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Sample records for accurate molecular diagnosis

  1. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology.

    PubMed

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10(-3) for bcr1 and bcr3 and 10(-)2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  2. Simple, rapid and accurate molecular diagnosis of acute promyelocytic leukemia by loop mediated amplification technology

    PubMed Central

    Spinelli, Orietta; Rambaldi, Alessandro; Rigo, Francesca; Zanghì, Pamela; D'Agostini, Elena; Amicarelli, Giulia; Colotta, Francesco; Divona, Mariadomenica; Ciardi, Claudia; Coco, Francesco Lo; Minnucci, Giulia

    2015-01-01

    The diagnostic work-up of acute promyelocytic leukemia (APL) includes the cytogenetic demonstration of the t(15;17) translocation and/or the PML-RARA chimeric transcript by RQ-PCR or RT-PCR. This latter assays provide suitable results in 3-6 hours. We describe here two new, rapid and specific assays that detect PML-RARA transcripts, based on the RT-QLAMP (Reverse Transcription-Quenching Loop-mediated Isothermal Amplification) technology in which RNA retrotranscription and cDNA amplification are carried out in a single tube with one enzyme at one temperature, in fluorescence and real time format. A single tube triplex assay detects bcr1 and bcr3 PML-RARA transcripts along with GUS housekeeping gene. A single tube duplex assay detects bcr2 and GUSB. In 73 APL cases, these assays detected in 16 minutes bcr1, bcr2 and bcr3 transcripts. All 81 non-APL samples were negative by RT-QLAMP for chimeric transcripts whereas GUSB was detectable. In 11 APL patients in which RT-PCR yielded equivocal breakpoint type results, RT-QLAMP assays unequivocally and accurately defined the breakpoint type (as confirmed by sequencing). Furthermore, RT-QLAMP could amplify two bcr2 transcripts with particularly extended PML exon 6 deletions not amplified by RQ-PCR. RT-QLAMP reproducible sensitivity is 10−3 for bcr1 and bcr3 and 10−2 for bcr2 thus making this assay particularly attractive at diagnosis and leaving RQ-PCR for the molecular monitoring of minimal residual disease during the follow up. In conclusion, PML-RARA RT-QLAMP compared to RT-PCR or RQ-PCR is a valid improvement to perform rapid, simple and accurate molecular diagnosis of APL. PMID:25815362

  3. Molecular systematics and diagnosis.

    PubMed

    Thompson, R C A; Zarlenga, D S; La Rosa, G; Pozio, E; Rosenthal, B; Bandi, C; Mortarino, M; Casiraghi, M; Genchi, C; Gasser, R B; Hu, M; Chilton, N B; Matthews, J B; Hodgkinson, J E

    2004-10-28

    This collection of articles provides an account of six presentations delivered at the 19th International Conference of the World Association for the Advancement of Veterinary Parasitology(WAAVP) (held in New Orleans, Louisiana, USA, from 10 to 14 August 2003) in a symposium session on Molecular Systematics and Diagnosis, organised and chaired by R.B. Gasser and D.S. Zarlenga. The focus was on recent advances in molecular tools for specific and genotypic identification,diagnosis, systematics and population genetics, with special emphasis on investigations of parasitic nematodes and protists.

  4. Molecular diagnosis of intrahepatic cholangiocarcinoma

    PubMed Central

    Haga, Hiroaki; Patel, Tushar

    2015-01-01

    Intrahepatic cholangiocarcinomas (iCCA) are primary intrahepatic malignancies originating from biliary epithelia. While both hepatocellular cancer and iCCA can present as mass lesions within the liver, these cancers are distinct in their morphology, etiology, pathology, natural history and response to therapy. There is a need for accurate and sensitive molecular markers for the diagnosis of iCCA. Recent advances in elucidating molecular and genetic characteristics of iCCA offer the potential of molecular-based diagnosis of iCCA. Specific genetic mutations of IDH1/2, BAP1, p53, and KRAS, FGFR gene fusions and alterations in microRNA have all been described in iCCA. Although there are no accurate serum or biliary biomarkers currently available for diagnosis of iCCA, several potential candidates have been identified. Knowledge of specific genetic or molecular abnormalities offers potential for individualized approaches for the treatment of patients with iCCA in the future. PMID:25267595

  5. Accurate diagnosis of Helicobacter pylori. Other tests.

    PubMed

    Bravos, E D; Gilman, R H

    2000-12-01

    The application of polymerase chain reaction (PCR) with respect to Helicobacter pylori is useful for molecular epidemiologic aspects and detection purposes. The authors address the current detection methods by PCR aimed at detecting H. pylori in clinical samples collected by less invasive methods, such as gastric juice, saliva, dental plaque, and feces. Enzyme immunoassay also is discussed.

  6. Accurate Molecular Polarizabilities Based on Continuum Electrostatics

    PubMed Central

    Truchon, Jean-François; Nicholls, Anthony; Iftimie, Radu I.; Roux, Benoît; Bayly, Christopher I.

    2013-01-01

    A novel approach for representing the intramolecular polarizability as a continuum dielectric is introduced to account for molecular electronic polarization. It is shown, using a finite-difference solution to the Poisson equation, that the Electronic Polarization from Internal Continuum (EPIC) model yields accurate gas-phase molecular polarizability tensors for a test set of 98 challenging molecules composed of heteroaromatics, alkanes and diatomics. The electronic polarization originates from a high intramolecular dielectric that produces polarizabilities consistent with B3LYP/aug-cc-pVTZ and experimental values when surrounded by vacuum dielectric. In contrast to other approaches to model electronic polarization, this simple model avoids the polarizability catastrophe and accurately calculates molecular anisotropy with the use of very few fitted parameters and without resorting to auxiliary sites or anisotropic atomic centers. On average, the unsigned error in the average polarizability and anisotropy compared to B3LYP are 2% and 5%, respectively. The correlation between the polarizability components from B3LYP and this approach lead to a R2 of 0.990 and a slope of 0.999. Even the F2 anisotropy, shown to be a difficult case for existing polarizability models, can be reproduced within 2% error. In addition to providing new parameters for a rapid method directly applicable to the calculation of polarizabilities, this work extends the widely used Poisson equation to areas where accurate molecular polarizabilities matter. PMID:23646034

  7. Molecular diagnosis of peanut allergy.

    PubMed

    Chan, Susan M H; Dumitru, Catalina; Turcanu, Victor

    2012-11-01

    Peanut allergy prevalence has increased in developed countries over the last few decades in the frame of the allergy epidemics, currently affecting 1-2% of children. While less frequent in developing countries, its prevalence is rising as these countries adopt a more westernized lifestyle. There is no curative treatment for peanut allergy at present so patient management relies on peanut avoidance, which requires an accurate diagnosis. Recent progress in peanut allergy diagnosis was made with the introduction of component resolved diagnosis that allows the assessment of IgE specific to individual peanut allergens. Component-resolved diagnosis needs to be interpreted in the context of clinical data but overall increases the diagnostic accuracy, as described in the typical cases that we present. Novel diagnostic tools have been proposed recently, such as the basophil activation test, mRNA expression and resonance magnetic evaluation of biomarkers. PMID:23249205

  8. Molecular diagnosis in lung diseases.

    PubMed

    Calabrese, Fiorella; Lunardi, Francesca; Popper, Helmut

    2015-01-01

    The development of different molecular biology techniques in the past decade has led to an explosion of new research in molecular pathology with consequent important applications to diagnosis, prognosis, and therapeutics, as well as a clearer concept of the disease pathogenesis. Many methods used in molecular pathology are now validated and used in several areas of pathological diagnosis, particularly on infectious and neoplastic diseases. The spectrum of infectious diseases, especially lung infective diseases, is now broadening and modifying, thus the pathologist is increasingly involved in the diagnosis of these pathologies. The precise tissue characterization of lung infections has an important impact on specific therapeutic treatment. Increased knowledge of significant alterations in lung cancer has led today to a better understanding of the pathogenic substrate underlying the development, progression and metastasis of neoplastic processes. Molecular tests are now routinely performed in different lung tumors allowing a more precise patient stratification in terms of prognosis and therapy. This review focuses on molecular pathology of the principal infective lung diseases and tumors.

  9. Molecular Diagnosis of Cystic Fibrosis.

    PubMed

    Deignan, Joshua L; Grody, Wayne W

    2016-01-01

    This unit describes a recommended approach to identifying causal genetic variants in an individual suspected of having cystic fibrosis. An introduction to the genetics and clinical presentation of cystic fibrosis is initially presented, followed by a description of the two main strategies used in the molecular diagnosis of cystic fibrosis: (1) an initial targeted variant panel used to detect only the most common cystic fibrosis-causing variants in the CFTR gene, and (2) sequencing of the entire coding region of the CFTR gene to detect additional rare causal CFTR variants. Finally, the unit concludes with a discussion regarding the analytic and clinical validity of these approaches.

  10. Molecular diagnosis of Helicobacter pylori.

    PubMed

    Westblom, T U

    1997-01-01

    H. pylori infection can be diagnosed with many different tests. If the patient is undergoing endoscopy with gastric biopsies, culture and histology remain the diagnostic methods of choice. Indirect tests include rapid urease tests, urea breath tests, and serology. Molecular methods such as PCR offer marginal improvements when done on biopsy material, but has the advantage of being able to accurately identify H. pylori in areas outside the stomach where cultures usually fail. PCR can detect low numbers of organisms in gastric juice, bile, stool and oral secretions. Because of its high sensitivity it can also be used for epidemiologic investigations of environmental sources. However, the largest role for PCR may be in molecular fingerprinting. Arbitrary Primer PCR (RAPD) on the whole bacterial genome can reliably and accurately distinguish between isolates. PCR-based RFLP analysis can separate isolates based on restriction fragment sizes in a smaller amplified genome segment. REP-PCR can group isolates into clusters that appear to have different clinical expressions. These methods promise to shed new light on the transmission and pathogenicity of H. pylori. PMID:9037621

  11. [Molecular diagnosis of mycobacterial infections].

    PubMed

    Fend, F; Langer, R; Hann von Weyhern, C W; Schulz, S; Miethke, T

    2007-01-01

    Tuberculosis remains a leading cause of morbidity and mortality worldwide. A rapid and reliable diagnosis and discrimination from infections with nontuberculous mycobacteria (NTM) is critical. Frequently, formalin-fixed, paraffin-embedded (FFPE) tissues remain the only source for detection of micro-organisms in suspected cases of mycobacterial infection. Recently, numerous methods, including PCR assays, in situ hybridization and immunohistochemistry have been developed for detection of mycobacteria in FFPE samples. PCR-based assays are directed either against M.tbc.-specific sequences, such as IS6110, or amplify regions common to many mycobacterial species, e.g. the 65 kDa antigen, and then require sequencing or restriction fragment length polymorphism for species identification. Whereas the detection of DNA of M.tbc. in the correct setting is always of clinical relevance, the presence of various NTM species has to be interpreted with great caution due to their ubiquitous nature. However, the routine application of molecular tests has demonstrated that NTM infections are more common than previously thought, even in non-immunosuppressed hosts. The introduction of real-time PCR technology allows precise quantification of mycobacterial DNA and can be used for species identification through melting point analysis or appropriate DNA probes. Application of these assays originally developed for clinical microbiology offer a great opportunity for diagnostic improvement in molecular pathology as compared to qualitative PCR, mainly due to an increased specificity and a lower risk of contamination. Given the clinical impact of a positive molecular result for M. tbc., future efforts have to be aimed at standardization and quality control. PMID:18314607

  12. [Molecular diagnosis of mycobacterial infections].

    PubMed

    Fend, F; Langer, R; Hann von Weyhern, C W; Schulz, S; Miethke, T

    2007-01-01

    Tuberculosis remains a leading cause of morbidity and mortality worldwide. A rapid and reliable diagnosis and discrimination from infections with nontuberculous mycobacteria (NTM) is critical. Frequently, formalin-fixed, paraffin-embedded (FFPE) tissues remain the only source for detection of micro-organisms in suspected cases of mycobacterial infection. Recently, numerous methods, including PCR assays, in situ hybridization and immunohistochemistry have been developed for detection of mycobacteria in FFPE samples. PCR-based assays are directed either against M.tbc.-specific sequences, such as IS6110, or amplify regions common to many mycobacterial species, e.g. the 65 kDa antigen, and then require sequencing or restriction fragment length polymorphism for species identification. Whereas the detection of DNA of M.tbc. in the correct setting is always of clinical relevance, the presence of various NTM species has to be interpreted with great caution due to their ubiquitous nature. However, the routine application of molecular tests has demonstrated that NTM infections are more common than previously thought, even in non-immunosuppressed hosts. The introduction of real-time PCR technology allows precise quantification of mycobacterial DNA and can be used for species identification through melting point analysis or appropriate DNA probes. Application of these assays originally developed for clinical microbiology offer a great opportunity for diagnostic improvement in molecular pathology as compared to qualitative PCR, mainly due to an increased specificity and a lower risk of contamination. Given the clinical impact of a positive molecular result for M. tbc., future efforts have to be aimed at standardization and quality control.

  13. Developing accurate molecular mechanics force fields for conjugated molecular systems.

    PubMed

    Do, Hainam; Troisi, Alessandro

    2015-10-14

    A rapid method to parameterize the intramolecular component of classical force fields for complex conjugated molecules is proposed. The method is based on a procedure of force matching with a reference electronic structure calculation. It is particularly suitable for those applications where molecular dynamics simulations are used to generate structures that are therefore analysed by electronic structure methods, because it is possible to build force fields that are consistent with electronic structure calculations that follow classical simulations. Such applications are commonly encountered in organic electronics, spectroscopy of complex systems and photobiology (e.g. photosynthetic systems). We illustrate the method by parameterizing the force fields of a molecule used in molecular semiconductors (2,2-dicyanovinyl-capped S,N-heteropentacene or DCV-SN5), a polymeric semiconductor (thieno[3,2-b]thiophene-diketopyrrolopyrrole TT-DPP) and a chromophore embedded in a protein environment (15,16-dihydrobiliverdin or DBV) where several hundreds of parameters need to be optimized in parallel.

  14. Accurate Documentation of Malnutrition Diagnosis Reflects Increased Healthcare Resource Utilization.

    PubMed

    Phillips, Wendy

    2015-10-01

    Nutrition support professionals often care for the sickest of hospitalized patients. An understanding of healthcare payment models can help the nutrition support professional know how documentation of nutrition status can ensure maximum resources are available to care for these patients. Medicare is the major funding source for many hospitals in the United States. Hospitals receive payments using the Acute Care Hospital Inpatient Prospective Payment System, which classifies patients into Medical Severity Diagnosis-Related Groups (MS-DRGs) to determine payment amounts. Documentation of comorbidities and complications can increase the payment hospitals receive to offset increased resource utilization. This article explains how malnutrition documentation and coding can influence the case mix index, an indicator of level of acuity of patients treated at the hospital, and the payment the hospital receives to care for the patient.

  15. A robust and accurate formulation of molecular and colloidal electrostatics

    NASA Astrophysics Data System (ADS)

    Sun, Qiang; Klaseboer, Evert; Chan, Derek Y. C.

    2016-08-01

    This paper presents a re-formulation of the boundary integral method for the Debye-Hückel model of molecular and colloidal electrostatics that removes the mathematical singularities that have to date been accepted as an intrinsic part of the conventional boundary integral equation method. The essence of the present boundary regularized integral equation formulation consists of subtracting a known solution from the conventional boundary integral method in such a way as to cancel out the singularities associated with the Green's function. This approach better reflects the non-singular physical behavior of the systems on boundaries with the benefits of the following: (i) the surface integrals can be evaluated accurately using quadrature without any need to devise special numerical integration procedures, (ii) being able to use quadratic or spline function surface elements to represent the surface more accurately and the variation of the functions within each element is represented to a consistent level of precision by appropriate interpolation functions, (iii) being able to calculate electric fields, even at boundaries, accurately and directly from the potential without having to solve hypersingular integral equations and this imparts high precision in calculating the Maxwell stress tensor and consequently, intermolecular or colloidal forces, (iv) a reliable way to handle geometric configurations in which different parts of the boundary can be very close together without being affected by numerical instabilities, therefore potentials, fields, and forces between surfaces can be found accurately at surface separations down to near contact, and (v) having the simplicity of a formulation that does not require complex algorithms to handle singularities will result in significant savings in coding effort and in the reduction of opportunities for coding errors. These advantages are illustrated using examples drawn from molecular and colloidal electrostatics.

  16. A robust and accurate formulation of molecular and colloidal electrostatics.

    PubMed

    Sun, Qiang; Klaseboer, Evert; Chan, Derek Y C

    2016-08-01

    This paper presents a re-formulation of the boundary integral method for the Debye-Hückel model of molecular and colloidal electrostatics that removes the mathematical singularities that have to date been accepted as an intrinsic part of the conventional boundary integral equation method. The essence of the present boundary regularized integral equation formulation consists of subtracting a known solution from the conventional boundary integral method in such a way as to cancel out the singularities associated with the Green's function. This approach better reflects the non-singular physical behavior of the systems on boundaries with the benefits of the following: (i) the surface integrals can be evaluated accurately using quadrature without any need to devise special numerical integration procedures, (ii) being able to use quadratic or spline function surface elements to represent the surface more accurately and the variation of the functions within each element is represented to a consistent level of precision by appropriate interpolation functions, (iii) being able to calculate electric fields, even at boundaries, accurately and directly from the potential without having to solve hypersingular integral equations and this imparts high precision in calculating the Maxwell stress tensor and consequently, intermolecular or colloidal forces, (iv) a reliable way to handle geometric configurations in which different parts of the boundary can be very close together without being affected by numerical instabilities, therefore potentials, fields, and forces between surfaces can be found accurately at surface separations down to near contact, and (v) having the simplicity of a formulation that does not require complex algorithms to handle singularities will result in significant savings in coding effort and in the reduction of opportunities for coding errors. These advantages are illustrated using examples drawn from molecular and colloidal electrostatics. PMID:27497538

  17. Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

    NASA Astrophysics Data System (ADS)

    Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

    2008-05-01

    Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

  18. Pollen Allergens for Molecular Diagnosis.

    PubMed

    Pablos, Isabel; Wildner, Sabrina; Asam, Claudia; Wallner, Michael; Gadermaier, Gabriele

    2016-04-01

    Pollen allergens are one of the main causes of type I allergies affecting up to 30% of the population in industrialized countries. Climatic changes affect the duration and intensity of pollen seasons and may together with pollution contribute to increased incidences of respiratory allergy and asthma. Allergenic grasses, trees, and weeds often present similar habitats and flowering periods compromising clinical anamnesis. Molecule-based approaches enable distinction between genuine sensitization and clinically mostly irrelevant IgE cross-reactivity due to, e. g., panallergens or carbohydrate determinants. In addition, sensitivity as well as specificity can be improved and lead to identification of the primary sensitizing source which is particularly beneficial regarding polysensitized patients. This review gives an overview on relevant pollen allergens and their usefulness in daily practice. Appropriate allergy diagnosis is directly influencing decisions for therapeutic interventions, and thus, reliable biomarkers are pivotal when considering allergen immunotherapy in the context of precision medicine.

  19. Molecular diagnosis of lymphoblastic leukemia.

    PubMed

    Goud, Kalal Iravathy; Dayakar, Seetha; Prasad, S V S S; Rao, Koteshwar N; Shaik, Amina; Vanjakshi, S

    2013-01-01

    The mixed lineage leukemia (MLL) gene at chromosome band 11q23 is commonly involved in reciprocal translocations that is detected in acute leukemia. The MLL gene, commonly known as mixed lineage leukemia or myeloid lymphoid leukemia, has been independently identified and cloned from the 11q23 breakpoint of acute leukemia. We describe a patient with acute lymphoblastic leukemia whose cells had shown reciprocal translocation between short arm (p21) of chromosome 2 and long arm (q23) of chromosome number 11 [t(2;11) (p21;q23)] by cytogenetic analysis. Fluorescence in situ hybridization analysis (FISH) was also performed for reconfirmation with a probe for MLL which showed split signals, hybridizing to both the derivative 2 and 11 chromosomes. Our study confirmed FISH as the most suitable assay for detecting MLL rearrangements because of its sensitivity and speed. It recommended that FISH should be used as complementary to conventional cytogenetic analysis. In conclusion, evaluation of the t(2;11)(p21;q23) was done by molecular clarification and flow cytometry. PMID:24125990

  20. Towards Accurate Molecular Modeling of Plastic Bonded Explosives

    NASA Astrophysics Data System (ADS)

    Chantawansri, T. L.; Andzelm, J.; Taylor, D.; Byrd, E.; Rice, B.

    2010-03-01

    There is substantial interest in identifying the controlling factors that influence the susceptibility of polymer bonded explosives (PBXs) to accidental initiation. Numerous Molecular Dynamics (MD) simulations of PBXs using the COMPASS force field have been reported in recent years, where the validity of the force field in modeling the solid EM fill has been judged solely on its ability to reproduce lattice parameters, which is an insufficient metric. Performance of the COMPASS force field in modeling EMs and the polymeric binder has been assessed by calculating structural, thermal, and mechanical properties, where only fair agreement with experimental data is obtained. We performed MD simulations using the COMPASS force field for the polymer binder hydroxyl-terminated polybutadiene and five EMs: cyclotrimethylenetrinitramine, 1,3,5,7-tetranitro-1,3,5,7-tetra-azacyclo-octane, 2,4,6,8,10,12-hexantirohexaazazisowurzitane, 2,4,6-trinitro-1,3,5-benzenetriamine, and pentaerythritol tetranitate. Predicted EM crystallographic and molecular structural parameters, as well as calculated properties for the binder will be compared with experimental results for different simulation conditions. We also present novel simulation protocols, which improve agreement between experimental and computation results thus leading to the accurate modeling of PBXs.

  1. Diagnosis and Molecular Classification of Lung Cancer.

    PubMed

    Rodriguez-Canales, Jaime; Parra-Cuentas, Edwin; Wistuba, Ignacio I

    2016-01-01

    Lung cancer is a complex disease composed of diverse histological and molecular types with clinical relevance. The advent of large-scale molecular profiling has been helpful to identify novel molecular targets that can be applied to the treatment of particular lung cancer patients and has helped to reshape the pathological classification of lung cancer. Novel directions include the immunotherapy revolution, which has opened the door for new opportunities for cancer therapy and is also redefining the classification of multiple tumors, including lung cancer. In the present chapter, we will review the main current basis of the pathological diagnosis and classification of lung cancer incorporating the histopathological and molecular dimensions of the disease. PMID:27535388

  2. Postmortem molecular diagnosis of sickle beta thalassaemia.

    PubMed

    Kutlar, F; Mirmow, D; Glendenning, M; Holley, L; Kutlar, A

    2005-05-01

    This report describes a case in which the diagnosis of sickle cell disease (SCD) was established after death. The diagnosis of sickle cell syndrome was made in a 68 year old black patient who was found to have sickled red blood cells in many organs at necropsy although the disease had not been diagnosed during her lifetime. DNA was isolated from a peripheral blood smear obtained on the day of the patient's death. The beta globin gene was polymerase chain reaction amplified and sequenced, revealing that the patient had S-beta(+) thalassaemia. This study shows that blood smears are a suitable source for retrospective DNA analysis studies. This case illustrates that relatively "mild" forms of SCD can be overlooked, despite symptomatology suggestive of a sickle syndrome, and demonstrates the feasibility of the postmortem molecular diagnosis of haemoglobinopathies in such cases.

  3. Molecular diagnosis of chronic granulomatous disease.

    PubMed

    Roos, D; de Boer, M

    2014-02-01

    Patients with chronic granulomatous disease (CGD) suffer from recurrent, life-threatening bacterial and fungal infections of the skin, the airways, the lymph nodes, liver, brain and bones. Frequently found pathogens are Staphylococcus aureus, Aspergillus species, Klebsiella species, Burkholderia cepacia and Salmonella species. CGD is a rare (∼1:250 000 births) disease caused by mutations in any one of the five components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. This enzyme generates superoxide and is essential for intracellular killing of pathogens by phagocytes. Molecular diagnosis of CGD involves measuring NADPH oxidase activity in phagocytes, measuring protein expression of NADPH oxidase components and mutation analysis of genes encoding these components. Residual oxidase activity is important to know for estimation of the clinical course and the chance of survival of the patient. Mutation analysis is mandatory for genetic counselling and prenatal diagnosis. This review summarizes the different assays available for the diagnosis of CGD, the precautions to be taken for correct measurements, the flow diagram to be followed, the assays for confirmation of the diagnosis and the determinations for carrier detection and prenatal diagnosis. PMID:24016250

  4. Molecular approaches in the diagnosis of dermatophytosis.

    PubMed

    Kanbe, Toshio

    2008-01-01

    Dermatophytosis is one of the most common infectious diseases in the world and can be caused by several dermatophyte species. These species are closely related in genetic structure in spite of different phenotypic and ecological features. The morphological similarity, variability, and polymorphism of dermatophytes have meant that species identification for dermatophytes is time consuming and requires a significant degree of knowledge and technological expertise. Molecular biology-based techniques have solved problems concerning the morphology-based identification of dermatophytes and have improved our knowledge on the epidemiology of dermatophytosis. Further development of molecular diagnosis of dermatophytosis requires the investigation of additional molecular markers for diagnostic tools targeting multiple loci as well as the improvement of techniques.

  5. Molecular diagnosis of orbital inflammatory disease.

    PubMed

    Rosenbaum, James T; Choi, Dongseok; Wilson, David J; Grossniklaus, Hans E; Sibley, Cailin H; Harrington, Christina A; Planck, Stephen R

    2015-04-01

    Orbital inflammatory diseases include thyroid eye disease (TED), granulomatosis with polyangiitis (GPA), sarcoidosis, and nonspecific orbital inflammation (NSOI). Histopathological diagnosis usually relies on the clinical context and is not always definitive. Gene expression profiling provides diagnostic and therapeutic information in several malignancies, but its role in evaluating nonmalignant disease is relatively untested. We hypothesized that gene expression profiling could provide diagnostic information for NSOI. We collected formalin-fixed, paraffin-embedded orbital biopsies from 10 institutions and 83 subjects including 25 with thyroid eye disease, 25 nonspecific orbital inflammation, 20 healthy controls, 6 with granulomatosis with polyangiitis, and 7 with sarcoidosis. Tissues were divided into discovery and validation sets. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays. A random forest statistical algorithm based on data from 39 probe sets identified controls, GPA, or TED with an average accuracy of 76% (p=0.02). Random forest analysis indicated that 52% of tissues from patients with nonspecific inflammation were consistent with a diagnosis of GPA. Molecular diagnosis by gene expression profiling will augment clinical data and histopathology in differentiating forms of orbital inflammatory disease.

  6. Advanced tests for early and accurate diagnosis of Creutzfeldt-Jakob disease.

    PubMed

    Zanusso, Gianluigi; Monaco, Salvatore; Pocchiari, Maurizio; Caughey, Byron

    2016-06-01

    Early and accurate diagnosis of Creutzfeldt-Jakob disease (CJD) is a necessary to distinguish this untreatable disease from treatable rapidly progressive dementias, and to prevent iatrogenic transmission. Currently, definitive diagnosis of CJD requires detection of the abnormally folded, CJD-specific form of protease-resistant prion protein (PrP(CJD)) in brain tissue obtained postmortem or via biopsy; therefore, diagnosis of sporadic CJD in clinical practice is often challenging. Supporting investigations, including MRI, EEG and conventional analyses of cerebrospinal fluid (CSF) biomarkers, are helpful in the diagnostic work-up, but do not allow definitive diagnosis. Recently, novel ultrasensitive seeding assays, based on the amplified detection of PrP(CJD), have improved the diagnostic process; for example, real-time quaking-induced conversion (RT-QuIC) is a sensitive method to detect prion-seeding activity in brain homogenate from humans with any subtype of sporadic CJD. RT-QuIC can also be used for in vivo diagnosis of CJD: its diagnostic sensitivity in detecting PrP(CJD) in CSF samples is 96%, and its specificity is 100%. Recently, we provided evidence that RT-QuIC of olfactory mucosa brushings is a 97% sensitive and 100% specific for sporadic CJD. These assays provide a basis for definitive antemortem diagnosis of prion diseases and, in doing so, improve prospects for reducing the risk of prion transmission. Moreover, they can be used to evaluate outcome measures in therapeutic trials for these as yet untreatable infections. PMID:27174240

  7. Odontoma-associated tooth impaction: accurate diagnosis with simple methods? Case report and literature review.

    PubMed

    Troeltzsch, Matthias; Liedtke, Jan; Troeltzsch, Volker; Frankenberger, Roland; Steiner, Timm; Troeltzsch, Markus

    2012-10-01

    Odontomas account for the largest fraction of odontogenic tumors and are frequent causes of tooth impaction. A case of a 13-year-old female patient with an odontoma-associated impaction of a mandibular molar is presented with a review of the literature. Preoperative planning involved simple and convenient methods such as clinical examination and panoramic radiography, which led to a diagnosis of complex odontoma and warranted surgical removal. The clinical diagnosis was confirmed histologically. Multidisciplinary consultation may enable the clinician to find the accurate diagnosis and appropriate therapy based on the clinical and radiographic appearance. Modern radiologic methods such as cone-beam computed tomography or computed tomography should be applied only for special cases, to decrease radiation.

  8. Bovine Ephemeral Fever in Iran: Diagnosis, Isolation and Molecular Characterization

    PubMed Central

    Bakhshesh, Mehran; Abdollahi, Darab

    2015-01-01

    Background: Bovine ephemeral fever (BEFV) is an arthropod-borne disease of cattle and water buffaloes. BEFV occurs seasonally in tropical, subtropical and temperate regions of Africa, Asia and Australia. It has been known for the past decades in Iran based on clinical signs but lack of an accurate diagnosis has made the real feature of disease obscured. This is the first scientific report on isolation and identification of the agent in which molecular diagnosis of BEFV was also set up with high sensitivity and specificity. Methods: The viral agent was successfully isolated through serial passages in brain of suckling mice and cell culture. In addition, the circulating virus during the autumn 2012 in Iran was molecularly characterized based on partial G gene. Results: Alignment of 3 virus sequences from different parts of Iran revealed that they are identical suggesting that the circulating viruses were most likely the same in this period. Phylogenetic analysis of the Iranian sequences with 17 sequences in the GenBank from the world showed that it is identical to the virus circulated in Turkey during the same period suggesting that the virus was circulated in a large geographic region. Conclusion: These results offer primary information about BEFV in Iran. To better understanding the epidemiology of the virus, further studies based on seroepidemiology, molecular epidemiology, entomology and meteorology together with finding the model of animal transportation in the region are necessary. PMID:26623431

  9. Molecular approach to allergy diagnosis and therapy.

    PubMed

    Ferreira, Fatima; Wolf, Martin; Wallner, Michael

    2014-07-01

    Presently, allergy diagnosis and therapy procedures are undergoing a transition phase in which allergen extracts are being step-by-step replaced by molecule-based products. The new developments will allow clinicians to obtain detailed information on sensitization patterns, more accurate interpretation of allergic symptoms, and thus improved patients' management. In this respect, recombinant technology has been applied to develop this new generation of molecule-based allergy products. The use of recombinant allergens allows full validation of identity, quantity, homogeneity, structure, aggregation, solubility, stability, IgE-binding and the biologic potency of the products. In contrast, such parameters are extremely difficult to assay and standardize for extract-based products. In addition to the possibility of bulk production of wild type molecules for diagnostic purposes, recombinant technology opened the possibility of developing safer and more efficacious products for allergy therapy. A number of molecule-based hypoallergenic preparations have already been successfully evaluated in clinical trials, bringing forward the next generation of allergy vaccines. In this contribution, we review the latest developments in allergen characterization, molecule-based allergy diagnosis, and the application of recombinant allergens in therapeutic setups. A comprehensive overview of clinical trials using recombinant allergens as well as synthetic peptides is presented.

  10. Molecular Approach to Allergy Diagnosis and Therapy

    PubMed Central

    Wolf, Martin; Wallner, Michael

    2014-01-01

    Presently, allergy diagnosis and therapy procedures are undergoing a transition phase in which allergen extracts are being step-by-step replaced by molecule-based products. The new developments will allow clinicians to obtain detailed information on sensitization patterns, more accurate interpretation of allergic symptoms, and thus improved patients' management. In this respect, recombinant technology has been applied to develop this new generation of molecule-based allergy products. The use of recombinant allergens allows full validation of identity, quantity, homogeneity, structure, aggregation, solubility, stability, IgE-binding and the biologic potency of the products. In contrast, such parameters are extremely difficult to assay and standardize for extract-based products. In addition to the possibility of bulk production of wild type molecules for diagnostic purposes, recombinant technology opened the possibility of developing safer and more efficacious products for allergy therapy. A number of molecule-based hypoallergenic preparations have already been successfully evaluated in clinical trials, bringing forward the next generation of allergy vaccines. In this contribution, we review the latest developments in allergen characterization, molecule-based allergy diagnosis, and the application of recombinant allergens in therapeutic setups. A comprehensive overview of clinical trials using recombinant allergens as well as synthetic peptides is presented. PMID:24954310

  11. Accurate diagnosis of axillary lymph node metastasis using contrast-enhanced ultrasonography with Sonazoid

    PubMed Central

    MATSUZAWA, FUMIHIKO; EINAMA, TAKAHIRO; ABE, HIRONORI; SUZUKI, TAKASHI; HAMAGUCHI, JUN; KAGA, TERUMI; SATO, MAMI; OOMURA, MASAKO; TAKATA, YUMIKO; FUJIBE, AYAKO; TAKEDA, CHIE; TAMURA, ETSUYA; TAKETOMI, AKINOBU; KYUNO, KENICHI

    2015-01-01

    Axillary lymph node enlargement following sentinel lymph node biopsy (SLNB) is often difficult to accurately diagnose. In keeping with the characteristically tortuous and aberrant pattern of tumor neovasculature, metastatic lymph nodes exhibit peripheral and mixed vascularity, resulting in a microvasculature that is often difficult to visualize. Contrast-enhanced ultrasonography (CEUS) with Sonazoid, a new generation contrast agent for ultrasonography, allows for the visualization of lymph node microvessels and may enable a more accurate evaluation of lymph node metastasis. This is a case report of axillary lymph node enlargement following SLNB, in which CEUS with Sonazoid resulted in an accurate diagnosis. On the basis of our experience with this case, we have initiated a clinical trial to evaluate the detection of lymph node metastasis through the use of CEUS in breast cancer patients. PMID:25798257

  12. Reporting Tumor Molecular Heterogeneity in Histopathological Diagnosis

    PubMed Central

    Mafficini, Andrea; Amato, Eliana; Fassan, Matteo; Simbolo, Michele; Antonello, Davide; Vicentini, Caterina; Scardoni, Maria; Bersani, Samantha; Gottardi, Marisa; Rusev, Borislav; Malpeli, Giorgio; Corbo, Vincenzo; Barbi, Stefano; Sikora, Katarzyna O.; Lawlor, Rita T.; Tortora, Giampaolo; Scarpa, Aldo

    2014-01-01

    Background Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples. Aim To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity. Methods 35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing. Results TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis. Conclusions TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy. PMID:25127237

  13. [Molecular diagnosis of Gaucher disease in Tunisia].

    PubMed

    Cherif, W; Ben Turkia, H; Ben Rhouma, F; Riahi, I; Chemli, J; Amaral, O; Sá Miranda, M C; Caillaud, C; Kaabachi, N; Tebib, N; Abdelhak, S; Ben Dridi, M F

    2013-04-01

    Gaucher disease is a lysosomal storage disorder caused by a deficiency of the enzyme acid β-glucosidase. In order to determine the mutation spectrum in Tunisia, we performed recurrent mutation screening in 30 Tunisian patients with Gaucher disease. Screening of recurrent mutation by PCR/RFLP and direct sequencing had shown that N370S was the most frequent mutation (22/50 mutant alleles, 44%), followed by L444P mutation, which is found in 16% (8/50 mutant alleles). The recombinant allele (RecNciI) represented 14%. Our findings revealed that the genotype N370S/RecNciI was mosst frequent in patients with childhood onset and it was associated with severe visceral involvement. The screening of these three mutations provided a simple tool for molecular diagnosis of Gaucher disease in Tunisian patients and allowed also genetic counselling for their family members.

  14. Improved internal control for molecular diagnosis assays.

    PubMed

    Vinayagamoorthy, T; Maryanski, Danielle; Vinayagamoorthy, Dilanthi; Hay, Katie S L; Yo, Jacob; Carter, Mark; Wiegel, Joseph

    2015-01-01

    The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1-4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step. •The analyte and internal control have the same PCR and sequencing annealing sequences.•This method assures for little or no false negatives and false positives due to the method's design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.•This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay.

  15. Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting

    PubMed Central

    Khan, Tarik A.; Friedensohn, Simon; de Vries, Arthur R. Gorter; Straszewski, Jakub; Ruscheweyh, Hans-Joachim; Reddy, Sai T.

    2016-01-01

    High-throughput antibody repertoire sequencing (Ig-seq) provides quantitative molecular information on humoral immunity. However, Ig-seq is compromised by biases and errors introduced during library preparation and sequencing. By using synthetic antibody spike-in genes, we determined that primer bias from multiplex polymerase chain reaction (PCR) library preparation resulted in antibody frequencies with only 42 to 62% accuracy. Additionally, Ig-seq errors resulted in antibody diversity measurements being overestimated by up to 5000-fold. To rectify this, we developed molecular amplification fingerprinting (MAF), which uses unique molecular identifier (UID) tagging before and during multiplex PCR amplification, which enabled tagging of transcripts while accounting for PCR efficiency. Combined with a bioinformatic pipeline, MAF bias correction led to measurements of antibody frequencies with up to 99% accuracy. We also used MAF to correct PCR and sequencing errors, resulting in enhanced accuracy of full-length antibody diversity measurements, achieving 98 to 100% error correction. Using murine MAF-corrected data, we established a quantitative metric of recent clonal expansion—the intraclonal diversity index—which measures the number of unique transcripts associated with an antibody clone. We used this intraclonal diversity index along with antibody frequencies and somatic hypermutation to build a logistic regression model for prediction of the immunological status of clones. The model was able to predict clonal status with high confidence but only when using MAF error and bias corrected Ig-seq data. Improved accuracy by MAF provides the potential to greatly advance Ig-seq and its utility in immunology and biotechnology. PMID:26998518

  16. 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

  17. 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.

  18. Blastocystis: Genetic diversity and molecular methods for diagnosis and epidemiology.

    PubMed

    Stensvold, Christen Rune

    2013-01-01

    Blastocystis, an unusual anaerobic, single-celled stramenopile, is a remarkably successful intestinal parasite of a vast array of host species including humans. Fecal Deoxyribonucleic acid (DNA) analysis by nucleic-acid based methods in particular has led to significant advances in Blastocystis diagnostics and research over the past few years enabling accurate identification of carriers and molecular characterization by high discriminatory power. Moreover, Blastocystis comprises a multitude of subtypes (STs) (arguably species) many of which have been identified only recently and molecular epidemiological studies have revealed a significant difference in the distribution of STs across host species and geographical regions. Having a cosmopolitan distribution, the parasite is a common laboratory finding in the stools of individuals with and without intestinal symptoms across the entire globe and while the parasite remains extremely difficult to eradicate and isolate in culture, appropriate molecular tools are now available to resolve important questions such as whether the clinical outcome of colonization is linked to ST and whether Blastocystis is transmitted zoonotically. This review summarizes some of the recent advances in the molecular diagnosis of Blastocystis and gives an introduction to Blastocystis STs, including a recommendation of subtyping methodology based on recent data and method comparisons. A few suggestions for future directions and research areas are given in the light of relevant technological advances and the availability of mitochondrial and nuclear genomes.

  19. Primary leiomyoma of the liver: accurate preoperative diagnosis on liver biopsy

    PubMed Central

    Sousa, Helena T; Portela, Francisco; Semedo, Luis; Furtado, Emanuel; Marinho, Carol; Cipriano, Maria A; Leitão, Maximino C

    2009-01-01

    Primary leiomyoma of the liver is an exceptionally rare tumour in non-immunocompromised patients. Preoperative diagnosis of the lesion is difficult as complete imaging of this type of lesion is scarcely defined and preoperative biopsy was not the practice in previously reported cases. We report a voluminous primary leiomyoma of the liver occurring in a healthy middle-aged woman where a preoperative diagnosis was accurately achieved on biopsy. Because of its size, surgery was undertaken for exclusion of malignancy. A 16-month uneventful follow-up has been completed. We discuss the advantage of a preoperative diagnosis and propose that an imaging-guided liver biopsy should be undertaken, provided malignancy features are absent. This could prevent liver surgery merely for diagnostic purposes. Finally, we report imaging features that have not been previously described, namely on magnetic resonance imaging, which may provide an insight about the nature of this particular lesion and, advantageously, contribute toward a non-invasive diagnosis. PMID:21686574

  20. MicroRNA-200 Family Profile: A Promising Ancillary Tool for Accurate Cancer Diagnosis.

    PubMed

    Liu, Xiaodong; Zhang, Jianhua; Xie, Botao; Li, Hao; Shen, Jihong; Chen, Jianheng

    2016-01-01

    Cancer is one of the most threatening diseases in the world and great interests have been paid to discover accurate and noninvasive methods for cancer diagnosis. The value of microRNA-200 (miRNA-200, miR-200) family has been revealed in many studies. However, the results from various studies were inconsistent, and thus a meta-analysis was designed and performed to assess the overall value of miRNA200 in cancer diagnosis. Relevant studies were searched electronically from the following databases: PubMed, Embase, Web of Science, the Cochrane Library, and Chinese National Knowledge Infrastructure. Keyword combined with "miR-200," "cancer," and "diagnosis" in any fields was used for searching relevant studies. Then, the pooled sensitivity, specificity, area under the curve (AUC), and partial AUC were calculated using the random-effects model. Heterogeneity among individual studies was also explored by subgroup analyses. A total of 28 studies from 18 articles with an overall sample size of 3676 subjects (2097 patients and 1579 controls) were included in this meta-analysis. The overall sensitivity and specificity with 95% confidence intervals (95% CIs) are 0.709 (95% CI: 0.657-0.755) and 0.667 (95% CI: 0.617-0.713), respectively. Additionally, AUC and partial AUC for the pooled data is 0.735 and 0.627, respectively. Subgroup analyses revealed that using miRNA-200 family for cancer diagnosis is more effective in white than in Asian ethnic groups. In addition, cancer diagnosis by miRNA using circulating specimen is more effective than that using noncirculating specimen. Finally, miRNA is more accurate in diagnosing endometrial cancer than other types of cancer, and some miRNA family members (miR-200b and miR-429) have superior diagnostic accuracy than other miR-200 family members. In conclusion, the profiling of miRNA-200 family is likely to be a valuable tool in cancer detection and diagnosis.

  1. Accurate Model Selection of Relaxed Molecular Clocks in Bayesian Phylogenetics

    PubMed Central

    Baele, Guy; Li, Wai Lok Sibon; Drummond, Alexei J.; Suchard, Marc A.; Lemey, Philippe

    2013-01-01

    Recent implementations of path sampling (PS) and stepping-stone sampling (SS) have been shown to outperform the harmonic mean estimator (HME) and a posterior simulation-based analog of Akaike’s information criterion through Markov chain Monte Carlo (AICM), in Bayesian model selection of demographic and molecular clock models. Almost simultaneously, a Bayesian model averaging approach was developed that avoids conditioning on a single model but averages over a set of relaxed clock models. This approach returns estimates of the posterior probability of each clock model through which one can estimate the Bayes factor in favor of the maximum a posteriori (MAP) clock model; however, this Bayes factor estimate may suffer when the posterior probability of the MAP model approaches 1. Here, we compare these two recent developments with the HME, stabilized/smoothed HME (sHME), and AICM, using both synthetic and empirical data. Our comparison shows reassuringly that MAP identification and its Bayes factor provide similar performance to PS and SS and that these approaches considerably outperform HME, sHME, and AICM in selecting the correct underlying clock model. We also illustrate the importance of using proper priors on a large set of empirical data sets. PMID:23090976

  2. Diagnosis of Neisseria gonorrhoeae using molecular beacon.

    PubMed

    Sachdev, Divya; 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.

  3. Molecular Diagnosis of Pathogenic Sporothrix Species

    PubMed Central

    Rodrigues, Anderson Messias; de Hoog, G. Sybren; de Camargo, Zoilo Pires

    2015-01-01

    Background Sporotrichosis is a chronic (sub)cutaneous infection caused by thermodimorphic fungi in the order, Ophiostomatales. These fungi are characterized by major differences in routes of transmission, host predilections, species virulence, and susceptibilities to antifungals. Sporothrix species emerge in the form of outbreaks. Large zoonoses and sapronoses are ongoing in Brazil and China, respectively. Current diagnostic methods based on morphology and physiology are inaccurate due to closely related phenotypes with overlapping components between pathogenic and non-pathogenic Sporothrix. There is a critical need for new diagnostic tools that are specific, sensitive, and cost-effective. Methodology We developed a panel of novel markers, based on calmodulin (CAL) gene sequences, for the large-scale diagnosis and epidemiology of clinically relevant members of the Sporothrix genus, and its relative, Ophiostoma. We identified specific PCR-based markers for S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and O. stenoceras. We employed a murine model of disseminated sporotrichosis to optimize a PCR assay for detecting Sporothrix in clinical specimens. Results Primer-BLAST searches revealed candidate sequences that were conserved within a single species. Species-specific primers showed no significant homology with human, mouse, or microorganisms outside the Sporothrix genus. The detection limit was 10–100 fg of DNA in a single round of PCR for identifying S. brasiliensis, S. schenckii, S. globosa, S. mexicana, and S. pallida. A simple, direct PCR assay, with conidia as a source of DNA, was effective for rapid, low-cost genotyping. Samples from a murine model of disseminated sporotrichosis confirmed the feasibility of detecting S. brasiliensis and S. schenckii DNA in spleen, liver, lungs, heart, brain, kidney, tail, and feces of infected animals. Conclusions This PCR-based method could successfully detect and identify a single species in samples

  4. Accurate diagnosis of thyroid follicular lesions from nuclear morphology using supervised learning.

    PubMed

    Ozolek, John A; Tosun, Akif Burak; Wang, Wei; Chen, Cheng; Kolouri, Soheil; Basu, Saurav; Huang, Hu; Rohde, Gustavo K

    2014-07-01

    Follicular lesions of the thyroid remain significant diagnostic challenges in surgical pathology and cytology. The diagnosis often requires considerable resources and ancillary tests including immunohistochemistry, molecular studies, and expert consultation. Visual analyses of nuclear morphological features, generally speaking, have not been helpful in distinguishing this group of lesions. Here we describe a method for distinguishing between follicular lesions of the thyroid based on nuclear morphology. The method utilizes an optimal transport-based linear embedding for segmented nuclei, together with an adaptation of existing classification methods. We show the method outputs assignments (classification results) which are near perfectly correlated with the clinical diagnosis of several lesion types' lesions utilizing a database of 94 patients in total. Experimental comparisons also show the new method can significantly outperform standard numerical feature-type methods in terms of agreement with the clinical diagnosis gold standard. In addition, the new method could potentially be used to derive insights into biologically meaningful nuclear morphology differences in these lesions. Our methods could be incorporated into a tool for pathologists to aid in distinguishing between follicular lesions of the thyroid. In addition, these results could potentially provide nuclear morphological correlates of biological behavior and reduce health care costs by decreasing histotechnician and pathologist time and obviating the need for ancillary testing.

  5. Accurate diagnosis of thyroid follicular lesions from nuclear morphology using supervised learning

    PubMed Central

    Ozolek, John A.; Tosun, Akif Burak; Wang, Wei; Chen, Cheng; Kolouri, Soheil; Basu, Saurav; Huang, Hu; Rohde, Gustavo K.

    2014-01-01

    Follicular lesions of the thyroid remain significant diagnostic challenges in surgical pathology and cytology. The diagnosis often requires considerable resources and ancillary tests including immunohistochemistry, molecular studies, and expert consultation. Visual analyses of nuclear morphological features, generally speaking, have not been helpful in distinguishing this group of lesions. Here we describe a method for distinguishing between follicular lesions of the thyroid based on nuclear morphology. The method utilizes an optimal transport-based linear embedding for segmented nuclei, together with an adaptation of existing classification methods. We show the method outputs assignments (classification results) which are near perfectly correlated with the clinical diagnosis of several lesion types' lesions utilizing a database of 94 patients in total. Experimental comparisons also show the new method can significantly outperform standard numerical feature-type methods in terms of agreement with the clinical diagnosis gold standard. In addition, the new method could potentially be used to derive insights into biologically meaningful nuclear morphology differences in these lesions. Our methods could be incorporated into a tool for pathologists to aid in distinguishing between follicular lesions of the thyroid. In addition, these results could potentially provide nuclear morphological correlates of biological behavior and reduce health care costs by decreasing histotechnician and pathologist time and obviating the need for ancillary testing. PMID:24835183

  6. Molecular diagnosis of α-thalassemias by the colorimetric nanogold.

    PubMed

    Chomean, Sirinart; Wangmaung, Nantawan; Sritongkham, Pornpimol; Promptmas, Chamras; Mas-Oodi, Sumana; Tanyong, Dalina; Ittarat, Wanida

    2014-02-21

    A new application of gold nanoparticles (AuNPs) as a colorimetric method for gene detection of α-thalassemia 1 (SEA deletion) is reported here for the first time. This technique is based on color changes from salt-induced aggregation of un-hybridized nanogold probes after hybridization with the target DNA. Specific DNA probes were synthesized, thiol modified and conjugated on the surface of AuNPs. The target DNA was amplified and hybridized with the AuNPs-immobilized probe. Salt solution (NaCl) was added to induce aggregation of the un-hybridized nanogold probes. The color changes were visualized either by the naked eye or by UV-vis spectrophotometry at 520 nm. By this nanogold colorimetric method samples carrying normal α-globin genes could be successfully identified from samples carrying α-globin genes causing α-thalassemia 1 (SEA deletion), either as a carrier or disease form. Results demonstrated that the new colorimetric nanogold method is a definite gene diagnosis of α-thalassemia. It is accurate, simple, rapid, specific, sensitive, and cost effective. It is also a promising point-of-care testing (POCT) method for thalassemias and other genetic disorders. The new colorimetric nanogold is a method of choice for areas where access to sophisticated molecular diagnosis is limited.

  7. The molecular mechanisms, diagnosis and management of congenital hyperinsulinism

    PubMed Central

    Senniappan, Senthil; Arya, Ved Bhushan; Hussain, Khalid

    2013-01-01

    Congenital hyperinsulinism (CHI) is the result of unregulated insulin secretion from the pancreatic β-cells leading to severe hypoglycaemia. In these patients it is important to make an accurate diagnosis and initiate the appropriate management so as to avoid hypoglycemic episodes and prevent the potentially associated complications like epilepsy, neurological impairment and cerebral palsy. At a genetic level abnormalities in eight different genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A and UCP2) have been reported with CHI. Loss of function mutations in ABCC8/KCNJ11 lead to the most severe forms of CHI which are usually medically unresponsive. At a histological level there are two major subgroups, diffuse and focal, each with a different genetic etiology. The focal form is sporadic in inheritance and is localized to a small region of the pancreas whereas the diffuse form is inherited in an autosomal recessive (or dominant) manner. Imaging using a specialized positron emission tomography scan with the isotope fluroine-18 L-3, 4-dihydroxyphenyalanine (18F-DOPA-PET-CT) is used to accurately locate the focal lesion pre-operatively and if removed can cure the patient from hypoglycemia. Understanding the molecular mechanisms, the histological basis, improvements in imaging modalities and surgical techniques have all improved the management of patients with CHI. PMID:23776849

  8. Barriers to accurate diagnosis and effective management of heart failure in primary care: qualitative study

    PubMed Central

    Fuat, Ahmet; Hungin, A Pali S; Murphy, Jeremy James

    2003-01-01

    Objective To ascertain the beliefs, current practices, and decision making of general practitioners in the diagnosis and management of suspected heart failure in primary care, with a view to identifying barriers to good care. Design A qualitative approach using focus groups with 30 general practitioners from four primary care groups. The sampling strategy was stratified and purposive. The contents of interviews were transcribed and analysed according to the principles of “pragmatic variant” grounded theory. Setting North east England. Results Three categories of difficulties contribute to variations in medical practice and to the reasons why general practitioners experience difficulties in diagnosing and managing heart failure. The first is uncertainty about clinical practice, including lack of confidence in establishing an accurate diagnosis and worries about using angiotensin converting enzyme inhibitors, β blockers, and spironolactone in patients who are often elderly and frail, with comorbidity and polypharmacy. The second is a lack of awareness of relevant research evidence in what was perceived to be a complex and rapidly changing therapeutic field. Doubts about the applicability of research findings in primary care, and fear of information overload also emerged. The third category consists of influences of individual preference and local organisational factors. Medical training, negative clinical experiences, and outside agencies influenced the behaviour of general practitioners and professional culture. Local factors included the availability of diagnostic services, resources (such as accessible cardiologists), and interactions between professionals in primary or secondary care, and they seemed to shape the practice and decision making processes in primary care. Conclusions The national service framework for coronary heart disease stresses that the substandard care of patients with heart failure is unacceptable. This study identified barriers to be

  9. Primary ciliary dyskinesia: From diagnosis to molecular mechanisms.

    PubMed

    Paff, Tamara; Daniels, Johannes M A; Pals, Gerard; Haarman, Eric G

    2014-06-01

    Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder affecting motile cilia. This can lead to neonatal respiratory distress, early onset upper and lower airway infections, laterality abnormalities and sub- or infertility. Although disease progression shows large individual variability, all adult patients eventually develop extensive bronchiectasis. As in cystic fibrosis, early diagnosis and frequent follow-up with microbiological control is the best therapeutic strategy, as other treatment options are lacking. PCD is underdiagnosed and diagnosed late due to clinical unawareness, limited availability of diagnostic tests and difficult interpretation of test results. Diagnosis is currently based on a combination of assessment of ciliary motion and ultrastructure by high-speed video microscopy and electron microscopy, respectively. As nasal nitric oxide is low in almost all PCD patients, these measurements can be used for screening. Although there are 26 PCD genes known so far, the genetic basis of the disease has not been unraveled in an estimated 30-40% of patients. However, the rapid discovery of novel PCD genes in recent years is expected to enable accurate genetic characterization of most patients in the near future. Large-scale use of next-generation sequencing and the availability of large ciliary proteomic and transcriptomic databases accelerate the identification of novel PCD genes, especially those that play a key role in cytoplasmic assembly of ciliary ultrastructural components. These genetic advances are revolutionizing the process of obtaining a molecular diagnosis for PCD as we speak and may ultimately lead to an increased understanding of ciliogenesis and function, providing novel handles for therapeutic interventions in PCD patients. PMID:27625868

  10. Primary ciliary dyskinesia: From diagnosis to molecular mechanisms

    PubMed Central

    Paff, Tamara; Daniels, Johannes M.A.; Pals, Gerard; Haarman, Eric G.

    2014-01-01

    Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder affecting motile cilia. This can lead to neonatal respiratory distress, early onset upper and lower airway infections, laterality abnormalities and sub- or infertility. Although disease progression shows large individual variability, all adult patients eventually develop extensive bronchiectasis. As in cystic fibrosis, early diagnosis and frequent follow-up with microbiological control is the best therapeutic strategy, as other treatment options are lacking. PCD is underdiagnosed and diagnosed late due to clinical unawareness, limited availability of diagnostic tests and difficult interpretation of test results. Diagnosis is currently based on a combination of assessment of ciliary motion and ultrastructure by high-speed video microscopy and electron microscopy, respectively. As nasal nitric oxide is low in almost all PCD patients, these measurements can be used for screening. Although there are 26 PCD genes known so far, the genetic basis of the disease has not been unraveled in an estimated 30–40% of patients. However, the rapid discovery of novel PCD genes in recent years is expected to enable accurate genetic characterization of most patients in the near future. Large-scale use of next-generation sequencing and the availability of large ciliary proteomic and transcriptomic databases accelerate the identification of novel PCD genes, especially those that play a key role in cytoplasmic assembly of ciliary ultrastructural components. These genetic advances are revolutionizing the process of obtaining a molecular diagnosis for PCD as we speak and may ultimately lead to an increased understanding of ciliogenesis and function, providing novel handles for therapeutic interventions in PCD patients.

