementia and that the disease represents a biological and clinical continuum of stages, from asymptomatic to severely impaired. Nevertheless, the sequence of the early molecular alterations and the interplay between them are incompletely understood. This review presents current knowledge about the oxidative stress-induced impairments and compromised oxidative stress defense mechanisms in AD brain and the cross-talk between various pathophysiological insults, with the focus on excessive reactive oxygen species (ROS) generation and Aβ overproduction at the early stages of the disease. Prospects for AD therapies targeting oxidant/antioxidant imbalance are being discussed, as well as for the development of novel oxidative stress-related, blood-based biomarkers for early, noninvasive AD diagnostics. PMID:29636850

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

PubMed

Perez-Guaita, David; Marzec, Katarzyna M; Hudson, Andrew; Evans, Corey; Chernenko, Tatyana; Matthäus, Christian; Miljkovic, Milos; Diem, Max; Heraud, Philip; Richards, Jack S; Andrew, Dean; Anderson, David A; Doerig, Christian; Garcia-Bustos, Jose; McNaughton, Don; Wood, Bayden R

2018-04-20

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

Molecular biology of Ganoderma pathogenicity and diagnosis in coconut seedlings.

PubMed

Kandan, A; Radjacommare, R; Ramanathan, A; Raguchander, T; Balasubramanian, P; Samiyappan, R

2009-01-01

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10-13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.

Mutation testing for directing upfront targeted therapy and post-progression combination therapy strategies in lung adenocarcinoma

PubMed Central

Salgia, Ravi

2016-01-01

ABSTRACT Introduction: Advances in the biology of non-small-cell lung cancer, especially adenocarcinoma, reveal multiple molecular subtypes driving oncogenesis. Accordingly, individualized targeted therapeutics are based on mutational diagnostics. Areas covered: Advances in strategies and techniques for individualized treatment, particularly of adenocarcinoma, are described through literature review. Approved therapies are established for some molecular subsets, with new driver mutations emerging that represent increasing proportions of patients. Actionable mutations are de novo oncogenic drivers or acquired resistance mediators, and mutational profiling is important for directing therapy. Patients should be monitored for emerging actionable resistance mutations. Liquid biopsy and associated multiplex diagnostics will be important means to monitor patients during treatment. Expert commentary: Outcomes with targeted agents may be improved by integrating mutation screens during treatment to optimize subsequent therapy. In order for this to be translated into impactful patient benefit, appropriate platforms and strategies need to be optimized and then implemented universally. PMID:27139190

Nanomedicine and personalised medicine: understanding the personalisation of health care in the molecular era.

PubMed

Noury, Mathieu; López, José

2017-05-01

Globally supported by public policy and investment, nanomedicine is presented as an ongoing medical revolution that will radically change the practice of health care from diagnostic to therapeutic, and everything in between. One of nanomedicine's major promises is that of personalised medicine, enabling diagnostics and therapeutics tailored to individual needs and developing a truly 'patient-friendly' medical approach. Based on qualitative interviews with nanomedicine researchers in Canada, this article explores the emerging concept of personalised medicine as it becomes entangled with nanomedical research. More precisely, drawing on insights from science studies and the sociology of expectations, it analyses researchers' perceptions of personalised medicine in the cutting edge of current nanomedicine research. Two perceptions of personalisation are identified; a molecular conception of individuality and a technical conception of personalisation. The article concludes by examining the relationship between the two conceptions and contrasts them with the normative reflex of a more expansive conception of personalised medicine. © 2016 Foundation for the Sociology of Health & Illness.

Costs of genetic testing: Supporting Brazilian Public Policies for the incorporating of molecular diagnostic technologies

PubMed Central

Schlatter, Rosane Paixão; Matte, Ursula; Polanczyk, Carisi Anne; Koehler-Santos, Patrícia; Ashton-Prolla, Patricia

2015-01-01

This study identifies and describes the operating costs associated with the molecular diagnosis of diseases, such as hereditary cancer. To approximate the costs associated with these tests, data informed by Standard Operating Procedures for various techniques was collected from hospital software and a survey of market prices. Costs were established for four scenarios of capacity utilization to represent the possibility of suboptimal use in research laboratories. Cost description was based on a single site. The results show that only one technique was not impacted by rising costs due to underutilized capacity. Several common techniques were considerably more expensive at 30% capacity, including polymerase chain reaction (180%), microsatellite instability analysis (181%), gene rearrangement analysis by multiplex ligation probe amplification (412%), non-labeled sequencing (173%), and quantitation of nucleic acids (169%). These findings should be relevant for the definition of public policies and suggest that investment of public funds in the establishment of centralized diagnostic research centers would reduce costs to the Public Health System. PMID:26500437

Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections.

PubMed

Verweij, Jaco J; Stensvold, C Rune

2014-04-01

Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

Molecular Testing for Clinical Diagnosis and Epidemiological Investigations of Intestinal Parasitic Infections

PubMed Central

Stensvold, C. Rune

2014-01-01

SUMMARY Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies. PMID:24696439

[Molecular biology for sarcoma: useful or necessary?].

PubMed

Neuville, Agnès; Coindre, Jean-Michel; Chibon, Frédéric

2015-01-01

Sarcomas are a heterogeneous group of tumors. Their diagnosis is based on morphology and immunohistochemical profile, with categories of tumors according to the type of tissue that they resemble. Nevertheless, for several tumors, cellular origin is unknown. Molecular analysis performed in recent years allowed, combining histophenotype and genomics, better classifying such sarcomas, individualizing new entities and grouping some tumors. Simple and recurrent genetic alterations, such as translocation, mutation, amplification, can be identified in one of two sarcomas and appear as new diagnostic markers. Their identification in specialized laboratories in molecular pathology of sarcomas is often useful and sometimes necessary for a good diagnosis, leading to a heavy and multidisciplinary multi-step treatment. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Quality Control of Direct Molecular Diagnostics for Methicillin-Resistant Staphylococcus aureus▿

PubMed Central

van Belkum, Alex; Niesters, Hubert G. M.; MacKay, William G.; van Leeuwen, Willem B.

2007-01-01

Ten samples containing various amounts of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus, methicillin-resistant Staphylococcus epidermidis (MRSE), and combinations thereof were distributed to 51 laboratories for molecular diagnostics testing. Samples containing 102 to 103 MRSA cells were frequently reported to be negative. MRSE samples were scored as negative by all commercial tests but by only two out of three in-house tests. PMID:17581936

Non-contact high resolution Bessel beam probe for diagnostic imaging of cornea and trabecular meshwork region in eye

NASA Astrophysics Data System (ADS)

Murukeshan, V. M.; Jesmond, Hong Xun J.; Shinoj, V. K.; Baskaran, M.; Tin, Aung

2015-07-01

Primary angle closure glaucoma is a major form of disease that causes blindness in Asia and worldwide. In glaucoma, irregularities in the ocular aqueous outflow system cause an elevation in intraocular pressure (IOP) with subsequent death of retinal ganglion cells, resulting in loss of vision. High resolution visualization of the iridocorneal angle region has great diagnostic value in understanding the disease condition which enables monitoring of surgical interventions that decrease IOP. None of the current diagnostic techniques such as goniophotography, ultrasound biomicroscopy (UBM), anterior segment optical coherence tomography (AS-OCT) and RetCam™ can image with molecular specificity and required spatial resolution that can delineate the trabecular meshwork structures. This paper in this context proposes new concepts and methodology using Bessel beams based illumination and imaging for such diagnostic ocular imaging applications. The salient features using Bessel beams instead of the conventional Gaussian beam, and the optimization challenges in configuring the probe system will be illustrated with porcine eye samples.

Design of a Tablet Computer App for Facilitation of a Molecular Blood Culture Test in Clinical Microbiology and Preliminary Usability Evaluation

PubMed Central

Pape-Haugaard, Louise; Meltzer, Michelle C; Fuchs, Martin; Schønheyder, Henrik C; Hejlesen, Ole