  11. ACE-I Angioedema: Accurate Clinical Diagnosis May Prevent Epinephrine-Induced Harm

    PubMed Central

    Curtis, R. Mason; Felder, Sarah; Borici-Mazi, Rozita; Ball, Ian

    2016-01-01

    Introduction Upper airway angioedema is a life-threatening emergency department (ED) presentation with increasing incidence. Angiotensin-converting enzyme inhibitor induced angioedema (AAE) is a non-mast cell mediated etiology of angioedema. Accurate diagnosis by clinical examination can optimize patient management and reduce morbidity from inappropriate treatment with epinephrine. The aim of this study is to describe the incidence of angioedema subtypes and the management of AAE. We evaluate the appropriateness of treatments and highlight preventable iatrogenic morbidity. Methods We conducted a retrospective chart review of consecutive angioedema patients presenting to two tertiary care EDs between July 2007 and March 2012. Results Of 1,702 medical records screened, 527 were included. The cause of angioedema was identified in 48.8% (n=257) of cases. The most common identifiable etiology was AAE (33.1%, n=85), with a 60.0% male predominance. The most common AAE management strategies included diphenhydramine (63.5%, n=54), corticosteroids (50.6%, n=43) and ranitidine (31.8%, n=27). Epinephrine was administered in 21.2% (n=18) of AAE patients, five of whom received repeated doses. Four AAE patients required admission (4.7%) and one required endotracheal intubation. Epinephrine induced morbidity in two patients, causing myocardial ischemia or dysrhythmia shortly after administration. Conclusion AAE is the most common identifiable etiology of angioedema and can be accurately diagnosed by physical examination. It is easily confused with anaphylaxis and mismanaged with antihistamines, corticosteroids and epinephrine. There is little physiologic rationale for epinephrine use in AAE and much risk. Improved clinical differentiation of mast cell and non-mast cell mediated angioedema can optimize patient management. PMID:27330660

  12. Immunological and molecular diagnosis of cysticercosis

    PubMed Central

    Rodriguez, Silvia; Wilkins, Patricia; Dorny, Pierre

    2012-01-01

    Cysticercosis, the infection with the larval stage of Taenia solium, is a cause of neurological symptoms including seizures, affecting the quality of life of patients and their families. Diagnosis focuses on brain imaging and serological tests are mostly used as confirmatory tools. Most cases, however, occur in poor endemic areas, where both kinds of diagnostic tools are poorly available. Development of point of care diagnostic tests is one of the most important priorities for cysticercosis researches today. The ideal point of care test would require detection of viable cysticercosis and hopefully identify cases with severe or progressive forms of neurocysticercosis, leading to referral of the patient for specialized medical attention. This manuscript describes the evolution of the serological diagnosis of cysticercosis over time, and the characteristics of the most common currently available tools, their advantages and disadvantages, and their potential use in future diagnostic tests. PMID:23265553

  13. Molecular markers in the epidemiology and diagnosis of coccidioidomycosis.

    PubMed

    Duarte-Escalante, Esperanza; Frías-De-León, María Guadalupe; Zúñiga, Gerardo; Martínez-Herrera, Erick; Acosta-Altamirano, Gustavo; Reyes-Montes, María Del Rocío

    2014-01-01

    The prevalence of coccidioidomycosis in endemic areas has been observed to increase daily. To understand the causes of the spread of the disease and design strategies for fungal detection in clinical and environmental samples, scientists have resorted to molecular tools that allow fungal detection in a natural environment, reliable identification in clinical cases and the study of biological characteristics, such as reproductive and genetic structure, demographic history and diversification. We conducted a review of the most important molecular markers in the epidemiology of Coccidioides spp. and the diagnosis of coccidioidomycosis. A literature search was performed for scientific publications concerning the application of molecular tools for the epidemiology and diagnosis of coccidioidomycosis. The use of molecular markers in the epidemiological study and diagnosis of coccidioidomycosis has allowed for the typing of Coccidioides spp. isolates, improved understanding of their mode of reproduction, genetic variation and speciation and resulted in the development specific, rapid and sensitive strategies for detecting the fungus in environmental and clinical samples. Molecular markers have revealed genetic variability in Coccidioides spp. This finding influences changes in the epidemiology of coccidioidomycosis, such as the emergence of more virulent or antifungal resistant genotypes. Furthermore, the molecular markers currently used to identify Coccidioides immitis and Coccidioides posadasii are specific and sensitive. However, they must be validated to determine their application in diagnosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  14. Genetics and cardiovascular disease: the impact of molecular diagnosis.

    PubMed

    Vengoechea, Jaime; McKelvey, Kent D

    2013-04-01

    Information technology is exponentially reducing the cost of genetic testing while multiple clinical applications emerge. Genetic diagnosis increasingly impacts prevention, diagnosis and treatment of disease. In cardiovascular medicine, the establishment of a specific genetic diagnosis may affect management of cardiomyopathy, arrhythmia, connective tissue and metabolic disease. Econometric studies have determined that genetic testing is cost-effective in hypertrophic cardiomyopathy and disease-specific interventions are now available for specific conditions. Identification of a specific genetic disorder now allows for more precise medicine in the affected individual and more accurate preventive care for asymptomatic family members.

  15. Molecular methods in the laboratory diagnosis of sexually transmitted infections.

    PubMed

    Muralidhar, Sumathi

    2015-01-01

    Sexually transmitted infections (STIs) are a public health problem, and their prevalence is rising even in developed nations, in the era of HIV/AIDS. While the consequences of STIs can be serious, the good news is that many of these complications are preventable if appropriate screening is done in high-risk individuals, when infection is strongly suspected. The diagnostic tests for STIs serve many purposes. Apart from aiding in the diagnosis of typical cases, they help diagnose atypical cases, asymptomatic infections and also multiple infections. But, the test methods used must fulfill the criteria of accuracy, affordability, accessibility, efficiency, sensitivity, specificity and ease of handling. The results must be rapid, cost-effective and reliable. Most importantly, they have to be less dependent on collection techniques. The existing diagnostic methods for STIs are fraught with several challenges, including delay in results, lack of sensitivity and specificity. With the rise of the machines in diagnostic microbiology, molecular methods offer increased sensitivity, specificity and speed. They are especially useful for microorganisms that cannot be, or are difficult to cultivate. With the newer diagnostic technologies, we are on the verge of a major change in the approach to STI control. When diagnostic methods are faster and results more accurate, they are bound to improve patient care. As automation and standardization increase and human error decreases, more laboratories will adopt molecular testing methods. An overview of these methods is given here, including a note on the point-of-care tests and their usefulness in the era of rapid diagnostic tests. PMID:26392648

  16. Molecular diagnosis of egg allergy: an update.

    PubMed

    Chokshi, Niti Y; Sicherer, Scott H

    2015-01-01

    Hen's egg allergy affects up to 2.5% of young children and is potentially life-threatening. Several phenotypes of egg allergy have been identified, including those who tolerate extensively heated egg in bakery products. Diagnosis and monitoring for resolution often requires oral food challenges, which can result in anaphylaxis. Newer approaches, such as component-resolved diagnostics, microarray analysis and epitope mapping, are being evaluated to determine if these strategies can replace or reduce the need for oral food challenges. Studies suggest that elevated levels of ovomucoid IgE indicate an inability to tolerate extensively heated forms of egg. Egg protein-specific IgE/IgG4 ratios may be helpful in predicting tolerance. Additionally, patients with conformational epitopes to hen's egg are more likely to resolve their allergy compared with those with IgE binding to sequential epitopes. The pairing of microarray technology to epitope mapping is a potential tool to improve diagnosis. This review examines the current body of literature on these tools. PMID:25975845

  17. Advanced fault diagnosis methods in molecular networks.

    PubMed

    Habibi, Iman; Emamian, Effat S; Abdi, Ali

    2014-01-01

    Analysis of the failure of cell signaling networks is an important topic in systems biology and has applications in target discovery and drug development. In this paper, some advanced methods for fault diagnosis in signaling networks are developed and then applied to a caspase network and an SHP2 network. The goal is to understand how, and to what extent, the dysfunction of molecules in a network contributes to the failure of the entire network. Network dysfunction (failure) is defined as failure to produce the expected outputs in response to the input signals. Vulnerability level of a molecule is defined as the probability of the network failure, when the molecule is dysfunctional. In this study, a method to calculate the vulnerability level of single molecules for different combinations of input signals is developed. Furthermore, a more complex yet biologically meaningful method for calculating the multi-fault vulnerability levels is suggested, in which two or more molecules are simultaneously dysfunctional. Finally, a method is developed for fault diagnosis of networks based on a ternary logic model, which considers three activity levels for a molecule instead of the previously published binary logic model, and provides equations for the vulnerabilities of molecules in a ternary framework. Multi-fault analysis shows that the pairs of molecules with high vulnerability typically include a highly vulnerable molecule identified by the single fault analysis. The ternary fault analysis for the caspase network shows that predictions obtained using the more complex ternary model are about the same as the predictions of the simpler binary approach. This study suggests that by increasing the number of activity levels the complexity of the model grows; however, the predictive power of the ternary model does not appear to be increased proportionally.

  18. Comparison of PCR, culturing and Pap smear microscopy for accurate diagnosis of genital Actinomyces.

    PubMed

    Kaya, Dilek; Demirezen, Şayeste; Hasçelik, Gülşen; Gülmez Kivanç, Dolunay; Beksaç, Mehmet Sinan

    2013-05-01

    Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

  19. Rapid diagnosis of medulloblastoma molecular subgroups

    PubMed Central

    Schwalbe, Ed C.; Lindsey, Janet C.; Straughton, Debbie; Hogg, Twala L.; Cole, Michael; Megahed, Hisham; Ryan, Sarra L.; Lusher, Meryl E.; Taylor, Michael D.; Gilbertson, Richard J.; Ellison, David W.; Bailey, Simon; Clifford, Steven C.

    2011-01-01

    PURPOSE Microarray studies indicate medulloblastoma comprises distinct molecular disease subgroups, which offer potential for improved clinical management. EXPERIMENTAL DESIGN Minimal mRNA expression signatures diagnostic for the Wnt/Wingless (WNT) and Sonic Hedgehog (SHH) subgroups were developed, validated and used to assign subgroup affiliation in 173 tumours from four independent cohorts, alongside a systematic investigation of subgroup clinical and molecular characteristics. RESULTS WNT tumours (12% (21/173)) were diagnosed >5 years of age (peak, 10 years), displayed classic histology, CTNNB1 mutation (19/20), associated chromosome 6 loss and have previously been associated with favourable prognosis. SHH cases (24% (42/173)) predominated in infants (<3 years) and showed an age-dependent relationship to desmoplastic/nodular pathology; all infant desmoplastic/nodular cases (previously associated with a good outcome) were SHH-positive, but these relationships broke down in non-infants. PTCH1 mutations were common (34%; 11/32), but PTCH1 exon1c hypermethylation, chromosome 9q and REN (KCTD11) genetic loss were not SHH-associated, and SMO or SUFU mutation, PTCH1 exon1a or SUFU hypermethylation did not play a role, indicating novel activating mechanisms in the majority of SHH cases. SHH tumours were associated with an absence of COL1A2 methylation. WNT/SHH-independent medulloblastomas (64% (110/173)) showed all histologies, peaked at 3-6 years, and were exclusively associated with chromosome 17p loss. CONCLUSIONS Medulloblastoma subgroups are characterised by distinct genomic, epigenomic and clinico-pathological features, and clinical outcomes. Validated array-independent gene expression assays for the rapid assessment of subgroup affiliation in small biopsies, provide a basis for their routine clinical application, in strategies including molecular disease-risk stratification and delivery of targeted therapeutics. PMID:21325292

  20. [Molecular spectral diagnosis of star forming regions].

    PubMed

    Xi, S; Qin, S; Deng, L; Yang, J

    2001-08-01

    Stars are the basic building blocks of our universe, therefore it is one of the most important research topics in astrophysics to understand the origin and the early evolution of these objects. The current picture is that stars are formed during the collapse of a large enough self-gravitating interstellar molecular cloud. The early collapse gives birth to a fetus of a star, which is surrounded by a rotating accretion disk. The proto-star accretes interstellar matter through the disk which in turn transfer the accumulated matter to the central proto-star, then the star gets weight during the process. Observation shows that gorgeous ejection of matter always come along with the accretion process. In the presence of disks, these outflows usually escape from the system along the axis of the disk, forming so called bipolar outflows. Typical tracers of these activities are rich molecules such as CO, SiC2, C3H, C3H2 etc. Observationally, such typical molecular outflows can be detected using Doppler effect by spectroscopic measurements. Using the 13.7 m radio telescope in Delingha station of Purple Mountain Observatory, we performed a survey for 12 low temperature IRAS objects, some of the sources show high velocity properties. Detailed analysis of the Doppler profiles of IRS34 is presented. Star forming activities are clearly seen in this field. PMID:12945260

  1. Race and Socioeconomic Status as Confounding Variables in the Accurate Diagnosis of Alcoholism.

    ERIC Educational Resources Information Center

    Luepnitz, Roy R.; And Others

    1982-01-01

    Studied the incidence of bias related to race and socioeconomic status which could confound the diagnosis of alcoholism. Graduate psychology students made a diagnosis based on videotapes. Results indicated lower socioeconomic class individuals were more often diagnosed correctly for alcoholism, and Blacks were diagnosed alcoholic more often than…

  2. Next-generation sequencing-based molecular diagnosis of 82 retinitis pigmentosa probands from Northern Ireland

    PubMed Central

    Zhao, Li; Wang, Feng; Wang, Hui; Li, Yumei; Alexander, Sharon; Wang, Keqing; Willoughby, Colin E.; Zaneveld, Jacques E.; Jiang, Lichun; Soens, Zachry T.; Earle, Philip; Simpson, David

    2015-01-01

    Retinitis pigmentosa (RP) is a group of inherited retinal disorders characterized by progressive photoreceptor degeneration. An accurate molecular diagnosis is essential for disease characterization and clinical prognoses. A retinal capture panel that enriches 186 known retinal disease genes, including 55 known RP genes, was developed. Targeted next-generation sequencing was performed for a cohort of 82 unrelated RP cases from Northern Ireland, including 46 simplex cases and 36 familial cases. Disease-causing mutations were identified in 49 probands, including 28 simplex cases and 21 familial cases, achieving a solving rate of 60 %. In total, 65 pathogenic mutations were found, and 29 of these were novel. Interestingly, the molecular information of 12 probands was neither consistent with their initial inheritance pattern nor clinical diagnosis. Further clinical reassessment resulted in a refinement of the clinical diagnosis in 11 patients. This is the first study to apply next-generation sequencing-based, comprehensive molecular diagnoses to a large number of RP probands from Northern Ireland. Our study shows that molecular information can aid clinical diagnosis, potentially changing treatment options, current family counseling and management. PMID:25472526

  3. Molecular cytogenetic diagnosis of Williams syndrome

    SciTech Connect

    Hirota, Hamao; Matsuoka, Rumiko; Kimura, Misa

    1996-08-23

    Williams syndrome (WS) is characterized by distinct facial changes, growth deficiency, mental retardation, and congenital heart defect (particularly supravalvular aortic stenosis), associated at times with infantile hypercalcemia. Molecular genetic studies have indicated that hemizygosity at the elastin locus (7q11.23) causes WS. The purpose of this study was to confirm that this regional deletion, involving the elastin locus, is the cause of WS in Japan, and to clarify the correlation between the phenotype and the elastin locus. Thirty-two patients with WS and thirty of their relatives were examined by fluorescent in situ hybridization (FISH), using the WS chromosome region (WSCR) probe. All patients had cardiovascular disease (100%), 30 had typical WS facial changes (94%), 31 had mental retardation or developmental delay (97%), 16 were small-for-date at birth (50%), 14 had short stature (44%), and 13 had dental anomalies (41%). No relatives showed any manifestation of WS. Hemizygosity for a region of 7q11.23, involving the elastin locus, was found in all WS patients, but was not found in the 30 relatives. 22 refs., 4 figs., 1 tab.

  4. Update on immunologic and molecular diagnosis of human strongyloidiasis.

    PubMed

    Levenhagen, Marcelo A; Costa-Cruz, Julia M

    2014-07-01

    Human strongyloidiasis is an intestinal parasitosis that may affect 100 million individuals. However, the prevalence rates of this infection may represent smaller values than the actual data, mainly due to difficulties in its diagnosis. The aim of this study was to update the immunological and molecular methods applied to the diagnosis of human strongyloidiasis. There is a great diversity of techniques used in the diagnosis of this parasitosis, such as immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), immunoblotting, luciferase immunoprecipitation system (LIPS), dispstick and polymerase chain reaction (PCR), all with advantages and disadvantages, and with unique features for specific purposes. Considering the magnitude of strongyloidiasis and the importance of early diagnosis, due to the possibility of chronicity and hyperinfection, this study analyzes the different methods currently employed, and demonstrates the necessity of developing innovative methodologies, which also maintain diagnostic accuracy, particularly for regions with limited technological resources.

  5. Accurate Diagnosis of Severe Hypospadias Using 2D and 3D Ultrasounds

    PubMed Central

    López Ramón y Cajal, Carlos; Marín Ortiz, Elena; Sarmiento Carrera, Nerea

    2016-01-01

    The hypospadias is the most common urogenital anomaly of male neonates but the prenatal diagnosis of this is often missed before birth. We present the prenatal diagnosis of a severe penoscrotal hypospadias using 2D and 3D ultrasounds. 3D sonography allowed us the best evaluation of the genitals and their anatomical relations. This ample detailed study allowed us to show the findings to the parents and the pediatric surgeon and to configure the best information about the prognosis and surgical treatment. PMID:27774326

  6. Machine Learning Predictions of Molecular Properties: Accurate Many-Body Potentials and Nonlocality in Chemical Space.

    PubMed

    Hansen, Katja; Biegler, Franziska; Ramakrishnan, Raghunathan; Pronobis, Wiktor; von Lilienfeld, O Anatole; Müller, Klaus-Robert; Tkatchenko, Alexandre

    2015-06-18

    Simultaneously accurate and efficient prediction of molecular properties throughout chemical compound space is a critical ingredient toward rational compound design in chemical and pharmaceutical industries. Aiming toward this goal, we develop and apply a systematic hierarchy of efficient empirical methods to estimate atomization and total energies of molecules. These methods range from a simple sum over atoms, to addition of bond energies, to pairwise interatomic force fields, reaching to the more sophisticated machine learning approaches that are capable of describing collective interactions between many atoms or bonds. In the case of equilibrium molecular geometries, even simple pairwise force fields demonstrate prediction accuracy comparable to benchmark energies calculated using density functional theory with hybrid exchange-correlation functionals; however, accounting for the collective many-body interactions proves to be essential for approaching the “holy grail” of chemical accuracy of 1 kcal/mol for both equilibrium and out-of-equilibrium geometries. This remarkable accuracy is achieved by a vectorized representation of molecules (so-called Bag of Bonds model) that exhibits strong nonlocality in chemical space. In addition, the same representation allows us to predict accurate electronic properties of molecules, such as their polarizability and molecular frontier orbital energies.

  7. Machine learning predictions of molecular properties: Accurate many-body potentials and nonlocality in chemical space

    SciTech Connect

    Hansen, Katja; Biegler, Franziska; Ramakrishnan, Raghunathan; Pronobis, Wiktor; von Lilienfeld, O. Anatole; Müller, Klaus -Robert; Tkatchenko, Alexandre

    2015-06-04

    Simultaneously accurate and efficient prediction of molecular properties throughout chemical compound space is a critical ingredient toward rational compound design in chemical and pharmaceutical industries. Aiming toward this goal, we develop and apply a systematic hierarchy of efficient empirical methods to estimate atomization and total energies of molecules. These methods range from a simple sum over atoms, to addition of bond energies, to pairwise interatomic force fields, reaching to the more sophisticated machine learning approaches that are capable of describing collective interactions between many atoms or bonds. In the case of equilibrium molecular geometries, even simple pairwise force fields demonstrate prediction accuracy comparable to benchmark energies calculated using density functional theory with hybrid exchange-correlation functionals; however, accounting for the collective many-body interactions proves to be essential for approaching the “holy grail” of chemical accuracy of 1 kcal/mol for both equilibrium and out-of-equilibrium geometries. This remarkable accuracy is achieved by a vectorized representation of molecules (so-called Bag of Bonds model) that exhibits strong nonlocality in chemical space. The same representation allows us to predict accurate electronic properties of molecules, such as their polarizability and molecular frontier orbital energies.

  8. Machine learning predictions of molecular properties: Accurate many-body potentials and nonlocality in chemical space

    DOE PAGES

    Hansen, Katja; Biegler, Franziska; Ramakrishnan, Raghunathan; Pronobis, Wiktor; von Lilienfeld, O. Anatole; Müller, Klaus -Robert; Tkatchenko, Alexandre

    2015-06-04

    Simultaneously accurate and efficient prediction of molecular properties throughout chemical compound space is a critical ingredient toward rational compound design in chemical and pharmaceutical industries. Aiming toward this goal, we develop and apply a systematic hierarchy of efficient empirical methods to estimate atomization and total energies of molecules. These methods range from a simple sum over atoms, to addition of bond energies, to pairwise interatomic force fields, reaching to the more sophisticated machine learning approaches that are capable of describing collective interactions between many atoms or bonds. In the case of equilibrium molecular geometries, even simple pairwise force fields demonstratemore » prediction accuracy comparable to benchmark energies calculated using density functional theory with hybrid exchange-correlation functionals; however, accounting for the collective many-body interactions proves to be essential for approaching the “holy grail” of chemical accuracy of 1 kcal/mol for both equilibrium and out-of-equilibrium geometries. This remarkable accuracy is achieved by a vectorized representation of molecules (so-called Bag of Bonds model) that exhibits strong nonlocality in chemical space. The same representation allows us to predict accurate electronic properties of molecules, such as their polarizability and molecular frontier orbital energies.« less

  9. Machine Learning Predictions of Molecular Properties: Accurate Many-Body Potentials and Nonlocality in Chemical Space

    PubMed Central

    2015-01-01

    Simultaneously accurate and efficient prediction of molecular properties throughout chemical compound space is a critical ingredient toward rational compound design in chemical and pharmaceutical industries. Aiming toward this goal, we develop and apply a systematic hierarchy of efficient empirical methods to estimate atomization and total energies of molecules. These methods range from a simple sum over atoms, to addition of bond energies, to pairwise interatomic force fields, reaching to the more sophisticated machine learning approaches that are capable of describing collective interactions between many atoms or bonds. In the case of equilibrium molecular geometries, even simple pairwise force fields demonstrate prediction accuracy comparable to benchmark energies calculated using density functional theory with hybrid exchange-correlation functionals; however, accounting for the collective many-body interactions proves to be essential for approaching the “holy grail” of chemical accuracy of 1 kcal/mol for both equilibrium and out-of-equilibrium geometries. This remarkable accuracy is achieved by a vectorized representation of molecules (so-called Bag of Bonds model) that exhibits strong nonlocality in chemical space. In addition, the same representation allows us to predict accurate electronic properties of molecules, such as their polarizability and molecular frontier orbital energies. PMID:26113956

  10. Emergence of Aureobasidium pullulans as human fungal pathogen and molecular assay for future medical diagnosis.

    PubMed

    Chan, Giek Far; Puad, Mohamad Safwan Ahmad; Chin, Chai Fung; Rashid, Noor Aini Abdul

    2011-09-01

    Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.

  11. Molecular diagnosis of sepsis: New aspects and recent developments

    PubMed Central

    Lehman, L.; Hunfeld, K.-P.; Kost, G.

    2014-01-01

    By shortening the time to pathogen identification and allowing for detection of organisms missed by blood culture, new molecular methods may provide clinical benefits for the management of patients with sepsis. While a number of reviews on the diagnosis of sepsis have recently been published we here present up-to-date new developments including multiplex PCR, mass spectrometry and array techniques. We focus on those techniques that are commercially available and for which clinical studies have been performed and published. PMID:24678402

  12. Accurate diagnosis of Helicobacter pylori. 13C-urea breath test.

    PubMed

    Graham, D Y; Klein, P D

    2000-12-01

    The preferred schema for management of Helicobacter pylori infection is diagnosis, treatment, and confirmation of cure. The 13C-urea breath test is ideal for active H. pylori infection for those in whom endoscopy is not required (e.g., those in whom cancer is not suspected) because it offers the combination of simplicity, accuracy, reliability, and absence of exposure to radioactivity. New versions of the test also offer increasing simplicity and lower costs. PMID:11190073

  13. Molecular diagnosis of endemic and invasive mycoses: advances and challenges.

    PubMed

    Gómez, Beatriz L

    2014-01-01

    The diagnosis of endemic and invasive fungal disease remains challenging. Molecular techniques for identification of fungi now play a significant and growing role in clinical mycology and offer distinct advantages as they are faster, more sensitive and more specific. The aim of this mini-review is to provide an overview of the state of the art of molecular diagnosis of endemic and invasive fungal diseases, and to emphasize the challenges and current need for standardization of the different methods. The European Aspergillus PCR Initiative (EAPCRI) has made significant progress in developing a standard for Aspergillus polymerase chain reaction (PCR), but recognizes that the process will not be finished until clinical utility has been established in formal and extensive clinical trials. Similar efforts should be implemented for the diagnosis of the other mycoses in order to fully validate the current methods or reinforce the need to design new ones. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

  14. Application of a cell microarray chip system for accurate, highly sensitive, and rapid diagnosis for malaria in Uganda.

    PubMed

    Yatsushiro, Shouki; Yamamoto, Takeki; Yamamura, Shohei; Abe, Kaori; Obana, Eriko; Nogami, Takahiro; Hayashi, Takuya; Sesei, Takashi; Oka, Hiroaki; Okello-Onen, Joseph; Odongo-Aginya, Emmanuel I; Alai, Mary Auma; Olia, Alex; Anywar, Dennis; Sakurai, Miki; Palacpac, Nirianne Mq; Mita, Toshihiro; Horii, Toshihiro; Baba, Yoshinobu; Kataoka, Masatoshi

    2016-01-01

    Accurate, sensitive, rapid, and easy operative diagnosis is necessary to prevent the spread of malaria. A cell microarray chip system including a push column for the recovery of erythrocytes and a fluorescence detector was employed for malaria diagnosis in Uganda. The chip with 20,944 microchambers (105 μm width and 50 μm depth) was made of polystyrene. For the analysis, 6 μl of whole blood was employed, and leukocytes were practically removed by filtration through SiO2-nano-fibers in a column. Regular formation of an erythrocyte monolayer in each microchamber was observed following dispersion of an erythrocyte suspension in a nuclear staining dye, SYTO 21, onto the chip surface and washing. About 500,000 erythrocytes were analyzed in a total of 4675 microchambers, and malaria parasite-infected erythrocytes could be detected in 5 min by using the fluorescence detector. The percentage of infected erythrocytes in each of 41 patients was determined. Accurate and quantitative detection of the parasites could be performed. A good correlation between examinations via optical microscopy and by our chip system was demonstrated over the parasitemia range of 0.0039-2.3438% by linear regression analysis (R(2) = 0.9945). Thus, we showed the potential of this chip system for the diagnosis of malaria. PMID:27445125

  15. Application of a cell microarray chip system for accurate, highly sensitive, and rapid diagnosis for malaria in Uganda

    PubMed Central

    Yatsushiro, Shouki; Yamamoto, Takeki; Yamamura, Shohei; Abe, Kaori; Obana, Eriko; Nogami, Takahiro; Hayashi, Takuya; Sesei, Takashi; Oka, Hiroaki; Okello-Onen, Joseph; Odongo-Aginya, Emmanuel I.; Alai, Mary Auma; Olia, Alex; Anywar, Dennis; Sakurai, Miki; Palacpac, Nirianne MQ; Mita, Toshihiro; Horii, Toshihiro; Baba, Yoshinobu; Kataoka, Masatoshi

    2016-01-01

    Accurate, sensitive, rapid, and easy operative diagnosis is necessary to prevent the spread of malaria. A cell microarray chip system including a push column for the recovery of erythrocytes and a fluorescence detector was employed for malaria diagnosis in Uganda. The chip with 20,944 microchambers (105 μm width and 50 μm depth) was made of polystyrene. For the analysis, 6 μl of whole blood was employed, and leukocytes were practically removed by filtration through SiO2-nano-fibers in a column. Regular formation of an erythrocyte monolayer in each microchamber was observed following dispersion of an erythrocyte suspension in a nuclear staining dye, SYTO 21, onto the chip surface and washing. About 500,000 erythrocytes were analyzed in a total of 4675 microchambers, and malaria parasite-infected erythrocytes could be detected in 5 min by using the fluorescence detector. The percentage of infected erythrocytes in each of 41 patients was determined. Accurate and quantitative detection of the parasites could be performed. A good correlation between examinations via optical microscopy and by our chip system was demonstrated over the parasitemia range of 0.0039–2.3438% by linear regression analysis (R2 = 0.9945). Thus, we showed the potential of this chip system for the diagnosis of malaria. PMID:27445125

  16. Molecular Simulation of Carbon Dioxide Capture by Montmorillonite Using an Accurate and Flexible Force Field

    SciTech Connect

    Romanov, V N; Cygan, R T; Myshakin, E M

    2012-06-21

    Naturally occurring clay minerals provide a distinctive material for carbon capture and carbon dioxide sequestration. Swelling clay minerals, such as the smectite variety, possess an aluminosilicate structure that is controlled by low-charge layers that readily expand to accommodate water molecules and, potentially, CO2. Recent experimental studies have demonstrated the efficacy of intercalating CO2 in the interlayer of layered clays, but little is known about the molecular mechanisms of the process and the extent of carbon capture as a function of clay charge and structure. A series of molecular dynamics simulations and vibrational analyses have been completed to assess the molecular interactions associated with incorporation of CO2 and H2O in the interlayer of montmorillonite clay and to help validate the models with experimental observation. An accurate and fully flexible set of interatomic potentials for CO2 is developed and combined with Clayff potentials to help evaluate the intercalation mechanism and examine the effect of molecular flexibility onthe diffusion rate of CO2 in water.

  17. Molecular Simulation of the Free Energy for the Accurate Determination of Phase Transition Properties of Molecular Solids

    NASA Astrophysics Data System (ADS)

    Sellers, Michael; Lisal, Martin; Brennan, John

    2015-06-01

    Investigating the ability of a molecular model to accurately represent a real material is crucial to model development and use. When the model simulates materials in extreme conditions, one such property worth evaluating is the phase transition point. However, phase transitions are often overlooked or approximated because of difficulty or inaccuracy when simulating them. Techniques such as super-heating or super-squeezing a material to induce a phase change suffer from inherent timescale limitations leading to ``over-driving,'' and dual-phase simulations require many long-time runs to seek out what frequently results in an inexact location of phase-coexistence. We present a compilation of methods for the determination of solid-solid and solid-liquid phase transition points through the accurate calculation of the chemical potential. The methods are applied to the Smith-Bharadwaj atomistic potential's representation of cyclotrimethylene trinitramine (RDX) to accurately determine its melting point (Tm) and the alpha to gamma solid phase transition pressure. We also determine Tm for a coarse-grain model of RDX, and compare its value to experiment and atomistic counterpart. All methods are employed via the LAMMPS simulator, resulting in 60-70 simulations that total 30-50 ns. Approved for public release. Distribution is unlimited.

  18. Fiber diffraction of skin and nails provides an accurate diagnosis of malignancies.

    PubMed

    James, Veronica J

    2009-07-01

    An early diagnosis of malignancies correlates directly with a better prognosis. Yet for many malignancies there are no readily available, noninvasive, cost-effective diagnostic tests with patients often presenting too late for effective treatment. This article describes for the first time the use of fiber diffraction patterns of skin or fingernails, using X-ray sources, as a biometric diagnostic method for detecting neoplastic disorders including but not limited to melanoma, breast, colon and prostate cancers. With suitable further development, an early low-cost, totally noninvasive yet reliable diagnostic test could be conducted on a regular basis in local radiology facilities, as a confirmatory test for other diagnostic procedures or as a mass screening test using suitable small angle X-ray beam-lines at synchrotrons.

  19. Fiber diffraction of skin and nails provides an accurate diagnosis of malignancies

    SciTech Connect

    James, Veronica J.

    2009-10-21

    An early diagnosis of malignancies correlates directly with a better prognosis. Yet for many malignancies there are no readily available, noninvasive, cost-effective diagnostic tests with patients often presenting too late for effective treatment. This article describes for the first time the use of fiber diffraction patterns of skin or fingernails, using X-ray sources, as a biometric diagnostic method for detecting neoplastic disorders including but not limited to melanoma, breast, colon and prostate cancers. With suitable further development, an early low-cost, totally noninvasive yet reliable diagnostic test could be conducted on a regular basis in local radiology facilities, as a confirmatory test for other diagnostic procedures or as a mass screening test using suitable small angle X-ray beam-lines at synchrotrons.

  20. Molecular pathological diagnosis for early esophageal cancer in Kazakh patients

    PubMed Central

    AWUT, IDIRIS; NIYAZ, MADINIYET; BIEKEMITOUFU, HADETI; ZHANG, ZHU; SHEYHEDIN, ILYAR; HAO, WEN

    2011-01-01

    Chromosome abnormalities in cancer cells occur early in carcinogenesis. We employed DNA probes for the detection of cancer cells in surgical specimens in Kazakh patients with suspected esophageal carcinoma, to analyze the application of this technique during the early diagnosis of esophageal cancer. Comparative analysis was used to compare the results of pathological diagnosis with the results of FISH. We performed esophagofiberscopic biopsy examinations in 50 Kazakh patients with suspected esophageal carcinoma, including 40 males and 10 females, with an average age of 56.8 years. The final diagnosis was esophageal squamous cell carcinoma in 47 patients, and adenocarcinoma, mucinous carcinoma and small cell carcinoma in one patient each. The pathological findings of the biopsy were positive in 45 cases, and false-negative in 5. The sensitivity and specificity of pathological diagnosis were 87.2 and 100%, respectively. Using FISH to examine the same tissues, we found that 48 cases showed aberrant copy numbers in either chromosome 3 or 17, and 2 cases were false-negative, with a sensitivity and specificity of 94.8 and 100%, respectively. The copy numbers of centromeres in chromosome 3 were significantly higher than the copy numbers of centromeres in chromosome 17 (P=0.0001). Compared with biopsy pathology, the FISH test was more sensitive. Being an objective and qualitative method, the technology of molecular pathological diagnosis may effectively increase the early diagnostic rate of esophageal cancer. In addition, the centromere probe in chromosome 3 may be the most sensitive probe for the diagnosis of esophageal cancer in Kazakh patients. PMID:22740949

  1. Clinical advances in molecular biomarkers for cancer diagnosis and therapy.

    PubMed

    Sethi, Seema; Ali, Shadan; Philip, Philip A; Sarkar, Fazlul H

    2013-07-16

    Cancer diagnosis is currently undergoing a paradigm shift with the incorporation of molecular biomarkers as part of routine diagnostic panel. The molecular alteration ranges from those involving the DNA, RNA, microRNAs (miRNAs) and proteins. The miRNAs are recently discovered small non-coding endogenous single-stranded RNAs that critically regulates the development, invasion and metastasis of cancers. They are altered in cancers and have the potential to serve as diagnostic markers for cancer. Moreover, deregulating their activity offers novel cancer therapeutic approaches. The availability of high throughput techniques for the identification of altered cellular molecules allowed their use in cancer diagnosis. Their application to a variety of body specimens from blood to tissues has been helpful for appreciating their use in the clinical context. The development of innovative antibodies for immunohistochemical detection of proteins also assists in diagnosis and risk stratification. Overall, the novel cancer diagnostic tools have extended their application as prognostic risk factors and can be used as targets for personalized medicine.

  2. A large catalog of accurate distances to molecular clouds from PS1 photometry

    SciTech Connect

    Schlafly, E. F.; Rix, H.-W.; Martin, N. F.; Green, G.; Finkbeiner, D. P.; Bell, E. F.; Burgett, W. S.; Chambers, K. C.; Hodapp, K. W.; Kaiser, N.; Magnier, E. A.; Tonry, J. L.; Draper, P. W.; Metcalfe, N.; Price, P. A.

    2014-05-01

    Distance measurements to molecular clouds are important but are often made separately for each cloud of interest, employing very different data and techniques. We present a large, homogeneous catalog of distances to molecular clouds, most of which are of unprecedented accuracy. We determine distances using optical photometry of stars along lines of sight toward these clouds, obtained from PanSTARRS-1. We simultaneously infer the reddenings and distances to these stars, tracking the full probability distribution function using a technique presented in Green et al. We fit these star-by-star measurements using a simple dust screen model to find the distance to each cloud. We thus estimate the distances to almost all of the clouds in the Magnani et al. catalog, as well as many other well-studied clouds, including Orion, Perseus, Taurus, Cepheus, Polaris, California, and Monoceros R2, avoiding only the inner Galaxy. Typical statistical uncertainties in the distances are 5%, though the systematic uncertainty stemming from the quality of our stellar models is about 10%. The resulting catalog is the largest catalog of accurate, directly measured distances to molecular clouds. Our distance estimates are generally consistent with available distance estimates from the literature, though in some cases the literature estimates are off by a factor of more than two.

  3. Indirect Terahertz Spectroscopy of Molecular Ions Using Highly Accurate and Precise Mid-Ir Spectroscopy

    NASA Astrophysics Data System (ADS)

    Mills, Andrew A.; Ford, Kyle B.; Kreckel, Holger; Perera, Manori; Crabtree, Kyle N.; McCall, Benjamin J.

    2009-06-01

    With the advent of Herschel and SOFIA, laboratory methods capable of providing molecular rest frequencies in the terahertz and sub-millimeter regime are increasingly important. As of yet, it has been difficult to perform spectroscopy in this wavelength region due to the limited availability of radiation sources, optics, and detectors. Our goal is to provide accurate THz rest frequencies for molecular ions by combining previously recorded microwave transitions with combination differences obtained from high precision mid-IR spectroscopy. We are constructing a Sensitive Resolved Ion Beam Spectroscopy setup which will harness the benefits of kinematic compression in a molecular ion beam to enable very high resolution spectroscopy. This ion beam is interrogated by continuous-wave cavity ringdown spectroscopy using a home-made widely tunable difference frequency laser that utilizes two near-IR lasers and a periodically-poled lithium niobate crystal. Here, we report our efforts to optimize our ion beam spectrometer and to perform high-precision and high-accuracy frequency measurements using an optical frequency comb. footnote

  4. Diagnosis of Whipple's disease using molecular biology techniques.

    PubMed

    Cosme, Ángel; Ojeda, Evelia; Muñagorri, Ana I; Gaminde, Eduardo; Bujanda, Luis; Larzabal, Mikel; Gil, Inés

    2011-04-01

    The diagnosis of Whipple's disease (WD) is based on the existence of clinical signs and symptoms compatible with the disease and in the presence of PAS-positive diastase-resistant granules in the macrophages of the small intestine. If there is suspicion of the disease but no histological findings or only isolated extraintestinal manifestations, species-specific PCR using different sequences of the T. whippleii genome from different tissue types and biological fluids is recommended.This study reports two cases: the first patient had diarrhea and the disease was suspected after an endoscopic examination of the ileum, while the second patient had multi-systemic manifestations,particularly abdominal, thoracic, and peripheral lymphadenopathies. In both cases, the diagnosis was confirmed using molecular biology techniques to samples from the small intestine or from a retroperineal lymph node, respectively. PMID:21526877

  5. Iofetamine I 123 single photon emission computed tomography is accurate in the diagnosis of Alzheimer's disease

    SciTech Connect

    Johnson, K.A.; Holman, B.L.; Rosen, T.J.; Nagel, J.S.; English, R.J.; Growdon, J.H. )

    1990-04-01

    To determine the diagnostic accuracy of iofetamine hydrochloride I 123 (IMP) with single photon emission computed tomography in Alzheimer's disease, we studied 58 patients with AD and 15 age-matched healthy control subjects. We used a qualitative method to assess regional IMP uptake in the entire brain and to rate image data sets as normal or abnormal without knowledge of subjects'clinical classification. The sensitivity and specificity of IMP with single photon emission computed tomography in AD were 88% and 87%, respectively. In 15 patients with mild cognitive deficits (Blessed Dementia Scale score, less than or equal to 10), sensitivity was 80%. With the use of a semiquantitative measure of regional cortical IMP uptake, the parietal lobes were the most functionally impaired in AD and the most strongly associated with the patients' Blessed Dementia Scale scores. These results indicated that IMP with single photon emission computed tomography may be a useful adjunct in the clinical diagnosis of AD in early, mild disease.

  6. Molecular karyotyping: array CGH quality criteria for constitutional genetic diagnosis.

    PubMed

    Vermeesch, Joris R; Melotte, Cindy; Froyen, Guy; Van Vooren, Steven; Dutta, Binita; Maas, Nicole; Vermeulen, Stefan; Menten, Björn; Speleman, Frank; De Moor, Bart; Van Hummelen, Paul; Marynen, Peter; Fryns, Jean-Pierre; Devriendt, Koen

    2005-03-01

    Array CGH (comparative genomic hybridization) enables the identification of chromosomal copy number changes. The availability of clone sets covering the human genome opens the possibility for the widespread use of array CGH for both research and diagnostic purposes. In this manuscript we report on the parameters that were critical for successful implementation of the technology, assess quality criteria, and discuss the potential benefits and pitfalls of the technology for improved pre- and postnatal constitutional genetic diagnosis. We propose to name the genome-wide array CGH "molecular karyotyping," in analogy with conventional karyotyping that uses staining methods to visualize chromosomes.

  7. Fast and accurate quantum molecular dynamics of dense plasmas across temperature regimes

    DOE PAGES

    Sjostrom, Travis; Daligault, Jerome

    2014-10-10

    Here, we develop and implement a new quantum molecular dynamics approximation that allows fast and accurate simulations of dense plasmas from cold to hot conditions. The method is based on a carefully designed orbital-free implementation of density functional theory. The results for hydrogen and aluminum are in very good agreement with Kohn-Sham (orbital-based) density functional theory and path integral Monte Carlo calculations for microscopic features such as the electron density as well as the equation of state. The present approach does not scale with temperature and hence extends to higher temperatures than is accessible in the Kohn-Sham method and lowermore » temperatures than is accessible by path integral Monte Carlo calculations, while being significantly less computationally expensive than either of those two methods.« less

  8. Fast and accurate quantum molecular dynamics of dense plasmas across temperature regimes

    SciTech Connect

    Sjostrom, Travis; Daligault, Jerome

    2014-10-10

    Here, we develop and implement a new quantum molecular dynamics approximation that allows fast and accurate simulations of dense plasmas from cold to hot conditions. The method is based on a carefully designed orbital-free implementation of density functional theory. The results for hydrogen and aluminum are in very good agreement with Kohn-Sham (orbital-based) density functional theory and path integral Monte Carlo calculations for microscopic features such as the electron density as well as the equation of state. The present approach does not scale with temperature and hence extends to higher temperatures than is accessible in the Kohn-Sham method and lower temperatures than is accessible by path integral Monte Carlo calculations, while being significantly less computationally expensive than either of those two methods.

  9. Accurate force fields and methods for modelling organic molecular crystals at finite temperatures.

    PubMed

    Nyman, Jonas; Pundyke, Orla Sheehan; Day, Graeme M

    2016-06-21

    We present an assessment of the performance of several force fields for modelling intermolecular interactions in organic molecular crystals using the X23 benchmark set. The performance of the force fields is compared to several popular dispersion corrected density functional methods. In addition, we present our implementation of lattice vibrational free energy calculations in the quasi-harmonic approximation, using several methods to account for phonon dispersion. This allows us to also benchmark the force fields' reproduction of finite temperature crystal structures. The results demonstrate that anisotropic atom-atom multipole-based force fields can be as accurate as several popular DFT-D methods, but have errors 2-3 times larger than the current best DFT-D methods. The largest error in the examined force fields is a systematic underestimation of the (absolute) lattice energy.

  10. TROP-2 immunohistochemistry: a highly accurate method in the differential diagnosis of papillary thyroid carcinoma.

    PubMed

    Bychkov, Andrey; Sampatanukul, Pichet; Shuangshoti, Shanop; Keelawat, Somboon

    2016-08-01

    We aimed to evaluate the diagnostic utility of the novel immunohistochemical marker TROP-2 on thyroid specimens (226 tumours and 207 controls). Whole slide immunohistochemistry was performed and scored by automated digital image analysis. Non-neoplastic thyroid, follicular adenomas, follicular carcinomas, and medullary carcinomas were negative for TROP-2 immunostaining. The majority of papillary thyroid carcinoma (PTC) specimens (94/114, 82.5%) were positive for TROP-2; however, the pattern of staining differed significantly between the histopathological variants. All papillary microcarcinomas (mPTC), PTC classic variant (PTC cv), and tall cell variant (PTC tcv) were TROP-2 positive, with mainly diffuse staining. In contrast, less than half of the PTC follicular variant specimens were positive for TROP-2, with only focal immunoreactivity. TROP-2 could identify PTC cv with 98.1% sensitivity and 97.5% specificity. ROC curve analysis found that the presence of >10% of TROP-2 positive cells in a tumour supported a diagnosis of PTC. The study of intratumoural heterogeneity showed that low-volume cytological samples of PTC cv could be adequately assessed by TROP-2 immunostaining. The TROP-2 H-score (intensity multiplied by proportion) was significantly associated with PTC variant and capsular invasion in encapsulated PTC follicular variant (p<0.001). None of the baseline (age, gender) and clinical (tumour size, nodal disease, stage) parameters were correlated with TROP-2 expression. In conclusion, TROP-2 membranous staining is a very sensitive and specific marker for PTC cv, PTC tcv, and mPTC, with high overall specificity for PTC. PMID:27311870

  11. [Molecular markers: an important tool in the diagnosis, treatment and epidemiology of invasive aspergillosis].

    PubMed

    Frías-de León, María Guadalupe; Acosta-Altamirano, Gustavo; Duarte-Escalante, Esperanza; Martínez-Hernández, José Enrique; Martínez-Rivera, María de Los Ángeles; Reyes-Montes, María Del Rocío

    2014-01-01

    Increase in the incidence of invasive aspergillosis has represented a difficult problem for management of patients with this infection due to its high rate of mortality, limited knowledge concerning its diagnosis, and therapeutic practice. The difficulty in management of patients with aspergillosis initiates with detection of the fungus in the specimens of immunosuppressed patients infected with Aspergillus fumigatus; in addition, difficulty exists in terms of the development of resistance to antifungals as a consequence of their indiscriminate use in prophylactic and therapeutic practice and to ignorance concerning the epidemiological data of aspergillosis. With the aim of resolving these problems, molecular markers is employed at present with specific and accurate results. However, in Mexico, the use of molecular markers has not yet been implemented in the routine of intrahospital laboratories; despite the fact that these molecular markers has been widely referred in the literature, it is necessary for it to validated and standardized to ensure that the results obtained in any laboratory would be reliable and comparable. In the present review, we present an update on the usefulness of molecular markers in accurate identification of A. fumigatus, detection of resistance to antifugal triazoles, and epidemiological studies for establishing the necessary measures for prevention and control of aspergillosis.

  12. Surface electron density models for accurate ab initio molecular dynamics with electronic friction

    NASA Astrophysics Data System (ADS)

    Novko, D.; Blanco-Rey, M.; Alducin, M.; Juaristi, J. I.

    2016-06-01

    Ab initio molecular dynamics with electronic friction (AIMDEF) is a valuable methodology to study the interaction of atomic particles with metal surfaces. This method, in which the effect of low-energy electron-hole (e-h) pair excitations is treated within the local density friction approximation (LDFA) [Juaristi et al., Phys. Rev. Lett. 100, 116102 (2008), 10.1103/PhysRevLett.100.116102], can provide an accurate description of both e-h pair and phonon excitations. In practice, its applicability becomes a complicated task in those situations of substantial surface atoms displacements because the LDFA requires the knowledge at each integration step of the bare surface electron density. In this work, we propose three different methods of calculating on-the-fly the electron density of the distorted surface and we discuss their suitability under typical surface distortions. The investigated methods are used in AIMDEF simulations for three illustrative adsorption cases, namely, dissociated H2 on Pd(100), N on Ag(111), and N2 on Fe(110). Our AIMDEF calculations performed with the three approaches highlight the importance of going beyond the frozen surface density to accurately describe the energy released into e-h pair excitations in case of large surface atom displacements.

  13. PyVCI: A flexible open-source code for calculating accurate molecular infrared spectra

    NASA Astrophysics Data System (ADS)

    Sibaev, Marat; Crittenden, Deborah L.

    2016-06-01

    The PyVCI program package is a general purpose open-source code for simulating accurate molecular spectra, based upon force field expansions of the potential energy surface in normal mode coordinates. It includes harmonic normal coordinate analysis and vibrational configuration interaction (VCI) algorithms, implemented primarily in Python for accessibility but with time-consuming routines written in C. Coriolis coupling terms may be optionally included in the vibrational Hamiltonian. Non-negligible VCI matrix elements are stored in sparse matrix format to alleviate the diagonalization problem. CPU and memory requirements may be further controlled by algorithmic choices and/or numerical screening procedures, and recommended values are established by benchmarking using a test set of 44 molecules for which accurate analytical potential energy surfaces are available. Force fields in normal mode coordinates are obtained from the PyPES library of high quality analytical potential energy surfaces (to 6th order) or by numerical differentiation of analytic second derivatives generated using the GAMESS quantum chemical program package (to 4th order).

  14. Molecular network topology and reliability for multipurpose diagnosis

    PubMed Central

    Jalil, MA; Moongfangklang, N; Innate, K; Mitatha, S; Ali, J; Yupapin, PP

    2011-01-01

    This investigation proposes the use of molecular network topology for drug delivery and diagnosis network design. Three modules of molecular network topologies, such as bus, star, and ring networks, are designed and manipulated based on a micro- and nanoring resonator system. The transportation of the trapping molecules by light in the network is described and the theoretical background is reviewed. The quality of the network is analyzed and calculated in terms of signal transmission (ie, signal to noise ratio and crosstalk effects). Results obtained show that a bus network has advantages over star and ring networks, where the use of mesh networks is possible. In application, a thin film network can be fabricated in the form of a waveguide and embedded in artificial bone, which can be connected to the required drug targets. The particular drug/nutrient can be transported to the required targets via the particular network used. PMID:22072875

  15. Molecular imaging agents: impact on diagnosis and therapeutics in oncology

    PubMed Central

    Seaman, Marc E.; Contino, Gianmarco; Bardeesy, Nabeel; Kelly, Kimberly A.

    2011-01-01

    Imaging has become a crucial tool in oncology throughout the course of disease detection and management and is an integral part of clinical trials. Anatomic and functional imaging led the way, providing valuable information used in the diagnosis of disease, including data regarding the size and location of the tumor and on physiologic processes such as blood flow and perfusion. As understanding of cancer pathogenesis has advanced through the identification of genetic, biochemical, and cellular alterations in evolving tumors, emphasis has been made on developing methods to detect and serially monitor such alterations. This class of approaches is referred to as molecular imaging. Molecular imaging offers the potential for increasingly sensitive and specific visualization and quantification of biological processes at the cellular and molecular level. These approaches have become established as essential tools for cancer research, early cancer detection and staging and monitoring and predicting response to targeted therapies. Here, we will discuss recent advances in the development of molecular imaging agents and their implementation in basic cancer research as well as in more rationalized approaches to cancer care. PMID:20633310

  16. The Importance of Accurate Atomic and Molecular Line-lists for Characterizing Exoplanetary Atmospheres

    NASA Astrophysics Data System (ADS)

    Madhusudhan, Nikku; Freedman, R.; Tennyson, J.

    2013-06-01

    Recent advancements in exoplanet observations are placing unprecedented constraints on the physical and chemical properties of exoplanetary atmospheres. Statistically significant constraints have been placed on the abundances of atomic and molecular species, elemental abundance ratios, temperature profiles, energy circulation, presence of hazes/clouds, and non-equilibrium chemistry, in several exoplanetary atmospheres, including gas giants, ice giants, as well as super-Earths, over a wide temperature range. The chemical constraints have also motivated new paradigms for classifying exoplanets and new efforts to constraint their formation conditions. Central to all interpretations of exoplanet spectra, however, is the accuracy of fundamental inputs in the models, primarily, the atomic and molecular opacities, which are derived from laboratory experiments and/or ab initio numerical calculations. In this talk, we will review the state-of-the-art in atomic and molecular line-lists as applied to studies of exoplanetary atmospheres. We will discuss examples where advances in laboratory astrophysics, experimental and computational, have addressed important problems in the area of exoplanetary atmospheres, as well as outstanding questions requiring new experiments and/or theoretical calculations. For example, recent studies are suggesting that high-temperature line-lists of hydrocarbons (CH4, C2H2, HCN, etc.), and several metal hydrides, in addition to refined line-lists of several well-studied molecules, are important to accurately interpret exoplanetary spectra. We will highlight several fundamental questions in the area that require new efforts in laboratory astrophysics. Besides their importance in interpreting observations with current instruments, the refined parameters are also critical in the assessment of future facilities for exoplanet characterization, such as JWST, GMT, etc.

  17. Clinical relevance of molecular diagnosis in pet allergy.

    PubMed

    Uriarte, S A; Sastre, J

    2016-07-01

    We describe the pattern of sensitisation to pet IgE components and its association with clinical symptoms. Hundred and fifty nine consecutive patients with rhinitis/asthma sensitised to dog, cat, and horse were recruited. Specific IgE to whole extracts and to pet recombinant allergens were performed. Only 5% of patients were monosensitised to animal allergens. Specific IgE to Can f 1 was significantly associated with persistent rhinitis, Can f 2 with asthma diagnosis, Can f 3 with moderate/severe rhinitis (M/S-R) and asthma diagnosis (AD), and Can f 5 with persistent and M/S-R. Positive IgE to Fel d 2 was significantly associated with M/S-R and AD, Equ c 1 with M/S-R and Equ c 3 with persistent rhinitis, AD and severe asthma. Sensitisation to ≥2 molecules or to pet albumins was associated with more severe respiratory symptoms. Molecular diagnosis in patients with pet allergy may also help clinicians to predict clinical symptoms and their severity. PMID:27108666

  18. Early diagnosis of complex diseases by molecular biomarkers, network biomarkers, and dynamical network biomarkers.

    PubMed

    Liu, Rui; Wang, Xiangdong; Aihara, Kazuyuki; Chen, Luonan

    2014-05-01

    Many studies have been carried out for early diagnosis of complex diseases by finding accurate and robust biomarkers specific to respective diseases. In particular, recent rapid advance of high-throughput technologies provides unprecedented rich information to characterize various disease genotypes and phenotypes in a global and also dynamical manner, which significantly accelerates the study of biomarkers from both theoretical and clinical perspectives. Traditionally, molecular biomarkers that distinguish disease samples from normal samples are widely adopted in clinical practices due to their ease of data measurement. However, many of them suffer from low coverage and high false-positive rates or high false-negative rates, which seriously limit their further clinical applications. To overcome those difficulties, network biomarkers (or module biomarkers) attract much attention and also achieve better performance because a network (or subnetwork) is considered to be a more robust form to characterize diseases than individual molecules. But, both molecular biomarkers and network biomarkers mainly distinguish disease samples from normal samples, and they generally cannot ensure to identify predisease samples due to their static nature, thereby lacking ability to early diagnosis. Based on nonlinear dynamical theory and complex network theory, a new concept of dynamical network biomarkers (DNBs, or a dynamical network of biomarkers) has been developed, which is different from traditional static approaches, and the DNB is able to distinguish a predisease state from normal and disease states by even a small number of samples, and therefore has great potential to achieve "real" early diagnosis of complex diseases. In this paper, we comprehensively review the recent advances and developments on molecular biomarkers, network biomarkers, and DNBs in particular, focusing on the biomarkers for early diagnosis of complex diseases considering a small number of samples and high

  19. Workshop on molecular methods for genetic diagnosis. Final technical report

    SciTech Connect

    Rinchik, E.M.

    1997-07-01

    The Sarah Lawrence College Human Genetics Program received Department of Energy funding to offer a continuing medical education workshop for genetic counselors in the New York metropolitan area. According to statistics from the National Society of Genetic Counselors, there are approximately 160 genetic counselors working in the tri-state area (New York, New Jersey, and Connecticut), and many of them had been working in the field for more than 10 years. Thus, there was a real need to offer these counselors an in-depth opportunity to learn the specifics of the major advances in molecular genetics, and, in particular, the new approaches to diagnostic testing for genetic disease. As a result of the DOE Award DE-FG02-95ER62048 ($20,583), in July 1995 we offered the {open_quotes}Workshop on Molecular Methods for Genetic Diagnosis{close_quotes} for 24 genetic counselors in the New York metropolitan area. The workshop included an initial review session on the basics of molecular biology, lectures and discussions on past and current topics in molecular genetics and diagnostic procedures, and, importantly, daily laboratory exercises. Each counselor gained not only background, but also firsthand experience, in the major techniques of biochemical and molecular methods for diagnosing genetic diseases as well as in mathematical and computational techniques involved in human genetics analyses. Our goal in offering this workshop was not to make genetic counselors experts in these laboratory diagnostic techniques, but to acquaint them, by hands-on experience, about some of the techniques currently in use. We also wanted to provide them a technical foundation upon which they can understand and appreciate new technical developments arising in the near future.

  20. Collagen, type XI, alpha 1: an accurate marker for differential diagnosis of breast carcinoma invasiveness in core needle biopsies.