2016-01-01

Background User mobility is an important aspect of the development of clinical information systems for health care professionals. Mobile phones and tablet computers have obtained widespread use by health care professionals, offering an opportunity for supporting the access to patient information through specialized applications (apps) while supporting the mobility of the users. The use of apps for mobile phones and tablet computers may support workflow of complex tasks, for example, molecular-based diagnostic tests in clinical microbiology. Multiplex Blood Culture Test (MuxBCT) is a molecular-based diagnostic test used for rapid identification of pathogens in positive blood cultures. To facilitate the workflow of the MuxBCT, a specialized tablet computer app was developed as an accessory to the diagnostic test. The app aims to reduce the complexity of the test by step-by-step guidance of microscopy and to assist users in reaching an exact bacterial or fungal diagnosis based on blood specimen observations and controls. Additionally, the app allows for entry of test results, and communication thereof to the laboratory information system (LIS). Objective The objective of the study was to describe the design considerations of the MuxBCT app and the results of a preliminary usability evaluation. Methods The MuxBCT tablet app was developed and set up for use in a clinical microbiology laboratory. A near-live simulation study was conducted in the clinical microbiology laboratory to evaluate the usability of the MuxBCT app. The study was designed to achieve a high degree of realism as participants carried out a scenario representing the context of use for the MuxBCT app. As the MuxBCT was under development, the scenario involved the use of molecular blood culture tests similar to the MuxBCT for identification of microorganisms from positive blood culture samples. The study participants were observed, and their interactions with the app were recorded. After the study, the participants were debriefed to clarify observations. Results Four medical laboratory technicians, for example, representative of end users of the app, participated in the clinical simulation study. Using the MuxBCT app, the study participants successfully identified and reported all microorganisms from the positive blood cultures examined. Three of the four participants reported that they found the app useful, while one study participant reported that she would prefer to make notes on paper and later enter them into the LIS. Conclusions The preliminary usability evaluation results indicate that use of the MuxBCT tablet app can facilitate the workflow of the MuxBCT diagnostic test. PMID:26993432

Alimentary Tract Bacteria Isolated and Identified with API-20E and Molecular Cloning Techniques from Australian Tropical Fruit Flies, Bactrocera cacuminata and B. tryoni

PubMed Central

Thaochan, N.; Drew, R. A. I.; Hughes, J. M.; Vijaysegaran, S.; Chinajariyawong, A.

2010-01-01

Bacteria were isolated from the crop and midgut of field collected Bactrocera cacuminata (Hering) and Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Two methods were used, firstly isolation onto two types of bacteriological culture media (PYEA and TSA) and identification using the API-20E diagnostic kit, and secondly, analysis of samples using the 16S rRNA gene molecular diagnostic method. Using the API-20E method, 10 genera and 17 species of bacteria in the family Enterobacteriaceae were identified from cultures growing on the nutrient agar. The dominant species in both the crop and midgut were Citrobacter freundii, Enterobacter cloacae and Klebsiella oxytoca. Providencia rettgeri, Klebsiella pneumoniae ssp ozaenae and Serratia marcescens were isolated from B. tryoni only. Using the molecular cloning technique that is based on 16S rRNA gene sequences, five bacteria classes were dignosed — Alpha-, Beta-, Gamma- and Delta- Proteobacteria and Firmicutes — including five families, Leuconostocaceae, Enterococcaceae, Acetobacteriaceae, Comamonadaceae and Enterobacteriaceae. The bacteria affiliated with Firmicutes were found mainly in the crop while the Gammaproteobacteria, especially the family Enterobacteriaceae, was dominant in the midgut. This paper presents results from the first known application of molecular cloning techniques to study bacteria within tephritid species and the first record of Firmicutes bacteria in these flies. PMID:20883132

75 FR 340 - Approval for Expansion of Subzone 22F, Abbott Molecular, Inc. (Pharmaceutical and Molecular...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-05

... DEPARTMENT OF COMMERCE Foreign-Trade Zones Board [Order No. 1654] Approval for Expansion of Subzone 22F, Abbott Molecular, Inc. (Pharmaceutical and Molecular Diagnostic Products), Chicago, IL, Area... manufacturing authority on behalf of Abbott Molecular, Inc., within FTZ 22F in Des Plaines and Elk Grove Village...

Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing

PubMed Central

Murphy, Danielle A.; Ely, Heather A.; Shoemaker, Robert; Boomer, Aaron; Culver, Brady P.; Hoskins, Ian; Haimes, Josh D.; Walters, Ryan D.; Fernandez, Diane; Stahl, Joshua A.; Lee, Jeeyun; Kim, Kyoung-Mee; Lamoureux, Jennifer

2017-01-01

Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens. PMID:27028240

Tuberculosis Diagnostics in 2015: Landscape, Priorities, Needs, and Prospects

PubMed Central

Pai, Madhukar; Schito, Marco

2015-01-01

In 2015, tuberculosis remains a major global health problem, and drug-resistant tuberculosis is a growing threat. Although tuberculosis diagnosis in many countries is still reliant on older tools, new diagnostics are changing the landscape. Stimulated, in part, by the success and roll out of Xpert MTB/RIF, there is now considerable interest in new technologies. The landscape looks promising, with a robust pipeline of new tools, particularly molecular diagnostics, and well over 50 companies actively engaged in product development. However, new diagnostics are yet to reach scale, and there needs to be greater convergence between diagnostics development and development of shorter-duration tuberculosis drug regimens. Another concern is the relative absence of non–sputum-based diagnostics in the pipeline for children and of biomarker tests for triage, cure, and progression of latent Mycobacterium tuberculosis infection. Several initiatives, described in this supplement, have been launched to further stimulate product development and policy, including assessment of needs and priorities, development of target product profiles, compilation of data on resistance-associated mutations, and assessment of market size and potential for new diagnostics. Advocacy is needed to increase funding for tuberculosis research and development, and governments in high-burden countries must invest more in tuberculosis control to meet post-2015 targets for care, control, and prevention. PMID:25765103

Impact of transition from microscopy to molecular screening for detection of intestinal protozoa in Dutch patients.

PubMed

Svraka-Latifovic, S; Bouter, S; Naus, H; Bakker, L J; Timmerman, C P; Dorigo-Zetsma, J W

2014-11-01

Detection of intestinal protozoa by PCR methods has been described as being sensitive and specific, and as improving the diagnostic yield. Here we present the outcome of the transition from microscopy to molecular screening for detection of a select group of intestinal protozoa in faeces in our laboratory. Introduction of molecular screening for intestinal protozoa resulted in higher sensitivity, reduced hands-on-time, reduced time-to-results, leading to improved diagnostic efficiency. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond

PubMed Central

Rogers, Toni-Maree; Fellowes, Andrew; Bell, Anthony; Fox, Stephen

2015-01-01

Somatic mutational profiling in cancer has revolutionized the practice of clinical oncology. The discovery of driver mutations in non-small cell lung cancer (NSCLC) is an example of this. Molecular testing of lung adenocarcinoma is now considered standard of care and part of the diagnostic algorithm. This article provides an overview of the workflow of molecular testing in a clinical diagnostic laboratory discussing in particular novel assays that are currently in use for somatic mutation detection in NSCLC focussing on epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK), ROS1 and RET rearrangements. PMID:25870795

[Evaluation of quality of HIV diagnostic procedures in Poland].

PubMed

Parczewski, Miłosz; Madaliński, Kazimierz; Leszczyszyn-Pynka, Magdalena; Boroń-Kaczmarska, Anna

2010-01-01

The aim of this work was quality assessment of HIV diagnostic procedures in Poland, including human and technical resources as well as laboratory practice. Sixty questionnaires were distributed among diagnostic centers to obtain qualitative data. Basing on the survey data serological control using coded panels of HIV-1/2 samples was performed. Thirty-one filled questionnaires were received (50.8%). Surveyed laboratories perform from 350 to 5500 serological screening tests per year. In most of laboratories fourth generation assays are available, while Blood Donation Centers screen the blood both with serological assays and by HIV-RNA detection. Sanitary and Epidemiological Stations and academic laboratories hold the ISO/IEC 17025 or IS0 9001:2001 accreditation, five of the surveyed centers participate in Labquality assurance and two in Quality Control in Molecular Diagnostics programs. Data of control serological testing were received from 21 centers. In the quality control assessment 194 analyses were performed with 91 true negative, 2 false negative, 96 true positive and 5 false positive results. False negative rate of % and false positive rate of 5.2% was noted for this study. Currently, virtually no guidelines related to the HIV-diagnostics quality assurance and control in Poland are in delineated. Development of the national unified quality control system, basing on the central institution is highly desirable. National certification within the frames of the quality control and assurance program should be mandatory for all the diagnostic labs, and aim at improvement of reliability of the result distributed among clinicians and patients.

Current and future molecular diagnostics in non-small-cell lung cancer.

PubMed

Li, Chun Man; Chu, Wing Ying; Wong, Di Lun; Tsang, Hin Fung; Tsui, Nancy Bo Yin; Chan, Charles Ming Lok; Xue, Vivian Wei Wen; Siu, Parco Ming Fai; Yung, Benjamin Yat Ming; Chan, Lawrence Wing Chi; Wong, Sze Chuen Cesar

2015-01-01

The molecular investigation of lung cancer has opened up an advanced area for the diagnosis and therapeutic management of lung cancer patients. Gene alterations in cancer initiation and progression provide not only information on molecular changes in lung cancer but also opportunities in advanced therapeutic regime by personalized targeted therapy. EGFR mutations and ALK rearrangement are important predictive biomarkers for the efficiency of tyrosine kinase inhibitor treatment in lung cancer patients. Moreover, epigenetic aberration and microRNA dysregulation are recent advances in the early detection and monitoring of lung cancer. Although a wide range of molecular tests are available, standardization and validation of assay protocols are essential for the quality of the test outcome. In this review, current and new advancements of molecular biomarkers for non-small-cell lung cancer will be discussed. Recommendations on future development of molecular diagnostic services will also be explored.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

PubMed

Mohammadi-Yeganeh, S; Paryan, M; Mirab Samiee, S; Kia, V; Rezvan, H

2012-06-01

Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella species and Entero-invasive Escherichia coli in the Netherlands.