    PubMed

    Freire, Javier; Domínguez-Hormaetxe, Saioa; Pereda, Saray; De Juan, Ana; Vega, Alfonso; Simón, Laureano; Gómez-Román, Javier

    2014-12-01

    Accurate diagnosis of invasive breast lesions, when analyzed by Core Needle Biopsy, may suppose a major challenge for the pathologist. Various markers of invasiveness such as laminin, S-100 protein, P63 or calponin have been described; however, none of them is completely reliable. The use of a specific marker of the infiltrating tumor microenvironment seems vital to support the diagnosis of invasive against in situ lesions. At this point, Collagen, type XI, alpha 1 (COL11A1), might be helpful since it has been described to be associated to cancer associated fibroblasts in other tumors such as lung, pancreas or colorectal. This paper aims to analyze the role of COL11A1 as a marker of invasiveness in breast tumor lesions. Two hundred and one breast Core Needle Biopsy samples were analyzed by immunohistochemistry against pro-COL11A1. The results show a significant difference (p < 0.0001) when comparing the expression in infiltrative tumors (93%) versus immunostaining of non-invasive lesions (4%). Forty cases of underestimated DCIS were also stained for COL11A1, presenting a sensitivity of 90% when compared with p63 and calponin which not tagged invasion. In conclusion, pro-COL11A1 expression is a promising marker of invasive breast lesions, and may be included in immunohistochemical panels aiming at identifying infiltration in problematic breast lesions.

  1. Accurate calculation of binding energies for molecular clusters - Assessment of different models

    NASA Astrophysics Data System (ADS)

    Friedrich, Joachim; Fiedler, Benjamin

    2016-06-01

    In this work we test different strategies to compute high-level benchmark energies for medium-sized molecular clusters. We use the incremental scheme to obtain CCSD(T)/CBS energies for our test set and carefully validate the accuracy for binding energies by statistical measures. The local errors of the incremental scheme are <1 kJ/mol. Since they are smaller than the basis set errors, we obtain higher total accuracy due to the applicability of larger basis sets. The final CCSD(T)/CBS benchmark values are ΔE = - 278.01 kJ/mol for (H2O)10, ΔE = - 221.64 kJ/mol for (HF)10, ΔE = - 45.63 kJ/mol for (CH4)10, ΔE = - 19.52 kJ/mol for (H2)20 and ΔE = - 7.38 kJ/mol for (H2)10 . Furthermore we test state-of-the-art wave-function-based and DFT methods. Our benchmark data will be very useful for critical validations of new methods. We find focal-point-methods for estimating CCSD(T)/CBS energies to be highly accurate and efficient. For foQ-i3CCSD(T)-MP2/TZ we get a mean error of 0.34 kJ/mol and a standard deviation of 0.39 kJ/mol.

  2. Hydration free energies of cyanide and hydroxide ions from molecular dynamics simulations with accurate force fields

    USGS Publications Warehouse

    Lee, M.W.; Meuwly, M.

    2013-01-01

    The evaluation of hydration free energies is a sensitive test to assess force fields used in atomistic simulations. We showed recently that the vibrational relaxation times, 1D- and 2D-infrared spectroscopies for CN(-) in water can be quantitatively described from molecular dynamics (MD) simulations with multipolar force fields and slightly enlarged van der Waals radii for the C- and N-atoms. To validate such an approach, the present work investigates the solvation free energy of cyanide in water using MD simulations with accurate multipolar electrostatics. It is found that larger van der Waals radii are indeed necessary to obtain results close to the experimental values when a multipolar force field is used. For CN(-), the van der Waals ranges refined in our previous work yield hydration free energy between -72.0 and -77.2 kcal mol(-1), which is in excellent agreement with the experimental data. In addition to the cyanide ion, we also study the hydroxide ion to show that the method used here is readily applicable to similar systems. Hydration free energies are found to sensitively depend on the intermolecular interactions, while bonded interactions are less important, as expected. We also investigate in the present work the possibility of applying the multipolar force field in scoring trajectories generated using computationally inexpensive methods, which should be useful in broader parametrization studies with reduced computational resources, as scoring is much faster than the generation of the trajectories.

  3. An efficient and accurate molecular alignment and docking technique using ab initio quality scoring

    PubMed Central

    Füsti-Molnár, László; Merz, Kenneth M.

    2008-01-01

    An accurate and efficient molecular alignment technique is presented based on first principle electronic structure calculations. This new scheme maximizes quantum similarity matrices in the relative orientation of the molecules and uses Fourier transform techniques for two purposes. First, building up the numerical representation of true ab initio electronic densities and their Coulomb potentials is accelerated by the previously described Fourier transform Coulomb method. Second, the Fourier convolution technique is applied for accelerating optimizations in the translational coordinates. In order to avoid any interpolation error, the necessary analytical formulas are derived for the transformation of the ab initio wavefunctions in rotational coordinates. The results of our first implementation for a small test set are analyzed in detail and compared with published results of the literature. A new way of refinement of existing shape based alignments is also proposed by using Fourier convolutions of ab initio or other approximate electron densities. This new alignment technique is generally applicable for overlap, Coulomb, kinetic energy, etc., quantum similarity measures and can be extended to a genuine docking solution with ab initio scoring. PMID:18624561

  4. A survey of factors contributing to accurate theoretical predictions of atomization energies and molecular structures

    NASA Astrophysics Data System (ADS)

    Feller, David; Peterson, Kirk A.; Dixon, David A.

    2008-11-01

    High level electronic structure predictions of thermochemical properties and molecular structure are capable of accuracy rivaling the very best experimental measurements as a result of rapid advances in hardware, software, and methodology. Despite the progress, real world limitations require practical approaches designed for handling general chemical systems that rely on composite strategies in which a single, intractable calculation is replaced by a series of smaller calculations. As typically implemented, these approaches produce a final, or "best," estimate that is constructed from one major component, fine-tuned by multiple corrections that are assumed to be additive. Though individually much smaller than the original, unmanageable computational problem, these corrections are nonetheless extremely costly. This study presents a survey of the widely varying magnitude of the most important components contributing to the atomization energies and structures of 106 small molecules. It combines large Gaussian basis sets and coupled cluster theory up to quadruple excitations for all systems. In selected cases, the effects of quintuple excitations and/or full configuration interaction were also considered. The availability of reliable experimental data for most of the molecules permits an expanded statistical analysis of the accuracy of the approach. In cases where reliable experimental information is currently unavailable, the present results are expected to provide some of the most accurate benchmark values available.

  5. Molecular Diagnosis of Eosinophilic Esophagitis by Gene Expression Profiling

    PubMed Central

    Wen, Ting; Stucke, Emily M.; Grotjan, Tommie M.; Kemme, Katherine A.; Abonia, J. Pablo; Putnam, Philip E.; Franciosi, James P.; Garza, Jose M.; Kaul, Ajay; King, Eileen C.; Collins, Margaret H.; Kushner, Jonathan P.; Rothenberg, Marc E.

    2014-01-01

    Background & Aims Gene expression profiling provides an opportunity for definitive diagnosis but has not yet been well applied to inflammatory diseases. Herein, we describe an approach for diagnosis of an emerging form of esophagitis, eosinophilic esophagitis (EoE), currently diagnosed by histology and clinical symptoms. Methods We developed an EoE diagnostic panel (EDP), comprising a 96-gene quantitative PCR array and an associated dual-algorithm that uses cluster analysis and dimensionality reduction, using a cohort of randomly selected esophageal biopsy samples from pediatric patients with EoE (n = 15) or without EoE (non-EoE controls, n = 14), subsequently vetted using a separate cohort of 194 pediatric and adult patient samples derived from both fresh or formalin-fixed paraffin embedded (FFPE) tissue: active EoE (n = 91), control (non-EoE and EoE remission, n = 57), histologically ambiguous (n = 34), and reflux (n = 12) samples. Results The EDP identified adult and pediatric patients with EoE with ~96% sensitivity and ~98% specificity, and distinguished patients with EoE in remission from controls, as well as identified patients exposed to swallowed glucorticoids. The EDP could be used with FFPE tissue RNA and distinguished patients with EoE from those with reflux esophagitis, identified by pH-impedance testing. Preliminary evidence showed that the EDP could identify patients likely to have disease relapse following treatment. Conclusions We developed a molecular diagnostic test (referred as the EDP) that identifies patients with esophagitis in a fast, objective, and mechanistic manner, offering an opportunity to improve diagnosis and treatment, and a platform approach for other inflammatory diseases. PMID:23978633

  6. First International External Quality Assessment Study on Molecular and Serological Methods for Yellow Fever Diagnosis

    PubMed Central

    Domingo, Cristina; Escadafal, Camille; Rumer, Leonid; Méndez, Jairo A.; García, Paquita; Sall, Amadou A.; Teichmann, Anette; Donoso-Mantke, Oliver; Niedrig, Matthias

    2012-01-01

    Objective We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis. Study Design For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included. Results Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols. Conclusion This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for

  7. Dawn of ocular gene therapy: implications for molecular diagnosis in retinal disease

    PubMed Central

    Jacques, ZANEVELD; Feng, WANG; Xia, WANG; Rui, CHEN

    2013-01-01

    Personalized medicine aims to utilize genomic information about patients to tailor treatment. Gene replacement therapy for rare genetic disorders is perhaps the most extreme form of personalized medicine, in that the patients’ genome wholly determines their treatment regimen. Gene therapy for retinal disorders is poised to become a clinical reality. The eye is an optimal site for gene therapy due to the relative ease of precise vector delivery, immune system isolation, and availability for monitoring of any potential damage or side effects. Due to these advantages, clinical trials for gene therapy of retinal diseases are currently underway. A necessary precursor to such gene therapies is accurate molecular diagnosis of the mutation(s) underlying disease. In this review, we discuss the application of Next Generation Sequencing (NGS) to obtain such a diagnosis and identify disease causing genes, using retinal disorders as a case study. After reviewing ocular gene therapy, we discuss the application of NGS to the identification of novel Mendelian disease genes. We then compare current, array based mutation detection methods against next NGS-based methods in three retinal diseases: Leber’s Congenital Amaurosis, Retinitis Pigmentosa, and Stargardt’s disease. We conclude that next-generation sequencing based diagnosis offers several advantages over array based methods, including a higher rate of successful diagnosis and the ability to more deeply and efficiently assay a broad spectrum of mutations. However, the relative difficulty of interpreting sequence results and the development of standardized, reliable bioinformatic tools remain outstanding concerns. In this review, recent advances NGS based molecular diagnoses are discussed, as well as their implications for the development of personalized medicine. PMID:23393028

  8. Fragile-X mental retardation: molecular diagnosis in Argentine patients.

    PubMed

    Florencia, Giliberto; Irene, Szijan; Veronica, Ferreiro

    2006-11-30

    Fragile-X-syndrome (FXS) is the most common type of inherited cognitive impairment. The underlying molecular alteration consists of a CGG-repeat amplification within the FMR-1 gene. The phenotype is only apparent once a threshold in the number of repeats has been exceeded (full mutation). The aim of this study was to characterize the FMR-1 CGG-repeat status in Argentine patients exhibiting mental retardation. A total of 330 blood samples from patients were analyzed by PCR and Southern blot analysis. Initially, DNA from 78 affected individuals were studied by PCR. Since this method is unable to detect high molecular weight alleles, however, we undertook a second approach using the Southern blotting technique to analyze the CGG repeat number and methylation status. Southern blot analysis showed an altered pattern in 14 out of 240 (6%) unrelated patients, with half of them presenting a mosaic pattern. Eight out of 17 families (47%) showed a (suggest deleting highlight). The characteristic FXS pattern was identified in 8/17 families (47%), and in 4 of these families 25% of the individuals presented with a mosaic model. The expansion from pre-mutation to full mutation was shown to occur both at the pre and post zygotic levels. The detection of FXS mutations has allowed us to offer more informed genetic counseling, prenatal diagnosis and reliable patient follow-up.

  9. Accurate molecular structure and spectroscopic properties for nucleobases: A combined computational - microwave investigation of 2-thiouracil as a case study

    PubMed Central

    Puzzarini, Cristina; Biczysko, Malgorzata; Barone, Vincenzo; Peña, Isabel; Cabezas, Carlos; Alonso, José L.

    2015-01-01

    The computational composite scheme purposely set up for accurately describing the electronic structure and spectroscopic properties of small biomolecules has been applied to the first study of the rotational spectrum of 2-thiouracil. The experimental investigation was made possible thanks to the combination of the laser ablation technique with Fourier Transform Microwave spectrometers. The joint experimental – computational study allowed us to determine accurate molecular structure and spectroscopic properties for the title molecule, but more important, it demonstrates a reliable approach for the accurate investigation of isolated small biomolecules. PMID:24002739

  10. [Present and future perspectives for the rapid molecular diagnosis of TB and MDR-TB].

    PubMed

    Tanasescu, Mihaela; Didilescu, Cristian; Marica, Constantin

    2013-01-01

    Tuberculosis is still one of the diseases with a major medical and social impact, and in terms of early diagnosis (which would imply a fair treatment and established at the time), difficulties related to the delay bacilli isolation in culture, decreased susceptibility testing methods to antituberculosis drugs, lack of methods for differentiation of M. Tuberculosis complex germs of non-TB Mycobacteria, may have important clinical implications. Traditional testing of anti-TB drug susceptibility on solid Löwenstein-Jensen medium (gold standard) or liquid media can only be performed using grown samples. Determining the time it takes up to 42 days on solid media and 12 days for liquid media. For MDR/XDR TB cases itis absolutely essential to reduce the detection time. In these cases rapid diagnostic methods prove their usefulness. Automatic testing in liquid medium, molecular hybridization methods are currently recommended by the current WHO guidelines. Rapid diagnosis of MDR-TBis extremely useful for the early establishment of an effective treatment tailored more accurately on the spectrum of sensitivity of the resistant strain (thus reducing the risk of developing additional resistance to other drugs) and control the spread of these strains. Genetic diagnostic methods, approved and recommended by the WHO, can reduce the time of diagnosis of TB case and, importantly, the case of MDR-TB. They do not replace the current standard diagnostic methods and resistance profile, but complete them in selected cases. PMID:24734352

  11. [Present and future perspectives for the rapid molecular diagnosis of TB and MDR-TB].

    PubMed

    Tanasescu, Mihaela; Didilescu, Cristian; Marica, Constantin

    2013-01-01

    Tuberculosis is still one of the diseases with a major medical and social impact, and in terms of early diagnosis (which would imply a fair treatment and established at the time), difficulties related to the delay bacilli isolation in culture, decreased susceptibility testing methods to antituberculosis drugs, lack of methods for differentiation of M. Tuberculosis complex germs of non-TB Mycobacteria, may have important clinical implications. Traditional testing of anti-TB drug susceptibility on solid Löwenstein-Jensen medium (gold standard) or liquid media can only be performed using grown samples. Determining the time it takes up to 42 days on solid media and 12 days for liquid media. For MDR/XDR TB cases itis absolutely essential to reduce the detection time. In these cases rapid diagnostic methods prove their usefulness. Automatic testing in liquid medium, molecular hybridization methods are currently recommended by the current WHO guidelines. Rapid diagnosis of MDR-TBis extremely useful for the early establishment of an effective treatment tailored more accurately on the spectrum of sensitivity of the resistant strain (thus reducing the risk of developing additional resistance to other drugs) and control the spread of these strains. Genetic diagnostic methods, approved and recommended by the WHO, can reduce the time of diagnosis of TB case and, importantly, the case of MDR-TB. They do not replace the current standard diagnostic methods and resistance profile, but complete them in selected cases.

  12. Exome sequencing is an efficient tool for variant late-infantile neuronal ceroid lipofuscinosis molecular diagnosis.

    PubMed

    Patiño, Liliana Catherine; Battu, Rajani; Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

    2014-01-01

    The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.

  13. Exome sequencing is an efficient tool for variant late-infantile neuronal ceroid lipofuscinosis molecular diagnosis.

    PubMed

    Patiño, Liliana Catherine; Battu, Rajani; Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

    2014-01-01

    The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease. PMID:25333361

  14. Molecular diagnosis in clinical parasitology: when and why?

    PubMed

    Wong, Samson S Y; Fung, Kitty S C; Chau, Sandy; Poon, Rosana W S; Wong, Sally C Y; Yuen, Kwok-Yung

    2014-11-01

    Microscopic detection and morphological identification of parasites from clinical specimens are the gold standards for the laboratory diagnosis of parasitic infections. The limitations of such diagnostic assays include insufficient sensitivity and operator dependence. Immunoassays for parasitic antigens are not available for most parasitic infections and have not significantly improved the sensitivity of laboratory detection. Advances in molecular detection by nucleic acid amplification may improve the detection in asymptomatic infections with low parasitic burden. Rapidly accumulating genomic data on parasites allow the design of polymerase chain reaction (PCR) primers directed towards multi-copy gene targets, such as the ribosomal and mitochondrial genes, which further improve the sensitivity. Parasitic cell or its free circulating parasitic DNA can be shed from parasites into blood and excreta which may allow its detection without the whole parasite being present within the portion of clinical sample used for DNA extraction. Multiplex nucleic acid amplification technology allows the simultaneous detection of many parasitic species within a single clinical specimen. In addition to improved sensitivity, nucleic acid amplification with sequencing can help to differentiate different parasitic species at different stages with similar morphology, detect and speciate parasites from fixed histopathological sections and identify anti-parasitic drug resistance. The use of consensus primer and PCR sequencing may even help to identify novel parasitic species. The key limitation of molecular detection is the technological expertise and expense which are usually lacking in the field setting at highly endemic areas. However, such tests can be useful for screening important parasitic infections in asymptomatic patients, donors or recipients coming from endemic areas in the settings of transfusion service or tertiary institutions with transplantation service. Such tests can also

  15. The clinical characteristics of Werner syndrome: molecular and biochemical diagnosis

    PubMed Central

    Muftuoglu, Meltem; Oshima, Junko; von Kobbe, Cayetano; Cheng, Wen-Hsing; Leistritz, Dru F.; Bohr, Vilhelm A.

    2015-01-01

    Werner syndrome (WS) is an adult onset segmental progeroid syndrome caused by mutations in the WRN gene. The WRN gene encodes a 180 kDa nuclear protein that possesses helicase and exonuclease activities. The absence of WRN protein leads to abnormalities in various DNA metabolic pathways such as DNA repair, replication and telomere maintenance. Individuals with WS generally develop normally until the third decade of life, when premature aging phenotypes and a series of age-related disorders begin to manifest. In Japan, where a founder effect has been described, the frequency of Werner heterozygotes appears to be as high as 1/180 in the general population. Due to the relatively non-specific nature of the symptoms and the lack of awareness of the condition, this disease may be under-diagnosed in other parts of the world. Genetic counseling of WS patients follows the path of other autosomal recessive disorders, with special attention needed for cancer surveillance in relatives. Molecular diagnosis of WS is made by nucleotide sequencing and, in some cases, protein analysis. It is also of potential interest to measure WRN activities in WS patients. More than 50 different disease-causing mutations in the WRN gene have been identified in WS patients from all over the world. All but one of these cases has mutations that result in the premature termination of the protein. Here we describe the clinical, molecular and biochemical characteristics of WS for use by medical professionals in a health care setting. Additional information is available through the International Registry of WS (http://www.wernersyndrome.org). PMID:18810497

  16. Molecular diagnosis and epidemiology of dengue virus infection.

    PubMed

    Chow, V T

    1997-11-01

    Early diagnosis of dengue fever contributes towards appropriate management of the disease and its potentially severe complications. Better and more rapid molecular procedures are increasingly available for detecting dengue viral RNA, antibodies and antigens. Using consensus primers based on the conserved non-structural-3 gene, the reverse transcription-polymerase chain reaction (RT-PCR) technique can amplify all four dengue virus types as well as certain flaviviruses. Consensus primers used together with four type-specific downstream primers in single-step or semi-nested RT-PCR formats can discriminate the specific dengue virus type by virtue of the diagnostic size of the RT-PCR target fragment on agarose gel electrophoresis. Alternatively, RT-PCR products may be labelled with digoxigenin and allowed to hybridise with individual biotinylated type-specific PCR primers which act as capture probes immobilised on solid phase via streptavidin-coated tubes. With automated instrumentation for enzyme-linked immunosorbent assay (ELISA), the hybridised RT-PCR products can be quantitated spectrophotometrically via anti-digoxigenin antibodies conjugated with an enzyme which reacts with colourimetric substrate. While RT-PCR is highly sensitive, specific and successfully identifies the dengue virus type in clinical serum samples and adult Aedes mosquitoes, it generally yields positive results in viraemic sera collected within 2 to 5 days of pyrexia. Sera obtained after the period of viraemia are more likely to be positive by serological tests such as IgM capture ELISA or the commercial Dengue Blot kit. The RT-PCR primers can also be utilised for direct cycle dideoxy DNA sequencing to monitor the molecular epidemiology and evolution of geographically and temporally separated virus strains. To exemplify this, nucleotide and amino acid sequence data as well as phylogenetic trees of several strains of dengue 1 and 2 viruses from patients and field-caught Aedes mosquitoes are

  17. Molecular diagnosis in clinical parasitology: when and why?

    PubMed

    Wong, Samson S Y; Fung, Kitty S C; Chau, Sandy; Poon, Rosana W S; Wong, Sally C Y; Yuen, Kwok-Yung

    2014-11-01

    Microscopic detection and morphological identification of parasites from clinical specimens are the gold standards for the laboratory diagnosis of parasitic infections. The limitations of such diagnostic assays include insufficient sensitivity and operator dependence. Immunoassays for parasitic antigens are not available for most parasitic infections and have not significantly improved the sensitivity of laboratory detection. Advances in molecular detection by nucleic acid amplification may improve the detection in asymptomatic infections with low parasitic burden. Rapidly accumulating genomic data on parasites allow the design of polymerase chain reaction (PCR) primers directed towards multi-copy gene targets, such as the ribosomal and mitochondrial genes, which further improve the sensitivity. Parasitic cell or its free circulating parasitic DNA can be shed from parasites into blood and excreta which may allow its detection without the whole parasite being present within the portion of clinical sample used for DNA extraction. Multiplex nucleic acid amplification technology allows the simultaneous detection of many parasitic species within a single clinical specimen. In addition to improved sensitivity, nucleic acid amplification with sequencing can help to differentiate different parasitic species at different stages with similar morphology, detect and speciate parasites from fixed histopathological sections and identify anti-parasitic drug resistance. The use of consensus primer and PCR sequencing may even help to identify novel parasitic species. The key limitation of molecular detection is the technological expertise and expense which are usually lacking in the field setting at highly endemic areas. However, such tests can be useful for screening important parasitic infections in asymptomatic patients, donors or recipients coming from endemic areas in the settings of transfusion service or tertiary institutions with transplantation service. Such tests can also

  18. A Novel Gene Signature for Molecular Diagnosis of Human Prostate Cancer by RT-qPCR

    PubMed Central

    Crafa, Pellegrino; Lazzaretti, Mirca; Remondini, Daniel; Ferretti, Stefania; Cortellini, Piero; Corti, Arnaldo; Bettuzzi, Saverio

    2008-01-01

    Background Prostate cancer (CaP) is one of the most relevant causes of cancer death in Western Countries. Although detection of CaP at early curable stage is highly desirable, actual screening methods present limitations and new molecular approaches are needed. Gene expression analysis increases our knowledge about the biology of CaP and may render novel molecular tools, but the identification of accurate biomarkers for reliable molecular diagnosis is a real challenge. We describe here the diagnostic power of a novel 8-genes signature: ornithine decarboxylase (ODC), ornithine decarboxylase antizyme (OAZ), adenosylmethionine decarboxylase (AdoMetDC), spermidine/spermine N(1)-acetyltransferase (SSAT), histone H3 (H3), growth arrest specific gene (GAS1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Clusterin (CLU) in tumour detection/classification of human CaP. Methodology/Principal Findings The 8-gene signature was detected by retrotranscription real-time quantitative PCR (RT-qPCR) in frozen prostate surgical specimens obtained from 41 patients diagnosed with CaP and recommended to undergo radical prostatectomy (RP). No therapy was given to patients at any time before RP. The bio-bank used for the study consisted of 66 specimens: 44 were benign-CaP paired from the same patient. Thirty-five were classified as benign and 31 as CaP after final pathological examination. Only molecular data were used for classification of specimens. The Nearest Neighbour (NN) classifier was used in order to discriminate CaP from benign tissue. Validation of final results was obtained with 10-fold crossvalidation procedure. CaP versus benign specimens were discriminated with (80±5)% accuracy, (81±6)% sensitivity and (78±7)% specificity. The method also correctly classified 71% of patients with Gleason score<7 versus ≥7, an important predictor of final outcome. Conclusions/Significance The method showed high sensitivity in a collection of specimens in which a significant

  19. Use of Targeted Exome Sequencing for Molecular Diagnosis of Skeletal Disorders

    PubMed Central

    Polla, Daniel L.; Cardoso, Maria T. O.; Silva, Mayara C. B.; Cardoso, Isabela C. C.; Medina, Cristina T. N.; Araujo, Rosenelle; Fernandes, Camila C.; Reis, Alessandra M. M.; de Andrade, Rosangela V.; Pereira, Rinaldo W.; Pogue, Robert

    2015-01-01

    Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known. PMID:26380986

  20. 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. PMID:19418253

  1. Molecular and Therapeutic Advances in the Diagnosis and Management of Malignant Pheochromocytomas and Paragangliomas

    PubMed Central

    Lowery, Aoife J.; Walsh, Siun; McDermott, Enda W.

    2013-01-01

    Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare catecholamine-secreting tumors derived from chromaffin cells originating in the neural crest. These tumors represent a significant diagnostic and therapeutic challenge because the diagnosis of malignancy is frequently made in retrospect by the development of metastatic or recurrent disease. Complete surgical resection offers the only potential for cure; however, recurrence can occur even after apparently successful resection of the primary tumor. The prognosis for malignant disease is poor because traditional treatment modalities have been limited. The last decade has witnessed exciting discoveries in the study of PCCs and PGLs; advances in molecular genetics have uncovered hereditary and germline mutations of at least 10 genes that contribute to the development of these tumors, and increasing knowledge of genotype-phenotype interactions has facilitated more accurate determination of malignant potential. Elucidating the molecular mechanisms responsible for malignant transformation in these tumors has opened avenues of investigation into targeted therapeutics that show promising results. There have also been significant advances in functional and radiological imaging and in the surgical approach to adrenalectomy, which remains the mainstay of treatment for PCC. In this review, we discuss the currently available diagnostic and therapeutic options for patients with malignant PCCs and PGLs and detail the molecular rationale and clinical evidence for novel and emerging diagnostic and therapeutic strategies. PMID:23576482

  2. A novel, integrated PET-guided MRS technique resulting in more accurate initial diagnosis of high-grade glioma.

    PubMed

    Kim, Ellen S; Satter, Martin; Reed, Marilyn; Fadell, Ronald; Kardan, Arash

    2016-06-01

    Glioblastoma multiforme (GBM) is the most common and lethal malignant glioma in adults. Currently, the modality of choice for diagnosing brain tumor is high-resolution magnetic resonance imaging (MRI) with contrast, which provides anatomic detail and localization. Studies have demonstrated, however, that MRI may have limited utility in delineating the full tumor extent precisely. Studies suggest that MR spectroscopy (MRS) can also be used to distinguish high-grade from low-grade gliomas. However, due to operator dependent variables and the heterogeneous nature of gliomas, the potential for error in diagnostic accuracy with MRS is a concern. Positron emission tomography (PET) imaging with (11)C-methionine (MET) and (18)F-fluorodeoxyglucose (FDG) has been shown to add additional information with respect to tumor grade, extent, and prognosis based on the premise of biochemical changes preceding anatomic changes. Combined PET/MRS is a technique that integrates information from PET in guiding the location for the most accurate metabolic characterization of a lesion via MRS. We describe a case of glioblastoma multiforme in which MRS was initially non-diagnostic for malignancy, but when MRS was repeated with PET guidance, demonstrated elevated choline/N-acetylaspartate (Cho/NAA) ratio in the right parietal mass consistent with a high-grade malignancy. Stereotactic biopsy, followed by PET image-guided resection, confirmed the diagnosis of grade IV GBM. To our knowledge, this is the first reported case of an integrated PET/MRS technique for the voxel placement of MRS. Our findings suggest that integrated PET/MRS may potentially improve diagnostic accuracy in high-grade gliomas.

  3. Molecular diagnosis of some common genetic diseases in Russia and the former USSR: present and future.

    PubMed Central

    Baranov, V S

    1993-01-01

    The current state of molecular diagnosis of some common genetic diseases, including cystic fibrosis, Duchenne muscular dystrophy, haemophilia A and B, phenylketonuria, and thalassaemia, in Russia and elsewhere in the former USSR is reviewed. Data on carrier detection and prenatal diagnosis are presented and some objective problems and obstacles hampering efficient molecular diagnosis in Russia are discussed. The necessity for molecular diagnosis of some other inherited diseases (for example, von Willebrand's disease, Martin-Bell syndrome, polycystic kidney disease, Huntington's disease, and myotonic dystrophy) is stressed. The need for establishing new diagnostic centres dealing with the most common diseases, as well as rare genetic diseases, is substantiated. Perspectives on the implementation of new molecular methods and new technical approaches (preimplantation embryo diagnosis, fetal cells selected from maternal blood) are briefly outlined. PMID:8445619

  4. Molecular biology in the diagnosis and prognosis of solid and lymphoid tumors.

    PubMed

    Lebovitz, R M; Albrecht, S

    1992-01-01

    The application of molecular biology to the study of human malignancies has led to tremendous gains in our understanding of their pathogenesis. Although their practical applications are still somewhat limited at this point, the use of molecular diagnostic tools is likely to grow at a very rapid rate as newer and more accurate prognostic markers are identified. The availability of reliable prognostic markers should allow earlier intervention in patients with aggressive disease but exhibiting only limited extent of disease at the time of initial diagnosis. Early intervention in such cases could realistically increase the probability of cure, since highly aggressive tumor cells are more likely to be eliminated by early institution of cytotoxic chemotherapy (4). The p53 tumor suppressor gene clearly represents the most promising potential prognostic marker at present, because of both the multiple phenotypic alterations caused by different p53 mutations and the high frequency of p53 mutations which have been observed in a variety of human cancers. Other prognostic markers related to oncogenes and tumor suppressor genes are almost certain to follow. Validation of new prognostic markers requires a knowledge of both histopathologic diagnostic criteria as well as the consequences for the patient of each diagnosis. There is bound to be some "shake-out" in the field of molecular diagnostics just as there was with other recently introduced techniques such as immunohistochemistry and flow cytometry which were found to provide additional useful information for some tumors and not for others. Since the clinical-pathologic studies needed for verification of putative prognostic markers require relatively long periods of follow up, progress in this area will almost certainly lag behind the ability of molecular biologists to identify new and potentially useful prognostic markers. Our collective ability to reap tangible gains in the clinical arena from our heavy investments in

  5. Recent advances in molecular medicine techniques for the diagnosis, prevention, and control of infectious diseases.

    PubMed

    França, R F O; da Silva, C C; De Paula, S O

    2013-06-01

    In recent years we have observed great advances in our ability to combat infectious diseases. Through the development of novel genetic methodologies, including a better understanding of pathogen biology, pathogenic mechanisms, advances in vaccine development, designing new therapeutic drugs, and optimization of diagnostic tools, significant infectious diseases are now better controlled. Here, we briefly describe recent reports in the literature concentrating on infectious disease control. The focus of this review is to describe the molecular methods widely used in the diagnosis, prevention, and control of infectious diseases with regard to the innovation of molecular techniques. Since the list of pathogenic microorganisms is extensive, we emphasize some of the major human infectious diseases (AIDS, tuberculosis, malaria, rotavirus, herpes virus, viral hepatitis, and dengue fever). As a consequence of these developments, infectious diseases will be more accurately and effectively treated; safe and effective vaccines are being developed and rapid detection of infectious agents now permits countermeasures to avoid potential outbreaks and epidemics. But, despite considerable progress, infectious diseases remain a strong challenge to human survival. PMID:23339016

  6. Recent advances in molecular medicine techniques for the diagnosis, prevention, and control of infectious diseases.

    PubMed

    França, R F O; da Silva, C C; De Paula, S O

    2013-06-01

    In recent years we have observed great advances in our ability to combat infectious diseases. Through the development of novel genetic methodologies, including a better understanding of pathogen biology, pathogenic mechanisms, advances in vaccine development, designing new therapeutic drugs, and optimization of diagnostic tools, significant infectious diseases are now better controlled. Here, we briefly describe recent reports in the literature concentrating on infectious disease control. The focus of this review is to describe the molecular methods widely used in the diagnosis, prevention, and control of infectious diseases with regard to the innovation of molecular techniques. Since the list of pathogenic microorganisms is extensive, we emphasize some of the major human infectious diseases (AIDS, tuberculosis, malaria, rotavirus, herpes virus, viral hepatitis, and dengue fever). As a consequence of these developments, infectious diseases will be more accurately and effectively treated; safe and effective vaccines are being developed and rapid detection of infectious agents now permits countermeasures to avoid potential outbreaks and epidemics. But, despite considerable progress, infectious diseases remain a strong challenge to human survival.

  7. What Is the Best NGS Enrichment Method for the Molecular Diagnosis of Monogenic Diabetes and Obesity?

    PubMed

    Philippe, Julien; Derhourhi, Mehdi; Durand, Emmanuelle; Vaillant, Emmanuel; Dechaume, Aurélie; Rabearivelo, Iandry; Dhennin, Véronique; Vaxillaire, Martine; De Graeve, Franck; Sand, Olivier; Froguel, Philippe; Bonnefond, Amélie

    2015-01-01

    Molecular diagnosis of monogenic diabetes and obesity is of paramount importance for both the patient and society, as it can result in personalized medicine associated with a better life and it eventually saves health care spending. Genetic clinical laboratories are currently switching from Sanger sequencing to next-generation sequencing (NGS) approaches but choosing the optimal protocols is not easy. Here, we compared the sequencing coverage of 43 genes involved in monogenic forms of diabetes and obesity, and variant detection rates, resulting from four enrichment methods based on the sonication of DNA (Agilent SureSelect, RainDance technologies), or using enzymes for DNA fragmentation (Illumina Nextera, Agilent HaloPlex). We analyzed coding exons and untranslated regions of the 43 genes involved in monogenic diabetes and obesity. We found that none of the methods achieves yet full sequencing of the gene targets. Nonetheless, the RainDance, SureSelect and HaloPlex enrichment methods led to the best sequencing coverage of the targets; while the Nextera method resulted in the poorest sequencing coverage. Although the sequencing coverage was high, we unexpectedly found that the HaloPlex method missed 20% of variants detected by the three other methods and Nextera missed 10%. The question of which NGS technique for genetic diagnosis yields the highest diagnosis rate is frequently discussed in the literature and the response is still unclear. Here, we showed that the RainDance enrichment method as well as SureSelect, which are both based on the sonication of DNA, resulted in a good sequencing quality and variant detection, while the use of enzymes to fragment DNA (HaloPlex or Nextera) might not be the best strategy to get an accurate sequencing. PMID:26599467

  8. COMPARISON OF MICROSCOPIC VERSUS MOLECULAR DIAGNOSIS OF CYCLOSPORA CAYETANENSIS

    EPA Science Inventory

    Objective: to investigate several ways to diagnose the food and waterborne protozoan parasite Cyclospora cayetanensis. Cyclospora cayetanensis is a protozoan parasite that infects human beings and causes gastroenteritis. Diagnosis of this parasite is complicated by the fact th...

  9. Accurate determination of the nuclear quadrupole moment of xenon from the molecular method

    NASA Astrophysics Data System (ADS)

    Canella, Guilherme A.; Santiago, Régis T.; Haiduke, Roberto L. A.

    2016-09-01

    This study provides a new determination of the nuclear electric quadrupole moment (NQM) for 131Xe, which is achieved by the molecular method. Dirac-Coulomb Coupled Cluster calculations with a Gaunt correction (DC+G-CC) of electric field gradients (EFGs) and experimental nuclear quadrupole coupling constants of six molecular systems (XeH+, XeCuF, XeCuCl, XeAgF, XeAgCl and XeAuF) were considered. The best NQM obtained by our DC+G-CCSD-T EFGs was -114.6(1.1) mbarn, which is recommended as the new reference value for this nuclide given the high level electron structure calculations done here.

  10. Utilizing fast multipole expansions for efficient and accurate quantum-classical molecular dynamics simulations.

    PubMed

    Schwörer, Magnus; Lorenzen, Konstantin; Mathias, Gerald; Tavan, Paul

    2015-03-14

    Recently, a novel approach to hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations has been suggested [Schwörer et al., J. Chem. Phys. 138, 244103 (2013)]. Here, the forces acting on the atoms are calculated by grid-based density functional theory (DFT) for a solute molecule and by a polarizable molecular mechanics (PMM) force field for a large solvent environment composed of several 10(3)-10(5) molecules as negative gradients of a DFT/PMM hybrid Hamiltonian. The electrostatic interactions are efficiently described by a hierarchical fast multipole method (FMM). Adopting recent progress of this FMM technique [Lorenzen et al., J. Chem. Theory Comput. 10, 3244 (2014)], which particularly entails a strictly linear scaling of the computational effort with the system size, and adapting this revised FMM approach to the computation of the interactions between the DFT and PMM fragments of a simulation system, here, we show how one can further enhance the efficiency and accuracy of such DFT/PMM-MD simulations. The resulting gain of total performance, as measured for alanine dipeptide (DFT) embedded in water (PMM) by the product of the gains in efficiency and accuracy, amounts to about one order of magnitude. We also demonstrate that the jointly parallelized implementation of the DFT and PMM-MD parts of the computation enables the efficient use of high-performance computing systems. The associated software is available online. PMID:25770527

  11. Utilizing fast multipole expansions for efficient and accurate quantum-classical molecular dynamics simulations

    SciTech Connect

    Schwörer, Magnus; Lorenzen, Konstantin; Mathias, Gerald; Tavan, Paul

    2015-03-14

    Recently, a novel approach to hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations has been suggested [Schwörer et al., J. Chem. Phys. 138, 244103 (2013)]. Here, the forces acting on the atoms are calculated by grid-based density functional theory (DFT) for a solute molecule and by a polarizable molecular mechanics (PMM) force field for a large solvent environment composed of several 10{sup 3}-10{sup 5} molecules as negative gradients of a DFT/PMM hybrid Hamiltonian. The electrostatic interactions are efficiently described by a hierarchical fast multipole method (FMM). Adopting recent progress of this FMM technique [Lorenzen et al., J. Chem. Theory Comput. 10, 3244 (2014)], which particularly entails a strictly linear scaling of the computational effort with the system size, and adapting this revised FMM approach to the computation of the interactions between the DFT and PMM fragments of a simulation system, here, we show how one can further enhance the efficiency and accuracy of such DFT/PMM-MD simulations. The resulting gain of total performance, as measured for alanine dipeptide (DFT) embedded in water (PMM) by the product of the gains in efficiency and accuracy, amounts to about one order of magnitude. We also demonstrate that the jointly parallelized implementation of the DFT and PMM-MD parts of the computation enables the efficient use of high-performance computing systems. The associated software is available online.

  12. Molecular Detection of Foodborne Pathogens: A Rapid and Accurate Answer to Food Safety.

    PubMed

    Mangal, Manisha; Bansal, Sangita; Sharma, Satish K; Gupta, Ram K

    2016-07-01

    Food safety is a global health concern. For the prevention and recognition of problems related to health and safety, detection of foodborne pathogen is of utmost importance at all levels of food production chain. For several decades, a lot of research has been targeted at the development of rapid methodology as reducing the time needed to complete pathogen detection tests has been the primary goal of food microbiologists. With the result, food microbiology laboratories now have a wide array of detection methods and automated technologies such as enzyme immunoassay, polymerase chain reaction, and microarrays, which can cut test times considerably. Nucleic acid amplification strategies and advances in amplicon detection methodologies have been the key factors in the progress of molecular microbiology. A comprehensive literature survey has been carried out to give an overview in the field of foodborne pathogen detection. In this paper, we describe the conventional methods, as well as recent developments in food pathogen detection, identification, and quantification, with a major emphasis on molecular detection methods.

  13. Accurate reaction-diffusion operator splitting on tetrahedral meshes for parallel stochastic molecular simulations

    NASA Astrophysics Data System (ADS)

    Hepburn, I.; Chen, W.; De Schutter, E.

    2016-08-01

    Spatial stochastic molecular simulations in biology are limited by the intense computation required to track molecules in space either in a discrete time or discrete space framework, which has led to the development of parallel methods that can take advantage of the power of modern supercomputers in recent years. We systematically test suggested components of stochastic reaction-diffusion operator splitting in the literature and discuss their effects on accuracy. We introduce an operator splitting implementation for irregular meshes that enhances accuracy with minimal performance cost. We test a range of models in small-scale MPI simulations from simple diffusion models to realistic biological models and find that multi-dimensional geometry partitioning is an important consideration for optimum performance. We demonstrate performance gains of 1-3 orders of magnitude in the parallel implementation, with peak performance strongly dependent on model specification.

  14. Accurate reaction-diffusion operator splitting on tetrahedral meshes for parallel stochastic molecular simulations.

    PubMed

    Hepburn, I; Chen, W; De Schutter, E

    2016-08-01

    Spatial stochastic molecular simulations in biology are limited by the intense computation required to track molecules in space either in a discrete time or discrete space framework, which has led to the development of parallel methods that can take advantage of the power of modern supercomputers in recent years. We systematically test suggested components of stochastic reaction-diffusion operator splitting in the literature and discuss their effects on accuracy. We introduce an operator splitting implementation for irregular meshes that enhances accuracy with minimal performance cost. We test a range of models in small-scale MPI simulations from simple diffusion models to realistic biological models and find that multi-dimensional geometry partitioning is an important consideration for optimum performance. We demonstrate performance gains of 1-3 orders of magnitude in the parallel implementation, with peak performance strongly dependent on model specification. PMID:27497550

  15. Accurate reaction-diffusion operator splitting on tetrahedral meshes for parallel stochastic molecular simulations.

    PubMed

    Hepburn, I; Chen, W; De Schutter, E

    2016-08-01

    Spatial stochastic molecular simulations in biology are limited by the intense computation required to track molecules in space either in a discrete time or discrete space framework, which has led to the development of parallel methods that can take advantage of the power of modern supercomputers in recent years. We systematically test suggested components of stochastic reaction-diffusion operator splitting in the literature and discuss their effects on accuracy. We introduce an operator splitting implementation for irregular meshes that enhances accuracy with minimal performance cost. We test a range of models in small-scale MPI simulations from simple diffusion models to realistic biological models and find that multi-dimensional geometry partitioning is an important consideration for optimum performance. We demonstrate performance gains of 1-3 orders of magnitude in the parallel implementation, with peak performance strongly dependent on model specification.

  16. A simple and accurate algorithm for path integral molecular dynamics with the Langevin thermostat.

    PubMed

    Liu, Jian; Li, Dezhang; Liu, Xinzijian

    2016-07-14

    We introduce a novel simple algorithm for thermostatting path integral molecular dynamics (PIMD) with the Langevin equation. The staging transformation of path integral beads is employed for demonstration. The optimum friction coefficients for the staging modes in the free particle limit are used for all systems. In comparison to the path integral Langevin equation thermostat, the new algorithm exploits a different order of splitting for the phase space propagator associated to the Langevin equation. While the error analysis is made for both algorithms, they are also employed in the PIMD simulations of three realistic systems (the H2O molecule, liquid para-hydrogen, and liquid water) for comparison. It is shown that the new thermostat increases the time interval of PIMD by a factor of 4-6 or more for achieving the same accuracy. In addition, the supplementary material shows the error analysis made for the algorithms when the normal-mode transformation of path integral beads is used.

  17. A simple and accurate algorithm for path integral molecular dynamics with the Langevin thermostat

    NASA Astrophysics Data System (ADS)

    Liu, Jian; Li, Dezhang; Liu, Xinzijian

    2016-07-01

    We introduce a novel simple algorithm for thermostatting path integral molecular dynamics (PIMD) with the Langevin equation. The staging transformation of path integral beads is employed for demonstration. The optimum friction coefficients for the staging modes in the free particle limit are used for all systems. In comparison to the path integral Langevin equation thermostat, the new algorithm exploits a different order of splitting for the phase space propagator associated to the Langevin equation. While the error analysis is made for both algorithms, they are also employed in the PIMD simulations of three realistic systems (the H2O molecule, liquid para-hydrogen, and liquid water) for comparison. It is shown that the new thermostat increases the time interval of PIMD by a factor of 4-6 or more for achieving the same accuracy. In addition, the supplementary material shows the error analysis made for the algorithms when the normal-mode transformation of path integral beads is used.

  18. A rapid and accurate method for calculation of stratospheric photolysis rates with molecular scattering

    NASA Technical Reports Server (NTRS)

    Boughner, Robert E.

    1986-01-01

    A method for calculating the photodissociation rates needed for photochemical modeling of the stratosphere, which includes the effects of molecular scattering, is described. The procedure is based on Sokolov's method of averaging functional correction. The radiation model and approximations used to calculate the radiation field are examined. The approximated diffuse fields and photolysis rates are compared with exact data. It is observed that the approximate solutions differ from the exact result by 10 percent or less at altitudes above 15 km; the photolysis rates differ from the exact rates by less than 5 percent for altitudes above 10 km and all zenith angles, and by less than 1 percent for altitudes above 15 km.

  19. Predicting accurate fluorescent spectra for high molecular weight polycyclic aromatic hydrocarbons using density functional theory

    NASA Astrophysics Data System (ADS)

    Powell, Jacob; Heider, Emily C.; Campiglia, Andres; Harper, James K.

    2016-10-01

    The ability of density functional theory (DFT) methods to predict accurate fluorescence spectra for polycyclic aromatic hydrocarbons (PAHs) is explored. Two methods, PBE0 and CAM-B3LYP, are evaluated both in the gas phase and in solution. Spectra for several of the most toxic PAHs are predicted and compared to experiment, including three isomers of C24H14 and a PAH containing heteroatoms. Unusually high-resolution experimental spectra are obtained for comparison by analyzing each PAH at 4.2 K in an n-alkane matrix. All theoretical spectra visually conform to the profiles of the experimental data but are systematically offset by a small amount. Specifically, when solvent is included the PBE0 functional overestimates peaks by 16.1 ± 6.6 nm while CAM-B3LYP underestimates the same transitions by 14.5 ± 7.6 nm. These calculated spectra can be empirically corrected to decrease the uncertainties to 6.5 ± 5.1 and 5.7 ± 5.1 nm for the PBE0 and CAM-B3LYP methods, respectively. A comparison of computed spectra in the gas phase indicates that the inclusion of n-octane shifts peaks by +11 nm on average and this change is roughly equivalent for PBE0 and CAM-B3LYP. An automated approach for comparing spectra is also described that minimizes residuals between a given theoretical spectrum and all available experimental spectra. This approach identifies the correct spectrum in all cases and excludes approximately 80% of the incorrect spectra, demonstrating that an automated search of theoretical libraries of spectra may eventually become feasible.

  20. Affine-response model of molecular solvation of ions: Accurate predictions of asymmetric charging free energies

    PubMed Central

    Bardhan, Jaydeep P.; Jungwirth, Pavel; Makowski, Lee

    2012-01-01

    Two mechanisms have been proposed to drive asymmetric solvent response to a solute charge: a static potential contribution similar to the liquid-vapor potential, and a steric contribution associated with a water molecule's structure and charge distribution. In this work, we use free-energy perturbation molecular-dynamics calculations in explicit water to show that these mechanisms act in complementary regimes; the large static potential (∼44 kJ/mol/e) dominates asymmetric response for deeply buried charges, and the steric contribution dominates for charges near the solute-solvent interface. Therefore, both mechanisms must be included in order to fully account for asymmetric solvation in general. Our calculations suggest that the steric contribution leads to a remarkable deviation from the popular “linear response” model in which the reaction potential changes linearly as a function of charge. In fact, the potential varies in a piecewise-linear fashion, i.e., with different proportionality constants depending on the sign of the charge. This discrepancy is significant even when the charge is completely buried, and holds for solutes larger than single atoms. Together, these mechanisms suggest that implicit-solvent models can be improved using a combination of affine response (an offset due to the static potential) and piecewise-linear response (due to the steric contribution). PMID:23020318

  1. Accurate and efficient integration for molecular dynamics simulations at constant temperature and pressure.

    PubMed

    Lippert, Ross A; Predescu, Cristian; Ierardi, Douglas J; Mackenzie, Kenneth M; Eastwood, Michael P; Dror, Ron O; Shaw, David E

    2013-10-28

    In molecular dynamics simulations, control over temperature and pressure is typically achieved by augmenting the original system with additional dynamical variables to create a thermostat and a barostat, respectively. These variables generally evolve on timescales much longer than those of particle motion, but typical integrator implementations update the additional variables along with the particle positions and momenta at each time step. We present a framework that replaces the traditional integration procedure with separate barostat, thermostat, and Newtonian particle motion updates, allowing thermostat and barostat updates to be applied infrequently. Such infrequent updates provide a particularly substantial performance advantage for simulations parallelized across many computer processors, because thermostat and barostat updates typically require communication among all processors. Infrequent updates can also improve accuracy by alleviating certain sources of error associated with limited-precision arithmetic. In addition, separating the barostat, thermostat, and particle motion update steps reduces certain truncation errors, bringing the time-average pressure closer to its target value. Finally, this framework, which we have implemented on both general-purpose and special-purpose hardware, reduces software complexity and improves software modularity.

  2. Accurate Gaussian basis sets for atomic and molecular calculations obtained from the generator coordinate method with polynomial discretization.

    PubMed

    Celeste, Ricardo; Maringolo, Milena P; Comar, Moacyr; Viana, Rommel B; Guimarães, Amanda R; Haiduke, Roberto L A; da Silva, Albérico B F

    2015-10-01

    Accurate Gaussian basis sets for atoms from H to Ba were obtained by means of the generator coordinate Hartree-Fock (GCHF) method based on a polynomial expansion to discretize the Griffin-Wheeler-Hartree-Fock equations (GWHF). The discretization of the GWHF equations in this procedure is based on a mesh of points not equally distributed in contrast with the original GCHF method. The results of atomic Hartree-Fock energies demonstrate the capability of these polynomial expansions in designing compact and accurate basis sets to be used in molecular calculations and the maximum error found when compared to numerical values is only 0.788 mHartree for indium. Some test calculations with the B3LYP exchange-correlation functional for N2, F2, CO, NO, HF, and HCN show that total energies within 1.0 to 2.4 mHartree compared to the cc-pV5Z basis sets are attained with our contracted bases with a much smaller number of polarization functions (2p1d and 2d1f for hydrogen and heavier atoms, respectively). Other molecular calculations performed here are also in very good accordance with experimental and cc-pV5Z results. The most important point to be mentioned here is that our generator coordinate basis sets required only a tiny fraction of the computational time when compared to B3LYP/cc-pV5Z calculations.

  3. Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

    PubMed Central

    Cazanave, Charles; Greenwood-Quaintance, Kerryl E.; Hanssen, Arlen D.; Karau, Melissa J.; Schmidt, Suzannah M.; Gomez Urena, Eric O.; Mandrekar, Jayawant N.; Osmon, Douglas R.; Lough, Lindsay E.; Pritt, Bobbi S.; Steckelberg, James M.

    2013-01-01

    We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee (n = 272) or hip (n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture (P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure. PMID:23658273

  4. Molecular diagnosis of hypophosphatasia and differential diagnosis by targeted Next Generation Sequencing.

    PubMed

    Taillandier, Agnès; Domingues, Christelle; De Cazanove, Clémence; Porquet-Bordes, Valérie; Monnot, Sophie; Kiffer-Moreira, Tina; Rothenbuhler, Agnès; Guggenbuhl, Pascal; Cormier, Catherine; Baujat, Geneviève; Debiais, Françoise; Capri, Yline; Cohen-Solal, Martine; Parent, Philippe; Chiesa, Jean; Dieux, Anne; Petit, Florence; Roume, Joelle; Isnard, Monica; Cormier-Daire, Valérie; Linglart, Agnès; Millán, José Luis; Salles, Jean-Pierre; Muti, Christine; Simon-Bouy, Brigitte; Mornet, Etienne

    2015-11-01

    Hypophosphatasia (HPP) is a rare inherited skeletal dysplasia due to loss of function mutations in the ALPL gene. The disease is subject to an extremely high clinical heterogeneity ranging from a perinatal lethal form to odontohypophosphatasia affecting only teeth. Up to now genetic diagnosis of HPP is performed by sequencing the ALPL gene by Sanger methodology. Osteogenesis imperfecta (OI) and campomelic dysplasia (CD) are the main differential diagnoses of severe HPP, so that in case of negative result for ALPL mutations, OI and CD genes had often to be analyzed, lengthening the time before diagnosis. We report here our 18-month experience in testing 46 patients for HPP and differential diagnosis by targeted NGS and show that this strategy is efficient and useful. We used an array including ALPL gene, genes of differential diagnosis COL1A1 and COL1A2 that represent 90% of OI cases, SOX9, responsible for CD, and 8 potentially modifier genes of HPP. Seventeen patients were found to carry a mutation in one of these genes. Among them, only 10 out of 15 cases referred for HPP carried a mutation in ALPL and 5 carried a mutation in COL1A1 or COL1A2. Interestingly, three of these patients were adults with fractures and/or low BMD. Our results indicate that HPP and OI may be easily misdiagnosed in the prenatal stage but also in adults with mild symptoms for these diseases. PMID:26432670

  5. Towards more accurate molecular dynamics calculation of thermal conductivity: Case study of GaN bulk crystals

    NASA Astrophysics Data System (ADS)

    Zhou, X. W.; Aubry, S.; Jones, R. E.; Greenstein, A.; Schelling, P. K.

    2009-03-01

    Significant differences exist among literature for thermal conductivity of various systems computed using molecular dynamics simulation. In some cases, unphysical results, for example, negative thermal conductivity, have been found. Using GaN as an example case and the direct nonequilibrium method, extensive molecular dynamics simulations and Monte Carlo analysis of the results have been carried out to quantify the uncertainty level of the molecular dynamics methods and to identify the conditions that can yield sufficiently accurate calculations of thermal conductivity. We found that the errors of the calculations are mainly due to the statistical thermal fluctuations. Extrapolating results to the limit of an infinite-size system tend to magnify the errors and occasionally lead to unphysical results. The error in bulk estimates can be reduced by performing longer time averages using properly selected systems over a range of sample lengths. If the errors in the conductivity estimates associated with each of the sample lengths are kept below a certain threshold, the likelihood of obtaining unphysical bulk values becomes insignificant. Using a Monte Carlo approach developed here, we have determined the probability distributions for the bulk thermal conductivities obtained using the direct method. We also have observed a nonlinear effect that can become a source of significant errors. For the extremely accurate results presented here, we predict a [0001] GaN thermal conductivity of 185W/Km at 300 K, 102W/Km at 500 K, and 74W/Km at 800 K. Using the insights obtained in the work, we have achieved a corresponding error level (standard deviation) for the bulk (infinite sample length) GaN thermal conductivity of less than 10W/Km , 5W/Km , and 15W/Km at 300 K, 500 K, and 800 K, respectively.

  6. First imported Plasmodium ovale malaria in Central America: case report of a Guatemalan soldier and a call to improve its accurate diagnosis.

    PubMed

    Castellanos, María Eugenia; Díaz, Sheilee; Parsons, Emily; Peruski, Leonard F; Enríquez, Fabiola; Ramírez, Juan Luis; Padilla, Norma

    2015-01-01

    The Mesoamerican Ministers of Health have set 2020 as the target for malaria elimination to be achieved in the region. Imported malaria cases are a potential threat to countries attempting elimination or working to prevent resurgence. We report the first imported Plasmodium ovale infection with molecular confirmation in Central America, which occurred in a Guatemalan soldier that had been deployed in Africa. The obstacles for its diagnosis using the standard microscopy technique and the need to improve its detection are discussed.

  7. Diagnosis of Wilson disease in young children: molecular genetic testing and a paradigm shift from the laboratory diagnosis.

    PubMed

    Seo, Jeong Kee

    2012-12-01

    Wilson disease (WD) is an autosomal recessive disorder of copper metabolism that results in accumulation of copper primarily in the liver, brain and cornea. Mutations in the WD gene, ATP7B, cause failure of copper excretion from hepatocyte into bile and a defective synthesis of ceruloplasmin. More than 500 mutations are now recognized, scattered throughout the ATP7B gene. Since WD has protean clinical presentations, awareness of WD in clinical practice is important for the early diagnosis and prevention of accumulated copper toxicity. Molecular genetic testing is playing an increasingly important role in the diagnosis of WD in uncertain cases and family screening. Siblings should be screened for WD once an index case has been diagnosed. Discrimination of heterozygotes from asymptomatic patients is essential to avoid inappropriate lifelong therapy for heterozygotes. Genetic testing, either by haplotype analysis or by mutation analysis, is the only definite solution for differentiating heterozygote carriers from affected asymptomatic patients. Routine genetic testing, because of the multitude of documented mutations, has been thought to be impractical until recently. However, genetic testing is now being more actively applied to the diagnosis of WD, particularly in young children in whom conventional biochemical diagnosis has much limitation and only genetic testing is able to confirm WD. Because advancement of modern biochemical technology now allows more rapid, easier, and less expensive mutation detection, direct DNA sequencing could be actively considered as the primary mode of diagnostic investigation rather than a supplementary test to the conventional biochemical tests. This review will focus on the recent advancement of molecular genetics and genetic diagnosis of WD in very young children on the basis of research data of the Seoul National University Children's Hospital and recent literature.

  8. Polyallelic structural variants can provide accurate, highly informative genetic markers focused on diagnosis and therapeutic targets: Accuracy vs. Precision.

    PubMed

    Roses, A D

    2016-02-01

    Structural variants (SVs) include all insertions, deletions, and rearrangements in the genome, with several common types of nucleotide repeats including single sequence repeats, short tandem repeats, and insertion-deletion length variants. Polyallelic SVs provide highly informative markers for association studies with well-phenotyped cohorts. SVs can influence gene regulation by affecting epigenetics, transcription, splicing, and/or translation. Accurate assays of polyallelic SV loci are required to define the range and allele frequency of variable length alleles. PMID:26517180

  9. Molecular Diagnosis of Diarrhea: Current Status and Future Potential

    PubMed Central

    Platts-Mills, James A; Operario, Darwin J

    2011-01-01

    Determining the microbiologic etiology of enteric infection remains an elusive goal. Conventional approaches, including culture, microscopy, and antigen-based tests have significant limitations such as limit of detection and the need for multiple procedures. Molecular diagnostics, especially PCR based tests, are rapidly changing research and practice in infectious diseases. Diarrheal disease, with its broad range of potential infectious etiologies, is well suited for multiplex molecular testing. This review highlights examples of currently employed molecular tests, as well as ways in which these tests can be applied in the future. The absence of a gold standard for the microbiologic cause of diarrhea means that the clinical significance of detected organisms may not always be clear. Conventional wisdom is that there should be one main pathogen causing diarrhea, however our thinking is challenged by increased detection of mixed infections. Thus, the successful incorporation of molecular diagnostics for diarrheal disease into practice will require both a careful understanding of the technical aspects and research to define their clinical utility. PMID:22116640

  10. Molecular methods of identification and diagnosis of fungi

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter presents detailed information about a wide range of molecular techniques (PCR- and nonPCR-based) that can be used for identifications or diagnoses of (mostly pathogenic) fungi affecting insects and other invertebrates. There are discussions of fungal genomes, of genomic fingerprinting t...

  11. Application of molecular techniques to the diagnosis of microsporidial infection.

    PubMed Central

    Fedorko, D. P.; Hijazi, Y. M.

    1996-01-01

    Microsporidia are now recognized as important pathogens of AIDS patients; the ability of these parasites to cause disease in immunocompetent persons is still being elucidated. Improved diagnostic tests for microsporidial infection are continually being sought for establishing diagnosis in order to avoid laborious electron microscopy studies that require invasively acquired biopsy specimens. Modified trichrome or chemofluorescent stains are useful for detecting microsporidia in bodily fluids and stool specimens, but they do not allow for speciation of microsporidia. Polymerase chain reaction with specific primers will allow the detection and speciation of microsporidia in biopsy tissue, bodily fluids, and stool specimens. PMID:8903228

  12. 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.

  13. Polymerase chain reaction-based molecular diagnosis of cutaneous infections in dermatopathology.

    PubMed

    Swick, Brian L

    2012-12-01

    Conventional methods, including microscopy, culture, and serologic studies, are a mainstay in the diagnosis of cutaneous infection. However, owing to limitations associated with these techniques, such as low sensitivity for standard microscopy and in the case of culture delay in diagnosis, polymerase chain-reaction based molecular techniques have taken on an expanding role in the diagnosis of infectious processes in dermatopathology. In particular, these assays are a useful adjunct in the diagnosis of cutaneous tuberculosis, atypical mycobacterial infection, leprosy, Lyme disease, syphilis, rickettsioses, leishmaniasis, and some fungal and viral infections. Already in the case of tuberculosis and atypical mycobacterial infection, standardized polymerase chain-reaction assays are commonly used for diagnostic purposes. With time, additional molecular-based techniques will decrease in cost and gain increased standardization, thus delivering rapid diagnostic confirmation for many difficult-to-diagnose cutaneous infections from standard formalin-fixed paraffin-embedded tissue specimens.

  14. Molecular diagnosis of infectious diarrhea: focus on enteric protozoa.

    PubMed

    Verkerke, Hans P; Sobuz, Shihab U; Petri, William A

    2014-11-01

    Robust detection of enteric protozoa is a critical step toward determining the etiology of diarrhea. Widespread use of conventional microscopy, culturing and antigen detection in both industrial and developing countries is limited by relatively low sensitivity and specificity. Refinements of these conventional approaches that reduce turnaround time and instrumentation have yielded strong alternatives for clinical and research use. However, advances in molecular diagnostics for protozoal, bacterial, viral and helminth infections offer significant advantages in studies seeking to understand pathogenesis, transmission and long-term consequences of infectious diarrhea. Quantitation of enteropathogen burden and highly multiplexed platforms for molecular detection dramatically improve predictive power in emerging models of diarrheal etiology, while eliminating the expense of multiple tests.

  15. Molecular diagnosis of Raoultella planticola infection of a surgical site.

    PubMed

    Wolcott, R; Dowd, S

    2010-08-01

    Raoultella planticola has been rarely diagnosed in clinical specimens. A case of a polymicrobial surgical site infection primarily caused by R. planticola in a 66-year-old Caucasian male with a fractured left tibia after an open reduction internal fixation of his left ankle is described and confirms this organism to be an opportunistic human pathogen. This pathogen was diagnosed with rapid clinical molecular pathogen diagnostic methods, which allowed an appropriate therapy to be implemented, thereby improving prognosis.

  16. Molecular Diagnosis of Human Taenia martis Eye Infection.

    PubMed

    Koch, Till; Schoen, Christoph; Muntau, Birgit; Addo, Marylyn; Ostertag, Helmut; Wiechens, Burkhard; Tappe, Dennis

    2016-05-01

    Taenia martis, a tapeworm harbored in the intestine of mustelids, is a rarely encountered zoonotic cysticercosis pathogen. The larval stage closely resembles the Taenia solium cysticercus, but the natural host and thus the epidemiology of the disease is different. We here report a human eye infection diagnosed molecularly in a previously healthy female German patient. The case represents the third human infection described worldwide; the two previous cases were also European, involving eye and brain. PMID:26928837

  17. Molecular beacons in biomedical detection and clinical diagnosis.

    PubMed

    Kim, Youngmi; Sohn, Dosung; Tan, Weihong

    2008-01-01

    Among the diverse nucleic acid probes, molecular beacons (MBs) have shown their excellent potential in a variety of basic researches and practical applications. Their excellent selectivity, sensitivity, and detection without separation have led them to be particularly useful in real-time intracellular monitoring of gene expression, development of biosensors, and clinical diagnostics. This paper will focus on the properties of various MBs and discuss their potential applications.

  18. Molecular Heterogeneity Within the Clinical Diagnosis of Pericentral Retinal Degeneration

    PubMed Central

    Matsui, Rodrigo; Cideciyan, Artur V.; Schwartz, Sharon B.; Sumaroka, Alexander; Roman, Alejandro J.; Swider, Malgorzata; Huang, Wei Chieh; Sheplock, Rebecca; Jacobson, Samuel G.

    2015-01-01

    Purpose To characterize in detail the phenotype and genotype of patients with pericentral retinal degeneration (PRD). Methods Patients were screened for an annular ring scotoma ranging from 3° to 40° (n = 28, ages 24–71) with kinetic perimetry. All patients had pigmentary retinopathy in the region of the dysfunction. Further studies included cross-sectional and en face imaging, static chromatic perimetry, and electroretinography. Molecular screening was performed. Results Genotypes of 14 of 28 PRD patients were identified: There were mutations in eight different genes previously associated with autosomal dominant or autosomal recessive RDs. Kinetic fields monitored in some patients over years to more than a decade could be stable or show increased extent of the scotoma. Electroretinograms were recordable but with different severities of dysfunction. Patterns of photoreceptor outer nuclear layer (ONL) loss corresponded to the distribution of visual dysfunction. Outer nuclear layer thickness topography and en face imaging indicated that the greatest disease expression was in the area of known highest rod photoreceptor density. Conclusions Molecular heterogeneity was a feature of the PRD phenotype. Many of the molecular causes were also associated with other phenotypes, such as maculopathies, typical retinitis pigmentosa (RP) and cone–rod dystrophy. The pericentral pattern of retinal degeneration is thus confirmed to be an uncommon phenotype of many different genotypes rather than a distinct disease entity. PMID:26393467

  19. Molecular diagnosis of autosomal dominant polycystic kidney disease.

    PubMed

    Torra Balcells, R; Ars Criach, E

    2011-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited renal disorder. Its estimated prevalence is 1 per 800 individuals. ADPKD patients constitute 8% of the population on dialysis or kidney transplantation. The disease can be diagnosed using radiological or genetic procedures. Direct genetic diagnosis of the disease can now be performed in Spain; however, it is not an easy or cheap test. This is why every case should be considered individually to determine whether genetic testing is appropriate, and to determine which genetic test is most adequate. Genetic testing in ADPKD is of special interest for living donors and neonatal and sporadic cases. Genetic testing offers the chance of performing prenatal or pre-implantation testing of embryos in families with severe cases of the disease. Also, this will enable the disease to be treated, when specific treatment becomes available, in cases that would not be candidates for treatment without genetic confirmation.

  20. Advances in Molecular Diagnosis of Neurofibromatosis Type 1.

    PubMed

    Shofty, Ben; Constantini, Shlomi; Ben-Shachar, Shay

    2015-12-01

    Neurofibromatosis 1 (NF1) is a common neurocutaneous and tumor predisposing genetic disorder with an autosomal dominant mode of inheritance. NF1 is solely caused by mutations in the NF1 gene, and disease-causing mutations can be found in more than 95% of individuals with a clinical diagnosis. Although NF1 has a distinctive clinical phenotype, it has a highly variable expression, even among individuals from the same family. Identifying the specific mutation does not usually assist in determining disease course and severity, and relatively few genotype-phenotype correlations have thus far been found. This review discusses the basic clinical aspects of NF1 and the current explanations for the high phenotypic variability, and provides the recently detected genotype-phenotype correlations. PMID:26706011

  1. NGS-based Molecular diagnosis of 105 eyeGENE(®) probands with Retinitis Pigmentosa.

    PubMed

    Ge, Zhongqi; Bowles, Kristen; Goetz, Kerry; Scholl, Hendrik P N; Wang, Feng; Wang, Xinjing; Xu, Shan; Wang, Keqing; Wang, Hui; Chen, Rui

    2015-12-15

    The National Ophthalmic Disease Genotyping and Phenotyping Network (eyeGENE(®)) was established in an effort to facilitate basic and clinical research of human inherited eye disease. In order to provide high quality genetic testing to eyeGENE(®)'s enrolled patients which potentially aids clinical diagnosis and disease treatment, we carried out a pilot study and performed Next-generation sequencing (NGS) based molecular diagnosis for 105 Retinitis Pigmentosa (RP) patients randomly selected from the network. A custom capture panel was designed, which incorporated 195 known retinal disease genes, including 61 known RP genes. As a result, disease-causing mutations were identified in 52 out of 105 probands (solving rate of 49.5%). A total of 82 mutations were identified, and 48 of them were novel. Interestingly, for three probands the molecular diagnosis was inconsistent with the initial clinical diagnosis, while for five probands the molecular information suggested a different inheritance model other than that assigned by the physician. In conclusion, our study demonstrated that NGS target sequencing is efficient and sufficiently precise for molecular diagnosis of a highly heterogeneous patient cohort from eyeGENE(®).

  2. Accurate crystal molecular dynamics simulations using particle-mesh-Ewald: RNA dinucleotides — ApU and GpC

    NASA Astrophysics Data System (ADS)

    Lee, Hsing; Darden, Thomas; Pedersen, Lee

    1995-09-01

    Long molecular dynamics (MD) simulations for two crystal RNA dinucleotides ApU (2.0 ns) and GpC (1.5 ns) were performed, starting from the crystallographic positions of all heavy atoms in the crystals. By employing the particle-mesh-Ewald algorithm [Darden et al., J. Chem. Phys. 98 (1993) 10089] to accommodate the long-range Coulomb interactions, highly accurate MD structures were obtained for both crystals. The instantaneous root-mean-square positional deviations of the heavy atoms equilibrate at approximately 0.4 Å for both systems, while the experimental and calculated temperature factors are comparable in size. These results describe the first successful crystal MD simulation of RNA molecules.

  3. Accurate CO{sub 2} laser frequencies and molecular constants of regular and new hot-band lines

    SciTech Connect

    Chou, Che-Chung; Shy, Jow-Tsong; Maki, A.G.

    1994-12-31

    A new, high-resolution, highly efficient, cw, CO{sub 2} laser oscillating on more than 250 lines including over 40 lines in the new 9 {mu}m hot band has been built at NIST, Boulder. The frequencies of the 9 and 10 {mu}m hot band lines and high J (to J=66) regular band lines of {sup 12}C{sup 16}O{sub 2} , which now fill the gap between the 9 and 10 {mu}m regions, have been locked to saturated fluorescence signals in CO{sub 2}, and measured. New molecular constants and more accurate frequencies of the four common isotopes of CO{sub 2} have been obtained.

  4. L-DNA molecular beacon: a safe, stable, and accurate intracellular nano-thermometer for temperature sensing in living cells.

    PubMed

    Ke, Guoliang; Wang, Chunming; Ge, Yun; Zheng, Nanfeng; Zhu, Zhi; Yang, Chaoyong James

    2012-11-21

    Noninvasive and accurate measurement of intracellular temperature is of great significance in biology and medicine. This paper describes a safe, stable, and accurate intracellular nano-thermometer based on an L-DNA molecular beacon (L-MB), a dual-labeled hairpin oligonucleotide built from the optical isomer of naturally occurring d-DNA. Relying on the temperature-responsive hairpin structure and the FRET signaling mechanism of MBs, the fluorescence of L-MBs is quenched below the melting temperature and enhanced with increasing temperature. Because of the excellent reversibility and tunable response range, L-MBs are very suitable for temperature sensing. More importantly, the non-natural L-DNA backbone prevents the L-MBs from binding to cellular nucleic acids and proteins as well as from being digested by nucleases inside the cells, thus ensuring excellent stability and accuracy of the nano-thermometer in a complex cellular environment. The L-MB nano-thermometer was used for the photothermal study of Pd nanosheets in living cells, establishing the nano-thermometer as a useful tool for intracellular temperature measurement.

  5. Bottom-up coarse-grained models that accurately describe the structure, pressure, and compressibility of molecular liquids

    SciTech Connect

    Dunn, Nicholas J. H.; Noid, W. G.

    2015-12-28

    The present work investigates the capability of bottom-up coarse-graining (CG) methods for accurately modeling both structural and thermodynamic properties of all-atom (AA) models for molecular liquids. In particular, we consider 1, 2, and 3-site CG models for heptane, as well as 1 and 3-site CG models for toluene. For each model, we employ the multiscale coarse-graining method to determine interaction potentials that optimally approximate the configuration dependence of the many-body potential of mean force (PMF). We employ a previously developed “pressure-matching” variational principle to determine a volume-dependent contribution to the potential, U{sub V}(V), that approximates the volume-dependence of the PMF. We demonstrate that the resulting CG models describe AA density fluctuations with qualitative, but not quantitative, accuracy. Accordingly, we develop a self-consistent approach for further optimizing U{sub V}, such that the CG models accurately reproduce the equilibrium density, compressibility, and average pressure of the AA models, although the CG models still significantly underestimate the atomic pressure fluctuations. Additionally, by comparing this array of models that accurately describe the structure and thermodynamic pressure of heptane and toluene at a range of different resolutions, we investigate the impact of bottom-up coarse-graining upon thermodynamic properties. In particular, we demonstrate that U{sub V} accounts for the reduced cohesion in the CG models. Finally, we observe that bottom-up coarse-graining introduces subtle correlations between the resolution, the cohesive energy density, and the “simplicity” of the model.

  6. Bottom-up coarse-grained models that accurately describe the structure, pressure, and compressibility of molecular liquids

    NASA Astrophysics Data System (ADS)

    Dunn, Nicholas J. H.; Noid, W. G.

    2015-12-01

    The present work investigates the capability of bottom-up coarse-graining (CG) methods for accurately modeling both structural and thermodynamic properties of all-atom (AA) models for molecular liquids. In particular, we consider 1, 2, and 3-site CG models for heptane, as well as 1 and 3-site CG models for toluene. For each model, we employ the multiscale coarse-graining method to determine interaction potentials that optimally approximate the configuration dependence of the many-body potential of mean force (PMF). We employ a previously developed "pressure-matching" variational principle to determine a volume-dependent contribution to the potential, UV(V), that approximates the volume-dependence of the PMF. We demonstrate that the resulting CG models describe AA density fluctuations with qualitative, but not quantitative, accuracy. Accordingly, we develop a self-consistent approach for further optimizing UV, such that the CG models accurately reproduce the equilibrium density, compressibility, and average pressure of the AA models, although the CG models still significantly underestimate the atomic pressure fluctuations. Additionally, by comparing this array of models that accurately describe the structure and thermodynamic pressure of heptane and toluene at a range of different resolutions, we investigate the impact of bottom-up coarse-graining upon thermodynamic properties. In particular, we demonstrate that UV accounts for the reduced cohesion in the CG models. Finally, we observe that bottom-up coarse-graining introduces subtle correlations between the resolution, the cohesive energy density, and the "simplicity" of the model.

  7. Choline metabolism-based molecular diagnosis of cancer: an update

    PubMed Central

    Glunde, Kristine; Penet, Marie-France; Jiang, Lu; Jacobs, Michael A; Bhujwalla, Zaver M

    2016-01-01

    Abnormal choline metabolism continues to be identified in multiple cancers. Molecular causes of abnormal choline metabolism are changes in choline kinase-α, ethanolamine kinase-α, phosphatidylcholine-specific phospholipase C and -D and glycerophosphocholine phosphodiesterases, as well as several choline transporters. The net outcome of these enzymatic changes is an increase in phosphocholine and total choline (tCho) and, in some cancers, a relative decrease of glycerophosphocholine. The increased tCho signal detected by 1H magnetic resonance spectroscopy is being evaluated as a diagnostic marker in multiple cancers. Increased expression and activity of choline transporters and choline kinase-α have spurred the development of radiolabeled choline analogs as PET imaging tracers. Both tCho 1H magnetic resonance spectroscopy and choline PET are being investigated to detect response to treatment. Enzymes mediating the abnormal choline metabolism are being explored as targets for cancer therapy. This review highlights recent molecular, therapeutic and clinical advances in choline metabolism in cancer. PMID:25921026

  8. The Role of Molecular Imaging in the Diagnosis and Management of Neuropsychiatric Disorders

    PubMed Central

    Shen, Lie-Hang; Tseng, Yu-Chin; Liao, Mei-Hsiu; Fu, Ying-Kai

    2011-01-01

    Neuropsychiatric disorders are becoming a major socioeconomic burden to modern society. In recent years, a dramatic expansion of tools has facilitated the study of the molecular basis of neuropsychiatric disorders. Molecular imaging has enabled the noninvasive characterization and quantification of biological processes at the cellular, tissue, and organism levels in intact living subjects. This technology has revolutionized the practice of medicine and has become critical to quality health care. New advances in research on molecular imaging hold promise for personalized medicine in neuropsychiatric disorders, with adjusted therapeutic doses, predictable responses, reduced adverse drug reactions, early diagnosis, and personal health planning. In this paper, we discuss the development of radiotracers for imaging dopaminergic, serotonergic, and noradrenergic systems and β-amyloid plaques. We will underline the role of molecular imaging technologies in various neuropsychiatric disorders, describe their unique strengths and limitations, and suggest future directions in the diagnosis and management of neuropsychiatric disorders. PMID:21541178

  9. Accurate static and dynamic properties of liquid electrolytes for Li-ion batteries from ab initio molecular dynamics.

    PubMed

    Ganesh, P; Jiang, De-en; Kent, P R C

    2011-03-31

    Lithium-ion batteries have the potential to revolutionize the transportation industry, as they did for wireless communication. A judicious choice of the liquid electrolytes used in these systems is required to achieve a good balance among high-energy storage, long cycle life and stability, and fast charging. Ethylene-carbonate (EC) and propylene-carbonate (PC) are popular electrolytes. However, to date, almost all molecular-dynamics simulations of these fluids rely on classical force fields, while a complete description of the functionality of Li-ion batteries will eventually require quantum mechanics. We perform accurate ab initio molecular-dynamics simulations of ethylene- and propylene-carbonate with LiPF(6) at experimental concentrations to build solvation models which explain available neutron scattering and nuclear magnetic resonance (NMR) results and to compute Li-ion solvation energies and diffusion constants. Our results suggest some similarities between the two liquids as well as some important differences. Simulations also provide useful insights into formation of solid-electrolyte interphases in the presence of electrodes in conventional Li-ion batteries.

  10. Accurate path integral molecular dynamics simulation of ab-initio water at near-zero added cost

    NASA Astrophysics Data System (ADS)

    Elton, Daniel; Fritz, Michelle; Soler, José; Fernandez-Serra, Marivi

    It is now established that nuclear quantum motion plays an important role in determining water's structure and dynamics. These effects are important to consider when evaluating DFT functionals and attempting to develop better ones for water. The standard way of treating nuclear quantum effects, path integral molecular dynamics (PIMD), multiplies the number of energy/force calculations by the number of beads, which is typically 32. Here we introduce a method whereby PIMD can be incorporated into a DFT molecular dynamics simulation at virtually zero cost. The method is based on the cluster (many body) expansion of the energy. We first subtract the DFT monomer energies, using a custom DFT-based monomer potential energy surface. The evolution of the PIMD beads is then performed using only the more-accurate Partridge-Schwenke monomer energy surface. The DFT calculations are done using the centroid positions. Various bead thermostats can be employed to speed up the sampling of the quantum ensemble. The method bears some resemblance to multiple timestep algorithms and other schemes used to speed up PIMD with classical force fields. We show that our method correctly captures some of key effects of nuclear quantum motion on both the structure and dynamics of water. We acknowledge support from DOE Award No. DE-FG02-09ER16052 (D.E.) and DOE Early Career Award No. DE-SC0003871 (M.V.F.S.).

  11. [Genetic Diagnosis and Molecular Therapies for Duchenne Muscular Dystrophy].

    PubMed

    Takeshima, Yasuhiro

    2015-10-01

    Duchenne muscular dystrophy (DMD) is the most common form of inherited muscle disease and is characterized by progressive muscle wasting, ultimately resulting in the death of patients in their twenties or thirties. DMD is characterized by a deficiency of the muscle dystrophin as a result of mutations in the dystrophin gene. Currently, no effective treatment for DMD is available. Promising molecular therapies which are mutation-specific have been developed. Transformation of an out-of-frame mRNA into an in-frame dystrophin message by inducing exon skipping is considered one of the approaches most likely to lead to success. We demonstrated that the intravenous administration of the antisense oligonucleotide against the splicing enhancer sequence results in exon skipping and production of the dystrophin protein in DMD case for the first time. After extensive studies, anti-sense oligonucleotides comprising different monomers have undergone clinical trials and provided favorable results, enabling improvements in ambulation of DMD patients. Induction of the read-through of nonsense mutations is expected to produce dystrophin in DMD patients with nonsense mutations, which are detected in 19% of DMD cases. The clinical effectiveness of gentamicin and PTC124 has been reported. We have demonstrated that arbekacin-mediated read-through can markedly ameliorate muscular dystrophy in vitro. We have already begun a clinical trial of nonsense mutation read-through therapy using arbekacin. Some of these drug candidates are planned to undergo submission for approval to regulatory agencies in the US and EU. We hope that these molecular therapies will contribute towards DMD treatment. PMID:26897856

  12. Recommendations for accurate CT diagnosis of suspected acute aortic syndrome (AAS)—on behalf of the British Society of Cardiovascular Imaging (BSCI)/British Society of Cardiovascular CT (BSCCT)

    PubMed Central

    Nicol, Edward; Morgan-Hughes, Gareth; Roobottom, Carl A; Roditi, Giles; Hamilton, Mark C K; Bull, Russell K; Pugliese, Franchesca; Williams, Michelle C; Stirrup, James; Padley, Simon; Taylor, Andrew; Davies, L Ceri; Bury, Roger; Harden, Stephen

    2016-01-01

    Accurate and timely assessment of suspected acute aortic syndrome is crucial in this life-threatening condition. Imaging with CT plays a central role in the diagnosis to allow expedited management. Diagnosis can be made using locally available expertise with optimized scanning parameters, making full use of recent advances in CT technology. Each imaging centre must optimize their protocols to allow accurate diagnosis, to optimize radiation dose and in particular to reduce the risk of false-positive diagnosis that may simulate disease. This document outlines the principles for the acquisition of motion-free imaging of the aorta in this context. PMID:26916280

  13. [The present and future prospects in rapid molecular diagnosis of tuberculosis and MDR-TB (First Part)].

    PubMed

    Tănăsescu, Mihaela; Didilescu, Cristian; Marica, Constantin

    2013-01-01

    Tuberculosis is still one of the diseases with a major medical and social impact, and in terms of early diagnosis (which would imply a fair treatment and established at the time), difficulties related to the delay bacilli isolation in culture, decreased susceptibility testing methods to antituberculosis drugs, lack of methods for differentiation of M. Tuberculosis complex germs of non TB Mycobacteria, may have important clinical implications. Traditional testing of anti-TB drug susceptibility on solid Löwenstein-Jensen medium (gold standard) or liquid media can only be performed using grown samples. Determining the time it takes up to 42 days on solid media and 12 days for liquid media. For MDR/XDR TB cases is absolutely essential to reduce the detection time. In these cases prove their usefulness rapid diagnostic methods. Automatic testing in liquid medium, molecular hybridization methods are currently recommended by the current WHO guidelines. Rapid diagnosis of MDR-TB is extremely useful for the early establishment of an effective treatment tailored more accurately on the spectrum of sensitivity of the resistant strain (thus reducing the risk of developing additional resistance to other drugs) and control the spread of these strains. Genetic diagnostic methods, approved and recommended by the WHO, can reduce the time of diagnosis of TB case and, importantly, the case of MDR TB. They do not replace the current standard diagnostic methods and resistance profile, but complete them in selected cases. PMID:24273995

  14. Development and Validation of a Mass Spectrometry-Based Assay for the Molecular Diagnosis of Mucin-1 Kidney Disease.

    PubMed

    Blumenstiel, Brendan; DeFelice, Matthew; Birsoy, Ozge; Bleyer, Anthony J; Kmoch, Stanislav; Carter, Todd A; Gnirke, Andreas; Kidd, Kendrah; Rehm, Heidi L; Ronco, Lucienne; Lander, Eric S; Gabriel, Stacey; Lennon, Niall J

    2016-07-01

    Mucin-1 kidney disease, previously described as medullary cystic kidney disease type 1 (MCKD1, OMIM 174000), is an autosomal dominant tubulointerstitial kidney disease recently shown to be caused by a single-base insertion within the variable number tandem repeat region of the MUC1 gene. Because of variable age of disease onset and often subtle signs and symptoms, clinical diagnosis of mucin-1 kidney disease and differentiation from other forms of hereditary kidney disease have been difficult. The causal insertion resides in a variable number tandem repeat region with high GC content, which has made detection by standard next-generation sequencing impossible to date. The inherently difficult nature of this mutation required an alternative method for routine detection and clinical diagnosis of the disease. We therefore developed and validated a mass spectrometry-based probe extension assay with a series of internal controls to detect the insertion event using 24 previously characterized positive samples from patients with mucin-1 kidney disease and 24 control samples known to be wild type for the variant. Validation results indicate an accurate and reliable test for clinically establishing the molecular diagnosis of mucin-1 kidney disease with 100% sensitivity and specificity across 275 tests called. PMID:27157321

  15. [The present and future prospects in rapid molecular diagnosis of tuberculosis and MDR-TB (First Part)].

    PubMed

    Tănăsescu, Mihaela; Didilescu, Cristian; Marica, Constantin

    2013-01-01

    Tuberculosis is still one of the diseases with a major medical and social impact, and in terms of early diagnosis (which would imply a fair treatment and established at the time), difficulties related to the delay bacilli isolation in culture, decreased susceptibility testing methods to antituberculosis drugs, lack of methods for differentiation of M. Tuberculosis complex germs of non TB Mycobacteria, may have important clinical implications. Traditional testing of anti-TB drug susceptibility on solid Löwenstein-Jensen medium (gold standard) or liquid media can only be performed using grown samples. Determining the time it takes up to 42 days on solid media and 12 days for liquid media. For MDR/XDR TB cases is absolutely essential to reduce the detection time. In these cases prove their usefulness rapid diagnostic methods. Automatic testing in liquid medium, molecular hybridization methods are currently recommended by the current WHO guidelines. Rapid diagnosis of MDR-TB is extremely useful for the early establishment of an effective treatment tailored more accurately on the spectrum of sensitivity of the resistant strain (thus reducing the risk of developing additional resistance to other drugs) and control the spread of these strains. Genetic diagnostic methods, approved and recommended by the WHO, can reduce the time of diagnosis of TB case and, importantly, the case of MDR TB. They do not replace the current standard diagnostic methods and resistance profile, but complete them in selected cases.

  16. Accurate diagnosis of myalgic encephalomyelitis and chronic fatigue syndrome based upon objective test methods for characteristic symptoms

    PubMed Central

    Twisk, Frank NM

    2015-01-01

    Although myalgic encephalomyelitis (ME) and chronic fatigue syndrome (CFS) are considered to be synonymous, the definitional criteria for ME and CFS define two distinct, partially overlapping, clinical entities. ME, whether defined by the original criteria or by the recently proposed criteria, is not equivalent to CFS, let alone a severe variant of incapacitating chronic fatigue. Distinctive features of ME are: muscle weakness and easy muscle fatigability, cognitive impairment, circulatory deficits, a marked variability of the symptoms in presence and severity, but above all, post-exertional “malaise”: a (delayed) prolonged aggravation of symptoms after a minor exertion. In contrast, CFS is primarily defined by (unexplained) chronic fatigue, which should be accompanied by four out of a list of 8 symptoms, e.g., headaches. Due to the subjective nature of several symptoms of ME and CFS, researchers and clinicians have questioned the physiological origin of these symptoms and qualified ME and CFS as functional somatic syndromes. However, various characteristic symptoms, e.g., post-exertional “malaise” and muscle weakness, can be assessed objectively using well-accepted methods, e.g., cardiopulmonary exercise tests and cognitive tests. The objective measures acquired by these methods should be used to accurately diagnose patients, to evaluate the severity and impact of the illness objectively and to assess the positive and negative effects of proposed therapies impartially. PMID:26140274

  17. EMQN best practice guidelines for the molecular genetic diagnosis of hereditary hemochromatosis (HH)

    PubMed Central

    Porto, Graça; Brissot, Pierre; Swinkels, Dorine W; Zoller, Heinz; Kamarainen, Outi; Patton, Simon; Alonso, Isabel; Morris, Michael; Keeney, Steve

    2016-01-01

    Molecular genetic testing for hereditary hemochromatosis (HH) is recognized as a reference test to confirm the diagnosis of suspected HH or to predict its risk. The vast majority (typically >90%) of patients with clinically characterized HH are homozygous for the p.C282Y variant in the HFE gene, referred to as HFE-related HH. Since 1996, HFE genotyping was implemented in diagnostic algorithms for suspected HH, allowing its early diagnosis and prevention. However, the penetrance of disease in p.C282Y homozygotes is incomplete. Hence, homozygosity for p.C282Y is not sufficient to diagnose HH. Neither is p.C282Y homozygosity required for diagnosis as other rare forms of HH exist, generally referred to as non-HFE-related HH. These pose significant challenges when defining criteria for referral, testing protocols, interpretation of test results and reporting practices. We present best practice guidelines for the molecular genetic diagnosis of HH where recommendations are classified, as far as possible, according to the level and strength of evidence. For clarification, the guidelines' recommendations are preceded by a detailed description of the methodology and results obtained with a series of actions taken in order to achieve a wide expert consensus, namely: (i) a survey on the current practices followed by laboratories offering molecular diagnosis of HH; (ii) a systematic literature search focused on some identified controversial topics; (iii) an expert Best Practice Workshop convened to achieve consensus on the practical recommendations included in the guidelines. PMID:26153218

  18. Differential diagnosis of lung carcinoma with three-dimensional quantitative molecular vibrational imaging

    NASA Astrophysics Data System (ADS)

    Gao, Liang; Hammoudi, Ahmad A.; Li, Fuhai; Thrall, Michael J.; Cagle, Philip T.; Chen, Yuanxin; Yang, Jian; Xia, Xiaofeng; Fan, Yubo; Massoud, Yehia; Wang, Zhiyong; Wong, Stephen T. C.

    2012-06-01

    The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.

  19. Molecular Diagnosis of Polycystic Echinococcosis Due to Echinococcus vogeli in a Paraguayan Immigrant in Argentina

    PubMed Central

    Frider, B.; Alvarez Rodriguez, J.; Amante, M.; Pestalardo, M. L.; Cazorla, A.; Bresson-Hadni, S.; Millon, L.

    2013-01-01

    Polycystic echinococcosis due to Echinococcus vogeli is a rare parasitic infection that occurs in rural areas of Central and South America. Only molecular identification performed on formalin-fixed paraffin-embedded liver tissue samples gave an unequivocal diagnosis of this disease in a Paraguayan immigrant in Argentina. PMID:23824768

  20. Molecular diagnosis of diphyllobothriasis in Spain, most presumably acquired via imported fish, or sojourn abroad

    PubMed Central

    Pastor-Valle, J; González, L M; Martín-Clemente, J P; Merino, F J; Gottstein, B; Gárate, T

    2014-01-01

    Human diphyllobothriasis is sporadically detected in Spain. Diphyllobothrium latum and Diplogonoporus balaenopterae have been identified. In the study, four cases of presumably imported diphyllobothriasis in Spanish patients were appraised. Molecular diagnosis allowed us to identify ‘exotic’ fish tapeworms such as Diplogonoporus balaenopterae in one patient and Diphyllobothrium pacificum in the others. PMID:25356331

  1. Pelizaeus-Merzbacher disease in patients with molecularly confirmed diagnosis.

    PubMed

    Mierzewska, H; Jamroz, E; Mazurczak, T; Hoffman-Zacharska, D; Szczepanik, E

    2016-01-01

    Pelizaeus-Merzbacher disease (PMD) is X-linked hypomyelinating leukodystrophy caused by mutations of the PLP1 gene, which codes the proteolipid protein 1. The result of mutations is abnormal myelination - hypomyelination and dysmyelination of cerebral white matter, and in some form of the disease hypomyelinating peripheral neuropathy. DNA samples from 68 patients suspected of PMD due to the clinical course and hypomyelination at magnetic resonance imaging (MRI) were analyzed. Medical history and detailed clinical course of PMD patients were also analyzed. Different mutations of the PLP1 gene were detected in 14 boys from 11 families (~20%). Amongst the molecularly confirmed patients, 13 presented classical PMD forms but clinical phenotypes varied in the severity even amongst siblings. One patient presented a severe connatal form. One mother, obligate carrier, presented complicated SPG2 (spastic paraparesis). There was no phenotype-genotype correlation in our material. In many cases PMD was suspected with a delay of many years, sometimes only after birth of another affected child in the family. Pelizaeus-Merzbacher disease was most frequently misdiagnosed as cerebral palsy. PMID:27179222

  2. Tylosis with oesophageal cancer: Diagnosis, management and molecular mechanisms.

    PubMed

    Ellis, Anthony; Risk, Janet M; Maruthappu, Thiviyani; Kelsell, David P

    2015-01-01

    Tylosis (hyperkeratosis palmaris et plantaris) is characterised by focal thickening of the skin of the hands and feet and is associated with a very high lifetime risk of developing squamous cell carcinoma of the oesophagus. This risk has been calculated to be 95% at the age of 65 in one large family, however the frequency of the disorder in the general population is not known and is likely to be less than one in 1,000,000. Oesophageal lesions appear as small (2-5 mm), white, polyploid lesions dotted throughout the oesophagus and oral leukokeratosis has also been described. Although symptoms of oesophageal cancer can include dysphagia, odynophagia, anorexia and weight loss, there may be an absence of symptoms in early disease, highlighting the importance of endoscopic surveillance in these patients. Oesophageal cancer associated with tylosis usually presents in middle to late life (from mid-fifties onwards) and shows no earlier development than the sporadic form of the disease. Tylosis with oesophageal cancer is inherited as an autosomal dominant trait with complete penetrance of the cutaneous features, usually by 7 to 8 years of age but can present as late as puberty. Mutations in RHBDF2 located on 17q25.1 have recently been found to be causative. A diagnosis of tylosis with oesophageal cancer is made on the basis of a positive family history, characteristic clinical features, including cutaneous and oesophageal lesions, and genetic analysis for mutations in RHBDF2. The key management goal is surveillance for early detection and treatment of oesophageal dysplasia. Surveillance includes annual gastroscopy with biopsy of any suspicious lesion together with quadratic biopsies from the upper, middle and lower oesophagus. This is coupled with dietary and lifestyle modification advice and symptom education. Symptomatic management of the palmoplantar keratoderma includes regular application of emollients, specialist footwear and early treatment of fissures and super

  3. Conservation of nucleotide sequences for molecular diagnosis of Middle East respiratory syndrome coronavirus, 2015.

    PubMed

    Furuse, Yuki; Okamoto, Michiko; Oshitani, Hitoshi

    2015-11-01

    Infection due to the Middle East respiratory syndrome coronavirus (MERS-CoV) is widespread. The present study was performed to assess the protocols used for the molecular diagnosis of MERS-CoV by analyzing the nucleotide sequences of viruses detected between 2012 and 2015, including sequences from the large outbreak in eastern Asia in 2015. Although the diagnostic protocols were established only 2 years ago, mismatches between the sequences of primers/probes and viruses were found for several of the assays. Such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. A slight modification in the primer design is suggested. Protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as MERS-CoV.

  4. The Role of Molecular Genetic Analysis in the Diagnosis of Primary Ciliary Dyskinesia

    PubMed Central

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

    2014-01-01

    Rationale: 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. Objectives: To determine the diagnostic yield of genetic testing of patients with a confirmed or suspected diagnosis of PCD in a multiethnic urban center. Methods: 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. Results: 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. Conclusions: 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. PMID:24498942

  5. Difficulties in the molecular diagnosis of Helicobacter rodent infections.

    PubMed

    Poynter, Scott; Phipps, Julian D; Naranjo-Pino, Angel; Sanchez-Morgado, Jose M

    2009-03-01

    Molecular diagnostic methods using the polymerase chain reaction (PCR) are the gold standard in Helicobacter diagnostics. Most rely on the amplification of parts of the 16S rRNA gene sequence. Therefore, the validity and accuracy of results depends heavily on the PCR design and the time of its publication because new sequences are continually being submitted to databases. Here we report the presence of helicobacter in commercially bred mice supposedly free of this infection. Furthermore, three out of six different commercial laboratories performing helicobacter testing on the same spiked faecal samples failed to detect and identify H. hepaticus. We designed a simple generic PCR assay that amplifies a 261bp amplicon spanning two of the seven variable regions in the 16S rRNA of helicobacter. Using this assay together with an established generic assay designed by Bohr [Bohr, U.R., Primus, A., Zagoura, A., Glasbrenner, B., Wex, T., Malfertheiner, P., 2002. A group-specific PCR assay for the detection of Helicobacteraceae in human gut. Helicobacter 7, 378-383] and then cloning and sequencing their products, we detected the H. hepaticus used in the study that three commercial laboratories failed to detect. We think these assays together could detect all the currently known species of helicobacter and hopefully the new ones as well. In addition, we have been able to identify different species of helicobacter and their relative proportions infecting a single animal. This information has also shown that some helicobacters may have a much broader host range than originally reported. PMID:18834679

  6. Comparative molecular approaches in Prader-Willi syndrome diagnosis.

    PubMed

    Botezatu, Anca; Puiu, Maria; Cucu, Natalia; Diaconu, Carmen C; Badiu, C; Arsene, C; Iancu, Iulia V; Plesa, Adriana; Anton, Gabriela

    2016-01-10

    Prader-Willi and Angelman syndromes are two distinct neurogenetic disorders caused by chromosomal deletions, uniparental disomy or loss of the imprinted gene expression in the 15q11-q13 region. PWS results from the lack of the paternally expressed gene contribution in the region. The aim of our study was to compare a new molecular approach based on the quantification of the expression of non-imprinted bi-allelic gene (NIPA1 and OCA2) with in house MS-PCR and the MS-MLPA test. Blood samples were collected from 12 patients, clinical criteria positives for Prader-Willi syndrome. DNA and RNA samples were isolated from white blood cells. Epigenetic changes at SNRPN gene locus were evaluated by MS-PCR technique. The expression levels of two non-imprinted genes (NIPA1 and OCA2) were evaluated in qReal-Time PCR, in order to identify type 1 and type 2 deletions. SALSA MS-MLPA kit ME028 was used to detect copy number changes and to analyze CpG islands methylation of the 15q11 region. MS-MLPA test confirmed that 8/12 patients presented different types of deletion at the SNRPN gene level (promoter, introns, and exons) and 4/8 displayed type 1 or type 2 deletion. In children with 15q11-13 deletions, the decreased level of NIPA1and OCA2 gene expression is related to chromosomal abnormality in the investigated area. The deletions were confirmed by MS-MLPA analysis, thus recommending NIPA1 and OCA2 gene expression as an alternate method to investigate deletions. PMID:26335514

  7. Theory of bi-molecular association dynamics in 2D for accurate model and experimental parameterization of binding rates

    NASA Astrophysics Data System (ADS)

    Yogurtcu, Osman N.; Johnson, Margaret E.

    2015-08-01

    The dynamics of association between diffusing and reacting molecular species are routinely quantified using simple rate-equation kinetics that assume both well-mixed concentrations of species and a single rate constant for parameterizing the binding rate. In two-dimensions (2D), however, even when systems are well-mixed, the assumption of a single characteristic rate constant for describing association is not generally accurate, due to the properties of diffusional searching in dimensions d ≤ 2. Establishing rigorous bounds for discriminating between 2D reactive systems that will be accurately described by rate equations with a single rate constant, and those that will not, is critical for both modeling and experimentally parameterizing binding reactions restricted to surfaces such as cellular membranes. We show here that in regimes of intrinsic reaction rate (ka) and diffusion (D) parameters ka/D > 0.05, a single rate constant cannot be fit to the dynamics of concentrations of associating species independently of the initial conditions. Instead, a more sophisticated multi-parametric description than rate-equations is necessary to robustly characterize bimolecular reactions from experiment. Our quantitative bounds derive from our new analysis of 2D rate-behavior predicted from Smoluchowski theory. Using a recently developed single particle reaction-diffusion algorithm we extend here to 2D, we are able to test and validate the predictions of Smoluchowski theory and several other theories of reversible reaction dynamics in 2D for the first time. Finally, our results also mean that simulations of reactive systems in 2D using rate equations must be undertaken with caution when reactions have ka/D > 0.05, regardless of the simulation volume. We introduce here a simple formula for an adaptive concentration dependent rate constant for these chemical kinetics simulations which improves on existing formulas to better capture non-equilibrium reaction dynamics from dilute

  8. Congenital neutropenia: diagnosis, molecular bases and patient management

    PubMed Central

    2011-01-01

    The term congenital neutropenia encompasses a family of neutropenic disorders, both permanent and intermittent, severe (<0.5 G/l) or mild (between 0.5-1.5 G/l), which may also affect other organ systems such as the pancreas, central nervous system, heart, muscle and skin. Neutropenia can lead to life-threatening pyogenic infections, acute gingivostomatitis and chronic parodontal disease, and each successive infection may leave permanent sequelae. The risk of infection is roughly inversely proportional to the circulating polymorphonuclear neutrophil count and is particularly high at counts below 0.2 G/l. When neutropenia is detected, an attempt should be made to establish the etiology, distinguishing between acquired forms (the most frequent, including post viral neutropenia and auto immune neutropenia) and congenital forms that may either be isolated or part of a complex genetic disease. Except for ethnic neutropenia, which is a frequent but mild congenital form, probably with polygenic inheritance, all other forms of congenital neutropenia are extremely rare and have monogenic inheritance, which may be X-linked or autosomal, recessive or dominant. About half the forms of congenital neutropenia with no extra-hematopoetic manifestations and normal adaptive immunity are due to neutrophil elastase (ELANE) mutations. Some patients have severe permanent neutropenia and frequent infections early in life, while others have mild intermittent neutropenia. Congenital neutropenia may also be associated with a wide range of organ dysfunctions, as for example in Shwachman-Diamond syndrome (associated with pancreatic insufficiency) and glycogen storage disease type Ib (associated with a glycogen storage syndrome). So far, the molecular bases of 12 neutropenic disorders have been identified. Treatment of severe chronic neutropenia should focus on prevention of infections. It includes antimicrobial prophylaxis, generally with trimethoprim-sulfamethoxazole, and also granulocyte

  9. An accurate metalloprotein-specific scoring function and molecular docking program devised by a dynamic sampling and iteration optimization strategy.

    PubMed

    Bai, Fang; Liao, Sha; Gu, Junfeng; Jiang, Hualiang; Wang, Xicheng; Li, Honglin

    2015-04-27

    Metalloproteins, particularly zinc metalloproteins, are promising therapeutic targets, and recent efforts have focused on the identification of potent and selective inhibitors of these proteins. However, the ability of current drug discovery and design technologies, such as molecular docking and molecular dynamics simulations, to probe metal-ligand interactions remains limited because of their complicated coordination geometries and rough treatment in current force fields. Herein we introduce a robust, multiobjective optimization algorithm-driven metalloprotein-specific docking program named MpSDock, which runs on a scheme similar to consensus scoring consisting of a force-field-based scoring function and a knowledge-based scoring function. For this purpose, in this study, an effective knowledge-based zinc metalloprotein-specific scoring function based on the inverse Boltzmann law was designed and optimized using a dynamic sampling and iteration optimization strategy. This optimization strategy can dynamically sample and regenerate decoy poses used in each iteration step of refining the scoring function, thus dramatically improving both the effectiveness of the exploration of the binding conformational space and the sensitivity of the ranking of the native binding poses. To validate the zinc metalloprotein-specific scoring function and its special built-in docking program, denoted MpSDockZn, an extensive comparison was performed against six universal, popular docking programs: Glide XP mode, Glide SP mode, Gold, AutoDock, AutoDock4Zn, and EADock DSS. The zinc metalloprotein-specific knowledge-based scoring function exhibited prominent performance in accurately describing the geometries and interactions of the coordination bonds between the zinc ions and chelating agents of the ligands. In addition, MpSDockZn had a competitive ability to sample and identify native binding poses with a higher success rate than the other six docking programs.

  10. Can optical diagnosis of small colon polyps be accurate? Comparing standard scope without narrow banding to high definition scope with narrow banding

    PubMed Central

    Ashktorab, Hassan; Etaati, Firoozeh; Rezaeean, Farahnaz; Nouraie, Mehdi; Paydar, Mansour; Namin, Hassan Hassanzadeh; Sanderson, Andrew; Begum, Rehana; Alkhalloufi, Kawtar; Brim, Hassan; Laiyemo, Adeyinka O

    2016-01-01

    AIM: To study the accuracy of using high definition (HD) scope with narrow band imaging (NBI) vs standard white light colonoscope without NBI (ST), to predict the histology of the colon polyps, particularly those < 1 cm. METHODS: A total of 147 African Americans patients who were referred to Howard University Hospital for screening or, diagnostic or follow up colonoscopy, during a 12-mo period in 2012 were prospectively recruited. Some patients had multiple polyps and total number of polyps was 179. Their colonoscopies were performed by 3 experienced endoscopists who determined the size and stated whether the polyps being removed were hyperplastic or adenomatous polyps using standard colonoscopes or high definition colonoscopes with NBI. The histopathologic diagnosis was reported by pathologists as part of routine care. RESULTS: Of participants in the study, 55 (37%) were male and median (interquartile range) of age was 56 (19-80). Demographic, clinical characteristics, past medical history of patients, and the data obtained by two instruments were not significantly different and two methods detected similar number of polyps. In ST scope 89% of polyps were < 1 cm vs 87% in HD scope (P = 0.7). The ST scope had a positive predictive value (PPV) and positive likelihood ratio (PLR) of 86% and 4.0 for adenoma compared to 74% and 2.6 for HD scope. There was a trend of higher sensitivity for HD scope (68%) compare to ST scope (53%) with almost the same specificity. The ST scope had a PPV and PLR of 38% and 1.8 for hyperplastic polyp (HPP) compared to 42% and 2.2 for HD scope. The sensitivity and specificity of two instruments for HPP diagnosis were similar. CONCLUSION: Our results indicated that HD scope was more sensitive in diagnosis of adenoma than ST scope. Clinical diagnosis of HPP with either scope is less accurate compared to adenoma. Colonoscopy diagnosis is not yet fully matched with pathologic diagnosis of colon polyp. However with the advancement of both

  11. Can optical diagnosis of small colon polyps be accurate? Comparing standard scope without narrow banding to high definition scope with narrow banding

    PubMed Central

    Ashktorab, Hassan; Etaati, Firoozeh; Rezaeean, Farahnaz; Nouraie, Mehdi; Paydar, Mansour; Namin, Hassan Hassanzadeh; Sanderson, Andrew; Begum, Rehana; Alkhalloufi, Kawtar; Brim, Hassan; Laiyemo, Adeyinka O

    2016-01-01

    AIM: To study the accuracy of using high definition (HD) scope with narrow band imaging (NBI) vs standard white light colonoscope without NBI (ST), to predict the histology of the colon polyps, particularly those < 1 cm. METHODS: A total of 147 African Americans patients who were referred to Howard University Hospital for screening or, diagnostic or follow up colonoscopy, during a 12-mo period in 2012 were prospectively recruited. Some patients had multiple polyps and total number of polyps was 179. Their colonoscopies were performed by 3 experienced endoscopists who determined the size and stated whether the polyps being removed were hyperplastic or adenomatous polyps using standard colonoscopes or high definition colonoscopes with NBI. The histopathologic diagnosis was reported by pathologists as part of routine care. RESULTS: Of participants in the study, 55 (37%) were male and median (interquartile range) of age was 56 (19-80). Demographic, clinical characteristics, past medical history of patients, and the data obtained by two instruments were not significantly different and two methods detected similar number of polyps. In ST scope 89% of polyps were < 1 cm vs 87% in HD scope (P = 0.7). The ST scope had a positive predictive value (PPV) and positive likelihood ratio (PLR) of 86% and 4.0 for adenoma compared to 74% and 2.6 for HD scope. There was a trend of higher sensitivity for HD scope (68%) compare to ST scope (53%) with almost the same specificity. The ST scope had a PPV and PLR of 38% and 1.8 for hyperplastic polyp (HPP) compared to 42% and 2.2 for HD scope. The sensitivity and specificity of two instruments for HPP diagnosis were similar. CONCLUSION: Our results indicated that HD scope was more sensitive in diagnosis of adenoma than ST scope. Clinical diagnosis of HPP with either scope is less accurate compared to adenoma. Colonoscopy diagnosis is not yet fully matched with pathologic diagnosis of colon polyp. However with the advancement of both

  12. Can simple tests performed in the primary care setting provide accurate and efficient diagnosis of benign prostatic hyperplasia? Rationale and design of the Diagnosis Improvement in Primary Care Trial.

    PubMed

    Carballido, J; Fourcade, R; Pagliarulo, A; Cricelli, C; Brenes, F; Pedromingo-Marino, A; Castro, R

    2009-08-01

    Effective treatment of benign prostatic hyperplasia (BPH) improves lower urinary tract symptoms (LUTS) and patient quality of life, and reduces the risk of complications arising from disease progression. However, treatment can only be initiated when men with BPH are identified by accurate diagnostic tests. Current evidence suggests that diagnostic procedures employed by primary care physicians vary widely across Europe. The expected increases in BPH prevalence accompanying the gradual aging of the population, coupled with greater use of medical therapy, mean that general practitioners (GPs) are likely to have an increasingly important role in managing the condition. The GP/primary care clinic is therefore an attractive target location for strategies designed to improve the accuracy of BPH diagnosis. The Diagnosis Improvement in Primary Care Trial (D-IMPACT) is a prospective, multicentre, epidemiological study that aims to identify the optimal subset of simple tests applied by GPs in the primary care setting to diagnose BPH in men who spontaneously report obstructive (voiding) and/or irritative (storage) LUTS. These tests comprise medical history, symptom assessment with the International Prostate Symptom Score questionnaire, urinalysis, measurement of serum levels of prostate-specific antigen and subjective GP diagnosis after completing all tests including digital rectal examination. GP diagnoses and all other tests will be compared with gold-standard diagnoses provided by specialist urologists following completion of additional diagnostic tests. D-IMPACT will establish the diagnostic performance using a non-subjective and reproducible algorithm. An adjusted and multivariate analysis of the results of D-IMPACT will allow identification of the most efficient combination of tests that facilitate accurate BPH diagnosis in the primary care setting. In addition, D-IMPACT will estimate the prevalence of BPH in patients who present spontaneously to GPs with LUTS. PMID

  13. Nonlinear Optical Properties of Fluorescent Dyes Allow for Accurate Determination of Their Molecular Orientations in Phospholipid Membranes.

    PubMed

    Timr, Štěpán; Brabec, Jiří; Bondar, Alexey; Ryba, Tomáš; Železný, Miloš; Lazar, Josef; Jungwirth, Pavel

    2015-07-30

    Several methods based on single- and two-photon fluorescence detected linear dichroism have recently been used to determine the orientational distributions of fluorescent dyes in lipid membranes. However, these determinations relied on simplified descriptions of nonlinear anisotropic properties of the dye molecules, using a transition dipole-moment-like vector instead of an absorptivity tensor. To investigate the validity of the vector approximation, we have now carried out a combination of computer simulations and polarization microscopy experiments on two representative fluorescent dyes (DiI and F2N12S) embedded in aqueous phosphatidylcholine bilayers. Our results indicate that a simplified vector-like treatment of the two-photon transition tensor is applicable for molecular geometries sampled in the membrane at ambient conditions. Furthermore, our results allow evaluation of several distinct polarization microscopy techniques. In combination, our results point to a robust and accurate experimental and computational treatment of orientational distributions of DiI, F2N12S, and related dyes (including Cy3, Cy5, and others), with implications to monitoring physiologically relevant processes in cellular membranes in a novel way.

  14. Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods

    PubMed Central

    Flynn, Jullien M; Brown, Emily A; Chain, Frédéric J J; MacIsaac, Hugh J; Cristescu, Melania E

    2015-01-01

    Metabarcoding has the potential to become a rapid, sensitive, and effective approach for identifying species in complex environmental samples. Accurate molecular identification of species depends on the ability to generate operational taxonomic units (OTUs) that correspond to biological species. Due to the sometimes enormous estimates of biodiversity using this method, there is a great need to test the efficacy of data analysis methods used to derive OTUs. Here, we evaluate the performance of various methods for clustering length variable 18S amplicons from complex samples into OTUs using a mock community and a natural community of zooplankton species. We compare analytic procedures consisting of a combination of (1) stringent and relaxed data filtering, (2) singleton sequences included and removed, (3) three commonly used clustering algorithms (mothur, UCLUST, and UPARSE), and (4) three methods of treating alignment gaps when calculating sequence divergence. Depending on the combination of methods used, the number of OTUs varied by nearly two orders of magnitude for the mock community (60–5068 OTUs) and three orders of magnitude for the natural community (22–22191 OTUs). The use of relaxed filtering and the inclusion of singletons greatly inflated OTU numbers without increasing the ability to recover species. Our results also suggest that the method used to treat gaps when calculating sequence divergence can have a great impact on the number of OTUs. Our findings are particularly relevant to studies that cover taxonomically diverse species and employ markers such as rRNA genes in which length variation is extensive. PMID:26078860

  15. Absorption spectroscopy of the rubidium dimer in an overheated vapor: An accurate check of molecular structure and dynamics

    SciTech Connect

    Beuc, R.; Movre, M.; Horvatic, V.; Vadla, C.; Dulieu, O.; Aymar, M.

    2007-03-15

    Experimental studies of the absorption spectrum of the Rb{sub 2} dimer are performed in the 600-1100 nm wavelength range for temperatures between 615 and 745 K. The reduced absorption coefficient is measured by spatially resolved white light absorption in overheated rubidium vapor with a radial temperature gradient, which enables simultaneous measurements at different temperatures. Semiclassical and quantum spectral simulations are obtained by taking into account all possible transitions involving the potential curves stemming from the 5 {sup 2}S+5 {sup 2}S and 5 {sup 2}S+5 {sup 2}P asymptotes. The most accurate experimental potential curves are used where available, and newly calculated potential curves and transition dipole moments otherwise. The overall consistency of the theoretical model with the experimental interpretation is obtained only if the radial dependence of both the calculated transition dipole moments and the spin-orbit coupling is taken into account. This highlights the low-resolution absorption spectroscopy as a valuable tool for checking the accuracy of molecular electronic structure calculations.