PubMed

van den Beld, Maaike J C; Friedrich, Alexander W; van Zanten, Evert; Reubsaet, Frans A G; Kooistra-Smid, Mirjam A M D; Rossen, John W A

2016-12-01

An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coli (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic methods and a sample panel, consisting of DNA-extracts and spiked stool samples with different concentrations of Shigella flexneri, was provided to each MML. The results of the trial showed an enormous variety in culture-dependent and molecular diagnostic techniques currently used among MMLs. Despite the various molecular procedures, 15 out of 16 MMLs were able to detect Shigella species or EIEC in all the samples provided, showing that the diversity of methods has no effect on the qualitative detection of Shigella flexneri. In contrast to semi quantitative analysis, the minimum and maximum values per sample differed by approximately five threshold cycles (Ct-value) between the MMLs included in the study. This indicates that defining a uniform Ct-value cut-off for notification to health authorities is not advisable. Copyright © 2016 Elsevier B.V. All rights reserved.

Verification of performance specifications of a molecular test: cystic fibrosis carrier testing using the Luminex liquid bead array.

PubMed

Lacbawan, Felicitas L; Weck, Karen E; Kant, Jeffrey A; Feldman, Gerald L; Schrijver, Iris

2012-01-01

The number of clinical laboratories introducing various molecular tests to their existing test menu is continuously increasing. Prior to offering a US Food and Drug Administration-approved test, it is necessary that performance characteristics of the test, as claimed by the company, are verified before the assay is implemented in a clinical laboratory. To provide an example of the verification of a specific qualitative in vitro diagnostic test: cystic fibrosis carrier testing using the Luminex liquid bead array (Luminex Molecular Diagnostics, Inc, Toronto, Ontario). The approach used by an individual laboratory for verification of a US Food and Drug Administration-approved assay is described. Specific verification data are provided to highlight the stepwise verification approach undertaken by a clinical diagnostic laboratory. Protocols for verification of in vitro diagnostic assays may vary between laboratories. However, all laboratories must verify several specific performance specifications prior to implementation of such assays for clinical use. We provide an example of an approach used for verifying performance of an assay for cystic fibrosis carrier screening.

Wound Healing Finally Enters the Age of Molecular Diagnostic Medicine

PubMed Central

Tatum, Owatha L.; Dowd, Scot E.

2012-01-01

Background Many wounds are difficult to heal because of the large, complex community of microbes present within the wound. The Problem Classical laboratory culture methods do not provide an accurate picture of the microbial interactions or representation of microorganisms within a wound. There is an inherent bias in diagnosis based upon classical culture stemming from the ability of certain organisms to thrive in culture while others are underrepresented or fail to be identified in culture altogether. Chronic wounds also contain polymicrobial infections existing as a cooperative community that is resistant to antibiotic therapy. Basic/Clinical Science Advances New methods in molecular diagnostic medicine allow the identification of nearly all organisms present in a wound irrespective of the ability of these organisms to be grown in culture. Advances in DNA analyses allow absolute identification of microorganisms from very small clinical specimens. These new methods also provide a quantitative representation of all microorganisms contributing to these polymicrobial infections. Clinical Care Relevance Technological advances in laboratory diagnostics can significantly shorten the time required to heal chronic wounds. Identification of the genetic signatures of organisms present within a wound allows clinicians to identify and treat the primary organisms responsible for nonhealing wounds. Conclusion Advanced genetic technologies targeting the specific needs of wound care patients are now accessible to all wound care clinicians. PMID:24527290

Exome Sequencing Frequently Reveals the Cause of Early-Onset Chronic Kidney Disease