  16. [Molecular diagnostic methods of respiratory infections. Has the scheme diagnosis changed?].

    PubMed

    Vila Estapé, Jordi; Zboromyrska, Yuliya; Vergara Gómez, Andrea; Alejo Cancho, Izaskun; Rubio García, Elisa; Álvarez-Martínez, Miriam José; la Bellacasa Brugada, Jorge Puig de; Marcos Maeso, M Ángeles

    2016-07-01

    Lower respiratory tract infections remain one of the most common causes of mortality worldwide, which is why early diagnosis is crucial. Traditionally the microbiological diagnosis of these infections has been based on conventional methods including culture on artificial media for isolation of bacteria and fungi and cell cultures for virus and antibody or antigen detection using antigen-antibody reactions. The main drawback of the above mentioned methods is the time needed for an etiological diagnosis of the infection. The techniques based on molecular biology have drawn much attention in recent decades as tools for rapid diagnosis of infections. Some techniques are very expensive, especially those that can detect various microorganisms in the same reaction, therefore the question that arises is whether the cost of such testing is justified by the information obtained and by the clinical impact that its implementation will determine. In this article we make a review of the various techniques of molecular biology applied to the diagnosis of pneumonia and focus primarily on analysing the impact they may have on the management of patients with acute respiratory tract infections.

  17. Rapid molecular prenatal diagnosis of spondyloepiphyseal dysplasia congenita by PCR-SSP assay.

    PubMed

    Cui, Ying-Xia; Xia, Xin-Yi; Bu, Ying; Zhou, Guo-Hua; Yang, Bin; Lu, Hong-Yong; Shi, Yi-Chao; Pan, Lian-Jun; Huang, Yu-Feng; Li, Xiao-Jun

    2008-12-01

    Heterozygous mutations of COL2A1 gene are responsible for type II collagenopathies. The common skeletal phenotypes include achondrogenesis type II, hypochondrogenesis, Stickler dysplasia, Kniest dysplasia, late onset spondyloepiphyseal dysplasia, and spondyloepiphyseal dysplasia congenita (SEDC). Prevention of SEDC can be achieved by prenatal diagnosis. This study reports the first rapid molecular prenatal diagnosis of SEDC performed in China by polymerase chain reaction sequence-specific primer (PCR-SSP) analysis. The pregnant woman we previously reported with SEDC carried the G to A substitution at nucleotide 1510 in exon 23 of COL2A1 gene, which caused a change from glycine to serine at codon 504 (G504S). By the time the woman got pregnant again, she had terminated two pregnancies and still had no child. In the first pregnancy, the molecular mutation of the family was not yet identified, and therefore prenatal diagnosis was unable to be performed by DNA analysis. In the second pregnancy, G504S mutation was found from fetal DNA. At the time of her third pregnancy, the woman and her husband became extremely worried about the potential SEDC for the fetus. For this reason, a quick and reliable molecular prenatal diagnosis of SEDC was performed by a PCR-SSP on an amniocyte sample collected at the 14th week of pregnancy. No mutation of the fetal DNA was identified. The result was obtained within 24 h after the sample was collected. The technique could be applied in confirmatory diagnosis and prenatal diagnosis for the affected family. PMID:19072565

  18. Staging of biliary atresia at diagnosis by molecular profiling of the liver

    PubMed Central

    2010-01-01

    Background Young age at portoenterostomy has been linked to improved outcome in biliary atresia, but pre-existing biological factors may influence the rate of disease progression. In this study, we aimed to determine whether molecular profiling of the liver identifies stages of disease at diagnosis. Methods We examined liver biopsies from 47 infants with biliary atresia enrolled in a prospective observational study. Biopsies were scored for inflammation and fibrosis, used for gene expression profiles, and tested for association with indicators of disease severity, response to surgery, and survival at 2 years. Results Fourteen of 47 livers displayed predominant histological features of inflammation (N = 9) or fibrosis (N = 5), with the remainder showing similar levels of both simultaneously. By differential profiling of gene expression, the 14 livers had a unique molecular signature containing 150 gene probes. Applying prediction analysis models, the probes classified 29 of the remaining 33 livers into inflammation or fibrosis. Molecular classification into the two groups was validated by the findings of increased hepatic population of lymphocyte subsets or tissue accumulation of matrix substrates. The groups had no association with traditional markers of liver injury or function, response to surgery, or complications of cirrhosis. However, infants with an inflammation signature were younger, while those with a fibrosis signature had decreased transplant-free survival. Conclusions Molecular profiling at diagnosis of biliary atresia uncovers a signature of inflammation or fibrosis in most livers. This signature may relate to staging of disease at diagnosis and has implications to clinical outcomes. PMID:20465800

  19. A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection.

    PubMed

    Otsuka, Nao; Gotoh, Kensei; Nishimura, Naoko; Ozaki, Takao; Nakamura, Yukitsugu; Haga, Kiyohito; Yamazaki, Makoto; Gondaira, Fumio; Okada, Kenji; Miyaji, Yusuke; Toyoizumi-Ajisaka, Hiromi; Shibayama, Keigo; Arakawa, Yoshichika; Kamachi, Kazunari

    2016-05-01

    An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

  20. Molecular Diagnostics

    PubMed Central

    Choe, Hyonmin; Deirmengian, Carl A.; Hickok, Noreen J.; Morrison, Tiffany N.; Tuan, Rocky S.

    2015-01-01

    Orthopaedic infections are complex conditions that require immediate diagnosis and accurate identification of the causative organisms to facilitate appropriate management. Conventional methodologies for diagnosis of these infections sometimes lack accuracy or sufficient rapidity. Current molecular diagnostics are an emerging area of bench-to-bedside research in orthopaedic infections. Examples of promising molecular diagnostics include measurement of a specific biomarker in the synovial fluid, polymerase chain reaction–based detection of bacterial genes, and metabolomic determination of responses to orthopaedic infection. PMID:25808967

  1. Intestinal Intraepithelial Lymphocyte Cytometric Pattern Is More Accurate than Subepithelial Deposits of Anti-Tissue Transglutaminase IgA for the Diagnosis of Celiac Disease in Lymphocytic Enteritis

    PubMed Central

    García-Puig, Roger; Rosinach, Mercè; González, Clarisa; Alsina, Montserrat; Loras, Carme; Salas, Antonio; Viver, Josep M.; Esteve, Maria

    2014-01-01

    Background & Aims An increase in CD3+TCRγδ+ and a decrease in CD3− intraepithelial lymphocytes (IEL) is a characteristic flow cytometric pattern of celiac disease (CD) with atrophy. The aim was to evaluate the usefulness of both CD IEL cytometric pattern and anti-TG2 IgA subepithelial deposit analysis (CD IF pattern) for diagnosing lymphocytic enteritis due to CD. Methods Two-hundred and five patients (144 females) who underwent duodenal biopsy for clinical suspicion of CD and positive celiac genetics were prospectively included. Fifty had villous atrophy, 70 lymphocytic enteritis, and 85 normal histology. Eight patients with non-celiac atrophy and 15 with lymphocytic enteritis secondary to Helicobacter pylori acted as control group. Duodenal biopsies were obtained to assess both CD IEL flow cytometric (complete or incomplete) and IF patterns. Results Sensitivity of IF, and complete and incomplete cytometric patterns for CD diagnosis in patients with positive serology (Marsh 1+3) was 92%, 85 and 97% respectively, but only the complete cytometric pattern had 100% specificity. Twelve seropositive and 8 seronegative Marsh 1 patients had a CD diagnosis at inclusion or after gluten free-diet, respectively. CD cytometric pattern showed a better diagnostic performance than both IF pattern and serology for CD diagnosis in lymphocytic enteritis at baseline (95% vs 60% vs 60%, p = 0.039). Conclusions Analysis of the IEL flow cytometric pattern is a fast, accurate method for identifying CD in the initial diagnostic biopsy of patients presenting with lymphocytic enteritis, even in seronegative patients, and seems to be better than anti-TG2 intestinal deposits. PMID:25010214

  2. Accurate molecular structure and spectroscopic properties of nucleobases: a combined computational-microwave investigation of 2-thiouracil as a case study.

    PubMed

    Puzzarini, Cristina; Biczysko, Malgorzata; Barone, Vincenzo; Peña, Isabel; Cabezas, Carlos; Alonso, José L

    2013-10-21

    The computational composite scheme purposely set up for accurately describing the electronic structure and spectroscopic properties of small biomolecules has been applied to the first study of the rotational spectrum of 2-thiouracil. The experimental investigation was made possible thanks to the combination of the laser ablation technique with Fourier transform microwave spectrometers. The joint experimental-computational study allowed us to determine the accurate molecular structure and spectroscopic properties of the title molecule, but more importantly, it demonstrates a reliable approach for the accurate investigation of isolated small biomolecules.

  3. MEGA-MD: molecular evolutionary genetics analysis software with mutational diagnosis of amino acid variation.

    PubMed

    Stecher, Glen; Liu, Li; Sanderford, Maxwell; Peterson, Daniel; Tamura, Koichiro; Kumar, Sudhir

    2014-05-01

    Computational diagnosis of amino acid variants in the human exome is the first step in assessing the disruptive impacts of non-synonymous single nucleotide variants (nsSNVs) on human health and disease. The Molecular Evolutionary Genetics Analysis software with mutational diagnosis (MEGA-MD) is a suite of tools developed to forecast the deleteriousness of nsSNVs using multiple methods and to explore nsSNVs in the context of the variability permitted in the long-term evolution of the affected position. In its graphical interface for use on desktops, it enables interactive computational diagnosis and evolutionary exploration of nsSNVs. As a web service, MEGA-MD is suitable for diagnosing variants on an exome scale. The MEGA-MD suite intends to serve the needs for conducting low- and high-throughput analysis of nsSNVs in diverse applications.

  4. A comparative analysis of different molecular targets using PCR for diagnosis of old world leishmaniasis.

    PubMed

    Koltas, Ismail S; Eroglu, Fadime; Uzun, Soner; Alabaz, Derya

    2016-05-01

    The different sensitivity values were obtained in each study conducted for the diagnosis of leishmaniasis with the polymerase chain reaction (PCR). However, a standardized PCR target for the diagnosis of leishmaniasis does not exist. The aim of the current study, the most ideal PCR target was determined for diagnosis of leishmaniasis. A total of 72 smear and 48 bone marrow samples were analyzed with six different molecular targets to determine their potential as a tool for the specific molecular diagnosis of leishmaniasis using PCR. The positivity-negativity value and the sensitivity-specificity of each PCR targets were calculated. The positivity value of PCR targets were sequenced in different levels in the diagnosis of leishmaniasis from highest to lowest in the order of kDNA-PCR > SSU rRNA-PCR > ITS2-PCR > ITS1-PCR > ME-PCR > HSP70-PCR. The sensitivities of PCR targets except ITS1-PCR, ME-PCR and HSP70-PCR were found to be 100% in cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) cases as compared to microscopic examination accepted as a gold standard. The sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 96.6%, 90.0% and 86.6%, respectively, in CL-cases. In addition, the sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 90.0%, 70.0% and 60.0%, respectively, in VL-cases. The kDNA genomic region was the most sensitive for routine diagnosis of leishmaniasis. ITS1-PCR restriction fragment length polymorphism, the alternative method for the identification of Old World Leishmania species, did not require culturing of the parasites.

  5. Comparison of molecular tests for the diagnosis of malaria in Honduras

    PubMed Central

    2012-01-01

    Background Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras. Methods A total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species. Results Of the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax

  6. Molecular-genetic analysis is essential for accurate classification of renal carcinoma resembling Xp11.2 translocation carcinoma.

    PubMed

    Hayes, Malcolm; Peckova, Kvetoslava; Martinek, Petr; Hora, Milan; Kalusova, Kristyna; Straka, Lubomir; Daum, Ondrej; Kokoskova, Bohuslava; Rotterova, Pavla; Pivovarčikova, Kristyna; Branzovsky, Jindrich; Dubova, Magdalena; Vesela, Pavla; Michal, Michal; Hes, Ondrej

    2015-03-01

    tumours can only be sub-classified accurately by multi-parameter molecular-genetic analysis. PMID:25544614

  7. Shwachman-Diamond syndrome: first molecular diagnosis in a Brazilian child.

    PubMed

    Alves, Cresio; Fernandes, Julia Constança; Sampaio, Silvana; Paiva, Raquel de Melo Alves; Calado, Rodrigo Tocantins

    2013-01-01

    Herein the first molecular diagnosis of a Brazilian child with Shwachman-Diamond Syndrome is reported. A 6-year-old boy was diagnosed with cystic fibrosis at the age of 15 months due to recurrent respiratory infections, diarrhea and therapeutic response to pancreatic enzymes. Three sweat tests were negative. At the age of 5 years, he began to experience pain in the lower limbs, laxity of joints, lameness and frequent falls. A radiological study revealed metaphyseal chondrodysplasia. A complete blood cell count showed leukopenia (leukocytes: 3.1-3.5 x 10(3)/µL), neutropenia (segmented neutrophils: 15-22%), but normal hemoglobin, hematocrit and platelet count. A molecular study revealed biallelic mutations in the Shwachman-Bodian-Diamond Syndrome gene (183-184TA-CT K62X in exon 2 and a 258+2T-C transition) confirming the diagnosis of Shwachman-Diamond Syndrome. A non-pathologic, silent nucleotide A to G transition at position 201 was also found in heterozygosis in the Shwachman-Bodian-Diamond Syndrome gene. This is the first report to describe a Brazilian child with molecular diagnosis of Shwachman-Diamond Syndrome, a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, intermittent or persistent neutropenia and skeletal changes. Other characteristics include immune system, hepatic and cardiac changes and predisposition to leukemia. Recurrent bacterial, viral and fungal infections are common. The possibility of Shwachman-Diamond Syndrome should be kept in mind when investigating children with a diagnosis of cystic fibrosis and normal sweat tests.

  8. Charcot-marie-tooth disease and related neuropathies: molecular basis for distinction and diagnosis.

    PubMed

    Pareyson, D

    1999-11-01

    Great advances have been made in understanding the molecular basis of Charcot-Marie-Tooth disease (CMT) and related neuropathies, namely Dejerine-Sottas disease (DSD), hereditary neuropathy with liability to pressure palsies (HNPP) and congenital hypomyelination (CH). The number of newly uncovered mutations and identified genetic loci is rapidly increasing, and, as a consequence, the classification of these disorders is becoming more complicated. Molecular genetics, animal models, and transfected cell studies are shedding light on function and dysfunction of proteins involved in hereditary myelinopathies-peripheral myelin protein 22 (PMP22), myelin protein zero (PO), connexin 32 (Cx32), and early growth response 2 (EGR2). Gene dosage effect, loss of function, gain of toxic function, and dominant negative effect are possible mechanisms whereby different gene mutations may exert their detrimental action on peripheral nerves. A tentative rational approach to clinical and molecular diagnosis based on genotype-phenotype correlation analysis is described. PMID:10514227

  9. Homing peptide guiding optical molecular imaging for the diagnosis of bladder cancer

    NASA Astrophysics Data System (ADS)

    Yang, Xiao-feng; Pang, Jian-zhi; Liu, Jie-hao; Zhao, Yang; Jia, Xing-you; Li, Jun; Liu, Reng-xin; Wang, Wei; Fan, Zhen-wei; Zhang, Zi-qiang; Yan, San-hua; Luo, Jun-qian; Zhang, Xiao-lei

    2014-11-01

    Background: The limitations of primary transurethral resection of bladder tumor (TURBt) have led the residual tumors rates as high as 75%. The intraoperative fluorescence imaging offers a great potential for improving TURBt have been confirmed. So we aim to distinguish the residual tumors and normal mucosa using fluorescence molecular imaging formed by conjugated molecule of the CSNRDARRC bladder cancer homing peptide with fluorescent dye. The conjugated molecule was abbreviated FIuo-ACP. In our study, we will research the image features of FIuo-ACP probe targeted bladder cancer for fluorescence molecular imaging diagnosis for bladder cancer in vivo and ex vivo. Methods: After the FIuo-ACP probe was synthetized, the binding sites, factors affecting binding rates, the specificity and the targeting of Fluo-ACP labeled with bladder cancer cells were studied respectively by laser scanning confocal microscope (LSCM), immunofluorescence and multispectral fluorescence ex vivo optical molecular imaging system. Results: The binding sites were located in nucleus and the binding rates were correlated linearly with the dose of probe and the grade of pathology. Moreover, the probe has a binding specificity with bladder cancer in vivo and ex vivo. Tumor cells being labeled by the Fluo-ACP, bright green spots were observed under LSCM. The tissue samples and tumor cells can be labeled and identified by fluorescence microscope. Optical molecular imaging of xenograft tumor tissues was exhibited as fluorescent spots under EMCCD. Conclusion: The CSNRDARRC peptides might be a useful bladder cancer targeting vector. The FIuo-ACP molecular probe was suitable for fluorescence molecular imaging diagnosis for bladder cancer in vivo and ex vivo.

  10. Toward molecular parasitologic diagnosis: enhanced diagnostic sensitivity for filarial infections in mobile populations.

    PubMed

    Fink, Doran L; Fahle, Gary A; Fischer, Steven; Fedorko, Daniel F; Nutman, Thomas B

    2011-01-01

    The diagnosis of filarial infections among individuals residing in areas where the disease is not endemic requires both strong clinical suspicion and expert training in infrequently practiced parasitological methods. Recently developed filarial molecular diagnostic assays are highly sensitive and specific but have limited availability and have not been closely evaluated for clinical use outside populations residing in areas of endemicity. In this study, we assessed the performance of a panel of real-time PCR assays for the four most common human filarial pathogens among blood and tissue samples collected from a cohort of patients undergoing evaluation for suspected filarial infections. Compared to blood filtration, real-time PCR was equally sensitive for the detection of microfilaremia due to Wuchereria bancrofti (2 of 46 samples positive by both blood filtration and PCR with no discordant results) and Loa loa (24 of 208 samples positive by both blood filtration and PCR, 4 samples positive by PCR only, and 3 samples positive by blood filtration only). Real-time PCR of skin snip samples was significantly more sensitive than microscopic examination for the detection of Onchocerca volvulus microfiladermia (2 of 218 samples positive by both microscopy and PCR and 12 samples positive by PCR only). The molecular assays required smaller amounts of blood and tissue than conventional methods and could be performed by laboratory personnel without specialized parasitology training. Taken together, these data demonstrate the utility of the molecular diagnosis of filarial infections in mobile populations. PMID:20980560

  11. Molecular epidemiology and diagnosis of PBG deaminase gene defects in acute intermittent porphyria.

    PubMed Central

    Puy, H; Deybach, J C; Lamoril, J; Robreau, A M; Da Silva, V; Gouya, L; Grandchamp, B; Nordmann, Y

    1997-01-01

    Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis. Images Figure 1 Figure 2 PMID:9199558

  12. An intra-body molecular communication networks framework for continuous health monitoring and diagnosis.

    PubMed

    Chahibi, Youssef; Balasingham, Ilangko

    2015-01-01

    Intra-body communication networks are designed to interconnect nano- or micro-sized sensors located inside the body for health monitoring and drug delivery. The most promising solutions are made of implanted nanosensors to timely monitor the body for the presence of specific diseases and pronounce a diagnosis without the intervention of a physician. In this manner, several deadly health conditions such as heart attacks are avoided through the early in vivo detection of their biomarkers. In reality, nanosensors are challenged by the individual specificities, molecular noise, limited durability, and low energy resources. In this paper, a framework is proposed for estimating and detecting diseases and localizing the nanosensors. This framework is based on molecular communication, a novel communication paradigm where information is conveyed through molecules. Through the case study of the shedding of endothelial cells as an early biomarker for heart attack, the intra-body molecular communication networks framework is shown to resolve major issues with in vivo nanosensors and lay the foundations of low-complexity biomedical signal processing algorithms for continuous disease monitoring and diagnosis. PMID:26737190

  13. An intra-body molecular communication networks framework for continuous health monitoring and diagnosis.

    PubMed

    Chahibi, Youssef; Balasingham, Ilangko

    2015-01-01

    Intra-body communication networks are designed to interconnect nano- or micro-sized sensors located inside the body for health monitoring and drug delivery. The most promising solutions are made of implanted nanosensors to timely monitor the body for the presence of specific diseases and pronounce a diagnosis without the intervention of a physician. In this manner, several deadly health conditions such as heart attacks are avoided through the early in vivo detection of their biomarkers. In reality, nanosensors are challenged by the individual specificities, molecular noise, limited durability, and low energy resources. In this paper, a framework is proposed for estimating and detecting diseases and localizing the nanosensors. This framework is based on molecular communication, a novel communication paradigm where information is conveyed through molecules. Through the case study of the shedding of endothelial cells as an early biomarker for heart attack, the intra-body molecular communication networks framework is shown to resolve major issues with in vivo nanosensors and lay the foundations of low-complexity biomedical signal processing algorithms for continuous disease monitoring and diagnosis.

  14. DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

    PubMed

    Bräunig, Patrícia; Portella, Luiza Pires; Cezar, Alfredo Skrebsky; Libardoni, Felipe; Sangioni, Luis Antonio; Vogel, Fernanda Silveira Flores; Gonçalves, Paulo Bayard Dias

    2016-10-01

    Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

  15. Molecular genetics of the sickling syndromes: evolution of new strategies for improved diagnosis.

    PubMed

    Benz, E J

    1984-01-01

    This survey is intended to illustrate some major areas relevant to the diagnosis and treatment of sickle cell syndromes that have benefited by the input of molecular genetic approaches. The development of gene mapping techniques has permitted the direct examination of the effect of co-inheritance of alpha-thalassemia and sickle cell anemia on clinical severity, providing, for the first time, a direct strategy for investigation of the clinical heterogeneity of these syndromes. In addition, antenatal diagnosis of these disorders is now best done by direct gene mapping whenever appropriate facilities are available. Treatment by manipulation of gamma-globin gene expression has been shown to be an effective means of achieving at least partial reversal of the hemoglobin F to hemoglobin A switch. Whether the magnitude of this reversal is sufficient to interfere with the clinical phenotype of sickle cell disease remains to be determined. Moreover, the agent currently available to accomplish this goal, 5-azacytidine, remains unsuitable for wide-spread clinical application for a variety of reasons. Nonetheless, the molecular geneticist has already demonstrated that desirable effects can be achieved by building on the knowledge of globin gene physiology. This knowledge is best acquired by application of the concepts and methodologies of molecular genetics.

  16. [Biochemical and molecular diagnosis of alpha 1 antitrypsin deficiency in a Tunisian family].

    PubMed

    Denden, S; Braham, W; Amri, F; Lakhdar, R; Lefranc, G; Knani, J; Ben Chibani, J; Haj Khelil, A

    2009-01-01

    Our study investigated alpha 1 antitrypsin deficiency (AATD) diagnosis in a family originated from central Tunisia and showing a familial history of asthma. Biochemical and genetic diagnosis for AATD was performed according to current diagnostic standards. AAT level quantification in affected individuals showed plasma AAT levels consistent with intermediate AATD (ranged from 0.91 to 1.04 g/L). The molecular analysis was assessed using the genotyping of the most prevalent PI*S and PI*Z SERPINA1 mutations and the sequencing of AAT coding exons for rare AATD variants detection. No PI*S or PI*Z deficient variants were seen in this family. Sequencing results showed the inheritance of the deficient rare variant PI*M(wurzburg) (P369S) at the heterozygous state in the mother and two affected siblings. However, AATD status remains unexplained in the third affected case, with no mutations detected in the AAT coding exons. PMID:19654085

  17. Genetics of hypertrophic cardiomyopathy: advances and pitfalls in molecular diagnosis and therapy

    PubMed Central

    Roma-Rodrigues, Catarina; Fernandes, Alexandra R

    2014-01-01

    Hypertrophic cardiomyopathy (HCM) is a primary disease of the cardiac muscle that occurs mainly due to mutations (>1,400 variants) in genes encoding for the cardiac sarcomere. HCM, the most common familial form of cardiomyopathy, affecting one in every 500 people in the general population, is typically inherited in an autosomal dominant pattern, and presents variable expressivity and age-related penetrance. Due to the morphological and pathological heterogeneity of the disease, the appearance and progression of symptoms is not straightforward. Most HCM patients are asymptomatic, but up to 25% develop significant symptoms, including chest pain and sudden cardiac death. Sudden cardiac death is a dramatic event, since it occurs without warning and mainly in younger people, including trained athletes. Molecular diagnosis of HCM is of the outmost importance, since it may allow detection of subjects carrying mutations on HCM-associated genes before development of clinical symptoms of HCM. However, due to the genetic heterogeneity of HCM, molecular diagnosis is difficult. Currently, there are mainly four techniques used for molecular diagnosis of HCM, including Sanger sequencing, high resolution melting, mutation detection using DNA arrays, and next-generation sequencing techniques. Application of these methods has proven successful for identification of mutations on HCM-related genes. This review summarizes the features of these technologies, highlighting their strengths and weaknesses. Furthermore, current therapeutics for HCM patients are correlated with clinically observed phenotypes and are based on the alleviation of symptoms. This is mainly due to insufficient knowledge on the mechanisms involved in the onset of HCM. Tissue engineering alongside regenerative medicine coupled with nanotherapeutics may allow fulfillment of those gaps, together with screening of novel therapeutic drugs and target delivery systems. PMID:25328416

  18. Comprehensive Molecular Diagnosis of Bardet-Biedl Syndrome by High-Throughput Targeted Exome Sequencing

    PubMed Central

    Huang, Na; Wu, Kun-Chao; Huang, Xiu-Feng; Huang, Fang; Tong, Yi; Pang, Chi-Pui; Qu, Jia; Jin, Zi-Bing

    2014-01-01

    Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder with significant genetic heterogeneity. BBS is linked to mutations in 17 genes, which contain more than 200 coding exons. Currently, BBS is diagnosed by direct DNA sequencing for mutations in these genes, which because of the large genomic screening region is both time-consuming and expensive. In order to develop a practical method for the clinic diagnosis of BBS, we have developed a high-throughput targeted exome sequencing (TES) for genetic diagnosis. Five typical BBS patients were recruited and screened for mutations in a total of 144 known genes responsible for inherited retinal diseases, a hallmark symptom of BBS. The genomic DNA of these patients and their families were subjected to high-throughput DNA re-sequencing. Deep bioinformatics analysis was carried out to filter the massive sequencing data, which were further confirmed through co-segregation analysis. TES successfully revealed mutations in BBS genes in each patient and family member. Six pathological mutations, including five novel mutations, were revealed in the genes BBS2, MKKS, ARL6, MKS1. This study represents the first report of targeted exome sequencing in BBS patients and demonstrates that high-throughput TES is an accurate and rapid method for the genetic diagnosis of BBS. PMID:24608809

  19. Molecular prenatal diagnosis of autosomal recessive childhood spinal muscular atrophies (SMAs).

    PubMed

    Essawi, Mona L; Al-Attribi, Ghada M; Gaber, Khaled R; El-Harouni, Ashraf A

    2012-11-01

    Autosomal recessive childhood spinal muscular atrophy (SMAs) is the second most common neuromuscular disorder and a common cause of infant disability and mortality. SMA patients are classified into three clinical types based on age of onset, and severity of symptoms. About 94% of patients have homozygous deletion of exon 7 in survival motor neuron (SMN1) gene. The neuronal apoptosis inhibitory protein (NAIP) gene was found to be more frequently deleted in the severest form of the disease. This study aimed to comment on the implementation of genetic counseling and prenatal diagnosis of SMAs for 85 fetuses from 75 Egyptian couples at risk of having an affected child. The homozygous deletion of exon 7 in SMN1 gene and the deletion of exon 5 of the NAIP gene were detected using PCR-REFLP and multiplex PCR methods respectively. Eighteen fetuses showed homozygous deletion of exon 7 in SMN1 gene and deletion of exon 5 in NAIP gene. In conclusion prenatal diagnosis is an important tool for accurate diagnosis and genetic counseling that help decision making in high risk families. PMID:22921322

  20. [Molecular-genetic approaches to diagnosis and intraspecific typing of causative agents of glanders and melioidosis].

    PubMed

    Antonov, V A; Iliukhin, V I

    2005-01-01

    Pathogenic Burkholderia--Burkholderia mallei and Burkholderia pseudomallei--are causative agents of glanders and melioidosis, severe infectious diseases of man and animals. They are regarded as potential agents of bioterrorism. The existing bacteriological and immunological methods of identification of B. mallei and B. pseudomallei are not efficient enough for the rapid diagnosis and typing of strains. Described in the paper are molecular methods of detection of the agents by PCR, hybridization and strain typing made on the basis of bacterial total cell protein profiles, RAPD, ribotyping as well as of plasmid and DNA microrestriction analyses.

  1. Clinical, cytogenetic, and molecular diagnosis of Angelman syndrome: Estimated prevalence rate in a Danish country

    SciTech Connect

    Petersen, M.B.; Brondum-Nielsen, K.; Hansen, L.K.; Wulff, K.

    1995-06-19

    Angelman syndrome (AS) was initially considered a rather rare abnormality, but in later years, with the possibilities for cytogenetic and molecular diagnosis an increasing number of patients have been reported. The incidence is quoted to be around 1:20,000. The etiology of AS is associated with the lack of maternal allele(s) of one or more loci at 15q11-q13, and is considered an effect of parental imprinting of that region, since a similar deficiency of paternal alleles leads to Prader-Willi syndrome. 9 refs., 1 tab.

  2. Molecular diagnosis of lobomycosis-like disease in a bottlenose dolphin in captivity.

    PubMed

    Esperón, Fernando; García-Párraga, Daniel; Bellière, Edwige Nina; Sánchez-Vizcaíno, José Manuel

    2012-01-01

    We report the diagnosis and molecular characterization of lobomycosis-like lesions in a captive bottlenose dolphin. The clinical picture and the absence of growth in conventional media resembled the features associated with Lacazia loboi. However sequencing of ribosomal DNA and further phylogenetic analyses showed a novel sequence more related to Paracoccidioides brasilensis than to L. loboi. Moreover, the morphology of the yeast cells differed from those L. loboi causing infections humans. These facts suggest that the dolphin lobomycosis-like lesions might have been be caused by different a different fungus clustered inside the order Onygenales. A successful treatment protocol based on topic and systemic terbinafine is also detailed. PMID:21838615

  3. The pinpoint promise of nanoparticle-based drug delivery and molecular diagnosis.

    PubMed

    Emerich, Dwaine F; Thanos, Christopher G

    2006-09-01

    Nanotechnology, or systems/device manufacture at the molecular level, is a multidisciplinary scientific field undergoing explosive development. The genesis of nanotechnology can be traced to the promise of revolutionary advances across medicine, communications, genomics and robotics. Without doubt one of the greatest values of nanotechnology will be in the development of new and effective medical treatments (i.e., nanomedicine). This review focuses on the potential of nanomedicine as it specifically relates to (1) the development of nanoparticles for enabling and improving the targeted delivery of therapeutic agents; (2) developing novel and more effective diagnostic and screening techniques to extend the limits of molecular diagnostics providing point-of-care diagnosis and more personalized medicine.

  4. Novel cytogenetic and molecular techniques in the diagnosis of congenital anomalies in newborns.

    PubMed

    Szczałuba, Krzysztof; Śmigiel, Robert

    2015-01-01

    Knowledge of what causes developmental disorders, including congenital structural defects/anomalies, in the newborn population, facilitates the choice of further investigations, therapy and rehabilitation, allows the use of appropriate prophylaxis against comorbidities, makes it possible to specify prognosis, as well as provide reliable family counselling (both pre- and postnatal). Attempting to formulate a clinical diagnosis of a specific congenital anomaly syndrome, with or without dysmorphic features, based on history and detailed physical examination, remains crucial for the selection of the right genetic testing. Modern methods of molecular cytogenetics and molecular biology are targeted in nature (microdeletion MLPA, single gene sequencing) or are capable of analyzing the genome as a whole (array CGH, newgeneration sequencing). Especially the latter techniques are now causing a rapid increase of diagnostic efficacy across different age groups, including newborns.

  5. Liver glycogen storage diseases due to phosphorylase system deficiencies: diagnosis thanks to non invasive blood enzymatic and molecular studies.

    PubMed

    Davit-Spraul, Anne; Piraud, Monique; Dobbelaere, Dries; Valayannopoulos, Vassili; Labrune, Philippe; Habes, Dalila; Bernard, Olivier; Jacquemin, Emmanuel; Baussan, Christiane

    2011-01-01

    Glycogen storage disease (GSD) due to a deficient hepatic phosphorylase system defines a genetically heterogeneous group of disorders that mainly manifests in children. We investigated 45 unrelated children in whom a liver GSD VI or IX was suspected on the basis of clinical symptoms including hepatomegaly, increased serum transaminases, postprandial lactatemia and/or mild fasting hypoglycemia. Liver phosphorylase and phosphorylase b kinase activities studied in peripheral blood cells allowed to suspect diagnosis in 37 cases but was uninformative in 5. Sequencing of liver phosphorylase genes was useful to establish an accurate diagnosis. Causative mutations were found either in the PYGL (11 patients), PHKA2 (26 patients), PHKG2 (three patients) or in the PHKB (three patients) genes. Eleven novel disease causative mutations, five missense (p.N188K, p.D228Y, p.P382L, p.R491H, p.L500R) and six truncating mutations (c.501_502ins361pb, c.528+2T>C, c.856-29_c.1518+614del, c.1620+1G>C, p.E703del and c.2313-1G>T) were identified in the PYGL gene. Seventeen novel disease causative mutations, ten missense (p.A42P, p.Q95R, p.G131D, p.G131V, p.Q134R, p.G187R, p.G300V, p.G300A, p.C326Y, p.W820G) and seven truncating (c.537+5G>A, p.G396DfsX28, p.Q404X, p.N653X, p.L855PfsX87, and two large deletions) were identified in the PHKA2 gene. Four novel truncating mutations (p.R168X, p.Q287X, p.I268PfsX12 and c.272-1G>C) were identified in the PHKG2 gene and three (c.573_577del, p.R364X, c.2427+3A>G) in the PHKB gene. Patients with PHKG2 mutations evolved towards cirrhosis. Molecular analysis of GSD VI or IX genes allows to confirm diagnosis suspected on the basis of enzymatic analysis and to establish diagnosis and avoid liver biopsy when enzymatic studies are not informative in blood cells.

  6. The status of and future research into Myalgic Encephalomyelitis and Chronic Fatigue Syndrome: the need of accurate diagnosis, objective assessment, and acknowledging biological and clinical subgroups

    PubMed Central

    Twisk, Frank N. M.

    2014-01-01

    Although Myalgic Encephalomyelitis (ME) and Chronic Fatigue Syndrome (CFS) are used interchangeably, the diagnostic criteria define two distinct clinical entities. Cognitive impairment, (muscle) weakness, circulatory disturbances, marked variability of symptoms, and, above all, post-exertional malaise: a long-lasting increase of symptoms after a minor exertion, are distinctive symptoms of ME. This latter phenomenon separates ME, a neuro-immune illness, from chronic fatigue (syndrome), other disorders and deconditioning. The introduction of the label, but more importantly the diagnostic criteria for CFS have generated much confusion, mostly because chronic fatigue is a subjective and ambiguous notion. CFS was redefined in 1994 into unexplained (persistent or relapsing) chronic fatigue, accompanied by at least four out of eight symptoms, e.g., headaches and unrefreshing sleep. Most of the research into ME and/or CFS in the last decades was based upon the multivalent CFS criteria, which define a heterogeneous patient group. Due to the fact that fatigue and other symptoms are non-discriminative, subjective experiences, research has been hampered. Various authors have questioned the physiological nature of the symptoms and qualified ME/CFS as somatization. However, various typical symptoms can be assessed objectively using standardized methods. Despite subjective and unclear criteria and measures, research has observed specific abnormalities in ME/CFS repetitively, e.g., immunological abnormalities, oxidative and nitrosative stress, neurological anomalies, circulatory deficits and mitochondrial dysfunction. However, to improve future research standards and patient care, it is crucial that patients with post-exertional malaise (ME) and patients without this odd phenomenon are acknowledged as separate clinical entities that the diagnosis of ME and CFS in research and clinical practice is based upon accurate criteria and an objective assessment of characteristic symptoms

  7. [Molecular diagnosis of hereditary neuropathies such as Charcot-Marie-Tooth disease].

    PubMed

    Pouget, J

    2004-02-01

    During the last decade, molecular biology has demonstrated the extraordinary heterogeneity of genetic abnormalities in Charcot-Marie-Tooth disease (CMT). The main phenotypes are either of the demyelinating or axonal type, transmitted with dominant or recessive autosomal inheritance. X-linked CMT is less rare than it was initially described and is often misdiagnosed as autosomal dominant type. Linked phenotypes are Dejerine-Sottas disease, congenital hypomyelinization and hereditary neuropathy with susceptibility to pressure palsies. Each phenotype can be due to different genotypes and concerned genes are numerous. Conversely, each genotype can express different phenotypes. Molecular diagnostic strategy of CMT is mainly baised on three elements: - phenotypic expertise which is based on the analysis of the inheritance mode and on electrophysiological data, which are peculiar in CMTX - knowledge of respective occurrence of the different genotypes and phenotypes which is increasing - technical feasibility of molecular biology methods which is important to consider, even though progress are fastly coming. According to these considerations, a strategy is proposed for molecular diagnosis of CMT. PMID:15034475

  8. Leishmaniases diagnosis: an update on the use of immunological and molecular tools.

    PubMed

    de Paiva-Cavalcanti, Milena; de Morais, Rayana Carla Silva; Pessoa-E-Silva, Rômulo; Trajano-Silva, Lays Adrianne Mendonça; Gonçalves-de-Albuquerque, Suênia da Cunha; Tavares, Diego de Hollanda Cavalcanti; Brelaz-de-Castro, Maria Carolina Accioly; Silva, Rafael de Freitas E; Pereira, Valéria Rêgo Alves

    2015-01-01

    Leishmaniases are caused by obligate intracellular protozoan parasites of the genus Leishmania. They cause a spectrum of diseases, most notably visceral (VL), cutaneous (CL), and mucosal (ML) leishmaniasis, which affect millions of people around the world, each year. Despite scientific advances, leishmaniases cases are expanding, constituting an important public health problem. Immunological and molecular diagnostic tools have been increasingly applied for the early detection of these parasitic infections, since the existence of limitations in clinical and parasitological examinations may provide false results, thus interfering in epidemiological research and diseases control. Although there is a great diversity of available immunological assays, important common deficiencies persist, which explains the current exploration of the molecular biology in research fields, especially the Polymerase Chain Reaction (PCR) and its variants, such as real-time quantitative PCR. However, in the last years, significant results have also been reached inside of immunological context (especially by Flow Cytometry), for humans and dogs, demonstrated by research works of the New and Old worlds. In spite of their potential to clarify and minimize the present global situation of the diseases, the implementation of molecular or immunological innovative reference assays for VL and CL at health services is still a challenge due to several reasons, including lack of standardization among laboratories and structural concerns. In this article we bring classical and current information about technological advances for the immunological and molecular leishmaniases diagnosis, their features, and applications.

  9. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

    PubMed

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa

    2016-09-01

    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. PMID:27422649

  10. Double-hairpin molecular-beacon-based amplification detection for gene diagnosis linked to cancer.

    PubMed

    Xu, Huo; Zhang, Rongbo; Li, Feng; Zhou, Yingying; Peng, Ting; Wang, Xuedong; Shen, Zhifa

    2016-09-01

    A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related KRAS gene detection based on the one-to-two stoichiometry. During target DNA detection, DHMB can execute signal transduction even if no any exogenous element is involved. Unlike the conventional molecular beacon based on the one-to-one interaction, one target DNA not only hybridizes with one DHMB and opens its hairpin but also promotes the interaction between two DHMBs, causing the separation of two fluorophores from quenchers. This leads to an enhanced fluorescence signal. As a result, the target KRAS gene is able to be detected within a wide dynamic range from 0.05 to 200 nM with the detection limit of 50 pM, indicating a dramatic improvement compared with traditional molecular beacons. Moreover, the point mutations existing in target DNAs can be easily screened. The potential application for target species in real samples was indicated by the analysis of PCR amplicons of DNAs from the DNA extracted from SW620 cell. Besides becoming a promising candidate probe for molecular biology research and clinical diagnosis of genetic diseases, the DHMB is expected to provide a significant insight into the design of DNA probe-based homogenous sensing systems. Graphical Abstract A powerful double-hairpin molecular beacon (DHMB) was developed for cancer-related gene KRAS detection based on the one-to-two stoichiometry. Without the help of any exogenous probe, the point mutation is easily screened, and the target DNA can be quantified down to 50 pM, indicating a dramatic improvement compared with traditional molecular beacons.

  11. Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

    PubMed

    Le Govic, Y; Guyot, K; Certad, G; Deschildre, A; Novo, R; Mary, C; Sendid, B; Viscogliosi, E; Favennec, L; Dei-Cas, E; Fréalle, E; Dutoit, E

    2016-01-01

    Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

  12. Molecular Diagnosis and Identification of Leishmania Species in Jordan from Saved Dry Samples.

    PubMed

    Hijjawi, Nawal; Kanani, Kalil A; Rasheed, Malak; Atoum, Manar; Abdel-Dayem, Mona; Irhimeh, Mohammad R

    2016-01-01

    Diagnosis of the endemic cutaneous leishmaniasis (CL) in Jordan relies on patient clinical presentation and microscopic identification. Studies toward improved identification of the causative Leishmania species, especially in regions where multiple species exist, and the introduction of these techniques into medical diagnosis is paramount. This study looked at the current epidemiology of CL in Jordan. Clinically diagnosed 41 patients with CL were tested for the presence of Leishmania parasite using both Giemsa staining from skin scraps on glass slides and ITS1-PCR from samples blotted onto storage cards (NucleoCards®). Microscopically, 28 out of the 41 (68.3%) collected samples were positive for amastigotes, whereas the molecular ITS1-PCR amplification successfully identified 30 of the 41 samples (73.2%). Furthermore, PCR-RFLP analysis allowed species identification which is impossible microscopically. Of the 30 PCR positive samples, 28 were Leishmania major positive and the other two samples were Leishmania tropica. This indicates that L. major is the most prevalent species in Jordan and the two L. tropica cases originated from Syria indicating possible future L. tropica outbreaks. Diagnosis of CL based on clinical presentation only may falsely increase its prevalence. Although PCR is more sensitive, it is still not available in our medical laboratories in Jordan. PMID:27403435

  13. Molecular Diagnosis of Hereditary Fructose Intolerance: Founder Mutation in a Community from India.

    PubMed

    Bijarnia-Mahay, Sunita; Movva, Sireesha; Gupta, Neerja; Sharma, Deepak; Puri, Ratna D; Kotecha, Udhaya; Saxena, Renu; Kabra, Madhulika; Mohan, Neelam; Verma, Ishwar C

    2015-01-01

    Hereditary fructose intolerance (HFI) is a difficult-to-confirm diagnosis, requiring either invasive liver biopsy-enzyme assay or potentially hazardous fructose challenge test or expensive molecular genetic analysis. Therefore, worldwide there has been a trend towards finding "common mutations" in distinct ethnic groups to simplify the process of diagnosis. The nonspecific presentation of the disease often leads to diagnostic confusion with other metabolic liver disorders such as glycogenoses, galactosemia, and tyrosinemia. This leads to much delay in diagnosis with consequent harm to the patient.We report mutations in the ALDOB gene, from eleven Indian patients, seven of whom belong to the Agarwal community. Six patients from the Agarwal community and two non-Agarwal patients harbored one novel mutation, c.324+1G>A (five homozygous and one heterozygous), in the ALDOB gene. Haplotyping performed in families confirmed a founder effect. The community has been known to harbor founder mutations in other genes such as the MLC1, PANK2, and CAPN3 genes, thus providing another evidence for a founder effect in the community in case of HFI. This may pave the path for a simpler and quicker test at least for this community in India. In addition to the founder mutation, we report four other novel mutations, c.112+1delG, c.380-1G>A, c.677G>A, and c.689delA, and a previously reported mutation, c.1013C>T, in the cohort from India.

  14. Molecular Diagnosis and Identification of Leishmania Species in Jordan from Saved Dry Samples

    PubMed Central

    Hijjawi, Nawal; Kanani, Kalil A.; Rasheed, Malak; Atoum, Manar; Abdel-Dayem, Mona; Irhimeh, Mohammad R.

    2016-01-01

    Diagnosis of the endemic cutaneous leishmaniasis (CL) in Jordan relies on patient clinical presentation and microscopic identification. Studies toward improved identification of the causative Leishmania species, especially in regions where multiple species exist, and the introduction of these techniques into medical diagnosis is paramount. This study looked at the current epidemiology of CL in Jordan. Clinically diagnosed 41 patients with CL were tested for the presence of Leishmania parasite using both Giemsa staining from skin scraps on glass slides and ITS1-PCR from samples blotted onto storage cards (NucleoCards®). Microscopically, 28 out of the 41 (68.3%) collected samples were positive for amastigotes, whereas the molecular ITS1-PCR amplification successfully identified 30 of the 41 samples (73.2%). Furthermore, PCR-RFLP analysis allowed species identification which is impossible microscopically. Of the 30 PCR positive samples, 28 were Leishmania major positive and the other two samples were Leishmania tropica. This indicates that L. major is the most prevalent species in Jordan and the two L. tropica cases originated from Syria indicating possible future L. tropica outbreaks. Diagnosis of CL based on clinical presentation only may falsely increase its prevalence. Although PCR is more sensitive, it is still not available in our medical laboratories in Jordan. PMID:27403435

  15. [Aberrant promoter methylation as biomarker for molecular cytological diagnosis of lung cancer].

    PubMed

    Grote, H J

    2006-01-01

    Aberrant promoter methylation represents a main mechanism of tumor suppressor gene inactivation and may serve as a new source for biomarker discovery. This study investigated its applicability as a molecular tool for lung cancer diagnostics on bronchial aspirates. A methylation assay was developed applying a quantitative methylation specific real-time PCR (QMSP). A total of 552 patients with the differential diagnosis of lung cancer were investigated. The QMSP findings on bronchial aspirates were compared with the methylation status of respective genes investigated in microdissected tumor tissues (QMSP, cloning and sequencing of promoter regions after bisulfite conversion). Among the genes tested a marker panel consisting of APC, p16(INK4a) and RASSF1A proved to be the best suited for lung cancer diagnostics. This panel allowed for a correct diagnosis of lung cancer in cases with an ambiguous or false negative conventional cytology. In a cohort study on 247 patients, the combination of histology (sensitivity 59 %), cytology (sensitivity 44 %) and QMSP-assay (sensitivity 53 %) raised the sensitivity of a single bronchoscopy for the diagnosis of lung cancer up to 81%. The methylation assay yielded its major diagnostic surplus with respect to peripheral tumors representing 59 % of all primaries detected. In patients without antecedent lung cancer its specificity considering malignancy was >99 %. Therefore, the QMSP-assay is a promising technique which could enhance the sensitivity and diagnostic impact of conventional cytology. The assay is applicable to residual material of regular diagnostic cytology even in retrospect.

  16. Folate Receptor-Targeting Gold Nanoclusters as Fluorescence Enzyme Mimetic Nanoprobes for Tumor Molecular Colocalization Diagnosis

    PubMed Central

    Hu, Dehong; Sheng, Zonghai; Fang, Shengtao; Wang, Yanan; Gao, Duyang; Zhang, Pengfei; Gong, Ping; Ma, Yifan; Cai, Lintao

    2014-01-01

    Nanoprobes with enzyme-like properties attracted a growing interest in early screening and diagnosis of cancer. To achieve high accuracy and specificity of tumor detection, the design and preparation of enzyme mimetic nanoprobes with high enzyme activity, tumor targeting and excellent luminescence property is highly desirable. Herein, we described a novel kind of fluorescence enzyme mimetic nanoprobe based on folate receptor-targeting Au nanoclusters. The nanoprobes exhibited excellent stability, low cytotoxicity, high fluorescence and enzyme activity. We demonstrated that the nanoprobes could be used for tumor tissues fluorescence/visualizing detection. For the same tumor tissue slice, the nanoprobes peroxidase staining and fluorescent staining were obtained simultaneously, and the results were mutually complementary. Therefore, the fluorescence enzyme mimetic nanoprobes could provide a molecular colocalization diagnosis strategy, efficiently avoid false-positive and false-negative results, and further improve the accuracy and specificity of cancer diagnoses. By examining different clinical samples, we demonstrated that the nanoprobes could distinguish efficiently cancerous cells from normal cells, and exhibit a clinical potential for cancer diagnosis. PMID:24465272

  17. Molecular diagnosis of African tick bite fever using eschar swabs in a traveller returning from Tanzania.

    PubMed

    Harrison, Nicole; Burgmann, Heinz; Forstner, Christina; Ramharter, Michael; Széll, Marton; Schötta, Anna-Margarita; Stanek, Gerold; Markowicz, Mateusz

    2016-08-01

    African tick bite fever is an emerging infectious disease among travellers caused by the pathogen Rickettsia africae. Most travel-associated cases have been reported from countries in southern Africa. So far it has rarely been reported among travellers to eastern Africa and our patient is one of the first described cases imported from Tanzania. A woman presented with fever, chills, headache, myalgia and a rickettsial eschar on her ankle after returning from Tanzania. The diagnosis of African tick bite fever is often based on clinical grounds due to a lack of reliable diagnostic tests at commencement of symptoms. In this patient direct molecular detection of R. africae was performed by PCR from a sample obtained non-invasively with a swab from the rickettsial eschar. A positive PCR result was achieved although the patient had already started antibiotic treatment with doxycycline. In conclusion, this non-invasive method enables early diagnosis of African tick bite fever by direct molecular detection of R. africae and might improve the management of undifferentiated fever in travellers from Africa. PMID:27488618

  18. Comprehensive Molecular Diagnosis of a Large Chinese Leber Congenital Amaurosis Cohort

    PubMed Central

    Wang, Hui; Wang, Xia; Zou, Xuan; Xu, Shan; Li, Hui; Soens, Zachry Tore; Wang, Keqing; Li, Yumei; Dong, Fangtian; Chen, Rui; Sui, Ruifang

    2015-01-01

    Purpose. Leber congenital amaurosis (LCA) is an inherited retinal disease that causes early-onset severe visual impairment. To evaluate the mutation spectrum in the Chinese population, we performed a mutation screen in 145 Chinese LCA families. Methods. First, we performed direct Sanger sequencing of 7 LCA disease genes in 81 LCA families. Next, we developed a capture panel that enriches the entire coding exons and splicing sites of 163 known retinal disease genes and other candidate retinal disease genes. The capture panel allowed us to quickly identify disease-causing mutations in a large number of genes at a relatively low cost. Thus, this method was applied to the 53 LCA families that were unsolved by direct Sanger sequencing of 7 LCA disease genes and an additional 64 LCA families. Systematic next-generation sequencing (NGS) data analysis, Sanger sequencing validation, and segregation analysis were used to identify pathogenic mutations. Results. Homozygous or compound heterozygous mutations were identified in 107 families, heterozygous autosomal dominant mutations were identified in 3 families and an X-linked mutation was found in 1 family, for a combined solving rate of 76.6%. In total, 136 novel pathogenic mutations were found in this study. In combination with two previous studies carried out in Chinese LCA patients, we concluded that the mutation spectrum in the Chinese population is distinct compared to that in the European population. After revisiting, we also refined the clinical diagnosis of 10 families based on their molecular diagnosis. Conclusions. Our results highlight the importance of a molecular diagnosis as an integral part of the clinical diagnotic process. PMID:26047050

  19. The emerging role of the molecular marker p27 in the differential diagnosis of adrenocortical tumors

    PubMed Central

    Pereira, Sofia S; Morais, Tiago; Costa, Madalena M; Monteiro, Mariana P; Pignatelli, Duarte

    2013-01-01

    Malignant adrenocortical tumors (ACTs) are rare and highly aggressive; conversely, benign tumors are common and frequently found incidentally (the so-called incidentalomas). Currently, the use of molecular markers in the diagnosis of ACTs is still controversial. The aim of this study was to analyze the molecular profile of different ACTs with the purpose of identifying markers useful for differentiating between these tumors. The ACTs that were studied (n=31) included nonfunctioning adenomas (ACAn)/incidentalomas (n=13), functioning adenomas with Cushing's syndrome (ACAc) (n=7), and carcinomas (n=11); normal adrenal glands (n=12) were used as controls. For each sample, the percentage area stained for the markers StAR, IGF2, IGF1R, p53, MDM2, p21, p27, cyclin D1, Ki-67, β-catenin, and E-cadherin was quantified using a morphometric computerized tool. IGF2, p27, cyclin D1, and Ki-67 were the markers for which the percentage of stained area was significantly higher in carcinoma samples than in adenoma samples. Ki-67 and p27 were the markers that exhibited the highest discriminative power for differential diagnosis between carcinomas and all type of adenomas, while IGF2 and StAR were only found to be useful for differentiating between carcinomas and ACAn and between carcinomas and ACAc respectively. The usefulness of Ki-67 has been recognized before in the differential diagnosis of malignant tumors. The additional use of p27 as an elective marker to distinguish benign ACTs from malignant ACTs should be considered. PMID:23925558

  20. Toward Accurate Reaction Energetics for Molecular Line Growth at Surface: Quantum Monte Carlo and Density Functional Theory Calculations

    SciTech Connect

    Kanai, Y; Takeuchi, N

    2009-10-14

    We revisit the molecular line growth mechanism of styrene on the hydrogenated Si(001) 2x1 surface. In particular, we investigate the energetics of the radical chain reaction mechanism by means of diffusion quantum Monte Carlo (QMC) and density functional theory (DFT) calculations. For the exchange correlation (XC) functional we use the non-empirical generalized-gradient approximation (GGA) and meta-GGA. We find that the QMC result also predicts the intra dimer-row growth of the molecular line over the inter dimer-row growth, supporting the conclusion based on DFT results. However, the absolute magnitudes of the adsorption and reaction energies, and the heights of the energy barriers differ considerably between the QMC and DFT with the GGA/meta-GGA XC functionals.

  1. Accurate and Scalable O(N) Algorithm for First-Principles Molecular-Dynamics Computations on Large Parallel Computers

    SciTech Connect

    Osei-Kuffuor, Daniel; Fattebert, Jean-Luc

    2014-01-01

    We present the first truly scalable first-principles molecular dynamics algorithm with O(N) complexity and controllable accuracy, capable of simulating systems with finite band gaps of sizes that were previously impossible with this degree of accuracy. By avoiding global communications, we provide a practical computational scheme capable of extreme scalability. Accuracy is controlled by the mesh spacing of the finite difference discretization, the size of the localization regions in which the electronic wave functions are confined, and a cutoff beyond which the components of the overlap matrix can be omitted when computing selected elements of its inverse. We demonstrate the algorithm's excellent parallel scaling for up to 101 952 atoms on 23 328 processors, with a wall-clock time of the order of 1 min per molecular dynamics time step and numerical error on the forces of less than 7x10-4 Ha/Bohr.

  2. Molecular Correlates and Recent Advancements in the Diagnosis and Screening of FMR1-Related Disorders

    PubMed Central

    Rajan-Babu, Indhu-Shree; Chong, Samuel S.

    2016-01-01

    Fragile X syndrome (FXS) is the most common monogenic cause of intellectual disability and autism. Molecular diagnostic testing of FXS and related disorders (fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS)) relies on a combination of polymerase chain reaction (PCR) and Southern blot (SB) for the fragile X mental retardation 1 (FMR1) CGG-repeat expansion and methylation analyses. Recent advancements in PCR-based technologies have enabled the characterization of the complete spectrum of CGG-repeat mutation, with or without methylation assessment, and, as a result, have reduced our reliance on the labor- and time-intensive SB, which is the gold standard FXS diagnostic test. The newer and more robust triplet-primed PCR or TP-PCR assays allow the mapping of AGG interruptions and enable the predictive analysis of the risks of unstable CGG expansion during mother-to-child transmission. In this review, we have summarized the correlation between several molecular elements, including CGG-repeat size, methylation, mosaicism and skewed X-chromosome inactivation, and the extent of clinical involvement in patients with FMR1-related disorders, and reviewed key developments in PCR-based methodologies for the molecular diagnosis of FXS, FXTAS and FXPOI, and large-scale (CGG)n expansion screening in newborns, women of reproductive age and high-risk populations. PMID:27754417

  3. The CHARMM-TURBOMOLE interface for efficient and accurate QM/MM molecular dynamics, free energies, and excited state properties.