PubMed Central

Vivante, Asaf; Hildebrandt, Friedhelm

2016-01-01

The primary causes of chronic kidney disease (CKD) in children differ from those of adult onset CKD. In the United States the most common diagnostic groups of CKD that manifests before 25 years of age are: i) congenital anomalies of the kidneys and urinary tract (CAKUT) (49.1%), ii) steroid-resistant nephrotic syndrome (SRNS) (10.4%), iii) chronic glomerulonephritis (8.1%), and iv) renal cystic ciliopathies (5.3 %), encompassing >70% of CKD together. Recent findings suggest that early-onset CKD is caused by mutations in any one of over 200 different monogenic genes. High-throughput sequencing has very recently rendered identification of causative mutations in this high number of genes feasible. Molecular genetic diagnostics in early onset-CKD (before the age of 25 years) will, i) provide patients and families with a molecular genetic diagnosis, ii) generate new insights into diseases mechanisms, iii) allow etiology-based classification of patient cohorts for clinical studies and, iv) may have consequences for personalized treatment and prevention of CKD. In this review, we will discuss the implications of next-generation sequencing for clinical genetic diagnostics and discovery of novel genes in early-onset CKD. We also delineate the resulting opportunities for deciphering disease mechanisms and therapeutic implications. PMID:26750453

An Integrated Quantum Dot Barcode Smartphone Optical Device for Wireless Multiplexed Diagnosis of Infected Patients

NASA Astrophysics Data System (ADS)

Ming, Kevin

Integrating mobile-cellular devices with multiplex molecular diagnostics can potentially provide the most powerful platform for tracking, managing and preventing the transmission of infectious diseases. With over 6.9 billion subscriptions globally, handheld mobile-cellular devices can be programmed to spatially map, temporally track, and transmit information on infections over wide geographical space and boundaries. Current cell phone diagnostic technologies have poor limit of detection, dynamic range, and cannot detect multiple pathogen targets simultaneously, limiting their utility to single infections with high load. Here we combined recent advances in quantum dot barcode technology for molecular detection with smartphones to engineer a simple and low-cost chip-based wireless multiplex diagnostic device. We validated our device using a variety of synthetic genomic targets for the respiratory virus and blood-borne pathogens, and demonstrated that it could detect clinical samples after simple amplification. More importantly, we confirmed that the device is capable of detecting patients infected with a single or multiple infectious pathogens (e.g., HIV and hepatitis B) in a single test. This device advances the capacity for global surveillance of infectious diseases and has the potential to accelerate knowledge exchange-transfer of emerging or exigent disease threats with healthcare and military organizations in real-time.

Applications of the oral scraped (exfoliative) cytology in oral cancer and precancer.

PubMed

Acha, Amelia; Ruesga, María T; Rodríguez, María J; Martínez de Pancorbo, María A; Aguirre, José M

2005-01-01

Scraped (exfoliative) cytology is a simple and harmless procedure, which has been a controversial technique according to its real validity in oral pathology. Lately it has re-emerged due to its application in oral precancer and cancer as a diagnostic and predictive method as well as for monitoring patients. New diagnostic techniques have been developed, such as "brush biopsy" and multiple molecular studies using the cells collected. In this review we are going to analyse the more novel aspects related with the applications of the scraped or exfoliative cytology in oral precancerous and cancerous pathology, specially focusing on molecular studies and their diagnostic and prognostic implications.

Array-on-a-disk? How Blu-ray technology can be applied to molecular diagnostics.

PubMed

Morais, Sergi; Tortajada-Genaro, Luis; Maquieira, Angel

2014-09-01

This editorial comments on the balance and perspectives of compact disk technology applied to molecular diagnostics. The development of sensitive, rapid and multiplex assays using Blu-ray technology for the determination of biomarkers, drug allergens, pathogens and detection of infections would have a direct impact on diagnostics. Effective tests for use in clinical, environmental and food applications require versatile and low-cost platforms as well as cost-effective detectors. Blu-ray technology accomplishes those requirements and advances on the concept of high density arrays for massive screening to achieve the demands of point of care or in situ analysis.

Polymerization-based signal amplification under ambient conditions with thirty-five second reaction times.

PubMed

Kaastrup, Kaja; Sikes, Hadley D

2012-10-21

Although polymerization-based amplification (PBA) has demonstrated promise as an inexpensive technique for use in molecular diagnostics, oxygen inhibition of radical photopolymerization has hindered its implementation in point-of-care devices. The addition of 0.3-0.7 μM eosin to an aqueous acrylate monomer solution containing a tertiary amine allows an interfacial polymerization reaction to proceed in air only near regions of a test surface where additional eosin initiators coupled to proteins have been localized as a function of molecular recognition events. The dose of light required for the reaction is inversely related to eosin concentration. This system achieves sensitivities comparable to those reported for inert gas-purged systems and requires significantly shorter reaction times. We provide several comparisons of this system with other implementations of polymerization-based amplification.