    PubMed

    Riahi, Saleh; Rowley, Christopher N

    2014-10-30

    The quantum mechanical (QM)/molecular mechanical (MM) interface between Chemistry at HARvard Molecular Mechanics (CHARMM) and TURBOMOLE is described. CHARMM provides an extensive set of simulation algorithms, like molecular dynamics (MD) and free energy perturbation, and support for mature nonpolarizable and Drude polarizable force fields. TURBOMOLE provides fast QM calculations using density functional theory or wave function methods and excited state properties. CHARMM-TURBOMOLE is well-suited for extended QM/MM MD simulations using first principles methods with large (triple-ζ) basis sets. We demonstrate these capabilities with a QM/MM simulation of Mg(2+) (aq), where the MM outer sphere water molecules are represented using the SWM4-NDP Drude polarizable force field and the ion and inner coordination sphere are represented using QM PBE, PBE0, and MP2 methods. The relative solvation free energies of Mg(2+) and Zn(2+) were calculated using thermodynamic integration. We also demonstrate the features for excited state properties. We calculate the time-averaged solution absorption spectrum of indole, the emission spectrum of the indole 1La excited state, and the electronic circular dichroism spectrum of an oxacepham.

  4. Development of an accurate molecular mechanics model for buckling behavior of multi-walled carbon nanotubes under axial compression.

    PubMed

    Safaei, B; Naseradinmousavi, P; Rahmani, A

    2016-04-01

    In the present paper, an analytical solution based on a molecular mechanics model is developed to evaluate the elastic critical axial buckling strain of chiral multi-walled carbon nanotubes (MWCNTs). To this end, the total potential energy of the system is calculated with the consideration of the both bond stretching and bond angular variations. Density functional theory (DFT) in the form of generalized gradient approximation (GGA) is implemented to evaluate force constants used in the molecular mechanics model. After that, based on the principle of molecular mechanics, explicit expressions are proposed to obtain elastic surface Young's modulus and Poisson's ratio of the single-walled carbon nanotubes corresponding to different types of chirality. Selected numerical results are presented to indicate the influence of the type of chirality, tube diameter, and number of tube walls in detailed. An excellent agreement is found between the present numerical results and those found in the literature which confirms the validity as well as the accuracy of the present closed-form solution. It is found that the value of critical axial buckling strain exhibit significant dependency on the type of chirality and number of tube walls.

  5. Presentation of Progressive Familial Intrahepatic Cholestasis Type 3 Mimicking Wilson Disease: Molecular Genetic Diagnosis and Response to Treatment

    PubMed Central

    Jain, Dhanpat; Schilsky, Michael L.

    2015-01-01

    Progressive familial intrahepatic cholestasis type 3 (PFIC3) is an autosomal recessive disorder of cholestasis of hepatocellular origin, typically seen in infancy or childhood caused by a defect in the ABCB4 located on chromosome 7. Here we report on an older patient, aged 15, who presented with biochemical testing that led to an initial consideration of a diagnosis of Wilson disease (WD) resulting in a delayed diagnosis of PFIC3. Diagnosis of PFIC3 was later confirmed by molecular studies that identified novel mutations in the ABCB4 gene. Cholestasis due to PFIC3 can cause elevated hepatic copper and increased urine copper excretion that overlap with current diagnostic criteria for WD. Molecular diagnostics are very useful for establishing the diagnosis of PFIC3. Ursodeoxycholic acid ameliorates cholestasis in PFIC3, and may help mediate a reduction in hepatic copper content in response to treatment. PMID:26473142

  6. 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

  7. Diagnosis of feline acute intermittent porphyria presenting with erythrodontia requires molecular analyses.

    PubMed

    Clavero, Sonia; Ahuja, Yuri; Bishop, David F; Kwait, Brittany; Haskins, Mark E; Giger, Urs; Desnick, Robert J

    2013-12-01

    Erythrodontia is the hallmark of human congenital erythropoietic porphyria (CEP), but is also a major phenotypic feature of acute intermittent porphyria (AIP) in cats. In this study, detailed biochemical and molecular analyses were performed on two unrelated cats with autosomal dominant AIP that presented with erythrodontia, yellow-brown urine and mild changes in erythrocytes. The cats had elevated concentrations of urinary 5-aminolevulinic acid and porphobilinogen, and half normal erythrocytic hydroxymethylbilane synthase (HMBS) activity. Two novel HMBS mutations were detected; one cat had a deletion (c.107_110delACAG) and one cat had a splicing alteration (c.826-1G>A), both leading to premature stop codons and truncated proteins (p.D36Vfs 6 and p.L276Efs 6, respectively). These studies highlight the importance of appropriate biochemical and molecular genetic analyses for the accurate diagnoses of porphyrias in cats and extend the molecular genetic heterogeneity of feline AIP. Thus, although erythrodontia is a classic sign of congenital erythropoietic porphyria in human beings, cats with erythrodontia may have acute intermittent porphyria, a hepatic porphyria.

  8. Accurate determination of genetic identity for a single cacao bean, using molecular markers with a nanofluidic system, ensures cocoa authentication.

    PubMed

    Fang, Wanping; Meinhardt, Lyndel W; Mischke, Sue; Bellato, Cláudia M; Motilal, Lambert; Zhang, Dapeng

    2014-01-15

    Cacao (Theobroma cacao L.), the source of cocoa, is an economically important tropical crop. One problem with the premium cacao market is contamination with off-types adulterating raw premium material. Accurate determination of the genetic identity of single cacao beans is essential for ensuring cocoa authentication. Using nanofluidic single nucleotide polymorphism (SNP) genotyping with 48 SNP markers, we generated SNP fingerprints for small quantities of DNA extracted from the seed coat of single cacao beans. On the basis of the SNP profiles, we identified an assumed adulterant variety, which was unambiguously distinguished from the authentic beans by multilocus matching. Assignment tests based on both Bayesian clustering analysis and allele frequency clearly separated all 30 authentic samples from the non-authentic samples. Distance-based principle coordinate analysis further supported these results. The nanofluidic SNP protocol, together with forensic statistical tools, is sufficiently robust to establish authentication and to verify gourmet cacao varieties. This method shows significant potential for practical application.

  9. The route to MBxNyCz molecular wheels: II. Results using accurate functionals and basis sets

    NASA Astrophysics Data System (ADS)

    Güthler, A.; Mukhopadhyay, S.; Pandey, R.; Boustani, I.

    2014-04-01

    Applying ab initio quantum chemical methods, molecular wheels composed of metal and light atoms were investigated. High quality basis sets 6-31G*, TZPV, and cc-pVTZ as well as exchange and non-local correlation functionals B3LYP, BP86 and B3P86 were used. The ground-state energy and structures of cyclic planar and pyramidal clusters TiBn (for n = 3-10) were computed. In addition, the relative stability and electronic structures of molecular wheels TiBxNyCz (for x, y, z = 0-10) and MBnC10-n (for n = 2 to 5 and M = Sc to Zn) were determined. This paper sustains a follow-up study to the previous one of Boustani and Pandey [Solid State Sci. 14 (2012) 1591], in which the calculations were carried out at the HF-SCF/STO3G/6-31G level of theory to determine the initial stability and properties. The results show that there is a competition between the 2D planar and the 3D pyramidal TiBn clusters (for n = 3-8). Different isomers of TiB10 clusters were also studied and a structural transition of 3D-isomer into 2D-wheel is presented. Substitution boron in TiB10 by carbon or/and nitrogen atoms enhances the stability and leads toward the most stable wheel TiB3C7. Furthermore, the computations show that Sc, Ti and V at the center of the molecular wheels are energetically favored over other transition metal atoms of the first row.

  10. Current Advances in the Application of Raman Spectroscopy for Molecular Diagnosis of Cervical Cancer

    PubMed Central

    Ramos, Inês Raquel Martins; Malkin, Alison; Lyng, Fiona Mary

    2015-01-01

    Raman spectroscopy provides a unique biochemical fingerprint capable of identifying and characterizing the structure of molecules, cells, and tissues. In cervical cancer, it is acknowledged as a promising biochemical tool due to its ability to detect premalignancy and early malignancy stages. This review summarizes the key research in the area and the evidence compiled is very encouraging for ongoing and further research. In addition to the diagnostic potential, promising results for HPV detection and monitoring treatment response suggest more than just a diagnosis prospective. A greater body of evidence is however necessary before Raman spectroscopy is fully validated for clinical use and larger comprehensive studies are required to fully establish the role of Raman spectroscopy in the molecular diagnostics of cervical cancer. PMID:26180802

  11. A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan

    PubMed Central

    2012-01-01

    Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue. PMID:22929369

  12. 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

  13. 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.

  14. Cutaneous tuberculosis diagnosis in an inhospitable Amazonian region by means of telemedicine and molecular biology.

    PubMed

    Angel, Diana I; Alfonso, Rosalba; Faizal, Michel; Ricaurte, Orlando; Baez, Julio A; Rojas, Andres; Barato, Paola; Patarroyo, Manuel E; Patarroyo, Manuel A

    2005-05-01

    We report on a 13-year-old boy who displayed a chronic granulomatous inflammatory reaction of 5 years duration. The lesion was resistant to different antibiotic schemes; his routine laboratory tests and chest radiographs were normal. Teledermatologic consultation and histopathologic study of skin biopsy suggested scrofulodermal tuberculosis. Polymerase chain reaction amplification of DNA extracted from lymph node biopsy was taken as starting material for dot-blot hybridization using Mtp-40 and IS 6110 as probes for detecting either Mycobacterium tuberculosis or any mycobacteria belonging to the M tuberculosis complex, respectively. Positive results in both hybridizations were further confirmed by culturing in BACTEC MGIT 960 system. The lesion greatly diminished following isoniazid, rifampin, and ethambutol treatment. Telemedicine allowed a cutaneous tuberculosis diagnosis to be made of a patient living in a remote town located in the Amazon jungle by using molecular biology techniques.

  15. Giant magnetoresistive biosensors for molecular diagnosis: surface chemistry and assay development

    NASA Astrophysics Data System (ADS)

    Yu, Heng; Osterfeld, Sebastian J.; Xu, Liang; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2008-08-01

    Giant magnetoresistive (GMR) biochips using magnetic nanoparticle as labels were developed for molecular diagnosis. The sensor arrays consist of GMR sensing strips of 1.5 μm or 0.75 μm in width. GMR sensors are exquisitely sensitive yet very delicate, requiring ultrathin corrosion-resistive passivation and efficient surface chemistry for oligonucleotide probe immobilization. A mild and stable surface chemistry was first developed that is especially suitable for modifying delicate electronic device surfaces, and a practical application of our GMR biosensors was then demonstrated for detecting four most common human papillomavirus (HPV) subtypes in plasmids. We also showed that the DNA hybridization time could potentially be reduced from overnight to about ten minutes using microfluidics.

  16. Emerging technologies for studying DNA methylation for the molecular diagnosis of cancer

    PubMed Central

    Marzese, Diego M.; Hoon, Dave S.B.

    2015-01-01

    DNA methylation is an epigenetic mechanism that plays a key role in regulating gene expression and other functions. Although this modification is seen in different sequence contexts, the most frequently detected DNA methylation in mammals involves cytosine-guanine dinucleotides. Pathological alterations in DNA methylation patterns are described in a variety of human diseases, including cancer. Unlike genetic changes, DNA methylation is heavily influenced by subtle modifications in the cellular microenvironment. In all cancers, aberrant DNA methylation is involved in the alteration of a large number of oncological pathways with relevant theranostic utility. Several technologies for DNA methylation mapping were recently developed and successfully applied in cancer studies. The scope of these technologies varies from assessing a single cytosine-guanine locus to genome-wide distribution of DNA methylation. Here, we review the strengths and weaknesses of these approaches in the context of clinical utility for the molecular diagnosis of human cancers. PMID:25797072

  17. An Omics Perspective on Molecular Biomarkers for Diagnosis, Prognosis, and Therapeutics of Cholangiocarcinoma

    PubMed Central

    Seeree, Pattaya; Pearngam, Phorutai; Kumkate, Supeecha; Janvilisri, Tavan

    2015-01-01

    Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy arising from the epithelial bile duct. The lack of early diagnostic biomarkers as well as therapeutic measures results in severe outcomes and poor prognosis. Thus, effective early diagnostic, prognostic, and therapeutic biomarkers are required to improve the prognosis and prolong survival rates in CCA patients. Recent advancement in omics technologies combined with the integrative experimental and clinical validations has provided an insight into the underlying mechanism of CCA initiation and progression as well as clues towards novel biomarkers. This work highlights the discovery and validation of molecular markers in CCA identified through omics approaches. The possible roles of these molecules in various cellular pathways, which render CCA carcinogenesis and progression, will also be discussed. This paper can serve as a reference point for further investigations to yield deeper understanding in the complex feature of this disease, potentially leading to better approaches for diagnosis, prognosis, and therapeutics. PMID:26421274

  18. Extending the molecular size in accurate quantum-chemical calculations: the equilibrium structure and spectroscopic properties of uracil.

    PubMed

    Puzzarini, Cristina; Barone, Vincenzo

    2011-04-21

    The equilibrium structure of uracil has been investigated using both theoretical and experimental data. With respect to the former, quantum-chemical calculations at the coupled-cluster level in conjunction with a triple-zeta basis set have been carried out. Extrapolation to the basis set limit, performed employing the second-order Møller-Plesset perturbation theory, and inclusion of core-correlation and diffuse-function corrections have also been considered. Based on the available rotational constants for various isotopic species together with corresponding computed vibrational corrections, the semi-experimental equilibrium structure of uracil has been determined for the first time. Theoretical and semi-experimental structures have been found in remarkably good agreement, thus pointing out the limitations of previous experimental determinations. Molecular and spectroscopic properties of uracil have then been studied by means of the composite computational approach introduced for the molecular structure evaluation. Among the results achieved, we mention the revision of the dipole moment. On the whole, it has been proved that the computational procedure presented is able to provide parameters with the proper accuracy to support experimental investigations of large molecules of biological interest.

  19. An accurate and scalable O(N) algorithm for First-Principles Molecular Dynamics computations on petascale computers and beyond

    NASA Astrophysics Data System (ADS)

    Osei-Kuffuor, Daniel; Fattebert, Jean-Luc

    2014-03-01

    We present a truly scalable First-Principles Molecular Dynamics algorithm with O(N) complexity and fully controllable accuracy, capable of simulating systems of sizes that were previously impossible with this degree of accuracy. By avoiding global communication, we have extended W. Kohn's condensed matter ``nearsightedness'' principle to a practical computational scheme capable of extreme scalability. Accuracy is controlled by the mesh spacing of the finite difference discretization, the size of the localization regions in which the electronic wavefunctions are confined, and a cutoff beyond which the components of the overlap matrix can be omitted when computing selected elements of its inverse. We demonstrate the algorithm's excellent parallel scaling for up to 100,000 atoms on 100,000 processors, with a wall-clock time of the order of one minute per molecular dynamics time step. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.

  20. Genetic analysis of Tunisian families with Usher syndrome type 1: toward improving early molecular diagnosis

    PubMed Central

    Ben-Rebeh, Imen; Bonnet, Crystel; Bouassida, Walid; Hadjamor, Imen; Ayadi, Hammadi; Ghorbel, Abdelmonem; Petit, Christine; Masmoudi, Saber

    2016-01-01

    Purpose Usher syndrome accounts for about 50% of all hereditary deaf-blindness cases. The most severe form of this syndrome, Usher syndrome type I (USH1), is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. Six USH1 genes have been identified, MYO7A, CDH23, PCDH15, USH1C, SANS, and CIB2, encoding myosin VIIA, cadherin-23, protocadherin-15, harmonin, scaffold protein containing ankyrin repeats and a sterile alpha motif (SAM) domain, and calcium- and integrin-binding member 2, respectively. Methods In the present study, we recruited four Tunisian families with a diagnosis of USH1, together with healthy unrelated controls. Affected members underwent detailed audiologic and ocular examinations. We used the North African Deafness (NADf) chip to search for known North African mutations associated with USH. Then, we selected microsatellite markers covering USH1 known loci to genotype the DNA samples. Finally, we performed DNA sequencing of three known USH1 genes: MYO7A, PCDH15, and USH1C. Results Four biallelic mutations, all single base changes, were found in the MYO7A, USH1C, and PCDH15 genes. These mutations consist of a previously reported splicing defect c.470+1G>A in MYO7A, three novel variants, including two nonsense (p.Arg3X and p.Arg134X) in USH1C and PCDH15, respectively, and one frameshift (p.Lys615Asnfs*6) in MYO7A. Conclusions We found a remarkable genetic heterogeneity in the studied families with USH1 with a variety of mutations, among which three were novel. These novel mutations will be included in the NADf mutation screening chip that will allow a higher diagnosis efficiency of this extremely genetically heterogeneous disease. Ultimately, efficient molecular diagnosis of USH in a patient’s early childhood is of utmost importance, allowing better educational and therapeutic management. PMID:27440999

  1. Comparison of serological and molecular test for diagnosis of infectious mononucleosis

    PubMed Central

    Salehi, Hassan; Salehi, Marziyeh; Roghanian, Rasoul; Bozari, Majid; Taleifard, Shirin; Salehi, Mohamad Mahdi; Salehi, Maryam

    2016-01-01

    Background: Epstein-Bar virus (EBV) is the main etiology of infectious mononucleosis (IM) syndrome that is characterized by fever, sore throat, and lymph adenopathy. Since, this virus could be associated with a number of malignancies, some hematologic disorders, and chronic fatigue syndrome, identification of IM is very important. The aim of study was to evaluate the specificity, as well as sensitivity of the two different methods that is, serology versus molecular diagnosis that are currently used for diagnosis of IM. Materials and Methods: In this study, during a period of 3.5 years, 100 suspected patients as case group and 100 healthy individuals as a control group were studied. Fifty samples in each group were tested by polymerase chain reaction (PCR) and all the samples including case group and control group were carried out by enzyme-linked immunosorbent assay (ELISA). Results: In 76% of patients and in 20% of the healthy individuals, samples were detected EBV DNA by PCR. On the other hand, 68.5% of the samples belong to the case group and 46% in the control group showed positivity by ELISA. Conclusion: By comparing the two methods, since PCR is very expensive and time consuming, and the percentages of difference ranges are narrow, ELISA could be applied as a first, easiest, and preliminary diagnostic test for IM. In addition, this test could be applied in various phases of the disease with a higher sensitivity comparing to PCR. Although PCR is routinely used for diagnosis of various infectious agents, it is considered as an expensive test and merely could be used after 1-2 weeks from the onset of the illness. PMID:27308267

  2. Diagnosis and molecular characterization of non-classic forms of Tay-Sachs disease in Brazil.

    PubMed

    Rozenberg, R; Kok, F; Burin, M G; Sá Miranda, M C; Vasques, C; Henriques-Souza, A M M; Giugliani, R; Vainzof, Mariz; Pereira, L V

    2006-06-01

    Molecular analysis of five Brazilian families, including eight patients presenting with nonclassic Tay-Sachs disease, was performed to identify frequent causative mutations and their correlation with clinical course. Three patients were affected by the B1 subacute variant and were shown to carry the R178H mutation (the DN allele), which is also common among Portuguese patients. Two of them were compound heterozygotes, whereas the third presented with the mutation in both alleles. Since Brazil was a Portuguese colony for over two centuries, common ancestry might be the probable explanation. The fourth patient presented with a juvenile phenotype and carries the R499H mutation, which has been reported only once worldwide and is associated with residual enzyme activity, responsible for a slower clinical course. The fifth family, of an Ashkenazi Jewish background, showed an extensive intrafamilial clinical variability among three affected sibs presenting with muscle atrophy, ataxia, and psychiatric symptoms. They were first diagnosed as having atypical spinal muscular atrophy and, subsequently, spinocerebellar ataxia, but, recently, the diagnosis of late-onset Tay-Sachs disease was confirmed based on reduced plasma hexosaminidase A activity and the G269S/InsTATC1278 genotype. It is therefore highly recommended to test patients with a similar clinical history for Tay-Sachs disease. In the same family, one first cousin committed suicide at the age of 24 years, presenting with a clinical phenotype that suggested an undiagnosed case and highlighting the effect of the intrafamilial clinical variability in delaying a prompt diagnosis. It is now recognized that his parents are, in fact, a carrier couple. Additionally, another relative had been previously identified as a heterozygote in a Tay-Sachs disease screening program, but the information was not shared among the family. Since this information might anticipate diagnosis and genetic counseling, it is advisable that

  3. EMQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies.

    PubMed

    Traeger-Synodinos, Joanne; Harteveld, Cornelis L; Old, John M; Petrou, Mary; Galanello, Renzo; Giordano, Piero; Angastioniotis, Michael; De la Salle, Barbara; Henderson, Shirley; May, Alison

    2015-04-01

    Haemoglobinopathies constitute the commonest recessive monogenic disorders worldwide, and the treatment of affected individuals presents a substantial global disease burden. Carrier identification and prenatal diagnosis represent valuable procedures that identify couples at risk for having affected children, so that they can be offered options to have healthy offspring. Molecular diagnosis facilitates prenatal diagnosis and definitive diagnosis of carriers and patients (especially 'atypical' cases who often have complex genotype interactions). However, the haemoglobin disorders are unique among all genetic diseases in that identification of carriers is preferable by haematological (biochemical) tests rather than DNA analysis. These Best Practice guidelines offer an overview of recommended strategies and methods for carrier identification and prenatal diagnosis of haemoglobinopathies, and emphasize the importance of appropriately applying and interpreting haematological tests in supporting the optimum application and evaluation of globin gene DNA analysis.

  4. EMQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies

    PubMed Central

    Traeger-Synodinos, Joanne; Harteveld, Cornelis L; Old, John M; Petrou, Mary; Galanello, Renzo; Giordano, Piero; Angastioniotis, Michael; De la Salle, Barbara; Henderson, Shirley; May, Alison

    2015-01-01

    Haemoglobinopathies constitute the commonest recessive monogenic disorders worldwide, and the treatment of affected individuals presents a substantial global disease burden. Carrier identification and prenatal diagnosis represent valuable procedures that identify couples at risk for having affected children, so that they can be offered options to have healthy offspring. Molecular diagnosis facilitates prenatal diagnosis and definitive diagnosis of carriers and patients (especially ‘atypical' cases who often have complex genotype interactions). However, the haemoglobin disorders are unique among all genetic diseases in that identification of carriers is preferable by haematological (biochemical) tests rather than DNA analysis. These Best Practice guidelines offer an overview of recommended strategies and methods for carrier identification and prenatal diagnosis of haemoglobinopathies, and emphasize the importance of appropriately applying and interpreting haematological tests in supporting the optimum application and evaluation of globin gene DNA analysis. PMID:25052315

  5. Direct Molecular Diagnosis of Aspergillosis and CYP51A Profiling from Respiratory Samples of French Patients

    PubMed Central

    Zhao, Yanan; Garnaud, Cécile; Brenier-Pinchart, Marie-Pierre; Thiébaut-Bertrand, Anne; Saint-Raymond, Christel; Camara, Boubou; Hamidfar, Rebecca; Cognet, Odile; Maubon, Danièle; Cornet, Muriel; Perlin, David S.

    2016-01-01

    Background: Microbiological diagnosis of aspergillosis and triazole resistance is limited by poor culture yield. To better estimate this shortcoming, we compared culture and molecular detection of A. fumigatus in respiratory samples from French patients at risk for aspergillosis. Methods: A total of 97 respiratory samples including bronchoalveolar lavages (BAL), bronchial aspirates (BA), tracheal aspirates, sputa, pleural fluids, and lung biopsy were collected from 33 patients having invasive aspergillosis (n = 12), chronic pulmonary aspergillosis (n = 3), allergic bronchopulmonary aspergillosis (n = 7), or colonization (n = 11) and 28 controls. Each specimen was evaluated by culture, pan-Aspergillus qPCR, and CYP51A PCR and sequencing. Results: One A. flavus and 19 A. fumigatus with one multiazole resistant strain (5.3%) were cultured from 20 samples. Culture positivity was 62.5, 75, 42.9, and 15.8% in ABPA, CPA, IA, and colonized patients, respectively. Aspergillus detection rate was significantly higher by pan-Aspergillus qPCR than by culture in IA (90.5 vs. 42.9%; P < 0.05) and colonization group (73.7 vs. 15.8%; P < 0.05). The CYP51A PCR found one TR34/L98H along with 5 novel cyp51A mutations (4 non-synonymous and 1 promoter mutations), yet no association can be established currently between these novel mutations and azole resistance. The analysis of 11 matched pairs of BA and BAL samples found that 9/11 BA carried greater fungal load than BAL and CYP51A detection was more sensitive in BA than in BAL. Conclusion: Direct molecular detection of Aspergillus spp. and azole resistance markers are useful adjunct tools for comprehensive aspergillosis diagnosis. The observed superior diagnostic value of BAs to BAL fluids warrants more in-depth study. PMID:27524978

  6. DIAGNOSIS OF Strongyloides stercoralis INFECTION IN IMMUNOCOMPROMISED PATIENTS BY SEROLOGICAL AND MOLECULAR METHODS

    PubMed Central

    de PAULA, Fabiana Martins; MALTA, Fernanda Mello; CORRAL, Marcelo Andreetta; MARQUES, Priscilla Duarte; GOTTARDI, Maiara; MEISEL, Dirce Mary Correia Lima; YAMASHIRO, Juliana; PINHO, João Renato Rebello; CASTILHO, Vera Lucia Pagliusi; GONÇALVES, Elenice Messias do Nascimento; GRYSCHEK, Ronaldo César Borges; CHIEFFI, Pedro Paulo

    2016-01-01

    SUMMARY Strongyloidiasis is a potentially serious infection in immunocompromised patients. Thus, the availability of sensitive and specific diagnostic methods is desirable, especially in the context of immunosuppressed patients in whom the diagnosis and treatment of strongyloidiasis is of utmost importance. In this study, serological and molecular tools were used to diagnose Strongyloides stercoralis infections in immunosuppressed patients. Serum and stool samples were obtained from 52 patients. Stool samples were first analyzed by Lutz, Rugai, and Agar plate culture methods, and then by a quantitative real time polymerase chain reaction (qPCR). Serum samples were evaluated by an enzyme-linked immunosorbent assay (ELISA) using a soluble (AS) or a membrane fractions antigen (AM) obtained from alkaline solutions of the filariform larvae of Strongyloides venezuelensis. Of the 52 immunosuppressed patients, three (5.8%) were positive for S. stercoralis by parasitological methods, compared to two patients (3.8%) and one patient (1.9%) who were detected by ELISA using the AS and the AM antigens, respectively. S. stercoralis DNA was amplified in seven (13.5%) stool samples by qPCR. These results suggest the utility of qPCR as an alternative diagnostic tool for the diagnosis of S. stercoralis infection in immunocompromised patients, considering the possible severity of this helminthiasis in this group of patients. PMID:27680168

  7. Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus.

    PubMed

    Lagerqvist, Nina; Hagström, Åsa; Lundahl, Malin; Nilsson, Elin; Juremalm, Mikael; Larsson, Inger; Alm, Erik; Bucht, Göran; Ahlm, Clas; Klingström, Jonas

    2016-05-01

    Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS. PMID:26962084

  8. Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus

    PubMed Central

    Lagerqvist, Nina; Hagström, Åsa; Lundahl, Malin; Nilsson, Elin; Juremalm, Mikael; Larsson, Inger; Alm, Erik; Bucht, Göran; Ahlm, Clas

    2016-01-01

    Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS. PMID:26962084

  9. Nucleic Acid Dipstick Test for Molecular Diagnosis of Pandemic H1N1 ▿

    PubMed Central

    Wu, Liang-Ta; Curran, Martin D.; Ellis, Joanna S.; Parmar, Surendra; Ritchie, Allyson V.; Sharma, Pia I.; Allain, Jean-Pierre; Jalal, Hamid; Zambon, Maria; Lee, Helen H.

    2010-01-01

    A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements. The amplification step is monitored with an internal control to ensure correct interpretation of test results. The clinical performance of this assay was evaluated using blinded RNA samples extracted from nasal/throat swab specimens from 262 patients exhibiting influenza-like illness. Compared with the United Kingdom National Standard Method, based on quantitative reverse transcription-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the new assay were 95.3% (95% confidence interval, 88.5 to 98.7%), 99.4% (95% confidence interval, 96.9 to 99.9%), 98.8% (95% confidence interval, 93.5 to 99.9%), and 97.8% (95% confidence interval, 94.4 to 99.4%), respectively. The SAMBA for pandemic H1N1 provides a new technology that could potentially facilitate timely diagnosis and management of infected individuals, thereby informing decision making with regard to patient isolation during a pandemic outbreak. PMID:20668123

  10. Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus.

    PubMed

    Lagerqvist, Nina; Hagström, Åsa; Lundahl, Malin; Nilsson, Elin; Juremalm, Mikael; Larsson, Inger; Alm, Erik; Bucht, Göran; Ahlm, Clas; Klingström, Jonas

    2016-05-01

    Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS.

  11. The Era of Molecular and Other Non-Culture-Based Methods in Diagnosis of Sepsis

    PubMed Central

    Mancini, Nicasio; Carletti, Silvia; Ghidoli, Nadia; Cichero, Paola; Burioni, Roberto; Clementi, Massimo

    2010-01-01

    Summary: Sepsis, a leading cause of morbidity and mortality throughout the world, is a clinical syndrome with signs and symptoms relating to an infectious event and the consequent important inflammatory response. From a clinical point of view, sepsis is a continuous process ranging from systemic inflammatory response syndrome (SIRS) to multiple-organ-dysfunction syndrome (MODS). Blood cultures are the current “gold standard” for diagnosis, and they are based on the detection of viable microorganisms present in blood. However, on some occasions, blood cultures have intrinsic limitations in terms of sensitivity and rapidity, and it is not expected that these drawbacks will be overcome by significant improvements in the near future. For these principal reasons, other approaches are therefore needed in association with blood culture to improve the overall diagnostic yield for septic patients. These considerations have represented the rationale for the development of highly sensitive and fast laboratory methods. This review addresses non-culture-based techniques for the diagnosis of sepsis, including molecular and other non-culture-based methods. In particular, the potential clinical role for the sensitive and rapid detection of bacterial and fungal DNA in the development of new diagnostic algorithms is discussed. PMID:20065332

  12. A Simple and Accurate Method To Calculate Free Energy Profiles and Reaction Rates from Restrained Molecular Simulations of Diffusive Processes.

    PubMed

    Ovchinnikov, Victor; Nam, Kwangho; Karplus, Martin

    2016-08-25

    A method is developed to obtain simultaneously free energy profiles and diffusion constants from restrained molecular simulations in diffusive systems. The method is based on low-order expansions of the free energy and diffusivity as functions of the reaction coordinate. These expansions lead to simple analytical relationships between simulation statistics and model parameters. The method is tested on 1D and 2D model systems; its accuracy is found to be comparable to or better than that of the existing alternatives, which are briefly discussed. An important aspect of the method is that the free energy is constructed by integrating its derivatives, which can be computed without need for overlapping sampling windows. The implementation of the method in any molecular simulation program that supports external umbrella potentials (e.g., CHARMM) requires modification of only a few lines of code. As a demonstration of its applicability to realistic biomolecular systems, the method is applied to model the α-helix ↔ β-sheet transition in a 16-residue peptide in implicit solvent, with the reaction coordinate provided by the string method. Possible modifications of the method are briefly discussed; they include generalization to multidimensional reaction coordinates [in the spirit of the model of Ermak and McCammon (Ermak, D. L.; McCammon, J. A. J. Chem. Phys. 1978, 69, 1352-1360)], a higher-order expansion of the free energy surface, applicability in nonequilibrium systems, and a simple test for Markovianity. In view of the small overhead of the method relative to standard umbrella sampling, we suggest its routine application in the cases where umbrella potential simulations are appropriate.

  13. Molecular Classification of Rhabdomyosarcoma—Genotypic and Phenotypic Determinants of Diagnosis

    PubMed Central

    Davicioni, Elai; Anderson, Michael J.; Finckenstein, Friedrich Graf; Lynch, James C.; Qualman, Stephen J.; Shimada, Hiroyuki; Schofield, Deborah E.; Buckley, Jonathan D.; Meyer, William H.; Sorensen, Poul H.B.; Triche, Timothy J.

    2009-01-01

    Rhabdomyosarcoma (RMS) in children occurs as two major histological subtypes, embryonal (ERMS) and alveolar (ARMS). ERMS is associated with an 11p15.5 loss of heterozygosity (LOH) and may be confused with nonmyogenic, non-RMS soft tissue sarcomas. ARMS expresses the product of a genomic translocation that fuses FOXO1 (FKHR) with either PAX3 or PAX7 (P-F); however, at least 25% of cases lack these translocations. Here, we describe a genomic-based classification scheme that is derived from the combined gene expression profiling and LOH analysis of 160 cases of RMS and non-RMS soft tissue sarcomas that is at variance with conventional histopathological schemes. We found that gene expression profiles and patterns of LOH of ARMS cases lacking P-F translocations are indistinguishable from conventional ERMS cases. A subset of tumors that has been histologically classified as RMS lack myogenic gene expression. However, classification based on gene expression is possible using as few as five genes with an estimated error rate of less than 5%. Using immunohistochemistry, we characterized two markers, HMGA2 and TFAP2ß, which facilitate the differential diagnoses of ERMS and P-F RMS, respectively, using clinical material. These objectively derived molecular classes are based solely on genomic analysis at the time of diagnosis and are highly reproducible. Adoption of these molecular criteria may offer a more clinically relevant diagnostic scheme, thus potentially improving patient management and therapeutic RMS outcomes. PMID:19147825

  14. [Molecular and serological diagnosis of a dengue outbreak in Coro, Falcón state, Venezuela].

    PubMed

    Moros, Zoila Caridad; Abad, María Jesús; Arsenak, Miriam; Martínez, Dilia; Cierco, Maria Magdalena; Costagliola, Asunta; Urbina, Leyda; Taylor, Peter; Liprandi, Ferdinando; Pujol, Flor Helene

    2003-09-01

    Dengue virus (DV) is responsible for a spectrum of diseases, from a self-limited fever disease (DF, dengue fever) to the more severe forms of hemorrhagic fever/dengue shock syndrome (DHF/DSS). The aim of this study was the serological and molecular confirmation of an outbreak of dengue in Falcon state, Venezuela. A total of 54 sera from patients with clinical diagnosis of DV infection were analyzed by an enzyme immunoassays developed in Venezuela (ELISA -IgM e -IgG) and by PCR. From them, 78% exhibited DV infection (PCR+ y/o IgM+), 48% exhibited viremia by PCR and 57% were positive to IgM. An interesting observation was the high percent (76%) of patients with past or secondary infection (IgG positive), which included all the patients exhibiting clinical symptoms of DHF (n = 8). From the PCR positive sera, serotype 1 was found in 27%, serotype 2 in 54% and serotype 4 in 19%. No serotype 3 was found circulating in this population, although this serotype was already circulating in the nearby island of Aruba. The combination of serological and molecular methods allow us to obtain a fairly precise information of this outbreak.

  15. Accurate Treatment of Electrostatics during Molecular Adsorption in Nanoporous Crystals without Assigning Point Charges to Framework Atoms

    SciTech Connect

    Watanabe, T; Manz, TA; Sholl, DS

    2011-03-24

    Molecular simulations have become an important complement to experiments for studying gas adsorption and separation in crystalline nanoporous materials. Conventionally, these simulations use force fields that model adsorbate-pore interactions by assigning point charges to the atoms of the adsorbent. The assignment of framework charges always introduces ambiguity because there are many different choices for defining point charges, even when the true electron density of a material is known. We show how to completely avoid such ambiguity by using the electrostatic potential energy surface (EPES) calculated from plane wave density functional theory (DFT). We illustrate this approach by simulating CO(2) adsorption in four metal-organic frameworks (MOFs): IRMOF-1, ZIE-8, ZIE-90, and Zn(nicotinate)(2). The resulting CO(2) adsorption isotherms are insensitive to the exchange-correlation functional used in the DFT calculation of the EPES but are sensitive to changes in the crystal structure and lattice parameters. Isotherms computed from the DFT EPES are compared to those computed from several point charge models. This comparison makes possible, for the first time, an unbiased assessment of the accuracy of these point charge models for describing adsorption in MOFs. We find an unusually high Henry's constant (109 mmol/g.bar) and intermediate isosteric heat of adsorption (34.9 kJ/mol) for Zn(nicotinate)(2), which makes it a potentially attractive mateiial for CO(2) adsorption applications.

  16. Accurate Treatment of Electrostatics during Molecular Adsorption in Nanoporous Crystals without Assigning Point Charges to Framework Atoms

    SciTech Connect

    Watanabe, Taku; Manz, Thomas A.; Sholl, David S.

    2011-02-28

    Molecular simulations have become an important complement to experiments for studying gas adsorption and separation in crystalline nanoporous materials. Conventionally, these simulations use force fields that model adsorbate-pore interactions by assigning point charges to the atoms of the adsorbent. The assignment of framework charges always introduces ambiguity because there are many different choices for defining point charges, even when the true electron density of a material is known. We show how to completely avoid such ambiguity by using the electrostatic potential energy surface (EPES) calculated from plane wave density functional theory (DFT). We illustrate this approach by simulating CO2 adsorption in four metal-organic frameworks (MOFs): IRMOF-1, ZIF-8, ZIF-90, and Zn(nicotinate)2. The resulting CO2 adsorption isotherms are insensitive to the exchange-correlation functional used in the DFT calculation of the EPES but are sensitive to changes in the crystal structure and lattice parameters. Isotherms computed from the DFT EPES are compared to those computed from several point charge models. This comparison makes possible, for the first time, an unbiased assessment of the accuracy of these point charge models for describing adsorption in MOFs. We find an unusually high Henry’s constant (109 mmol/g·bar) and intermediate isosteric heat of adsorption (34.9 kJ/mol) for Zn(nicotinate)2, which makes it a potentially attractive material for CO2 adsorption applications.

  17. Avoiding fractional electrons in subsystem DFT based ab-initio molecular dynamics yields accurate models for liquid water and solvated OH radical

    NASA Astrophysics Data System (ADS)

    Genova, Alessandro; Ceresoli, Davide; Pavanello, Michele

    2016-06-01

    In this work we achieve three milestones: (1) we present a subsystem DFT method capable of running ab-initio molecular dynamics simulations accurately and efficiently. (2) In order to rid the simulations of inter-molecular self-interaction error, we exploit the ability of semilocal frozen density embedding formulation of subsystem DFT to represent the total electron density as a sum of localized subsystem electron densities that are constrained to integrate to a preset, constant number of electrons; the success of the method relies on the fact that employed semilocal nonadditive kinetic energy functionals effectively cancel out errors in semilocal exchange-correlation potentials that are linked to static correlation effects and self-interaction. (3) We demonstrate this concept by simulating liquid water and solvated OH• radical. While the bulk of our simulations have been performed on a periodic box containing 64 independent water molecules for 52 ps, we also simulated a box containing 256 water molecules for 22 ps. The results show that, provided one employs an accurate nonadditive kinetic energy functional, the dynamics of liquid water and OH• radical are in semiquantitative agreement with experimental results or higher-level electronic structure calculations. Our assessments are based upon comparisons of radial and angular distribution functions as well as the diffusion coefficient of the liquid.

  18. Madura foot in Europe: diagnosis of an autochthonous case by molecular approach and review of the literature.

    PubMed

    Mencarini, Jessica; Antonelli, Alberto; Scoccianti, Guido; Bartolini, Laura; Roselli, Giuliana; Capanna, Rodolfo; Rossolini, Gian Maria; Bartoloni, Alessandro; Bartalesi, Filippo

    2016-04-01

    Madura foot is a chronic granulomatous infection of the soft-tissue of the foot and it is endemic in tropical and subtropical countries. Some cases have also been reported in local people or migrants in temperate countries. The microbiological diagnosis requires prolonged bacterial cultures in aerobic and anaerobic conditions, but the use of the molecular approach could be helpful for an early and rapid diagnosis. We describe an autochthonous case of Actinomadura madurae foot infection in an Italian woman. The diagnosis was achieved 36 months after symptoms onset by PCR detection and sequencing of 16S rDNA directly on biopsy. She started therapy with rifampin, trimethoprim-sulfamethoxazole, and amikacin. After 3 months the pain had disappeared and the swelling subsided. We reviewed the literature on Madura foot due to bacterial causative agents in Europe and observed that the median time from onset to diagnosis is high, possibly due to several factors like the difficulties of the microbiological and radiological diagnosis. Our case report and the review of literature point out that the implementation of a surveillance system, the involvement of an infectious diseases specialist, with experience in tropical diseases, and the availability of a microbiology unit to perform feasible and rapid molecular diagnostic tests could result in an earlier diagnosis and an optimal antibiotic therapy of this rare but difficult-to-treat and, above all, difficult-to-diagnose infection. PMID:27196558

  19. Gene Expression Profile of Peripheral Blood Monocytes: A Step towards the Molecular Diagnosis of Celiac Disease?

    PubMed Central

    Cielo, Donatella; Morelli, Marinita; Gambino, Giuseppina; Zanzi, Delia; Strisciuglio, Caterina; Sperandeo, Maria Pia; Greco, Luigi; Auricchio, Renata

    2013-01-01

    Aim Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls. Participants Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn’s disease patients) were enrolled. Results Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%. Conclusions The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease. PMID:24069342

  20. Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study.

    PubMed

    Robert-Gangneux, Florence; Sterkers, Yvon; Yera, Hélène; Accoceberry, Isabelle; Menotti, Jean; Cassaing, Sophie; Brenier-Pinchart, Marie-Pierre; Hennequin, Christophe; Delhaes, Laurence; Bonhomme, Julie; Villena, Isabelle; Scherer, Emeline; Dalle, Frédéric; Touafek, Feriel; Filisetti, Denis; Varlet-Marie, Emmanuelle; Pelloux, Hervé; Bastien, Patrick

    2015-05-01

    Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV(+); the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.

  1. Molecular techniques in the diagnosis and management of infectious diseases: do they have a role in bacteriology?

    PubMed

    Poxton, Ian R

    2005-01-01

    The diagnosis and management of bacterial diseases has been done by traditional methods for a century or more. With the advent of molecular methods, however, these traditional approaches are being challenged. This review examines the pros and cons of traditional versus modern methods and tries to answer the question: when are molecular methods useful or essential? The following topics are addressed with appropriate examples: diagnosis; identification, typing and fingerprinting; pathogenesis; patient management; susceptibility to disease, and resistance to antimicrobial agents. It was concluded that there is still a place for both traditional and modern molecular methods, and training of staff must include both methodologies. Innovation is encouraged--but new technologies must be thoroughly tested before introduction into the routine lab. Liaison between laboratory scientist and physician is important, but above all experience is paramount.

  2. Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

    PubMed

    Singleton, Jered; Osborn, Jennifer L; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

  3. A tool for diagnosis of Dicrocoelium dendriticum infection: hatching eggs and molecular identification of the miracidium.

    PubMed

    Sandoval, H; Manga-González, M Yolanda; Castro, José M

    2013-04-01

    DNA primers were designed from the 18S rRNA sequence from the relevant digenean trematode Dicrocoelium dendriticum to evaluate a polymerase chain reaction-based diagnostic method of this parasite from its eggs in faeces of naturally and experimentally infected sheep. In order to get DNA from D. dendriticum eggs, several hatching mechanisms were studied. Successful results were obtained when the eggs were frozen to -80 °C and/or in liquid nitrogen and then defrosted. This method allowed the opening of the egg operculum and the liberation of the miracidium. DNA from D. dendriticum adults and from hatching egg miracidia was obtained and an amplification single band of 1.95 kb was observed using primers designed for the total 18S rRNA sequence in both cases as well as when the template DNA was from adults of the closely related parasite Fasciola hepatica; in addition, a single and specific 0.8-kb band was obtained when primers based on an internal partial 18S rRNA sequence were used. The method showed to be useful not only in samples coming from adults, but in eggs from gall bladder and faeces as well. F. hepatica internal 18S rRNA primers were also designed and used as a negative control to prove that the eggs in faeces came from D. dendriticum and not from F. hepatica. A molecular tool able to detect a minimum of about 40 D. dendriticum eggs in one of the definitive host faeces has been developed for the first time and could provide a useful molecular tool to improve the conventional coprological diagnosis for detecting D. dendriticum eggs. PMID:23385970

  4. Electricity-Free Amplification and Detection for Molecular Point-of-Care Diagnosis of HIV-1

    PubMed Central

    Singleton, Jered; Osborn, Jennifer L.; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens. PMID:25426953

  5. Pitfalls in molecular diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia

    PubMed Central

    Kolahdouz, Mahsa; Mohammadi, Zahra; Kolahdouz, Parisa; Tajamolian, Masoud; Khanahmad, Hossein

    2015-01-01

    Congenital adrenal hyperplasia (CAH) is a putative error of metabolism with autosomal recessive heredity pattern. The main manifestations of classic form of CAH are salt-wasting, dehydration and simple virilization in both sexes and ambiguous genitalia in female gender. 21-hyroxylase (CYP21A2) impairment with prevalence value of 1 in 10,000–15,000 live births is the most common etiology of CAH. Because of consanguineous marriages, the frequency of the CAH in Iran is very high. A wide range of mutations diversity exists in CYP21A2 gene and a large number of these mutations derived from a highly homologous pseudogene, CYP21A1P, through gene conversion. In addition, new mutations such as small and large deletion and point mutations can also result in enzyme deficiency. Various methods for mutation detection were performed. The main obstacle in molecular diagnosis of CAH is amplification of pseudogene during polymerase chain reaction of CYP21A2. All attempts focus on discrimination of pseudogene from gene; that is why, there is the majority of mutations on pseudogene, and if we have contamination with the pseudogene, the result will be unreliable. Here, we discuss this methods and advantage and disadvantage of those. PMID:26605228

  6. Improving molecular diagnosis in epilepsy by a dedicated high-throughput sequencing platform.

    PubMed

    Della Mina, Erika; Ciccone, Roberto; Brustia, Francesca; Bayindir, Baran; Limongelli, Ivan; Vetro, Annalisa; Iascone, Maria; Pezzoli, Laura; Bellazzi, Riccardo; Perotti, Gianfranco; De Giorgis, Valentina; Lunghi, Simona; Coppola, Giangennaro; Orcesi, Simona; Merli, Pietro; Savasta, Salvatore; Veggiotti, Pierangelo; Zuffardi, Orsetta

    2015-03-01

    We analyzed by next-generation sequencing (NGS) 67 epilepsy genes in 19 patients with different types of either isolated or syndromic epileptic disorders and in 15 controls to investigate whether a quick and cheap molecular diagnosis could be provided. The average number of nonsynonymous and splice site mutations per subject was similar in the two cohorts indicating that, even with relatively small targeted platforms, finding the disease gene is not an univocal process. Our diagnostic yield was 47% with nine cases in which we identified a very likely causative mutation. In most of them no interpretation would have been possible in absence of detailed phenotype and familial information. Seven out of 19 patients had a phenotype suggesting the involvement of a specific gene. Disease-causing mutations were found in six of these cases. Among the remaining patients, we could find a probably causative mutation only in three. None of the genes affected in the latter cases had been suspected a priori. Our protocol requires 8-10 weeks including the investigation of the parents with a cost per patient comparable to sequencing of 1-2 medium-to-large-sized genes by conventional techniques. The platform we used, although providing much less information than whole-exome or whole-genome sequencing, has the advantage that can also be run on 'benchtop' sequencers combining rapid turnaround times with higher manageability.

  7. Performance limitations of label-free sensors in molecular diagnosis using complex samples

    NASA Astrophysics Data System (ADS)

    Varma, Manoj

    2016-03-01

    Label-free biosensors promised a paradigm involving direct detection of biomarkers from complex samples such as serum without requiring multistep sample processing typical of labelled methods such as ELISA or immunofluorescence assays. Label-free sensors have witnessed decades of development with a veritable zoo of techniques available today exploiting a multitude of physical effects. It is appropriate now to critically assess whether label-free technologies have succeeded in delivering their promise with respect to diagnostic applications, particularly, ambitious goals such as early cancer detection using serum biomarkers, which require low limits of detection (LoD). Comparison of nearly 120 limits of detection (LoD) values reported by labelled and label-free sensing approaches over a wide range of detection techniques and target molecules in serum revealed that labeled techniques achieve 2-3 orders of magnitude better LoDs. Data from experiments where labelled and label-free assays were performed simultaneously using the same assay parameters also confirm that the LoD achieved by labelled techniques is 2 to 3 orders of magnitude better than that by label-free techniques. Furthermore, label-free techniques required significant signal amplification, for e.g. using nanoparticle conjugated secondary antibodies, to achieve LoDs comparable to labelled methods substantially deviating from the original "direct detection" paradigm. This finding has important implications on the practical limits of applying label-free detection methods for molecular diagnosis.

  8. Improving molecular diagnosis in epilepsy by a dedicated high-throughput sequencing platform

    PubMed Central

    Mina, Erika Della; Ciccone, Roberto; Brustia, Francesca; Bayindir, Baran; Limongelli, Ivan; Vetro, Annalisa; Iascone, Maria; Pezzoli, Laura; Bellazzi, Riccardo; Perotti, Gianfranco; De Giorgis, Valentina; Lunghi, Simona; Coppola, Giangennaro; Orcesi, Simona; Merli, Pietro; Savasta, Salvatore; Veggiotti, Pierangelo; Zuffardi, Orsetta

    2015-01-01

    We analyzed by next-generation sequencing (NGS) 67 epilepsy genes in 19 patients with different types of either isolated or syndromic epileptic disorders and in 15 controls to investigate whether a quick and cheap molecular diagnosis could be provided. The average number of nonsynonymous and splice site mutations per subject was similar in the two cohorts indicating that, even with relatively small targeted platforms, finding the disease gene is not an univocal process. Our diagnostic yield was 47% with nine cases in which we identified a very likely causative mutation. In most of them no interpretation would have been possible in absence of detailed phenotype and familial information. Seven out of 19 patients had a phenotype suggesting the involvement of a specific gene. Disease-causing mutations were found in six of these cases. Among the remaining patients, we could find a probably causative mutation only in three. None of the genes affected in the latter cases had been suspected a priori. Our protocol requires 8–10 weeks including the investigation of the parents with a cost per patient comparable to sequencing of 1–2 medium-to-large-sized genes by conventional techniques. The platform we used, although providing much less information than whole-exome or whole-genome sequencing, has the advantage that can also be run on ‘benchtop' sequencers combining rapid turnaround times with higher manageability. PMID:24848745

  9. Application of imaging mass spectrometry for the molecular diagnosis of human breast tumors.

    PubMed

    Mao, Xinxin; He, Jiuming; Li, Tiegang; Lu, Zhaohui; Sun, Jian; Meng, Yunxiao; Abliz, Zeper; Chen, Jie

    2016-01-01

    Distinguishing breast invasive ductal carcinoma (IDC) and breast ductal carcinoma in situ (DCIS) is a key step in breast surgery, especially to determine whether DCIS is associated with tumor cell micro-invasion. However, there is currently no reliable method to obtain molecular information for breast tumor analysis during surgery. Here, we present a novel air flow-assisted ionization (AFAI) mass spectrometry imaging method that can be used in ambient environments to differentiate breast cancer by analyzing lipids. In this study, we demonstrate that various subtypes and histological grades of IDC and DCIS can be discriminated using AFAI-MSI: phospholipids were more abundant in IDC than in DCIS, whereas fatty acids were more abundant in DCIS than in IDC. The classification of specimens in the subtype and grade validation sets showed 100% and 78.6% agreement with the histopathological diagnosis, respectively. Our work shows the rapid classification of breast cancer utilizing AFAI-MSI. This work suggests that this method could be developed to provide surgeons with nearly real-time information to guide surgical resections. PMID:26868906

  10. Molecular heterogeneity of beta-thalassaemia in Malaysia: a practical approach to diagnosis.

    PubMed

    Thong, M K; Law, H Y; Ng, I S

    1996-01-01

    The beta-thalassaemia mutations in 20 Malaysian children with beta-thalassaemia major were characterised by using a multi-modal approach, consisting of a slot-blot hybridisation with selected allele-specific oligonucleotides (ASO), followed by reverse dot-blot assay (RDB), amplification refractory mutation system (ARMS) and genomic sequencing. This strategy yielded a 94.4% mutation detection rate. The 6 most common mutations were codons 41/42 (-TTCT), IVS II nt 654(C --> T), IVS I nt 5(G --> C), IVS I nt 1(G -->T), codon 35 (-C) and codon 19 (A --> G), which accounted for 83.3% of all mutations detected. A strategy of initial screening with the above 6 selected ASOs for slot-blot hybridisation followed by RDB assay for the less common Asian mutations would give a mutation identification of 91.7%. Another feasible approach would be to analyse alleles from a particular racial group, by a judicious selection of 4 ASOs common to that particular subpopulation and then supplement this with RDB assay. This could yield a 100% coverage for the Chinese subpopulation in Malaysia. With these strategies, a practical approach has been identified to overcome the pitfalls posed by the molecular heterogeneity of beta-thalassaemia to enable prenatal diagnosis and carrier screening to be carried out. Regional collaborative studies are to be encouraged as an indispensable tool in providing better health care services to our patients.

  11. [Comparison of direct microscopy, culture, ELISA and molecular methods for diagnosis of Entamoeba histolytica].

    PubMed

    Tüzemen, Nazmiye Ulkü; Doğan, Nihal

    2014-01-01

    Amebiasis, a parasitic infection caused by Entamoeba histolytica, is one of the most common parasitic infections worldwide. Since it is still an important public health problem in developing countries, rapid differential diagnosis of amebiasis is crucial in terms of treatment. The most frequently used method for laboratory diagnosis is direct microscopy, however more reliable and specific methods are needed in order to differentiate the apathogenic Entamoeba dispar under the microscope. This study was conducted to compare the results of different methods namely, direct microscopy, culture, ELISA and PCR for the detection of E.histolytica in stool samples and to evaluate the performances of those methods. A total of 1049 stool samples collected from pediatric and adult patients who were admitted to hospital with diarrhea complaint between January 2011-March 2013, and randomly selected samples from primary school children, were included in the study. Direct microscopic examination was performed by native-lugol, physiological saline, modified formol-ethyl acetate sedimentation and trichrome staining methods. The stool samples were also inoculated into TYI-S-33 media for axenic cultivation of amoeba. The presence of amebic antigens in the samples were screened by a commercial ELISA kit (TechLab, E.histolytica II, USA). For the molecular diagnosis, a multiplex tandem real-time PCR (MT-PCR) kit (AusDiagnostics Pty Ltd, Australia) was used, after the extraction of DNAs with QIAamp DNA Stool Mini Kit (Qiagen, USA). A total of 354 samples which could be evaluated by all of the methods, were included in the study. Of the 354 stool samples, 84 (23.7%) were found E.histolytica/E.dispar positive by direct microscopy, 61 (17.2%) by trichrome staining, 46 (12.9%) by culture, 31 (8.7%) by ELISA and 9 (2.5%) by MT-PCR. Of direct microscopy positive samples 54.7% (46/84) were also positive with trichrome staining, 39.3% (33/84) with culture, 15.5% (13/84) with ELISA and 7.1% (6

  12. Communication: Rate coefficients of the H + CH4 → H2 + CH3 reaction from ring polymer molecular dynamics on a highly accurate potential energy surface.

    PubMed

    Meng, Qingyong; Chen, Jun; Zhang, Dong H

    2015-09-14

    The ring polymer molecular dynamics (RPMD) calculations are performed to calculate rate constants for the title reaction on the recently constructed potential energy surface based on permutation invariant polynomial (PIP) neural-network (NN) fitting [J. Li et al., J. Chem. Phys. 142, 204302 (2015)]. By inspecting convergence, 16 beads are used in computing free-energy barriers at 300 K ≤ T ≤ 1000 K, while different numbers of beads are used for transmission coefficients. The present RPMD rates are in excellent agreement with quantum rates computed on the same potential energy surface, as well as with the experimental measurements, demonstrating further that the RPMD is capable of producing accurate rates for polyatomic chemical reactions even at rather low temperatures.

  13. Communication: Rate coefficients of the H + CH4 → H2 + CH3 reaction from ring polymer molecular dynamics on a highly accurate potential energy surface

    NASA Astrophysics Data System (ADS)

    Meng, Qingyong; Chen, Jun; Zhang, Dong H.

    2015-09-01

    The ring polymer molecular dynamics (RPMD) calculations are performed to calculate rate constants for the title reaction on the recently constructed potential energy surface based on permutation invariant polynomial (PIP) neural-network (NN) fitting [J. Li et al., J. Chem. Phys. 142, 204302 (2015)]. By inspecting convergence, 16 beads are used in computing free-energy barriers at 300 K ≤ T ≤ 1000 K, while different numbers of beads are used for transmission coefficients. The present RPMD rates are in excellent agreement with quantum rates computed on the same potential energy surface, as well as with the experimental measurements, demonstrating further that the RPMD is capable of producing accurate rates for polyatomic chemical reactions even at rather low temperatures.