Giant magnetoimpedance-based microchannel system for quick and parallel genotyping of human papilloma virus type 16/18

NASA Astrophysics Data System (ADS)

Yang, Hao; Chen, Lei; Lei, Chong; Zhang, Ju; Li, Ding; Zhou, Zhi-Min; Bao, Chen-Chen; Hu, Heng-Yao; Chen, Xiang; Cui, Feng; Zhang, Shuang-Xi; Zhou, Yong; Cui, Da-Xiang

2010-07-01

Quick and parallel genotyping of human papilloma virus (HPV) type 16/18 is carried out by a specially designed giant magnetoimpedance (GMI) based microchannel system. Micropatterned soft magnetic ribbon exhibiting large GMI ratio serves as the biosensor element. HPV genotyping can be determined by the changes in GMI ratio in corresponding detection region after hybridization. The result shows that this system has great potential in future clinical diagnostics and can be easily extended to other biomedical applications based on molecular recognition.

Development and validation of the JAX Cancer Treatment Profile™ for detection of clinically actionable mutations in solid tumors

PubMed Central

Ananda, Guruprasad; Mockus, Susan; Lundquist, Micaela; Spotlow, Vanessa; Simons, Al; Mitchell, Talia; Stafford, Grace; Philip, Vivek; Stearns, Timothy; Srivastava, Anuj; Barter, Mary; Rowe, Lucy; Malcolm, Joan; Bult, Carol; Karuturi, Radha Krishna Murthy; Rasmussen, Karen; Hinerfeld, Douglas

2015-01-01

Background The continued development of targeted therapeutics for cancer treatment has required the concomitant development of more expansive methods for the molecular profiling of the patient’s tumor. We describe the validation of the JAX Cancer Treatment Profile™ (JAX-CTP™), a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable mutations in solid tumors to inform the selection of targeted therapeutics for cancer treatment. Methods NGS libraries are generated from DNA extracted from formalin fixed paraffin embedded tumors. Using hybrid capture, the genes of interest are enriched and sequenced on the Illumina HiSeq 2500 or MiSeq sequencers followed by variant detection and functional and clinical annotation for the generation of a clinical report. Results The JAX-CTP™ detects actionable variants, in the form of single nucleotide variations and small insertions and deletions (≤50bp) in 190 genes in specimens with a neoplastic cell content of ≥10%. The JAX-CTP™ is also validated for the detection of clinically actionable gene amplifications. Conclusions There is a lack of consensus in the molecular diagnostics field on the best method for the validation of NGS-based assays in oncology, thus the importance of communicating methods, as contained in this report. The growing number of targeted therapeutics and the complexity of the tumor genome necessitates continued development and refinement of advanced assays for tumor profiling to enable precision cancer treatment. PMID:25562415

Accelerated Photobleaching of a Cyanine Dye in the Presence of a Ternary Target DNA, PNA Probe, Dye Catalytic Complex: A Molecular Diagnostic

PubMed Central

Wang, M.; Holmes-Davis, R.; Rafinski, Z.; Jedrzejewska, B.; Choi, K. Y.; Zwick, M.; Bupp, C.; Izmailov, A.; Paczkowski, J.; Warner, B.; Koshinsky, H.

2009-01-01

In many settings, molecular testing is needed but unavailable due to complexity and cost. Simple, rapid, and specific DNA detection technologies would provide important alternatives to existing detection methods. Here we report a novel, rapid nucleic acid detection method based on the accelerated photobleaching of the light-sensitive cyanine dye, 3,3′-diethylthiacarbocyanine iodide (DiSC2(3) I−), in the presence of a target genomic DNA and a complementary peptide nucleic acid (PNA) probe. On the basis of the UV–vis, circular dichroism, and fluorescence spectra of DiSC2(3) with PNA–DNA oligomer duplexes and on characterization of a product of photolysis of DiSC2(3) I−, a possible reaction mechanism is proposed. We propose that (1) a novel complex forms between dye, PNA, and DNA, (2) this complex functions as a photosensitizer producing 1O2, and (3) the 1O2 produced promotes photobleaching of dye molecules in the mixture. Similar cyanine dyes (DiSC3(3), DiSC4(3), DiSC5(3), and DiSCpy(3)) interact with preformed PNA–DNA oligomer duplexes but do not demonstrate an equivalent accelerated photobleaching effect in the presence of PNA and target genomic DNA. The feasibility of developing molecular diagnostic assays based on the accelerated photobleaching (the smartDNA assay) that results from the novel complex formed between DiSC2(3) and PNA–DNA is under way. PMID:19231844

Sex chromosomal abnormalities associated with equine infertility: validation of a simple molecular screening tool in the Purebred Spanish Horse.