  14. Towards a Better Molecular Diagnosis of FMR1-Related Disorders-A Multiyear Experience from a Reference Lab.

    PubMed

    Rzońca, Sylwia Olimpia; Gos, Monika; Szopa, Daniel; Sielska-Rotblum, Danuta; Landowska, Aleksandra; Szpecht-Potocka, Agnieszka; Milewski, Michał; Czekajska, Jolanta; Abramowicz, Anna; Obersztyn, Ewa; Maciejko, Dorota; Mazurczak, Tadeusz; Bal, Jerzy

    2016-01-01

    The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5'UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of FXS-related disorders. The new methods were subsequently implemented in our laboratory. This allowed the significance of the Southern blot technique to be decreased until its complete withdrawal. Our experience points out the necessity of implementation of the GeneScan based methods to simplify the testing procedure as well as to obtain more information for the patient, especially if TP-PCR based methods are used. PMID:27598204

  15. Towards a Better Molecular Diagnosis of FMR1-Related Disorders—A Multiyear Experience from a Reference Lab

    PubMed Central

    Rzońca, Sylwia Olimpia; Gos, Monika; Szopa, Daniel; Sielska-Rotblum, Danuta; Landowska, Aleksandra; Szpecht-Potocka, Agnieszka; Milewski, Michał; Czekajska, Jolanta; Abramowicz, Anna; Obersztyn, Ewa; Maciejko, Dorota; Mazurczak, Tadeusz; Bal, Jerzy

    2016-01-01

    The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5′UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of FXS-related disorders. The new methods were subsequently implemented in our laboratory. This allowed the significance of the Southern blot technique to be decreased until its complete withdrawal. Our experience points out the necessity of implementation of the GeneScan based methods to simplify the testing procedure as well as to obtain more information for the patient, especially if TP-PCR based methods are used. PMID:27598204

  16. The accurate calculation of the band gap of liquid water by means of GW corrections applied to plane-wave density functional theory molecular dynamics simulations.

    PubMed

    Fang, Changming; Li, Wun-Fan; Koster, Rik S; Klimeš, Jiří; van Blaaderen, Alfons; van Huis, Marijn A

    2015-01-01

    Knowledge about the intrinsic electronic properties of water is imperative for understanding the behaviour of aqueous solutions that are used throughout biology, chemistry, physics, and industry. The calculation of the electronic band gap of liquids is challenging, because the most accurate ab initio approaches can be applied only to small numbers of atoms, while large numbers of atoms are required for having configurations that are representative of a liquid. Here we show that a high-accuracy value for the electronic band gap of water can be obtained by combining beyond-DFT methods and statistical time-averaging. Liquid water is simulated at 300 K using a plane-wave density functional theory molecular dynamics (PW-DFT-MD) simulation and a van der Waals density functional (optB88-vdW). After applying a self-consistent GW correction the band gap of liquid water at 300 K is calculated as 7.3 eV, in good agreement with recent experimental observations in the literature (6.9 eV). For simulations of phase transformations and chemical reactions in water or aqueous solutions whereby an accurate description of the electronic structure is required, we suggest to use these advanced GW corrections in combination with the statistical analysis of quantum mechanical MD simulations.

  17. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology

    PubMed Central

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K. P.; Woo, Patrick C. Y.

    2015-01-01

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10–49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n = 2), Pichia (Candida) norvegensis (n = 2), Candida tropicalis (n = 1) and Saccharomyces cerevisiae (n = 1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study. PMID:26506340

  18. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

    PubMed

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

    2015-10-22

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

  19. Postmortem CT is more accurate than clinical diagnosis for identifying the immediate cause of death in hospitalized patients: a prospective autopsy-based study.

    PubMed

    Inai, Kunihiro; Noriki, Sakon; Kinoshita, Kazuyuki; Sakai, Toyohiko; Kimura, Hirohiko; Nishijima, Akihiko; Iwasaki, Hiromichi; Naiki, Hironobu

    2016-07-01

    Despite 75 to 90 % physician accuracy in determining the underlying cause of death, precision of determination of the immediate cause of death is approximately 40 %. In contrast, two thirds of immediate causes of death in hospitalized patients are correctly diagnosed by postmortem computed tomography (CT). Postmortem CT might provide an alternative approach to verifying the immediate cause of death. To evaluate the effectiveness of postmortem CT as an alternative method to determine the immediate cause of death in hospitalized patients, an autopsy-based prospective study was performed. Of 563 deaths from September 2011 to August 2013, 50 consecutive cadavers undergoing hospital autopsies with consent for additional postmortem CT at the University of Fukui were enrolled. The accuracy of determination of the immediate cause of death by postmortem CT was evaluated in these patients. Diagnostic discrepancy was also compared between radiologists and attending physicians. The immediate cause of death was correctly diagnosed in 37 of 50 subjects using postmortem CT (74 %), concerning 29 cases of respiratory failure, 4 of hemorrhage, 3 of liver failure and 1 of septic shock. Six cases of organ failure involving 13 patients were not identified as the cause of death by postmortem CT. Regarding the immediate cause of death, accuracy of clinical diagnosis was significantly lower than that of postmortem CT (46 vs 74 %, P < 0.01). Postmortem CT may be more useful than clinical diagnosis for identifying the immediate cause of death in hospitalized patients not undergoing autopsy. PMID:27085336

  20. Nanosized multifunctional liposomes for tumor diagnosis and molecular imaging by SPECT/CT.

    PubMed

    Silindir, Mine; Erdoğan, Suna; Özer, A Yekta; Doğan, A Lale; Tuncel, Murat; Uğur, Ömer; Torchilin, Vladimir P

    2013-03-01

    Among currently used cancer imaging methods, nuclear medicine modalities provide metabolic information, whereas modalities in radiology provide anatomical information. However, different modalities, having different acquisition times in separate machines, decrease the specificity and accuracy of images. To solve this problem, hybrid imaging modalities were developed as a new era, especially in the cancer imaging field. With widespread usage of hybrid imaging modalities, specific contrast agents are essentially needed to use in both modalities, such as single-photon emission computed tomography/computed tomography (SPECT/CT). Liposomes are one of the most desirable drug delivery systems, depending on their suitable properties. The aim of this study was to develop a liposomal contrast agent for the diagnosis and molecular imaging of tumor by SPECT/CT. Liposomes were prepared nanosized, coated with polyethylene glycol to obtain long blood circulation, and modified with monoclonal antibody 2C5 for specific tumor targeting. Although DTPA-PE and DTPA-PLL-NGPE (polychelating amphilic polymers; PAPs) were loaded onto liposomes for stable radiolabeling for SPECT imaging, iopromide was encapsulated into liposomes for CT imaging. Liposomes [(DPPC:PEG(2000)-PE:Chol:DTPA-PE), (PL 90G:PEG(2000)-PE:Chol:DTPA-PE), (DPPC:PEG(2000)-PE:Chol:PAPs), (PL 90G:PEG(2000)-PE:Chol:PAPs), (60:0.9:39:0.1% mol ratio)] were characterized in terms of entrapment efficiency, particle size, physical stability, and release kinetics. Additionally, in vitro cell-binding studies were carried out on two tumor cell lines (MCF-7 and EL 4) by counting radioactivity. Tumor-specific antibody-modified liposomes were found to be effective multimodal contrast agents by designating almost 3-8 fold more uptake than nonmodified ones in different tumor cell lines. These results could be considered as an important step in the development of tumor-targeted SPECT/CT contrast agents for cancer imaging. PMID:23078019

  1. Molecular diagnosis and anti-microbial resistance patterns among Shigella spp. isolated from patients with diarrhea

    PubMed Central

    Hosseini Nave, Hossein; Mansouri, Shahla; Sadeghi, Amin; Moradi, Mohammad

    2016-01-01

    Aim: This study aims to determine the serogroup distribution and molecular diagnosis, as well as antimicrobial resistance profiles among Shigella spp. isolated from patients with diarrhea in Kerman, southeast of Iran. Background: Shigella species are frequent cause of bacterial dysentery worldwide. Previous studies have been reported that S. sonnei and S. flexneri are the most prevalent serogroups in various parts of Iran. Patients and methods: A total of 624 stool samples were randomly collected from patients with diarrhea from June 2013 to August 2014. Biochemical and serological characterizations were performed for identifying Shigella spp. In addition, the multiplex PCR assay was carried out for the detection and differentiation of three pathogenic Shigella spp. Antibiotic susceptibility testing was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Results: Fifty six (9%) Shigella strains were isolated from stool samples. The most common species were S. flexneri 31(55.4%), followed by S .sonnei 18(32.1%) and S. boydii 7(12.5%). S. dysentery was not detected in the present study. All the isolates that identified by serological test as Shigella spp. were confirmed by the multiplex PCR method. The highest rate of resistance was observed for ampicillin and trimethoprim-sulphamethoxazole antibiotics with 52(92.9%) resistant, followed by tetracycline 44(78.6%) and cefotaxime 33(58.9%). All Shigella isolates were susceptible to ciprofloxacin. A significant relationship was found between the Shigella species and cefotaxime resistance (p<0.05). Conclusion: S. flexneri was found as the most prevalent serogroup causing shigellosis. The high rate of resistance to third-generation cephalosporins limits the treatment options available for the management of shigellosis in Kerman, Iran. PMID:27458513

  2. Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis

    PubMed Central

    Martin, Hilary C.; Kim, Grace E.; Pagnamenta, Alistair T.; Murakami, Yoshiko; Carvill, Gemma L.; Meyer, Esther; Copley, Richard R.; Rimmer, Andrew; Barcia, Giulia; Fleming, Matthew R.; Kronengold, Jack; Brown, Maile R.; Hudspith, Karl A.; Broxholme, John; Kanapin, Alexander; Cazier, Jean-Baptiste; Kinoshita, Taroh; Nabbout, Rima; Bentley, David; McVean, Gil; Heavin, Sinéad; Zaiwalla, Zenobia; McShane, Tony; Mefford, Heather C.; Shears, Deborah; Stewart, Helen; Kurian, Manju A.; Scheffer, Ingrid E.; Blair, Edward; Donnelly, Peter; Kaczmarek, Leonard K.; Taylor, Jenny C.

    2014-01-01

    In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders. PMID:24463883

  3. Recognizing the link between CKD and CVD in the primary care setting: accurate and early diagnosis for timely and appropriate intervention.

    PubMed

    Basile, Jan N

    2007-05-01

    Chronic kidney disease (CKD), which is becoming increasingly prevalent in the US and worldwide, eventually progresses to end-stage renal disease (ESRD), requiring renal replacement therapy. Diabetes and hypertension, the two leading causes of CKD, are themselves reaching near epidemic proportions. Hypertension can cause both the development and progression of CKD, and CKD is a significant risk factor for the development of cardiovascular disease. Indeed, CKD patients are more likely to die of cardiovascular complications than progress to ESRD. However, data indicate that early recognition and management of CKD can have a significant positive impact on disease outcome. This creates an important interventional opportunity for the primary care physician. This report describes the major risk factors and comorbidities associated with the development and progression of CKD and offers suggestions for timely diagnosis and management of CKD in the primary care setting.

  4. Next-generation sequencing for molecular diagnosis of autosomal recessive polycystic kidney disease.

    PubMed

    Edrees, Burhan M; Athar, Mohammad; Al-Allaf, Faisal A; Taher, Mohiuddin M; Khan, Wajahatullah; Bouazzaoui, Abdellatif; Al-Harbi, Naffaa; Safar, Ramzia; Al-Edressi, Howaida; Alansary, Khawala; Anazi, Abulkareem; Altayeb, Naji; Ahmed, Muawia A; Abduljaleel, Zainularifeen

    2016-10-10

    Autosomal recessive polycystic kidney disease (ARPKD) a rare genetic disorder, described by formation of cysts in the kidney. A targeted customized sequencing of genes implicated in ARPKD phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified likely pathogenic disease causing variants during the validation process. Four potential pathogenic variants [c.4870C>T, p.(Arg1624Trp)], [c.5725C>T, p.(Arg1909Trp)], c.1736C>T, p.(Thr579Met)] and [(c.10628T>G), p.(Leu3543Trp)] were observed in PKHD1 gene among 12 out of 18 samples. The rest of the patient samples also showed few variants in ADPKD (Autosomal Dominant Polycystic Kidney Disease) disease causing genes PKD1 and PKD2 i.e. [c.12433G>A, p.(Val4145Ile)] and [c.1445T>G, p.(Phe482Cys)], respectively. All causative variants were validated by capillary sequencing, confirming the presence of a novel homozygous variants [c.10628T>G, p.(Leu3543Trp)] found in exon 61 of a male proband. All potentially deleterious variants identified in PKHD1, PKD1, and PKD2 gene, also exhibited pathologically or clinically significance based on the computational predictions involved in predicting the impact of non-synonymous SNPs (nsSNPs) on protein function such as Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen2). SIFT classified 50% of our nsSNPs as "deleterious", while PolyPhen2 identified 45% of our nsSNPs as "Probably damaged" and the results from both programs were largely complementary. Taken together, these results suggest that the NGS strategies provide a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in targeted genes sequence analysis. PMID:27401137

  5. Next-generation sequencing for molecular diagnosis of autosomal recessive polycystic kidney disease.

    PubMed

    Edrees, Burhan M; Athar, Mohammad; Al-Allaf, Faisal A; Taher, Mohiuddin M; Khan, Wajahatullah; Bouazzaoui, Abdellatif; Al-Harbi, Naffaa; Safar, Ramzia; Al-Edressi, Howaida; Alansary, Khawala; Anazi, Abulkareem; Altayeb, Naji; Ahmed, Muawia A; Abduljaleel, Zainularifeen

    2016-10-10

    Autosomal recessive polycystic kidney disease (ARPKD) a rare genetic disorder, described by formation of cysts in the kidney. A targeted customized sequencing of genes implicated in ARPKD phenotype was performed to identify candidate variants using the Ion torrent PGM next-generation sequencing. The results identified likely pathogenic disease causing variants during the validation process. Four potential pathogenic variants [c.4870C>T, p.(Arg1624Trp)], [c.5725C>T, p.(Arg1909Trp)], c.1736C>T, p.(Thr579Met)] and [(c.10628T>G), p.(Leu3543Trp)] were observed in PKHD1 gene among 12 out of 18 samples. The rest of the patient samples also showed few variants in ADPKD (Autosomal Dominant Polycystic Kidney Disease) disease causing genes PKD1 and PKD2 i.e. [c.12433G>A, p.(Val4145Ile)] and [c.1445T>G, p.(Phe482Cys)], respectively. All causative variants were validated by capillary sequencing, confirming the presence of a novel homozygous variants [c.10628T>G, p.(Leu3543Trp)] found in exon 61 of a male proband. All potentially deleterious variants identified in PKHD1, PKD1, and PKD2 gene, also exhibited pathologically or clinically significance based on the computational predictions involved in predicting the impact of non-synonymous SNPs (nsSNPs) on protein function such as Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen2). SIFT classified 50% of our nsSNPs as "deleterious", while PolyPhen2 identified 45% of our nsSNPs as "Probably damaged" and the results from both programs were largely complementary. Taken together, these results suggest that the NGS strategies provide a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in targeted genes sequence analysis.

  6. KRT9 gene mutation as a reliable indicator in the prenatal molecular diagnosis of epidermolytic palmoplantar keratoderma.

    PubMed

    Ke, Hai-Ping; Jiang, Hu-Ling; Lv, Ya-Su; Huang, Yi-Zhou; Liu, Rong-Rong; Chen, Xiao-Ling; Du, Zhen-Fang; Luo, Yu-Qin; Xu, Chen-Ming; Fan, Qi-Hui; Zhang, Xian-Ning

    2014-08-01

    Epidermolytic palmoplantar keratoderma (EPPK) is the most frequent form of such keratodermas. It is inherited in an autosomal dominant pattern and is clinically characterized by diffuse yellowish thickening of the skin on the palms and soles with erythematous borders during the first weeks or months after birth. EPPK is generally caused by mutations of the KRT9 gene. More than 26 KRT9 gene mutations responsible for EPPK have been described (Human Intermediate Filament Database, www.interfil.org), and many of these variants are located within the highly-conserved coil 1A region of the α-helical rod domain of keratin 9. Unfortunately, there is no satisfactory treatment for EPPK. Thus, prenatal molecular diagnosis or pre-pregnancy diagnosis is crucial and benefits those affected who seek healthy descendants. In the present study, we performed amniotic fluid-DNA-based prenatal testing for three at-risk pregnant EPPK women from three unrelated southern Chinese families who carried the KRT9 missense mutations p.Arg163Trp and p.Arg163Gln, and successfully helped two families to bear normal daughters. We suggest that before the successful application of preimplantation genetic diagnosis (PGD), and noninvasive prenatal diagnosis of EPPK that analyzes fetal cells or cell-free DNA in maternal blood, prenatal genetic diagnosis by amniocentesis or chorionic villus sampling (CVS) offers a quite acceptable option for EPPK couples-at-risk to avoid the birth of affected offspring, especially in low- and middle-income countries.

  7. A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement

    PubMed Central

    Prasad, Megana K; Geoffroy, Véronique; Vicaire, Serge; Jost, Bernard; Dumas, Michael; Le Gras, Stéphanie; Switala, Marzena; Gasse, Barbara; Laugel-Haushalter, Virginie; Paschaki, Marie; Leheup, Bruno; Droz, Dominique; Dalstein, Amelie; Loing, Adeline; Grollemund, Bruno; Muller-Bolla, Michèle; Lopez-Cazaux, Séréna; Minoux, Maryline; Jung, Sophie; Obry, Frédéric; Vogt, Vincent; Davideau, Jean-Luc; Davit-Beal, Tiphaine; Kaiser, Anne-Sophie; Moog, Ute; Richard, Béatrice; Morrier, Jean-Jacques; Duprez, Jean-Pierre; Odent, Sylvie; Bailleul-Forestier, Isabelle; Rousset, Monique Marie; Merametdijan, Laure; Toutain, Annick; Joseph, Clara; Giuliano, Fabienne; Dahlet, Jean-Christophe; Courval, Aymeric; El Alloussi, Mustapha; Laouina, Samir; Soskin, Sylvie; Guffon, Nathalie; Dieux, Anne; Doray, Bérénice; Feierabend, Stephanie; Ginglinger, Emmanuelle; Fournier, Benjamin; de la Dure Molla, Muriel; Alembik, Yves; Tardieu, Corinne; Clauss, François; Berdal, Ariane; Stoetzel, Corinne; Manière, Marie Cécile; Dollfus, Hélène; Bloch-Zupan, Agnès

    2016-01-01

    Background Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. Methods We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. Results We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. Conclusions We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. Trial registration numbers NCT01746121 and NCT02397824. PMID:26502894

  8. Molecular subtyping of diffuse large B-cell lymphoma: update on biology, diagnosis and emerging platforms for practising pathologists.

    PubMed

    Gifford, Grace K; Gill, Anthony J; Stevenson, William S

    2016-01-01

    Molecular classification of diffuse large B-cell lymphoma (DLBCL) is critical. Numerous methodologies have demonstrated that DLBCL is biologically heterogeneous despite morphological similarities. This underlies the disparate outcomes of treatment response or failure in this common non-Hodgkin lymphoma. This review will summarise historical approaches to lymphoma classifications, current diagnosis of DLBCL, molecular techniques that have primarily been used in the research setting to distinguish and subclassify DLBCL, evaluate contemporary diagnostic methodologies that seek to translate lymphoma biology into clinical practice, and introduce novel diagnostic platforms that may overcome current issues. The review concludes with an overview of key molecular lesions currently identified in DLBCL, all of which are potential targets for drug treatments that may improve survival and cure.

  9. State-of-the-art molecular imaging in esophageal cancer management: implications for diagnosis, prognosis, and treatment

    PubMed Central

    Lin, Jolinta; Kligerman, Seth; Goel, Rakhi; Sajedi, Payam; Suntharalingam, Mohan

    2015-01-01

    Molecular imaging techniques are increasingly being used in addition to standard imaging methods such as endoscopic ultrasound (EUS) and computed tomography (CT) for many cancers including those of the esophagus. In this review, we will discuss the utility of the most widely used molecular imaging technique, 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET). 18F-FDG PET has a variety of potential applications ranging from improving staging accuracy at the time of initial diagnosis to assisting in radiation target volume delineation. Furthermore, 18F-FDG PET can be used to evaluate treatment response after completion of neoadjuvant therapy or potentially during neoadjuvant therapy. Finally, we will also discuss other novel molecular imaging techniques that have potential to further improve cancer care. PMID:25642333

  10. Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing

    PubMed Central

    Redin, Claire; Gérard, Bénédicte; Lauer, Julia; Herenger, Yvan; Muller, Jean; Quartier, Angélique; Masurel-Paulet, Alice; Willems, Marjolaine; Lesca, Gaétan; El-Chehadeh, Salima; Le Gras, Stéphanie; Vicaire, Serge; Philipps, Muriel; Dumas, Michaël; Geoffroy, Véronique; Feger, Claire; Haumesser, Nicolas; Alembik, Yves; Barth, Magalie; Bonneau, Dominique; Colin, Estelle; Dollfus, Hélène; Doray, Bérénice; Delrue, Marie-Ange; Drouin-Garraud, Valérie; Flori, Elisabeth; Fradin, Mélanie; Francannet, Christine; Goldenberg, Alice; Lumbroso, Serge; Mathieu-Dramard, Michèle; Martin-Coignard, Dominique; Lacombe, Didier; Morin, Gilles; Polge, Anne; Sukno, Sylvie; Thauvin-Robinet, Christel; Thevenon, Julien; Doco-Fenzy, Martine; Genevieve, David; Sarda, Pierre; Edery, Patrick; Isidor, Bertrand; Jost, Bernard; Olivier-Faivre, Laurence; Mandel, Jean-Louis; Piton, Amélie

    2014-01-01

    Background Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. Methods We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. Results We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients’ clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. Conclusions With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes

  11. Polymerase chain reaction amplifying mycobacterial DNA from aspirates obtained by endoscopic ultrasound allows accurate diagnosis of mycobacterial disease in HIV-positive patients with abdominal lymphadenopathy.

    PubMed

    Nieuwoudt, Martin; Lameris, Roeland; Corcoran, Craig; Rossouw, Theresa M; Slavik, Tomas; Du Plessis, Johannie; Omoshoro-Jones, Jones A O; Stivaktas, Paraskevi; Potgieter, Fritz; Van der Merwe, Schalk W

    2014-09-01

    Abdominal lymphadenopathy in human immunodeficiency virus (HIV) infection remains a diagnostic challenge. We performed a prospective cohort study by recruiting 31 symptomatic HIV + patients with abdominal lymphadenopathy and assessing the diagnostic yield of endoscopic ultrasound fine-needle aspiration (EUS-FNA). Mean age was 38 years; 52% were female; and mean CD4 count and viral load were 124 cells/μL and 4 log, respectively. EUS confirmed additional mediastinal nodes in 26%. The porta hepatis was the most common abdominal site. Aspirates obtained by EUS-FNA were subjected to cytology, culture and polymerase chain reaction (PCR) analysis. Mycobacterial infections were confirmed in 67.7%, and 31% had reactive lymphadenopathy. Cytology and culture had low sensitivity, whereas PCR identified 90% of mycobacterial infections. By combining the appearance of aspirates obtained by EUS-FNA and cytologic specimens, we developed a diagnostic algorithm to indicate when analysis with PCR would be useful. PCR performed on material obtained by EUS-FNA was highly accurate in confirming mycobacterial disease and determining genotypic drug resistance.

  12. Ab initio molecular dynamics of liquid water using embedded-fragment second-order many-body perturbation theory towards its accurate property prediction

    PubMed Central

    Willow, Soohaeng Yoo; Salim, Michael A.; Kim, Kwang S.; Hirata, So

    2015-01-01

    A direct, simultaneous calculation of properties of a liquid using an ab initio electron-correlated theory has long been unthinkable. Here we present structural, dynamical, and response properties of liquid water calculated by ab initio molecular dynamics using the embedded-fragment spin-component-scaled second-order many-body perturbation method with the aug-cc-pVDZ basis set. This level of theory is chosen as it accurately and inexpensively reproduces the water dimer potential energy surface from the coupled-cluster singles, doubles, and noniterative triples with the aug-cc-pVQZ basis set, which is nearly exact. The calculated radial distribution function, self-diffusion coefficient, coordinate number, and dipole moment, as well as the infrared and Raman spectra are in excellent agreement with experimental results. The shapes and widths of the OH stretching bands in the infrared and Raman spectra and their isotropic-anisotropic Raman noncoincidence, which reflect the diverse local hydrogen-bond environment, are also reproduced computationally. The simulation also reveals intriguing dynamic features of the environment, which are difficult to probe experimentally, such as a surprisingly large fluctuation in the coordination number and the detailed mechanism by which the hydrogen donating water molecules move across the first and second shells, thereby causing this fluctuation. PMID:26400690

  13. Ab initio molecular dynamics of liquid water using embedded-fragment second-order many-body perturbation theory towards its accurate property prediction.

    PubMed

    Willow, Soohaeng Yoo; Salim, Michael A; Kim, Kwang S; Hirata, So

    2015-01-01

    A direct, simultaneous calculation of properties of a liquid using an ab initio electron-correlated theory has long been unthinkable. Here we present structural, dynamical, and response properties of liquid water calculated by ab initio molecular dynamics using the embedded-fragment spin-component-scaled second-order many-body perturbation method with the aug-cc-pVDZ basis set. This level of theory is chosen as it accurately and inexpensively reproduces the water dimer potential energy surface from the coupled-cluster singles, doubles, and noniterative triples with the aug-cc-pVQZ basis set, which is nearly exact. The calculated radial distribution function, self-diffusion coefficient, coordinate number, and dipole moment, as well as the infrared and Raman spectra are in excellent agreement with experimental results. The shapes and widths of the OH stretching bands in the infrared and Raman spectra and their isotropic-anisotropic Raman noncoincidence, which reflect the diverse local hydrogen-bond environment, are also reproduced computationally. The simulation also reveals intriguing dynamic features of the environment, which are difficult to probe experimentally, such as a surprisingly large fluctuation in the coordination number and the detailed mechanism by which the hydrogen donating water molecules move across the first and second shells, thereby causing this fluctuation.

  14. Ring polymer molecular dynamics fast computation of rate coefficients on accurate potential energy surfaces in local configuration space: Application to the abstraction of hydrogen from methane

    NASA Astrophysics Data System (ADS)

    Meng, Qingyong; Chen, Jun; Zhang, Dong H.

    2016-04-01

    To fast and accurately compute rate coefficients of the H/D + CH4 → H2/HD + CH3 reactions, we propose a segmented strategy for fitting suitable potential energy surface (PES), on which ring-polymer molecular dynamics (RPMD) simulations are performed. On the basis of recently developed permutation invariant polynomial neural-network approach [J. Li et al., J. Chem. Phys. 142, 204302 (2015)], PESs in local configuration spaces are constructed. In this strategy, global PES is divided into three parts, including asymptotic, intermediate, and interaction parts, along the reaction coordinate. Since less fitting parameters are involved in the local PESs, the computational efficiency for operating the PES routine is largely enhanced by a factor of ˜20, comparing with that for global PES. On interaction part, the RPMD computational time for the transmission coefficient can be further efficiently reduced by cutting off the redundant part of the child trajectories. For H + CH4, good agreements among the present RPMD rates and those from previous simulations as well as experimental results are found. For D + CH4, on the other hand, qualitative agreement between present RPMD and experimental results is predicted.

  15. How Iron-Containing Proteins Control Dioxygen Chemistry: A Detailed Atomic Level Description Via Accurate Quantum Chemical and Mixed Quantum Mechanics/Molecular Mechanics Calculations.

    SciTech Connect

    Friesner, Richard A.; Baik, Mu-Hyun; Gherman, Benjamin F.; Guallar, Victor; Wirstam, Maria E.; Murphy, Robert B.; Lippard, Stephen J.

    2003-03-01

    Over the past several years, rapid advances in computational hardware, quantum chemical methods, and mixed quantum mechanics/molecular mechanics (QM/MM) techniques have made it possible to model accurately the interaction of ligands with metal-containing proteins at an atomic level of detail. In this paper, we describe the application of our computational methodology, based on density functional (DFT) quantum chemical methods, to two diiron-containing proteins that interact with dioxygen: methane monooxygenase (MMO) and hemerythrin (Hr). Although the active sites are structurally related, the biological function differs substantially. MMO is an enzyme found in methanotrophic bacteria and hydroxylates aliphatic C-H bonds, whereas Hr is a carrier protein for dioxygen used by a number of marine invertebrates. Quantitative descriptions of the structures and energetics of key intermediates and transition states involved in the reaction with dioxygen are provided, allowing their mechanisms to be compared and contrasted in detail. An in-depth understanding of how the chemical identity of the first ligand coordination shell, structural features, electrostatic and van der Waals interactions of more distant shells control ligand binding and reactive chemistry is provided, affording a systematic analysis of how iron-containing proteins process dioxygen. Extensive contact with experiment is made in both systems, and a remarkable degree of accuracy and robustness of the calculations is obtained from both a qualitative and quantitative perspective.

  16. A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

    PubMed

    Lee, Sang Wook; Hosokawa, Kazuo; Kim, Soyoun; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas; Maeda, Mizuo

    2015-05-22

    Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

  17. Molecular diagnosis and genotype analysis of Giardia duodenalis in asymptomatic children from a rural area in central Colombia.

    PubMed

    Ramírez, Juan David; Heredia, Rubén Darío; Hernández, Carolina; León, Cielo M; Moncada, Ligia Inés; Reyes, Patricia; Pinilla, Análida Elizabeth; Lopez, Myriam Consuelo

    2015-06-01

    Giardiasis is a parasitic infection that affects around 200 million people worldwide. This parasite presents a remarkable genetic variability observed in 8 genetic clusters named as 'assemblages' (A-H). These assemblages are host restricted and could be zoonotic where A and B infect humans and animals around the globe. The knowledge of the molecular epidemiology of human giardiasis in South-America is scarce and also the usefulness of PCR to detect this pathogen in fecal samples remains controversial. The aim of this study was to conduct a cross-sectional study to compare the molecular targets employed for the molecular diagnosis of Giardia DNA and to discriminate the parasite assemblages circulating in the studied population. We analyzed 181 fecal samples from Children at La Virgen, Cundinamarca, Colombia that were DNA-extracted and analyzed by SSU rDNA, tpi and gdh loci. We observed positivity by microscopy of 13% and by PCR around 76-80% depending on the molecular marker. Additionally, a lack of statistical concordance between microscopy and PCR was detected. Regarding the genetic assemblages, we detected assemblage A (3%), assemblage B (90%) and mixed infections assemblages A+B (7%). Hence, the sub-assemblages were typed as AI, AII, BIII and BIV across the population. This study represents a reliable attempt to understand the molecular epidemiology of giardiasis in Colombia and the use of PCR to detect cryptic infections. The epidemiological implications are herein discussed.

  18. [Molecular diagnosis and phylogenetic analysis of the first MERS case in Turkey].

    PubMed

    Bayrakdar, Fatma; Altaş, Ayşe Başak; Korukluoğlu, Gülay; Topal, Selmur

    2015-07-01

    Coronaviruses (CoV) are enveloped, spherical, single-stranded positive-sense RNA viruses causing mainly respiratory and intestinal infections in animals and humans. Until recently five types of human coronaviruses (HCoV-OC43, HCoV-HKU1, HCoV-NL63, HCoV-229E, SARS-CoV) have been known, however a novel CoV has been identified in 2012 in Saudi Arabia. This virus, namely MERS-CoV (Middle East Respiratory Syndrome Coronavirus), was classified within Coronaviridae family, Coronavirinae sub-family, Betacoronavirus genus, clade C. It causes acute respiratory infections in humans and transmits via respiratory route and close contact between humans. The aim of this study was to present the first MERS case from Turkey identified by molecular methods and the results of viral sequence analysis. A 42-year-old male Turkish citizen who worked as an employee in Jeddah, Kingdom of Saudi Arabia, admitted to hospital with the complaints of fever and malaise on 25-26 September 2014. Since his symptoms went on and got worse, he returned to Turkey, and hospitalized in a hospital's intensive care unit in Hatay on 6th of October with the symptoms of fever, malaise, sweating, cough and respiratory distress. He transferred to a university hospital on 8th of October and died on 11th October. The tracheal aspirate sample obtained before he died was sent to Virology Unit of Reference Laboratories of the Turkish Public Health Institution. Detection of viral RNA was performed by using a commercial real-time PCR kit (hCoV-EMC Real-Time RT-PCR, Fast Track Diagnostics, Luxembourg) targeting the MERS-CoV E protein (upE), ORF1a and ORF1b gene regions. The reference method Superscript III One Step RT-PCR (Invitrogen, USA) recommended by World Health Organization (WHO) was also applied for confirmation. Both of the methods yielded positive results for MERS-CoV RNA. For the amplification of nucleocapsid (N) and RNA-dependent RNA polymerase (RdRp) genes, hemi-nested PCR (Invitrogen, ABD) was conducted

  19. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis

    PubMed Central

    Sun, Dawei; Nakao, Shintaro; Xie, Fang; Zandi, Souska; Bagheri, Abouzar; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Soheili, Zahra-Soheila; Frimmel, Sonja; Zhang, Zhongyu; Ablonczy, Zsolt; Ahmadieh, Hamid; Hafezi-Moghadam, Ali

    2014-01-01

    Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumulation of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR-2) in retinal and choroidal vessels of diabetic animals (P<0.01), compared to normal controls. More than 80% of the VEGFR-2 in the diabetic retina was in the capillaries, compared to 47% in normal controls (P<0.01). Angiography in rabbit retinas revealed microvascular capillaries to be the location for VEGF-A-induced leakage, as expressed by significantly higher rate of fluorophore spreading with VEGF-A injection when compared to vehicle control (26±2 vs. 3±1 μm/s, P<0.05). Immunohistochemistry showed VEGFR-2 expression in capillaries of diabetic animals but not in normal controls. Macular vessels from diabetic patients (n=7) showed significantly more VEGFR-2 compared to nondiabetic controls (n=5) or peripheral retinal regions of the same retinas (P<0.01 in both cases). Here we introduce a new approach for early diagnosis of DR and VEGFR-2 as a molecular marker. VEGFR-2 could become a key diagnostic target, one that might help to prevent retinal vascular leakage and proliferation in diabetic patients.—Sun, D., Nakao, S., Xie, F., Zandi, S., Bagheri, A., Kanavi, M. R., Samiei, S., Soheili, Z.-S., Frimmel, S., Zhang, Z., Ablonczy, Z., Ahmadieh, H., Hafezi-Moghadam, A. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis. PMID:24903276

  20. Translation of recent advances and discoveries in molecular biology and immunology in the diagnosis and treatment of pancreatic cancer.

    PubMed

    Albo, Daniel; Farrow, Buckminster; Berger, David H

    2008-04-01

    Recent advances in understanding the molecular mechanisms of cancer progression have allowed for targeted approaches to the diagnosis and treatment of pancreatic cancer. New biologic markers are emerging that may improve the ability to detect these tumors earlier. Targeted biologic cancer therapies promise more effective and less toxic systemic treatment options. Although a clear "magic bullet" has yet to emerge, this type of targeted approach offers hope in the management of this dreadful disease. This article offers an update on these promising diagnostic and treatment modalities.

  1. Molecular Biomarkers of Pancreatic Intraepithelial Neoplasia and Their Implications in Early Diagnosis and Therapeutic Intervention of Pancreatic Cancer

    PubMed Central

    Guo, Junli; Xie, Keping; Zheng, Shaojiang

    2016-01-01

    Lack of early detection and effective interventions is a major reason for the poor prognosis and dismal survival rates for pancreatic cancer. Pancreatic intraepithelial neoplasia (PanIN) is the most common precursor of invasive pancreatic ductal adenocarcinoma (PDAC). Each stage in the progression from PanIN to PDAC is well characterized by multiple significant genetic alterations affecting signaling pathways. Understanding the biological behavior and molecular alterations in the progression from PanIN to PDAC is crucial to the identification of noninvasive biomarkers for early detection and diagnosis and the development of preventive and therapeutic strategies for control of pancreatic cancer progression. This review focuses on molecular biomarkers of PanIN and their important roles in early detection and treatment of pancreatic cancer. PMID:26929736

  2. Molecular Diagnosis of Chagas Disease in Colombia: Parasitic Loads and Discrete Typing Units in Patients from Acute and Chronic Phases

    PubMed Central

    Hernández, Carolina; Cucunubá, Zulma; Flórez, Carolina; Olivera, Mario; Valencia, Carlos; Zambrano, Pilar; León, Cielo; Ramírez, Juan David

    2016-01-01

    Background The diagnosis of Chagas disease is complex due to the dynamics of parasitemia in the clinical phases of the disease. The molecular tests have been considered promissory because they detect the parasite in all clinical phases. Trypanosoma cruzi presents significant genetic variability and is classified into six Discrete Typing Units TcI-TcVI (DTUs) with the emergence of foreseen genotypes within TcI as TcIDom and TcI Sylvatic. The objective of this study was to determine the operating characteristics of molecular tests (conventional and Real Time PCR) for the detection of T. cruzi DNA, parasitic loads and DTUs in a large cohort of Colombian patients from acute and chronic phases. Methodology/Principal Findings Samples were obtained from 708 patients in all clinical phases. Standard diagnosis (direct and serological tests) and molecular tests (conventional PCR and quantitative PCR) targeting the nuclear satellite DNA region. The genotyping was performed by PCR using the intergenic region of the mini-exon gene, the 24Sa, 18S and A10 regions. The operating capabilities showed that performance of qPCR was higher compared to cPCR. Likewise, the performance of qPCR was significantly higher in acute phase compared with chronic phase. The median parasitic loads detected were 4.69 and 1.33 parasite equivalents/mL for acute and chronic phases. The main DTU identified was TcI (74.2%). TcIDom genotype was significantly more frequent in chronic phase compared to acute phase (82.1% vs 16.6%). The median parasitic load for TcIDom was significantly higher compared with TcI Sylvatic in chronic phase (2.58 vs.0.75 parasite equivalents/ml). Conclusions/Significance The molecular tests are a precise tool to complement the standard diagnosis of Chagas disease, specifically in acute phase showing high discriminative power. However, it is necessary to improve the sensitivity of molecular tests in chronic phase. The frequency and parasitemia of TcIDom genotype in chronic

  3. CML patients in the molecular era – report of five years experience of diagnosis and treatment in a single center

    PubMed Central

    Voican, I; Vladareanu, AM; Bumbea, H; Barsan, L; Vasile, D

    2013-01-01

    Chronic myeloid leukemia (CML) represents about 15% of all leukemia cases. Although the incidence of the disease is rather low, the therapeutic progress of the last decade has dramatically changed the evolution of this disease, whose survival considerably increased and in whom we now speak even about cure. The success of the therapy is strongly connected to the precocity of the diagnosis and molecular targeted therapy that implies a close monitoring of the patient. The specific molecular assay, that developed a lot in the last years, became an important tool in the management of these patients, providing the possibility of efficient changing in therapy. The purpose of our study was to identify the characteristics of our CML patients in terms of clinical and biological behavior. We analyzed 21 patients diagnosed between October 2007 and December 2010 and compared the data with a historical group of patients, also diagnosed in our department between March 2005 and September 2007. We found a better outcome and overall survival in the study group, due to improved diagnosis and monitoring techniques as well as to better access to therapy. Abbreviations accelerated phase (AP), blast phase (BP), Chronic myeloid leukemia (CML), chronic phase (CP), complete cytogenetic response (CCyR), complete hematologic response (CHR), complete molecular response (CMolR), European LeukemiaNet (ELN), Imatinib mesylate (IM) , major molecular response (MMolR), minimal cytogenetic response (minCyR), partial cytogenetic response (PCyR), polymerase-chain-reaction (PCR), Qualitative Polymerase-Chain-Reaction (Q-PCR), Quantitative Real-Time Polymerase-Chain-Reaction (RT-PCR), tyrosine kinase inhibitors (TKI) PMID:24146695

  4. Molecular Diagnosis of Abdominal Armillifer grandis Pentastomiasis in the Democratic Republic of Congo.

    PubMed

    Tappe, Dennis; Sulyok, Mihály; Rózsa, Lajos; Muntau, Birgit; Haeupler, Alexandra; Bodó, Imre; Hardi, Richard

    2015-07-01

    Pentastomiasis is an emerging snake-borne parasitic zoonosis in the tropics. We describe a molecular and morphological study to diagnose a cluster of asymptomatic abdominal human infections caused by Armillifer grandis. The findings may indicate a silent epidemic in a rural area where severe symptomatic ocular cases with the same parasite species have recently surfaced. Molecular diagnostics are of increasing importance when patient material from remote areas cannot be thoroughly examined locally for logistic reasons. PMID:25948609

  5. Comparison of rapid immunodiagnosis assay kit with molecular and immunopathological approaches for diagnosis of rabies in cattle

    PubMed Central

    Ahmad, Ajaz; Singh, C. K.

    2016-01-01

    Aim: Presently, diagnosis of rabies is primarily based on, conventional fluorescent antibody technique (FAT), immunopathological and molecular techniques. Recently, rapid immunodiagnostic assay (RIDA) - A monoclonal antibody-based technique has been introduced for rapid diagnosis of rabies. The present investigation is envisaged to study the efficacy of RIDA kit for the diagnosis of rabies in cattle. Materials and Methods: About 11 brain samples from cattle, clinically suspected for rabies, were screened by the FAT, Heminested reverse transcriptase polymerase chain reaction (HnRT-PCR), Immunohistochemistry (IHC), and RIDA. Results: The sensitivity for detection of rabies from brain tissue by RIDA was 85.7% as compared to 100% by IHC as well as HnRT-PCR. The accuracy of detection of rabies by RIDA was 91.6% as compared to 100% that of IHC and HnRT-PCR, whereas specificity of RIDA was 100% like that of the IHC and HnRT-PCR. Conclusion: Despite a comparatively low-sensitivity and accuracy of RIDA, latter can still be useful in screening a large number of field samples promptly. However, it is recommended that negative results with RIDA in cattle need to be authenticated by suitable alternative diagnostic approaches. PMID:27051193

  6. High volume molecular genetic identification of single nucleotide polymorphisms using Genetic Bit Analysis Application to human genetic diagnosis

    SciTech Connect

    Boyce-Jacino, M.T.; Reynolds, J.; Nikiforov, T.

    1994-09-01

    The most common type of genetic disease-associated mutation is the single nucleotide polymorphism (SNP). Because most genetic diseases can be caused by multiple SNPs in the same gene, effective routine diagnosis of complex genetic diseases is dependent on a simple and reliable method of interrogating SNP sites. Molecular Tool`s solid phase assay capable of direct genotyping (single base sequencing) of SNP sites, Genetic Bit Analysis (GBA), involves hybridization-capture of a single-stranded PCR product to a sequence-specific, microtiter plate-bound oligonucleotide primer. The captured PCR product then acts as template for single-base extension of the capture primer across the polymorphic site, enabling direct determination of the base composition of the polymorphism through a simple colormetric assay. Genotyping in a high volume, semi-automated, processing system with a current capacity of 100 SNP interrogations per technician per day enables the screening of candidate mutations rapidly and cost-effectively, critically important to comprehensive genetic diagnosis. Using this gel-free technology, we have developed prototype diagnostic tests for CFTR and ApoE polymorphisms which enable direct sequencing of the polymorphic base at each site of interest. Routine clinical diagnosis of genetically complex diseases such as cystic fibrosis is dependent on this combination of robust biochemistry and simple format. Additionally, the ability to transfer the format and biochemistry to any disease gene of interest enables the broad application of this technology to clinical diagnostics, especially for genetically complex diseases.

  7. Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia.

    PubMed

    Chandrasekharappa, Settara C; Lach, Francis P; Kimble, Danielle C; Kamat, Aparna; Teer, Jamie K; Donovan, Frank X; Flynn, Elizabeth; Sen, Shurjo K; Thongthip, Supawat; Sanborn, Erica; Smogorzewska, Agata; Auerbach, Arleen D; Ostrander, Elaine A

    2013-05-30

    Current methods for detecting mutations in Fanconi anemia (FA)-suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA.

  8. Massively parallel sequencing, aCGH, and RNA-Seq technologies provide a comprehensive molecular diagnosis of Fanconi anemia

    PubMed Central

    Lach, Francis P.; Kimble, Danielle C.; Kamat, Aparna; Teer, Jamie K.; Donovan, Frank X.; Flynn, Elizabeth; Sen, Shurjo K.; Thongthip, Supawat; Sanborn, Erica; Smogorzewska, Agata; Ostrander, Elaine A.

    2013-01-01

    Current methods for detecting mutations in Fanconi anemia (FA)–suspected patients are inefficient and often miss mutations. We have applied recent advances in DNA sequencing and genomic capture to the diagnosis of FA. Specifically, we used custom molecular inversion probes or TruSeq-enrichment oligos to capture and sequence FA and related genes, including introns, from 27 samples from the International Fanconi Anemia Registry at The Rockefeller University. DNA sequencing was complemented with custom array comparative genomic hybridization (aCGH) and RNA sequencing (RNA-seq) analysis. aCGH identified deletions/duplications in 4 different FA genes. RNA-seq analysis revealed lack of allele specific expression associated with a deletion and splicing defects caused by missense, synonymous, and deep-in-intron variants. The combination of TruSeq-targeted capture, aCGH, and RNA-seq enabled us to identify the complementation group and biallelic germline mutations in all 27 families: FANCA (7), FANCB (3), FANCC (3), FANCD1 (1), FANCD2 (3), FANCF (2), FANCG (2), FANCI (1), FANCJ (2), and FANCL (3). FANCC mutations are often the cause of FA in patients of Ashkenazi Jewish (AJ) ancestry, and we identified 2 novel FANCC mutations in 2 patients of AJ ancestry. We describe here a strategy for efficient molecular diagnosis of FA. PMID:23613520

  9. Molecular analysis of the Duchenne muscular dystrophy gene in Spanish individuals: Deletion detection and familial diagnosis

    SciTech Connect

    Patino, A.; Garcia-Delgado, M.; Narbona, J.

    1995-11-06

    Deletion studies were performed in 26 Duchenne muscular dystrophy (DMD) patients through amplification of nine different exons by multiplex polymerase chain reaction (PCR). DNA from paraffin-embedded muscle biopsies was analyzed in 12 of the 26 patients studied. Optimization of this technique is of great utility because it enables analysis of material stored in pathology archives. PCR deletion detection, useful in DMD-affected boys, is problematic in determining the carrier state in female relatives. For this reason, to perform familial linkage diagnosis, we made use of a dinucleotide repeat polymorphism (STRP, or short tandem repeat polymorphism) located in intron 49 of the gene. We designed a new pair of primers that enabled the detection of 22 different alleles in relatives in the 14 DMD families studied. The use of this marker allowed familial diagnosis in 11 of the 14 DMD families and detection of de novo deletions in 3 of the probands. 8 refs., 5 figs., 2 tabs.

  10. Molecular diagnosis of subcutaneous Pythium insidiosum infection by use of PCR screening and DNA sequencing.

    PubMed

    Salipante, Stephen J; Hoogestraat, Daniel R; SenGupta, Dhruba J; Murphey, Donald; Panayides, Kyriacos; Hamilton, Emma; Castañeda-Sánchez, Irene; Kennedy, Jason; Monsaas, Peter W; Mendoza, Leonel; Stephens, Karen; Dunn, James J; Cookson, Brad T

    2012-04-01

    Pythium insidiosum is an emerging human pathogen classified among brown algae and diatoms that can cause significant morbidity and mortality in otherwise healthy individuals. Here we describe a pediatric patient with pythiosis acquired in the southern United States, diagnosed by molecular screening and DNA sequencing of internal transcribed spacer region 1. PMID:22205808

  11. Molecular Diagnosis of Actinomadura madurae Infection by 16S rRNA Deep Sequencing

    PubMed Central

    SenGupta, Dhruba J.; Hoogestraat, Daniel R.; Cummings, Lisa A.; Bryant, Bronwyn H.; Natividad, Catherine; Thielges, Stephanie; Monsaas, Peter W.; Chau, Mimosa; Barbee, Lindley A.; Rosenthal, Christopher; Cookson, Brad T.; Hoffman, Noah G.

    2013-01-01

    Next-generation DNA sequencing can be used to catalog individual organisms within complex, polymicrobial specimens. Here, we utilized deep sequencing of 16S rRNA to implicate Actinomadura madurae as the cause of mycetoma in a diabetic patient when culture and conventional molecular methods were overwhelmed by overgrowth of other organisms. PMID:24108607

  12. Molecular Diagnosis of Subcutaneous Pythium insidiosum Infection by Use of PCR Screening and DNA Sequencing

    PubMed Central

    Hoogestraat, Daniel R.; SenGupta, Dhruba J.; Murphey, Donald; Panayides, Kyriacos; Hamilton, Emma; Castañeda-Sánchez, Irene; Kennedy, Jason; Monsaas, Peter W.; Mendoza, Leonel; Stephens, Karen; Dunn, James J.; Cookson, Brad T.

    2012-01-01

    Pythium insidiosum is an emerging human pathogen classified among brown algae and diatoms that can cause significant morbidity and mortality in otherwise healthy individuals. Here we describe a pediatric patient with pythiosis acquired in the southern United States, diagnosed by molecular screening and DNA sequencing of internal transcribed spacer region 1. PMID:22205808

  13. Molecular diagnosis of animal diseases: some experiences over the past decade.

    PubMed

    Belák, S; Thorén, P

    2001-11-01

    The experiences of a veterinary research and diagnostic laboratory are summarized on the development and application of the PCR to diagnose a wide range of viral diseases in animals. The group started the routine diagnostic application of the PCR as early as 1988 and today a total of 35 nested PCR assays are in routine use for the detection of 15 DNA and 20 RNA viruses. Special tools and laboratory practice were applied to avoid false-positive results, while false-negatives are avoided by internal controls (mimics). At present, the classical nested PCR methods are being replaced by real-time TaqMan and molecular beacon assays and the multiplex real-time PCR detection of viruses is also under development. By direct sequencing of the PCR products, phylogeny studies are performed and molecular epizootiology results are provided for rapid and exact identification of virus variants. Molecular epizootiology also contributes to trace the routes of virus spreading on large geographic areas. Recently, large efforts have been made to follow the recommendations of Office International des Epizooties for the standardization and international harmonization of the molecular diagnostic assays.

  14. Molecular diagnosis of toxoplasmosis: value of the buffy coat for the detection of circulating Toxoplasma gondii.

    PubMed

    Brenier-Pinchart, Marie-Pierre; Capderou, Elodie; Bertini, Rose-Laurence; Bailly, Sébastien; Fricker-Hidalgo, Hélène; Varlet-Marie, Emmanuelle; Murat, Jean-Benjamin; Sterkers, Yvon; Touafek, Fériel; Bastien, Patrick; Pelloux, Hervé

    2015-08-01

    Early detection of Toxoplasma tachyzoites circulating in blood using PCR is recommended for immunosuppressed patients at high risk for disseminated toxoplasmosis. Using a toxoplasmosis mouse model, we show that the sensitivity of detection is higher using buffy coat isolated from a large blood volume than using whole blood for this molecular monitoring.

  15. Molecular and serological methods for the diagnosis of viruses in potato tubers.

    PubMed

    Lacomme, Christophe; Holmes, Ross; Evans, Fiona

    2015-01-01

    Viruses cause important diseases to potato crops. Monitoring virus content in plant material for quarantine or seed certification scheme purposes is essential to prevent the spread of viruses and to minimize the impact of viral diseases. There are currently two main methods for virus diagnosis in potato tubers: growing-on ELISA testing which requires breaking tuber dormancy followed by an ELISA test on grown plantlets and direct real-time RT-PCR testing on tubers. This chapter will describe both methods that can be adapted for large-scale virus testing activities.

  16. Molecular Diagnosis of Subcutaneous Spirometra erinaceieuropaei Sparganosis in a Japanese Immigrant

    PubMed Central

    Tappe, Dennis; Berger, Luise; Haeupler, Alexandra; Muntau, Birgit; Racz, Paul; Harder, Yves; Specht, Katja; Prazeres da Costa, Clarissa; Poppert, Sven

    2013-01-01

    We report a case of subcutaneous sparganosis in a 68-year-old female Japanese immigrant in Germany. The patient complained of a painless erythema caudal of the umbilicus with a palpable subcutaneous cherry-sized lump. Polymerase chain reaction on formalin-fixed parasite tissue identified Spirometra erinaceieuropaei as the causative agent; the proliferative form of sparganosis, which is caused by the branching and disseminating Sparganum proliferum, could, thus, be excluded. From the excised sparganum, an immunofluorescence test was established and revealed an antibody response directed against the parasite's tegument. Histological key features of the plerocercoid that facilitate diagnosis with different stains are presented. PMID:23166198

  17. Clinical spectrum and molecular diagnosis of Angelman and Prader-Willi syndrome patients with an imprinting mutation

    SciTech Connect

    Saitoh, S.; Cassidy, S.B.; Conroy, J.M.

    1997-01-20

    Recent studies have identified a new class of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients who have biparental inheritance, but neither the typical deletion nor uniparental disomy (UPD) or translocation. However, these patients have uniparental DNA methylation throughout 15q11-q13, and thus appear to have a mutation in the imprinting process for this region. Here we describe detailed clinical findings of five AS imprinting mutation patients (three families) and two PWS imprinting mutation patients (one new family). All these patients have essentially the classical clinical phenotype for the respective syndrome, except that the incidence of microcephaly is lower in imprinting mutation AS patients than in deletion AS patients. Furthermore, imprinting mutation AS and PWS patients do not typically have hypopigmentation, which is commonly found in patients with the usual large deletion. Molecular diagnosis of these cases is initially achieved by DNA methylation analyses of the DN34/ZNF127, PW71 (D15S63), and SNRPN loci. The latter two probes have clear advantages in the simple molecular diagnostic analysis of PWS and AS patients with an imprinting mutation, as has been found for typical deletion or UPD PWS and AS cases. With the recent finding of inherited microdeletions in PWS and AS imprinting mutation families, our studies define a new class of these two syndromes. The clinical and molecular identification of these PWS and AS patients has important genetic counseling consequences. 49 refs., 4 figs., 3 tabs.

  18. Molecular-based isothermal tests for field diagnosis of malaria and their potential contribution to malaria elimination.

    PubMed

    Oriero, Eniyou C; Jacobs, Jan; Van Geertruyden, Jean-Pierre; Nwakanma, Davis; D'Alessandro, Umberto

    2015-01-01

    In countries where malaria transmission has decreased substantially, thanks to the scale-up of control interventions, malaria elimination may be feasible. Nevertheless, this goal requires new strategies such as the active detection and treatment of infected individuals. As the detection threshold for the currently used diagnostic methods is 100 parasites/μL, most low-density, asymptomatic infections able to maintain transmission cannot be detected. Identifying them by molecular methods such as PCR is a possible option but the field deployment of these tests is problematic. Isothermal amplification of nucleic acids (at a constant temperature) offers the opportunity of addressing some of the challenges related to the field deployment of molecular diagnostic methods. One of the novel isothermal amplification methods for which a substantial amount of work has been done is the loop-mediated isothermal amplification (LAMP) assay. The present review describes LAMP and several other isothermal nucleic acid amplification methods, such as thermophilic helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification and nucleic acid sequence-based amplification, and explores their potential use as high-throughput, field-based molecular tests for malaria diagnosis.