PubMed

Anaya, G; Molina, A; Valera, M; Moreno-Millán, M; Azor, P; Peral-García, P; Demyda-Peyrás, S

2017-08-01

Chromosomal abnormalities in the sex chromosome pair (ECAX and ECAY) are widely associated with reproductive problems in horses. However, a large proportion of these abnormalities remains undiagnosed due to the lack of an affordable diagnostic tool that allows for avoiding karyotyping tests. Hereby, we developed an STR (single-tandem-repeat)-based molecular method to determine the presence of the main sex chromosomal abnormalities in horses in a fast, cheap and reliable way. The frequency of five ECAX-linked (LEX026, LEX003, TKY38, TKY270 and UCDEQ502) and two ECAY-linked (EcaYH12 and SRY) markers was characterized in 261 Purebred Spanish Horses to determine the efficiency of the methodology developed to be used as a chromosomal diagnostic tool. All the microsatellites analyzed were highly polymorphic, with a sizeable number of alleles (polymorphic information content > 0.5). Based on this variability, the methodology showed 100% sensitivity and 99.82% specificity to detect the most important sex chromosomal abnormalities reported in horses (chimerism, Turner's syndrome and sex reversal syndromes). The method was also validated with 100% efficiency in 10 individuals previously diagnosed as chromosomally aberrant. This STR screening panel is an efficient and reliable molecular-cytogenetic tool for the early detection of sex chromosomal abnormalities in equines that could be included in breeding programs to save money, effort and time of veterinary practitioners and breeders. © 2017 Stichting International Foundation for Animal Genetics.

Quantum Dot Enabled Molecular Sensing and Diagnostics

PubMed Central

Zhang, Yi; Wang, Tza-Huei

2012-01-01

Since its emergence, semiconductor nanoparticles known as quantum dots (QDs) have drawn considerable attention and have quickly extended their applicability to numerous fields within the life sciences. This is largely due to their unique optical properties such as high brightness and narrow emission band as well as other advantages over traditional organic fluorophores. New molecular sensing strategies based on QDs have been developed in pursuit of high sensitivity, high throughput, and multiplexing capabilities. For traditional biological applications, QDs have already begun to replace traditional organic fluorophores to serve as simple fluorescent reporters in immunoassays, microarrays, fluorescent imaging applications, and other assay platforms. In addition, smarter, more advanced QD probes such as quantum dot fluorescence resonance energy transfer (QD-FRET) sensors, quenching sensors, and barcoding systems are paving the way for highly-sensitive genetic and epigenetic detection of diseases, multiplexed identification of infectious pathogens, and tracking of intracellular drug and gene delivery. When combined with microfluidics and confocal fluorescence spectroscopy, the detection limit is further enhanced to single molecule level. Recently, investigations have revealed that QDs participate in series of new phenomena and exhibit interesting non-photoluminescent properties. Some of these new findings are now being incorporated into novel assays for gene copy number variation (CNV) studies and DNA methylation analysis with improved quantification resolution. Herein, we provide a comprehensive review on the latest developments of QD based molecular diagnostic platforms in which QD plays a versatile and essential role. PMID:22916072

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value.

PubMed

Farhat, Maha R; Sultana, Razvan; Iartchouk, Oleg; Bozeman, Sam; Galagan, James; Sisk, Peter; Stolte, Christian; Nebenzahl-Guimaraes, Hanna; Jacobson, Karen; Sloutsky, Alexander; Kaur, Devinder; Posey, James; Kreiswirth, Barry N; Kurepina, Natalia; Rigouts, Leen; Streicher, Elizabeth M; Victor, Tommie C; Warren, Robin M; van Soolingen, Dick; Murray, Megan

2016-09-01

The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value

PubMed Central

Sultana, Razvan; Iartchouk, Oleg; Bozeman, Sam; Galagan, James; Sisk, Peter; Stolte, Christian; Nebenzahl-Guimaraes, Hanna; Jacobson, Karen; Sloutsky, Alexander; Kaur, Devinder; Posey, James; Kreiswirth, Barry N.; Kurepina, Natalia; Rigouts, Leen; Streicher, Elizabeth M.; Victor, Tommie C.; Warren, Robin M.; van Soolingen, Dick; Murray, Megan

2016-01-01

Rationale: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance–conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. Objectives: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. Methods: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. Measurements and Main Results: The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. Conclusions: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs. PMID:26910495