  19. Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

    PubMed

    Giugliani, Roberto; Brusius-Facchin, Ana-Carolina; Pasqualim, Gabriela; Leistner-Segal, Sandra; Riegel, Mariluce; Matte, Ursula

    2016-01-01

    Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)]. PMID:26567866

  20. The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation.

    PubMed

    Dittrich, Sabine; Rudgard, William E; Woods, Kate L; Silisouk, Joy; Phuklia, Weerawat; Davong, Viengmon; Vongsouvath, Manivanh; Phommasone, Koukeo; Rattanavong, Sayaphet; Knappik, Michael; Craig, Scott B; Weier, Steven L; Tulsiani, Suhella M; Dance, David A B; Newton, Paul N

    2016-04-01

    Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used. PMID:26880775

  1. Telomerase in Acute Myeloid Leukemia: A Molecular Update on Diagnosis, Prognosis, and Treatment.

    PubMed

    Fonseka, Lakshan N; Tirado, Carlos A

    2016-01-01

    It is expected that 10,460 patients will die from acute myeloid leukemia (AML) in the United States in 2016. Despite progress in clinical management, AML patients still have a 25.9% survival rate in the U.S. Researchers have sought to further understand this hematological malignancy and a number of studies have focused on unveiling the role of telomerase in disease initiation, progression, and maintenance. Though the role of telomerase in diagnosis has remained relatively static, its role in prognosis and treatment has become much clearer. While variants in TERT and TERC have been associated with worse clinical outcomes, telomerase and survivin co-expression can predict improved clinical outcomes. In regards to treatment, novel therapies such as mesoindigo and sodium metaarsenite provide new insights in clinical management. The use of leukemic stem cells in mouse models has shown promising results as well. Herein, we provide an update on the role of telomerase in AML through a survey of recent literature, focusing on the diagnosis, prognosis, and treatment of AML. PMID:27606805

  2. Clinical, radiological and molecular diagnosis correlation in serum samples from patients with osteoarticular tuberculosis

    PubMed Central

    García-Elorriaga, Guadalupe; Martínez-Elizondo, Olga; del Rey-Pineda, Guillermo; González-Bonilla, César

    2014-01-01

    Objective To assess the role of polymerase chain reaction (PCR) in serum samples, in the diagnosis of osteoarticular tuberculosis (OTB) in a setting where only clinical and imaging diagnoses determine the treatment. Methods A total of 44 consecutive serum specimens were collected from clinically suspected OTB patients, based on clinical and radiological [X-ray or magnetic resonance imaging/computed tomography] features. They were screened by in-house nested PCR. In addition, a few specimens were examined by Gram stain, acid-fast bacilli stain, histopathology and routine bacterial culture. A total of 39 specimens were collected from patients suffering from other bone diseases of nontuberculous origin and included as negative controls. Results Of the 44 clinically suspected OTB patients, in-house nested PCR was positive in 40 (91%) cases; PCR was negative in 38 (97%) negative controls. Sensitivity and specificity of our in-house nested PCR was 90.9% and 97.4%, respectively. The PCR report was available within 48 h. It was possible to standardize serum PCR technique and in positive cases, a good correlation was observed in terms of an adequate treatment response. Conclusions Nested PCR in serum samples is a rapid, highly sensitive and specific modality for OTB detection. PCR should be performed in addition to clinical evaluation, imaging studies, acid-fast bacilli staining, culture and histopathology diagnosis, if possible. PMID:25183281

  3. Toward Best Practice in Using Molecular Diagnosis to Guide Medical Management, Are We There Yet?

    PubMed Central

    Tsai, Anne Chun-Hui; Liu, Xuezhong

    2015-01-01

    Molecular genetics testing has made several huge breakthroughs in the past two decades and many molecular technologies have been applied to our daily medical progress. However, the clinical utility has not reach a consensus by the medical and genetic peers as well as third party payers. The predictive value and clinical applications are variable from one condition to the other. Numerous questions remain including technology deficits, data interpretation and unpredicted phenotypes in complex disorders. In this commentary, the authors reviewed the historical perspective of genetic testing and summarized the current technical deficit, clinical dilemma and suggested a few critical threshold to overcome before the implementation of useful genetic information in standard health care can become a reality. PMID:26191339

  4. Making the most of pathological specimens: molecular diagnosis in formalin-fixed, paraffin embedded tissue.

    PubMed

    Fairley, Jennifer A; Gilmour, Katelyn; Walsh, Kathy

    2012-11-01

    The development of commercial reagents designed specifically for use with formalin-fixed paraffin-embedded (FFPE) tissue has unlocked the diagnostic potential of this prolific resource. The availability of archival FFPE tissue and tissue from current patients make it an ideal resource for molecular testing. Despite its stability and ability to preserve morphological information, FFPE provides a number of technical challenges to the study of biomolecules. In particular, the cross-linking and processing present problems in the extraction and isolation of DNA, RNA and protein and affect their use in downstream analysis. Here we will discuss some of the problems of FFPE tissue, how they can be overcome and how FFPE material can be used within clinical molecular diagnostics.

  5. 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.

  6. Molecular diagnosis of resistance to antimalarial drugs during epidemics and in war zones.

    PubMed

    Djimdé, Abdoulaye A; Dolo, Amagana; Ouattara, Amed; Diakité, Sira; Plowe, Christopher V; Doumbo, Ogobara K

    2004-08-15

    Plasmodium falciparum mutations pfcrt K76T and the dhfr/dhps "quintuple mutant" are molecular markers of resistance to chloroquine and sulfadoxine-pyrimethamine, respectively. During an epidemic of P. falciparum malaria in an area of political unrest in northern Mali, where standard efficacy studies have been impossible, we measured the prevalence of these markers in a cross-sectional survey. In 80% of cases of infection, pfcrt K76T was detected, but none of the cases carried the dhfr/dhps quintuple mutant. On the basis of these results, chloroquine was replaced by sulfadoxine-pyrimethamine in control efforts. This example illustrates how molecular markers for drug resistance can provide timely data that inform malaria-control policy during epidemics and other emergency situations.

  7. 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. PMID:26891612

  8. [Usefulness of the molecular profile in the diagnosis, prognosis and treatment of hepatocellular carcinoma].

    PubMed

    Bruix, Jordi

    2014-07-01

    Hepatocellular carcinoma is one of the most significant causes of death from cancer and is currently the main cause of death in patients with hepatic cirrhosis. Various scientific societies have compiled evidence-based clinical practice guidelines. Homogeneous criteria are therefore available for establishing and predicting the prognosis and directing the most favorable treatment option for each evolutionary stage. In all of these aspects, patient assessment is based on conventional instruments: clinical assessment, laboratory tests and examinations using imaging techniques. For now, research into molecular profiles has not resulted in this information impacting the clinical decision. Numerous investigations have provided relevant information on the possible classification of tumors based on genetic abnormalities and on the possible identification of molecular targets. In the future, this type of data will be incorporated into conventional practice in a similar manner as has occurred with breast, lung, colorectal and melanoma. PMID:25087717

  9. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    PubMed

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management. PMID:27318568

  10. A molecular-beacon-based asymmetric PCR assay for easy visualization of amplicons in the diagnosis of trichomoniasis.

    PubMed

    Sonkar, Subash C; Sachdev, Divya; Mishra, Prashant K; Kumar, Anita; Mittal, Pratima; Saluja, Daman

    2016-12-15

    The currently available nucleic acid amplification tests (NAATs) for trichomoniasis are accurate, quick and confirmative with superior sensitivity than traditional culture-based microbiology assays. However, these assays are associated with problems of carry over contamination, false positive results, requirement of technical expertise for performance and detection of end product. Hence, a diagnostic assay with easy visualization of the amplified product will be profitable. An in-house, rapid, sensitive, specific molecular-beacon-based PCR assay, using primers against pfoB gene of Trichomonas vaginalis, was developed and evaluated using dry ectocervical swabs (n=392) from symptomatic females with vaginal discharge. Total DNA was isolated and used as template for the PCR assays. The performance and reproducibility of PCR assay was evaluated by composite reference standard (CRS). For easy visualization of the amplified product, molecular-beacon was designed and amplicons were visualized directly using fluorescent handheld dark reader or by Micro-Plate Reader. Molecular-beacons are single-stranded hairpin shaped nucleic acid probes composed of a stem, with fluorophore/quencher pair and a loop region complementary to the desired DNA. The beacon-based PCR assay designed in the present study is highly specific as confirmed by competition experiments and extremely sensitive with detection limit of 20fg of genomic DNA (3-4 pathogens). The minimum infrastructure requirement and ease to perform the assay makes this method highly useful for resource poor countries for better disease management.

  11. Molecular features assisting in diagnosis, surgery, and treatment decision making in low-grade gliomas.

    PubMed

    Chen, Ricky; Ravindra, Vijay M; Cohen, Adam L; Jensen, Randy L; Salzman, Karen L; Prescot, Andrew P; Colman, Howard

    2015-03-01

    The preferred management of suspected low-grade gliomas (LGGs) has been disputed, and the implications of molecular changes for medical and surgical management of LGGs are important to consider. Current strategies that make use of molecular markers and imaging techniques and therapeutic considerations offer additional options for management of LGGs. Mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes suggest a role for this abnormal metabolic pathway in the pathogenesis and progression of these primary brain tumors. Use of magnetic resonance spectroscopy can provide preoperative detection of IDH-mutated gliomas and affect surgical planning. In addition, IDH1 and IDH2 mutation status may have an effect on surgical resectability of gliomas. The IDH-mutated tumors exhibit better prognosis throughout every grade of glioma, and mutation may be an early genetic event, preceding lineage-specific secondary and tertiary alterations that transform LGGs into secondary glioblastomas. The O6-methylguanine-DNAmethyltransferase (MGMT) promoter methylation and 1p19q codeletion status can predict sensitivity to chemotherapy and radiation in low- and intermediate-grade gliomas. Thus, these recent advances, which have led to a better understanding of how molecular, genetic, and epigenetic alterations influence the pathogenicity of the different histological grades of gliomas, can lead to better prognostication and may lead to specific targeted surgical interventions and medical therapies. PMID:25727224

  12. A novel asymmetric-loop molecular beacon-based two-phase hybridization assay for accurate and high-throughput detection of multiple drug resistance-conferring point mutations in Mycobacterium tuberculosis.

    PubMed

    Chen, Qinghai; Wu, Nan; Xie, Meng; Zhang, Bo; Chen, Ming; Li, Jianjun; Zhuo, Lisha; Kuang, Hong; Fu, Weiling

    2012-04-01

    The accurate and high-throughput detection of drug resistance-related multiple point mutations remains a challenge. Although the combination of molecular beacons with bio-immobilization technology, such as microarray, is promising, its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface. Here, we propose a novel asymmetric-loop molecular beacon in which the loop consists of 2 parts. One is complementary to a target, while the other is complementary to an oligonucleotide probe immobilized on the chip surface. With this novel probe, a two-phase hybridization assay can be used for simultaneously detecting multiple point mutations. This assay will have advantages, such as easy probe availability, multiplex detection, low background, and high-efficiency hybridization, and may provide a new avenue for the immobilization of molecular beacons and high-throughput detection of point mutations.

  13. Molecular diagnosis of Baylisascaris schroederi infections in giant panda (Ailuropoda melanoleuca) feces using PCR.

    PubMed

    Zhou, Xuan; Yu, Hua; Wang, Ning; Xie, Yue; Liang, Yi-nan; Li, De-sheng; Wang, Cheng-dong; Chen, Si-jie; Yan, Yu-bo; Gu, Xiao-bin; Wang, Shu-xian; Peng, Xue-rong; Yang, Guang-you

    2013-10-01

    The helminth Baylisascaris schroederi is one of the most harmful parasites infecting giant pandas (Ailuropoda melanoleuca). It is therefore important to develop an exact diagnostic technique to detect this parasite. Using a known number (1, 2, 3, 4, 5, 10, 25, 50, 100) of feces-isolated B. schroederi egg and adult DNA, we developed a PCR to detect a portion of the mitochondrial 12S rRNA and applied it to giant panda fecal samples. The method was sufficiently sensitive to detect B. schroederi DNA from isolated eggs in a fecal sample with a detection threshold of one egg. We detected B. schroederi in 88% of fecal samples, 30% higher than the conventional flotation technique. No cross-reactivity with other common nematode DNA was detected. Our PCR assay may constitute a valuable alternative for the diagnosis of B. schroederi infections.

  14. Molecular diagnosis of Baylisascaris schroederi infections in giant panda (Ailuropoda melanoleuca) feces using PCR.

    PubMed

    Zhou, Xuan; Yu, Hua; Wang, Ning; Xie, Yue; Liang, Yi-nan; Li, De-sheng; Wang, Cheng-dong; Chen, Si-jie; Yan, Yu-bo; Gu, Xiao-bin; Wang, Shu-xian; Peng, Xue-rong; Yang, Guang-you

    2013-10-01

    The helminth Baylisascaris schroederi is one of the most harmful parasites infecting giant pandas (Ailuropoda melanoleuca). It is therefore important to develop an exact diagnostic technique to detect this parasite. Using a known number (1, 2, 3, 4, 5, 10, 25, 50, 100) of feces-isolated B. schroederi egg and adult DNA, we developed a PCR to detect a portion of the mitochondrial 12S rRNA and applied it to giant panda fecal samples. The method was sufficiently sensitive to detect B. schroederi DNA from isolated eggs in a fecal sample with a detection threshold of one egg. We detected B. schroederi in 88% of fecal samples, 30% higher than the conventional flotation technique. No cross-reactivity with other common nematode DNA was detected. Our PCR assay may constitute a valuable alternative for the diagnosis of B. schroederi infections. PMID:24502740

  15. Differential diagnosis of breast cancer using quantitative, label-free and molecular vibrational imaging.

    PubMed

    Yang, Yaliang; Li, Fuhai; Gao, Liang; Wang, Zhiyong; Thrall, Michael J; Shen, Steven S; Wong, Kelvin K; Wong, Stephen T C

    2011-08-01

    We present a label-free, chemically-selective, quantitative imaging strategy to identify breast cancer and differentiate its subtypes using coherent anti-Stokes Raman scattering (CARS) microscopy. Human normal breast tissue, benign proliferative, as well as in situ and invasive carcinomas, were imaged ex vivo. Simply by visualizing cellular and tissue features appearing on CARS images, cancerous lesions can be readily separated from normal tissue and benign proliferative lesion. To further distinguish cancer subtypes, quantitative disease-related features, describing the geometry and distribution of cancer cell nuclei, were extracted and applied to a computerized classification system. The results show that in situ carcinoma was successfully distinguished from invasive carcinoma, while invasive ductal carcinoma (IDC) and invasive lobular carcinoma were also distinguished from each other. Furthermore, 80% of intermediate-grade IDC and 85% of high-grade IDC were correctly distinguished from each other. The proposed quantitative CARS imaging method has the potential to enable rapid diagnosis of breast cancer.

  16. Prenatal Diagnosis of Congenital Lipoid Adrenal Hyperplasia (CLAH) by Molecular Genetic Testing in Korean Siblings

    PubMed Central

    Ko, Hyun Sun; Lee, Seungok; Chae, Hyojin; Choi, Sae Kyung; Kim, Myungshin; Park, In Yang; Suh, Byung Kyu

    2011-01-01

    Congenital lipoid adrenal hyperplasia (CLAH) is caused by mutations to the steroidogenic acute regulatory protein (StAR) gene associated with the inability to synthesize all adrenal and gonadal steroids. Inadequate treatment in an infant with this condition may result in sudden death from an adrenal crisis. We report a case in which CLAH developed in Korean siblings; the second child was prenatally diagnosed because the first child was affected and low maternal serum estriol was detected in a prenatal screening test. To our knowledge, this is the first prenatal diagnosis of the Q258X StAR mutation, which is the only consistent genetic cluster identified to date in Japanese and Korean populations. PMID:22028173

  17. Novel Interpretation of Molecular Diagnosis of Congenital Toxoplasmosis According to Gestational Age at the Time of Maternal Infection

    PubMed Central

    Sterkers, Yvon; Pratlong, Francine; Albaba, Sahar; Loubersac, Julie; Picot, Marie-Christine; Pretet, Vanessa; Issert, Eric; Boulot, Pierre

    2012-01-01

    From a prospective cohort of 344 women who seroconverted for toxoplasmosis during pregnancy, 344 amniotic fluid, 264 placenta, and 216 cord blood samples were tested for diagnosis of congenital toxoplasmosis using the same PCR assay. The sensitivity and negative predictive value of the PCR assay using amniotic fluid were 86.3% and 97.2%, respectively, and both specificity and positive predictive value were 100%. Using placenta and cord blood, sensitivities were 79.5% and 21.2%, and specificities were 92% and 100%, respectively. In addition, the calculation of pretest and posttest probabilities and the use of logistic regression allowed us to obtain curves that give a dynamic interpretation of the risk of congenital toxoplasmosis according to gestational age at maternal infection, as represented by the three sample types (amniotic fluid, placenta, and cord blood). Two examples are cited here: for a maternal infection at 25 weeks of amenorrhea, a negative result of prenatal diagnosis allowed estimation of the probability of congenital toxoplasmosis at 5% instead of an a priori (pretest) risk estimate of 33%. For an infection at 10 weeks of amenorrhea associated with a pretest congenital toxoplasmosis risk of 7%, a positive PCR result using placenta at birth yields a risk increase to 43%, while a negative result damps down the risk to 0.02%. Thus, with a molecular diagnosis performing at a high level, and in spite of the persistence of false negatives, posttest risk curves using both negative and positive results prove highly informative, allowing a better assessment of the actual risk of congenital toxoplasmosis and finally an improved decision guide to treatment. PMID:23035201

  18. Early molecular diagnosis of acute Chagas disease after transplantation with organs from Trypanosoma cruzi-infected donors.

    PubMed

    Cura, C I; Lattes, R; Nagel, C; Gimenez, M J; Blanes, M; Calabuig, E; Iranzo, A; Barcan, L A; Anders, M; Schijman, A G

    2013-12-01

    Organ transplantation (TX) is a novel transmission modality of Chagas disease. The results of molecular diagnosis and characterization of Trypanosoma cruzi acute infection in naïve TX recipients transplanted with organs from infected deceased donors are reported. Peripheral blood and cerebrospinal fluid samples from the TX recipients of organs from infected donors were prospectively and sequentially studied for detection of T. cruzi by means of kinetoplastid DNA polymerase chain reaction (kDNA-PCR). In positive blood samples, a PCR algorithm for identification of T. cruzi Discrete Typing Units (DTUs) and quantitative real-time PCR (qPCR) to quantify parasitic loads were performed. Minicircle signatures of T. cruzi infecting populations were also analyzed using restriction fragment length polymorphism (RFLP)-PCR. Eight seronegative TX recipients from four infected donors were studied. In five, the infection was detected at 68.4 days post-TX (36-98 days). In one case, it was transmitted to two of three TX recipients. The comparison of the minicircle signatures revealed nearly identical RFLP-PCR profiles, confirming a common source of infection. The five cases were infected by DTU TcV. This report reveals the relevance of systematic monitoring of TX recipients using PCR strategies in order to provide an early diagnosis allowing timely anti-trypanosomal treatment.

  19. Carrier detection and prenatal molecular diagnosis in a Duchenne muscular dystrophy family without any affected relative available.

    PubMed

    Alcántara, M A; García-Cavazos, R; Hernández-U, E; González-del Angel, A; Carnevale, A; Orozco, L

    2001-01-01

    In this paper we report a family where the affected DMD patients were not available for study and a molecular strategy was used for female carriers detection and for prenatal diagnosis. Linkage analysis was performed with two markers within the DMD gene, in all family members screened. DMD markers used (pERT87.8/Taq1 and pERT87.15/Xmn1) seemed not to be informative because the propositas mother (II-2) was homozygous for the minor allele at each marker (T2 and X2), however, the proposita and one sister carried only the major allele, which was inherited from the father. These results suggested that a deletion involving both markers could be present, and was inherited from the mother to both daughters. Quantitative multiplex PCR confirmed the deletion in female carriers, involving at least exons 12 to 17. DNA studies of cultured amniotic fluid cells at 14 weeks gestation, by amplification of specific Y-chromosome sequences, followed by multiplex PCR, lead to the diagnosis of a male fetus affected by DMD.

  20. Genetics of Tinnitus: An Emerging Area for Molecular Diagnosis and Drug Development

    PubMed Central

    Lopez-Escamez, Jose A.; Bibas, Thanos; Cima, Rilana F. F.; Van de Heyning, Paul; Knipper, Marlies; Mazurek, Birgit; Szczepek, Agnieszka J.; Cederroth, Christopher R.

    2016-01-01

    Subjective tinnitus is the perception of sound in the absence of external or bodily-generated sounds. Chronic tinnitus is a highly prevalent condition affecting over 70 million people in Europe. A wide variety of comorbidities, including hearing loss, psychiatric disorders, neurodegenerative disorders, and temporomandibular joint (TMJ) dysfunction, have been suggested to contribute to the onset or progression of tinnitus; however, the precise molecular mechanisms of tinnitus are not well understood and the contribution of genetic and epigenetic factors remains unknown. Human genetic studies could enable the identification of novel molecular therapeutic targets, possibly leading to the development of novel pharmaceutical therapeutics. In this article, we briefly discuss the available evidence for a role of genetics in tinnitus and consider potential hurdles in designing genetic studies for tinnitus. Since multiple diseases have tinnitus as a symptom and the supporting genetic evidence is sparse, we propose various strategies to investigate the genetic underpinnings of tinnitus, first by showing evidence of heritability using concordance studies in twins, and second by improving patient selection according to phenotype and/or etiology in order to control potential biases and optimize genetic data output. The increased knowledge resulting from this endeavor could ultimately improve the drug development process and lead to the preventive or curative treatment of tinnitus. PMID:27594824

  1. Serological, Bacteriological, and Molecular Diagnosis of Brucellosis in Domestic Animals in Croatia

    PubMed Central

    Špičić, Silvio; Zdelar-Tuk, Maja; Račić, Ivana; Duvnjak, Sanja; Cvetnić, Željko

    2010-01-01

    Aim To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular methods for diagnostics of brucellosis in domestic animals. Methods We serologically tested 42 785 cattle serums, 22 686 sheep and goat serums, and 28 520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10 173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples. Results B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 herds in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties. Conclusions In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease. PMID:20718085

  2. Genetics of Tinnitus: An Emerging Area for Molecular Diagnosis and Drug Development

    PubMed Central

    Lopez-Escamez, Jose A.; Bibas, Thanos; Cima, Rilana F. F.; Van de Heyning, Paul; Knipper, Marlies; Mazurek, Birgit; Szczepek, Agnieszka J.; Cederroth, Christopher R.

    2016-01-01

    Subjective tinnitus is the perception of sound in the absence of external or bodily-generated sounds. Chronic tinnitus is a highly prevalent condition affecting over 70 million people in Europe. A wide variety of comorbidities, including hearing loss, psychiatric disorders, neurodegenerative disorders, and temporomandibular joint (TMJ) dysfunction, have been suggested to contribute to the onset or progression of tinnitus; however, the precise molecular mechanisms of tinnitus are not well understood and the contribution of genetic and epigenetic factors remains unknown. Human genetic studies could enable the identification of novel molecular therapeutic targets, possibly leading to the development of novel pharmaceutical therapeutics. In this article, we briefly discuss the available evidence for a role of genetics in tinnitus and consider potential hurdles in designing genetic studies for tinnitus. Since multiple diseases have tinnitus as a symptom and the supporting genetic evidence is sparse, we propose various strategies to investigate the genetic underpinnings of tinnitus, first by showing evidence of heritability using concordance studies in twins, and second by improving patient selection according to phenotype and/or etiology in order to control potential biases and optimize genetic data output. The increased knowledge resulting from this endeavor could ultimately improve the drug development process and lead to the preventive or curative treatment of tinnitus.

  3. The importance of molecular markers for diagnosis and selection of targeted treatments in patients with cancer.

    PubMed

    Tobin, N P; Foukakis, T; De Petris, L; Bergh, J

    2015-12-01

    The past 30 years have seen the introduction of a number of cancer therapies with the aim of restricting the growth and spread of primary and metastatic tumours. A shared commonality among these therapies is their targeting of various aspects of the cancer hallmarks, that is traits that are essential to successful tumour propagation and dissemination. The evolution of molecular-scale technology has been central to the identification of new cancer targets, and it is not a coincidence that improved therapies have emerged at the same time as gene expression arrays and DNA sequencing have enhanced our understanding of cancer genetics. Modern tumour pathology is now viewed at the molecular level ranging from IHC biomarkers, to gene signature classifiers and gene mutations, all of which provide crucial information about which patients will respond to targeted therapy regimens. In this review, we briefly discuss the general types of targeted therapies used in a clinical setting and provide a short background on immunohistochemical, gene expression and DNA sequencing technologies, before focusing on three tumour types: breast, lung and colorectal cancers. For each of these cancer types, we provide a background to the disease along with an overview of the current standard therapies and then focus on the relevant targeted therapies and the pathways they inhibit. Finally, we highlight several strategies that are pivotal to the successful development of targeted anti-cancer drugs.

  4. Genetics of Tinnitus: An Emerging Area for Molecular Diagnosis and Drug Development.

    PubMed

    Lopez-Escamez, Jose A; Bibas, Thanos; Cima, Rilana F F; Van de Heyning, Paul; Knipper, Marlies; Mazurek, Birgit; Szczepek, Agnieszka J; Cederroth, Christopher R

    2016-01-01

    Subjective tinnitus is the perception of sound in the absence of external or bodily-generated sounds. Chronic tinnitus is a highly prevalent condition affecting over 70 million people in Europe. A wide variety of comorbidities, including hearing loss, psychiatric disorders, neurodegenerative disorders, and temporomandibular joint (TMJ) dysfunction, have been suggested to contribute to the onset or progression of tinnitus; however, the precise molecular mechanisms of tinnitus are not well understood and the contribution of genetic and epigenetic factors remains unknown. Human genetic studies could enable the identification of novel molecular therapeutic targets, possibly leading to the development of novel pharmaceutical therapeutics. In this article, we briefly discuss the available evidence for a role of genetics in tinnitus and consider potential hurdles in designing genetic studies for tinnitus. Since multiple diseases have tinnitus as a symptom and the supporting genetic evidence is sparse, we propose various strategies to investigate the genetic underpinnings of tinnitus, first by showing evidence of heritability using concordance studies in twins, and second by improving patient selection according to phenotype and/or etiology in order to control potential biases and optimize genetic data output. The increased knowledge resulting from this endeavor could ultimately improve the drug development process and lead to the preventive or curative treatment of tinnitus. PMID:27594824

  5. Molecular Characterisation and Diagnosis of Root-Knot Nematodes (Meloidogyne spp.) from Turfgrasses in North Carolina, USA

    PubMed Central

    Ye, Weimin; Zeng, Yongsan; Kerns, James

    2015-01-01

    Root-knot nematodes (Meloidogyne spp.) are the most common and destructive plant-parasitic nematode group worldwide and adversely influence both crop quality and yield. In this study, a total of 51 root-knot nematode populations from turfgrasses were tested, of which 44 were from North Carolina, 6 from South Carolina and 1 from Virginia. Molecular characterisation was performed on these samples by DNA sequencing on the ribosomal DNA 18S, ITS and 28S D2/D3. Species-specific primers were developed to identify turfgrass root-knot nematode through simplex or duplex PCR. Four species were identified, including M. marylandi Jepson & Golden in Jepson, 1987, M. graminis (Sledge & Golden, 1964) Whitehead, 1968, M. incognita (Kofoid & White, 1919) Chitwood, 1949 and M. naasi Franklin, 1965 through a combined analysis of DNA sequencing and PCR by species-specific primers. M. marylandi has been reported from North Carolina and South Carolina for the first time. Molecular diagnosis using PCR by species-specific primers provides a rapid and cheap species identification approach for turfgrass root-knot nematodes. PMID:26599462

  6. Next-generation sequencing-based molecular diagnosis of 12 inherited retinal disease probands of Uyghur ethnicity

    PubMed Central

    Tajiguli, Abulikemu; Xu, Mingchu; Fu, Qing; Yiming, Rouzimaimaiti; Wang, Keqing; Li, Yumei; Eblimit, Aiden; Sui, Ruifang; Chen, Rui; Aisa, Haji Akber

    2016-01-01

    Inherited retinal disease (IRD) is a category of genetic disorders affecting retina. Understanding the molecular basis of IRD is vital for clinical and genetic classification of patients. Uyghur people is an isolated ethnic group mainly residing in northwestern China with genetic admixture from Europeans and East Asians. The genetic etiology of IRD in this specific population still remains unknown. Here, by next-generation sequencing (NGS), we screened mutations in over 200 known retinal disease genes in a cohort of 12 unrelated Uyghur IRD probands. Out of the 12 probands, six are solved with high confidence, two with low confidence, while the remaining four are unsolved. We identified known disease-causing alleles in this cohort that suggest ancient Uyghur migration and also discovered eight novel disease-associated variants. Our results showed NGS-based mutation screening as a reliable approach for molecular diagnosis. In addition, this approach can also be applied to reveal the genetic history of a specific ethnic group. PMID:26856745

  7. Diagnosis and Molecular Variability of an Argentinean Population of Nacobbus aberrans with Some Observations on Histopathology in Tomato

    PubMed Central

    Vovlas, N.; Nico, A. I.; De Luca, F.; De Giorgi, C.; Castillo, P.

    2007-01-01

    Diagnosis of an Argentinean population of Nacobbus sp. infecting sweet pepper (lamuyo) was carried out including morphology, scanning electron microscopy, and molecular studies. In light of our morphometric, molecular and host-range results, we consider the studied population to belong to N. aberrans s. l., and by host range tests the population is assigned to the "sugar beet group." ITS-PCR analysis on individual male and immature female specimens of this population yielded amplification products of approximately 922 bp. RFLP profiles and sequencing of the ITS region revealed that, in addition to the host group, the present population can be assigned to the "Argentina 2" group. Disease development and histopathology were investigated with glasshouse observations using tomato, pepper, sugar beet and potato seedlings exposed to nematode infection for 45 days at 28 ± 2°C. Histopathology of tomato roots confirmed that all immature stages and young females and males are migratory, whereas mature females are obligate sedentary endoparasites. Rather than syncytia, large regions of cortical necrosis and cavities were detected in tomato swellings infected by juveniles. However, syncytia were associated only with adult females. Large root galls, hyperplasia, abnormal proliferation of lateral roots and asymmetry of root structure were common anatomical changes induced by the nematode feeding in tomato roots. PMID:19259470

  8. High Molecular Weight Proteins of Trypanosoma cruzi Reduce Cross-Reaction with Leishmania spp. in Serological Diagnosis Tests

    PubMed Central

    Cervantes-Landín, Alejandra Yunuen; Martínez, Ignacio; Schabib, Muslim; Espinoza, Bertha

    2014-01-01

    Chagas disease is caused by the parasite Trypanosoma cruzi. Because of its distribution throughout Latin America, sometimes it can overlap with other parasitic diseases, such as leishmaniasis, caused by Leishmania spp. This might represent a problem when performing serological diagnosis, because both parasites share antigens, resulting in cross-reactions. In the present work we evaluated Mexican sera samples: 83.8% of chagasic patients recognized at least one antigen of high molecular weight (>95 kDa) when evaluated by Western blot. Proteins of 130 kDa and 160 kDa are predominantly being recognized by asymptomatic chagasic patients. When the proteins were extracted using Triton X-100 detergent, a larger number of specific T. cruzi proteins were obtained. This protein fraction can be used to increase specificity to 100% in Western blot assays without losing sensitivity of the test. High molecular weight proteins of T. cruzi include glycoproteins with a great amount of αMan (α-mannose), αGlc (α-glucose), GlcNAc (N-acetylglucosamine), and αGal (α-galactose) content and these structures play an essential role in antigens recognition by antibodies present in patients' sera. PMID:25136581

  9. Molecular Probes for Diagnosis of Clinically Relevant Bacterial Infections in Blood Cultures▿

    PubMed Central

    Hansen, Wendy L. J.; Beuving, Judith; Bruggeman, Cathrien A.; Wolffs, Petra F. G.

    2010-01-01

    Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided into Pseudomonas species, Pseudomonas aeruginosa, Escherichia coli, and other Gram-negative species. Within the Gram-positive species, probes were designed for Staphylococcus species, Staphylococcus aureus, Enterococcus species, Streptococcus species, and Streptococcus pneumoniae. The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targeting Pseudomonas spp., P. aeruginosa, E. coli, Streptococcus spp., S. pneumoniae, Enterococcus spp., and Staphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific for S. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections. PMID:20962139

  10. The molecular basis of Tay-Sachs disease: mutation identification and diagnosis.

    PubMed

    Mahuran, D J; Triggs-Raine, B L; Feigenbaum, A J; Gravel, R A

    1990-10-01

    Tay-Sachs disease is the prototype of lysosomal storage disease. While it was first described over a century ago, the defective enzyme was not identified until 1969, making possible the development of enzyme-based diagnostic and carrier screening techniques. This led to the establishment of the successful international Tay-Sachs screening program, primarily for the high risk Ashkenazi Jewish population. In the past five years the development of recombinant DNA technology has allowed researchers to characterize 95-99% of the mutations causing Tay-Sachs disease in this high risk ethnic group. Knowledge of the exact mutations responsible for the disease coupled with the powerful polymerase chain reaction technique has now made DNA-based screening and diagnosis possible. While the enzyme-based test has proven to be reliable and economical, it cannot differentiate variant phenotypes and requires the presence of specialized testing centers. Although the DNA-based test is presently less economical, it can provide carrier couples with their exact genotype and thus, predict the general phenotype of an unborn child. Furthermore, as the catalogue of mutations leading to human disease increases, more economical DNA methodologies will be developed. In the future it would be expected that a laboratory using a single DNA-based technology could diagnose and screen for a myriad of human diseases including Tay-Sachs disease.

  11. Pitt-Hopkins Syndrome and Differential Diagnosis: A Molecular and Clinical Challenge.

    PubMed

    Marangi, Giuseppe; Zollino, Marcella

    2015-09-01

    Pitt-Hopkins syndrome is an emerging neurodevelopmental disorder caused by haploinsufficiency of the TCF4 gene on chromosome 18q21. It is characterized by severe intellectual disability, seizures, microcephaly, constipation and a distinctive facial gestalt. Although the overlapping phenotype of microcephaly, epilepsy, absent speech and constipation represents a challenge for the differential diagnosis with Angelman syndrome, Rett syndrome and Mowat-Wilson syndrome, distinctive of Pitt-Hopkins syndrome are breathing abnormalities, that can occur as either hyperventilation episodes or apnea crises, and a typical facial dysmorphism, including bitemporal narrowing, squared forehead, deep-set eyes, peculiar nose conformation, with broad nasal bridge, down-turned nasal tip and flaring nostrils, typical shape of the mouth, with a tented and M shaped upper lip, and widely spaced teeth. The occurrence of these signs in variable association of uncoordinated movements, microcephaly of postnatal onset, eye abnormalities, constipation, epilepsy and subtle brain abnormalities is highly predictive of a TCF4 mutation, making it possible to plan a genetic test of choice among severe encephalopathies. Angelman syndrome represents the nosological condition closest to Pitt-Hopkins syndrome. PMID:27617128

  12. Differential diagnosis of breast cancer using quantitative, label-free and molecular vibrational imaging

    PubMed Central

    Yang, Yaliang; Li, Fuhai; Gao, Liang; Wang, Zhiyong; Thrall, Michael J.; Shen, Steven S.; Wong, Kelvin K.; Wong, Stephen T. C.

    2011-01-01

    We present a label-free, chemically-selective, quantitative imaging strategy to identify breast cancer and differentiate its subtypes using coherent anti-Stokes Raman scattering (CARS) microscopy. Human normal breast tissue, benign proliferative, as well as in situ and invasive carcinomas, were imaged ex vivo. Simply by visualizing cellular and tissue features appearing on CARS images, cancerous lesions can be readily separated from normal tissue and benign proliferative lesion. To further distinguish cancer subtypes, quantitative disease-related features, describing the geometry and distribution of cancer cell nuclei, were extracted and applied to a computerized classification system. The results show that in situ carcinoma was successfully distinguished from invasive carcinoma, while invasive ductal carcinoma (IDC) and invasive lobular carcinoma were also distinguished from each other. Furthermore, 80% of intermediate-grade IDC and 85% of high-grade IDC were correctly distinguished from each other. The proposed quantitative CARS imaging method has the potential to enable rapid diagnosis of breast cancer. PMID:21833355

  13. Degradation diagnosis of ultrahigh-molecular weight polyethylene with terahertz-time-domain spectroscopy

    SciTech Connect

    Yamamoto, Kohji; Yamaguchi, Mariko; Tani, Masahiko; Hangyo, Masanori; Teramura, Satoshi; Isu, Toshiro; Tomita, Naohide

    2004-11-29

    We investigated ultrahigh-molecular-weight-polyethylene (UHMWPE) samples prepared by various conditions with terahertz-time-domain spectroscopy (THz-TDS). Degradation of the virgin UHMWPE samples by {gamma} irradiation induced a drastic increase of the absorption ranging continuously over the THz region. The increase of the absorption continuum is interpreted to originate in the oxidation of the amorphous region within the sample. Only slight THz spectral changes induced by the {gamma} irradiation were, however, observed for the UHMWPE samples doped with 0.1 and 0.3 wt % vitamin E. This result agrees with the earlier indication that vitamin E has an antidegradation effect on UHMWPE. The present result shows that the THz-TDS can be used for the quality control of UHMWPE by monitoring the absorption continuum in the THz region.

  14. Molecular diagnosis of maturity onset diabetes of the young in India

    PubMed Central

    Nair, Veena V.; Chapla, Aaron; Arulappan, Nishanth; Thomas, Nihal

    2013-01-01

    Diabetes is highly prevalent in India and the proportion of younger patients developing diabetes is on the increase. Apart from the more universally known type 1 diabetes and obesity related type 2 diabetes, monogenic forms of diabetes are also suspected to be prevalent in many young diabetic patients. The identification of the genetic basis of the disease not only guides in therapeutic decision making, but also aids in genetic counselling and prognostication. Genetic testing may establish the occurrence and frequency of early diabetes in our population. This review attempts to explore the utilities and horizons of molecular genetics in the field of maturity onset diabetes of the young (MODY), which include the commoner forms of monogenic diabetes. PMID:23869298

  15. Microbiological diagnosis and molecular typing of Legionella strains during an outbreak of legionellosis in Southern Germany.

    PubMed

    Essig, Andreas; von Baum, Heike; Gonser, Theodor; Haerter, Georg; Lück, Christian

    2016-02-01

    An explosive outbreak of Legionnaires' disease with 64 reported cases occurred in Ulm/Neu-Ulm in the South of Germany in December 2009/January 2010 caused by Legionella (L.) pneumophila serogroup 1, monoclonal (mAb) subtype Knoxville, sequence type (ST) 62. Here we present the clinical microbiological results from 51 patients who were diagnosed at the University hospital of Ulm, the results of the environmental investigations and of molecular typing of patients and environmental strains. All 50 patients from whom urine specimens were available were positive for L. pneumophila antigen when an enzyme-linked immunosorbent assay (EIA) was used following concentration of those urine samples that tested initially negative. The sensitivity of the BinaxNow rapid immunographic assay (ICA), after 15 min reading and after 60 min reading were 70% and 84%, respectively. Direct typing confirmed the monoclonal subtype Knoxville in 5 out of 8 concentrated urine samples. Real time PCR testing of respiratory tract specimens for L. pneumophila was positive in 15 out of 25 (60%) patients. Direct nested sequence based typing (nSBT) in some of these samples allowed partial confirmation of ST62. L. pneumophila serogroup 1, monoclonal subtype Knoxville ST62, defined as the epidemic strain was isolated from 8 out of 31 outbreak patients (26%) and from one cooling tower confirming it as the most likely source of the outbreak. While rapid detection of Legionella antigenuria was crucial for the recognition and management of the outbreak, culture and molecular typing of the strains from patients and environmental specimens was the clue for the rapid identification of the source of infection. PMID:26868659

  16. Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis.

    PubMed

    Tan, Susanna K; Burgener, Elizabeth B; Waggoner, Jesse J; Gajurel, Kiran; Gonzalez, Sarah; Chen, Sharon F; Pinsky, Benjamin A

    2016-01-01

    Background.  Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods.  Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results.  Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was performed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the comprehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions.  Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis.

  17. Molecular and Culture-Based Bronchoalveolar Lavage Fluid Testing for the Diagnosis of Cytomegalovirus Pneumonitis

    PubMed Central

    Tan, Susanna K.; Burgener, Elizabeth B.; Waggoner, Jesse J.; Gajurel, Kiran; Gonzalez, Sarah; Chen, Sharon F.; Pinsky, Benjamin A.

    2016-01-01

    Background. Cytomegalovirus (CMV) is a major cause of morbidity and mortality in immunocompromised patients, with CMV pneumonitis among the most severe manifestations of infection. Although bronchoalveolar lavage (BAL) samples are frequently tested for CMV, the clinical utility of such testing remains uncertain. Methods. Retrospective analysis of adult patients undergoing BAL testing via CMV polymerase chain reaction (PCR), shell vial culture, and conventional viral culture between August 2008 and May 2011 was performed. Cytomegalovirus diagnostic methods were compared with a comprehensive definition of CMV pneumonitis that takes into account signs and symptoms, underlying host immunodeficiency, radiographic findings, and laboratory results. Results. Seven hundred five patients underwent 1077 bronchoscopy episodes with 1090 BAL specimens sent for CMV testing. Cytomegalovirus-positive patients were more likely to be hematopoietic cell transplant recipients (26% vs 8%, P < .0001) and less likely to have an underlying condition not typically associated with lung disease (3% vs 20%, P < .0001). Histopathology was performed in only 17.3% of CMV-positive bronchoscopy episodes. When CMV diagnostic methods were evaluated against the comprehensive definition, the sensitivity and specificity of PCR, shell vial culture, and conventional culture were 91.3% and 94.6%, 54.4% and 97.4%, and 28.3% and 96.5%, respectively. Compared with culture, PCR provided significantly higher sensitivity and negative predictive value (P ≤ .001), without significantly lower positive predictive value. Cytomegalovirus quantitation did not improve test performance, resulting in a receiver operating characteristic curve with an area under the curve of 0.53. Conclusions. Cytomegalovirus PCR combined with a comprehensive clinical definition provides a pragmatic approach for the diagnosis of CMV pneumonitis. PMID:26885542

  18. [Evaluation of a newly-developed ready-to-use commercial PCR kit for the molecular diagnosis of Francisella tularensis].

    PubMed

    Celebi, Bekir; Kılıç, Selçuk; Yeşilyurt, Murat; Acar, Bülent

    2014-01-01

    Tularemia is a rare zoonotic infection, however, considerations of tularemia as a biological weapon and several recent major epidemics have caused renewed interest in this disease. Laboratory diagnosis of tularemia is done in the presence of appropriate epidemiological data, by the demonstration of specific antibodies in the serum samples obtained with 1-2 week intervals following the development of symptoms. It is an a posteriori analysis with limited use for prompt diagnosis of the patient during the early symptomatic phase and deliberate release of biological agents. Limitations in both culture and serology have led to substantial research in the development of new diagnostic techniques. Several PCR methods for tularemia have been developed, both for conventional and real-time polymerase chain reaction (rtPCR). However, PCR methods are hard to be deployed in remote endemic areas that lack sufficient infrastructure. Recently a "Toolbox" which includes all instruments, equipments and solutions [DNA4U® Bacteria Genomic DNA Isolation Kit, CubeCycler® (Personal Thermal Cycler), PCR4U® Bioterrorism Agents Detection Kit, electrophoresis tank, power supply, ready-agarose gel and electrophoresis buffer] necessary for conventional PCR, was developed for the identification of bioterrorism agents in the field. In this study we aimed to evaluate the efficacy of a ready-to-use commercial PCR kit (Nanobiz, Ankara, Turkey) targeting the tul4 gene, for the diagnosis of tularemia and to compare the results with an in-house conventional PCR and a rtPCR test. We applied the assay to a collection of four F.tularensis standard strains, 15 field isolates (from humans, animals, water), 13 non-Francisella strains which are phylogenetically related to F.tularensis and a total of 60 lymph node aspirates obtained from suspected tularemia cases. Compared to the in-house PCR method used in our laboratory, the sensitivity, specificity, positive and negative predictive values of Nanobiz PCR

  19. Next-generation sequencing-based molecular diagnosis of neonatal hypotonia in Chinese Population

    PubMed Central

    Wang, Yan; Peng, Wei; Guo, Hong-Yan; Li, Hui; Tian, Jie; Shi, Yu-Jing; Yang, Xiao; Yang, Yao; Zhang, Wan-Qiao; Liu, Xin; Liu, Guan-Nan; Deng, Tao; Sun, Yi-Min; Xing, Wan-li; Cheng, Jing; Feng, Zhi-Chun

    2016-01-01

    Neonatal hypotonia is extremely challenging to diagnose because numerous disorders present similar clinical manifestations. Two panels for diagnosing neonatal hypotonia were developed, which enriches 35 genes corresponding to 61 neonatal hypotonia-related disorders. A cohort of 214 neonates with hypotonia was recruited from 2012 to 2014 in China for this study. Of these subjects, twenty-eight neonates with hypotonia were eliminated according to exclusion criteria and 97 were confirmed using traditional detection methods. The clinical diagnoses of the remaining 89 neonates with hypotonia were approached by targeted next-generation sequencing (NGS). Among the 89 tested neonates, 25 potentially pathogenic variants in nine genes (RYR1, MECP2, MUT, CDKL5, MPZ, PMM2, MTM1, LAMA2 and DMPK) were identified in 22 patients. Six of these pathogenic variants were novel. Of the 186 neonates with hypotonia, we identified the genetic causes for 117 neonates by the traditional detection methods and targeted NGS, achieving a high solving rate of 62.9%. In addition, we found seven neonates with RETT syndrome carrying five mutations, thus expanding the mutation profiles in Chinese neonates with hypotonia. Our study highlights the utility of comprehensive molecular genetic testing, which provides the advantage of speed and diagnostic specificity without invasive procedures. PMID:27353517

  20. Inherited pancreatic endocrine tumor syndromes: advances in molecular pathogenesis, diagnosis, management and controversies

    PubMed Central

    Jensen, Robert T.; Berna, Marc J.; Bingham, David B; Norton, Jeffrey A.

    2008-01-01

    Pancreatic endocrine tumors (PETs) can occur in as part of four inherited disorders including: Multiple Endocrine Neoplasia type 1 (MEN1), von Hippel-Lindau disease (VHL), neurofibromatosis 1(NF-1) [von Recklinghausen’s disease] and the tuberous sclerosis complex (TSC). The relative frequency with which patients with these disorders develop PETs is MEN1>VHL>NF-1>TSC. Over the last few years there have been major advances in the understanding of the genetics and molecular pathogenesis of these disorders as well in the localization, medical and surgical treatment of the PETs in these patients. The study of the PETs in these disorders has not only provided insights into the possible pathogenesis of sporadic PETs, but have also presented a number of unique management and treatment issues, some of which are applicable to patients with sporadic PETs. Therefore the study of PETs in these uncommon disorders has provided valuable insights that in many cases are applicable to the general group of patients with sporadic PETs. In this article these areas are briefly reviewed as well as the current state of knowledge of the PETs in these disorders and the controversies that exist in their management are briefly summarized and discussed. PMID:18798544

  1. Pituitary tumors in childhood: update of diagnosis, treatment and molecular genetics.

    PubMed

    Keil, Margaret F; Stratakis, Constantine A

    2008-04-01

    Pituitary tumors are rare in childhood and adolescence, with a reported prevalence of up to one per 1 million children. Only 2-6% of surgically treated pituitary tumors occur in children. Although pituitary tumors in children are almost never malignant and hormonal secretion is rare, these tumors may result in significant morbidity. Tumors within the pituitary fossa are mainly of two types: craniopharyngiomas and adenomas. Craniopharyngiomas cause symptoms by compressing normal pituitary, causing hormonal deficiencies and producing mass effects on surrounding tissues and the brain; adenomas produce a variety of hormonal conditions such as hyperprolactinemia, Cushing disease and acromegaly or gigantism. Little is known about the genetic causes of sporadic lesions, which comprise the majority of pituitary tumors, but in children, more frequently than in adults, pituitary tumors may be a manifestation of genetic conditions such as multiple endocrine neoplasia type 1, Carney complex, familial isolated pituitary adenoma and McCune-Albright syndrome. The study of pituitary tumorigenesis in the context of these genetic syndromes has advanced our knowledge of the molecular basis of pituitary tumors and may lead to new therapeutic developments. PMID:18416659

  2. Pituitary Tumors in Childhood: an update in their diagnosis, treatment and molecular genetics

    PubMed Central

    Keil, Margaret F.; Stratakis, Constantine A.

    2009-01-01

    Pituitary tumors are rare in childhood and adolescence, with a reported prevalence of up to 1 per million children. Only 2 - 6% of surgically treated pituitary tumors occur in children. Although pituitary tumors in children are almost never malignant and hormonal secretion is rare, these tumors may result in significant morbidity. Tumors within the pituitary fossa are of two types mainly, craniopharyngiomas and adenomas; craniopharyngiomas cause symptoms by compressing normal pituitary, causing hormonal deficiencies and producing mass effects on surrounding tissues and the brain; adenomas produce a variety of hormonal conditions such as hyperprolactinemia, Cushing disease and acromegaly or gigantism. Little is known about the genetic causes of sporadic lesions, which comprise the majority of pituitary tumors, but in children, more frequently than in adults, pituitary tumors may be a manifestation of genetic conditions such as multiple endocrine neoplasia type 1 (MEN 1), Carney complex, familial isolated pituitary adenoma (FIPA), and McCune-Albright syndrome. The study of pituitary tumorigenesis in the context of these genetic syndromes has advanced our knowledge of the molecular basis of pituitary tumors and may lead to new therapeutic developments. PMID:18416659

  3. Evaluation of Four Commercial Multiplex Molecular Tests for the Diagnosis of Acute Respiratory Infections

    PubMed Central

    Salez, Nicolas; Vabret, Astrid; Leruez-Ville, Marianne; Andreoletti, Laurent; Carrat, Fabrice; Renois, Fanny; de Lamballerie, Xavier

    2015-01-01

    Acute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden’s index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86–1.00; PPV:78.95–100.00; Sp:97.32–100.00] & B [YI:0.44–1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50–0.99; PPV:85.71–100.00; Sp:99.38–100.00], hMPV [YI:0.71–1.00; PPV:83.33–100.00; Sp:99.37–100.00], EV/hRV [YI:0.62–0.82; PPV:93.33–100.00; Sp:94.48–100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20–0.80; PPV:57.14–100.00; Sp:98.14–100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections. PMID:26107509

  4. Prenatal diagnosis of chromosome 15 abnormalities in the Prader-Willi/Angelman syndrome region by traditional and molecular cytogenetics

    SciTech Connect

    Toth-Fejel, S.; Magenis, R.E.; Leff, S.

    1995-02-13

    With improvements in culturing and banding techniques, amniotic fluid studies now achieve a level of resolution at which the Prader-Willi syndrome (PWS) and Angelman syndrome (AS) region may be questioned. Chromosome 15 heteromorphisms, detected with Q- and R-banding and used in conjunction with PWS/AS region-specific probes, can confirm a chromosome deletion and establish origin to predict the clinical outcome. We report four de novo cases of an abnormal-appearing chromosome 15 in amniotic fluid samples referred for advanced maternal age or a history of a previous chromosomally abnormal child. The chromosomes were characterized using G-, Q-, and R-banding, as well as isotopic and fluorescent in situ hybridization of DNA probes specific for the proximal chromosome 15 long arm. In two cases, one chromosome 15 homolog showed a consistent deletion of the ONCOR PWS/AS region A and B. In the other two cases, one of which involved an inversion with one breakpoint in the PWS/AS region, all of the proximal chromosome 15 long arm DNA probes used in the in situ hybridization were present on both homologs. Clinical follow-up was not available on these samples, as in all cases the parents chose to terminate the pregnancies. These cases demonstrate the ability to prenatally diagnose chromosome 15 abnormalities associated with PWS/AS. In addition, they highlight the need for a better understanding of this region for accurate prenatal diagnosis. 41 refs., 5 figs.

  5. Accurate calculation of chemical shifts in highly dynamic H2@C60 through an integrated quantum mechanics/molecular dynamics scheme.

    PubMed

    Jiménez-Osés, Gonzalo; García, José I; Corzana, Francisco; Elguero, José

    2011-05-20

    A new protocol combining classical MD simulations and DFT calculations is presented to accurately estimate the (1)H NMR chemical shifts of highly mobile guest-host systems and their thermal dependence. This strategy has been successfully applied for the hydrogen molecule trapped into C(60) fullerene, an unresolved and challenging prototypical case for which experimental values have never been reproduced. The dependence of the final values on the theoretical method and their implications to avoid over interpretation of the obtained results are carefully described.

  6. Accurate quantum chemical calculations

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Langhoff, Stephen R.; Taylor, Peter R.

    1989-01-01

    An important goal of quantum chemical calculations is to provide an understanding of chemical bonding and molecular electronic structure. A second goal, the prediction of energy differences to chemical accuracy, has been much harder to attain. First, the computational resources required to achieve such accuracy are very large, and second, it is not straightforward to demonstrate that an apparently accurate result, in terms of agreement with experiment, does not result from a cancellation of errors. Recent advances in electronic structure methodology, coupled with the power of vector supercomputers, have made it possible to solve a number of electronic structure problems exactly using the full configuration interaction (FCI) method within a subspace of the complete Hilbert space. These exact results can be used to benchmark approximate techniques that are applicable to a wider range of chemical and physical problems. The methodology of many-electron quantum chemistry is reviewed. Methods are considered in detail for performing FCI calculations. The application of FCI methods to several three-electron problems in molecular physics are discussed. A number of benchmark applications of FCI wave functions are described. Atomic basis sets and the development of improved methods for handling very large basis sets are discussed: these are then applied to a number of chemical and spectroscopic problems; to transition metals; and to problems involving potential energy surfaces. Although the experiences described give considerable grounds for optimism about the general ability to perform accurate calculations, there are several problems that have proved less tractable, at least with current computer resources, and these and possible solutions are discussed.

  7. Molecular diagnosis of pediatric patients with citrin deficiency in China: SLC25A13 mutation spectrum and the geographic distribution

    PubMed Central

    Lin, Wei-Xia; Zeng, Han-Shi; Zhang, Zhan-Hui; Mao, Man; Zheng, Qi-Qi; Zhao, Shu-Tao; Cheng, Ying; Chen, Feng-Ping; Wen, Wang-Rong; Song, Yuan-Zong

    2016-01-01

    Citrin deficiency (CD) is a Mendelian disease due to biallelic mutations of SLC25A13 gene. Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is the major pediatric CD phenotype, and its definite diagnosis relies on SLC25A13 genetic analysis. China is a vast country with a huge population, but the SLC25A13 genotypic features of CD patients in our country remains far from being well clarified. Via sophisticated molecular analysis, this study diagnosed 154 new CD patients in mainland China and identified 9 novel deleterious SLC25A13 mutations, i.e. c.103A > G, [c.329 − 154_c.468 + 2352del2646; c.468 + 2392_c.468 + 2393ins23], c.493C > T, c.755 − 1G > C, c.845_c.848 + 1delG, c.933_c.933 + 1insGCAG, c.1381G > T, c.1452 + 1G > A and c.1706_1707delTA. Among the 274 CD patients diagnosed by our group thus far, 41 SLC25A13 mutations/variations were detected. The 7 mutations c.775C > T, c.851_854del4, c.1078C > T, IVS11 + 1G > A, c.1364G > T, c.1399C > T and IVS16ins3kb demonstrated significantly different geographic distribution. Among the total 53 identified genotypes, only c.851_854del4/c.851_854del4 and c.851_854del4/c.1399C > T presented different geographic distribution. The northern population had a higher level of SLC25A13 allelic heterogeneity than those in the south. These findings enriched the SLC25A13 mutation spectrum and brought new insights into the geographic distribution of the variations and genotypes, providing reliable evidences for NICCD definite diagnosis and for the determination of relevant molecular targets in different Chinese areas. PMID:27405544

  8. Early diagnosis of fungal infections using piezomicrogravimetric and electric chemosensors based on polymers molecularly imprinted with d-arabitol.

    PubMed

    Dabrowski, Marcin; Sharma, Piyush Sindhu; Iskierko, Zofia; Noworyta, Krzysztof; Cieplak, Maciej; Lisowski, Wojciech; Oborska, Sylwia; Kuhn, Alexander; Kutner, Wlodzimierz

    2016-05-15

    An elevated concentration of d-arabitol in urine, especially compared to that of l-arabitol or creatinine, is indicative of a fungal infection. For that purpose, we devised, fabricated, and tested chemical sensors determining d-arabitol. These chemosensors comprised the quartz crystal resonator (QCR) or extended-gate field-effect transistor (EG-FET) transducers integrated with molecularly imprinted polymer (MIP) film recognition units. To this end, we successfully applied a covalent approach to molecular imprinting, which involved formation of weak reversible covalent bonds between vicinal hydroxyl groups of arabitol and boronic acid substituents of the bithiophene functional monomer used. The MIP films were synthesized and simultaneously deposited on gold electrodes of quartz crystal resonators (Au-QCRs) or Au-glass slides by oxidative potentiodynamic electropolymerization. With the QCR and EG-FET chemosensors, the d-arabitol concentration was determined under flow-injection analysis and stagnant-solution binding conditions, respectively. Selectivity with respect to common interferences, and l-arabitol in particular, of the devised chemosensors was superior. Limits of detection and linear dynamic concentration ranges of the QCR and EG-FET chemosensors were 0.15 mM and 0.15 to 1.25 mM as well as 0.12 mM and 0.12 to 1.00 mM, respectively, being lower than the d-arabitol concentrations in urine of patients with invasive candidiasis (>220 μM). Therefore, the devised chemosensors are suitable for early diagnosis of fungal infections caused by Candida sp. yeasts. PMID:26761618

  9. Size of the exon 1-CAG repeats of the androgen receptor gene employed as a molecular marker in the diagnosis of Turner syndrome in girls with short stature.

    PubMed

    Figueiredo, C C; Kochi, C; Longui, C A; Rocha, M N; Richeti, F; Evangelista, N M A; Calliari, L E P; Monte, O

    2008-01-01

    Turner syndrome (TS) is one of the most common chromosomal abnormalities among girls. Complete monosomy of X chromosome is responsible for almost 50% of all cases of TS, and mosaicism and X anomaly are detected in the other half. It has already been demonstrated that early diagnosis of these children allows appropriate growth hormone treatment with better final height prognosis and introduction of estrogen at an ideal chronological age. Sixty-four short-stature girls were selected and the clinical data obtained were birth weight and height, weight and height at the first medical visit and target height. Other clinical data including cardiac and renal abnormalities, otitis, Hashimoto thyroiditis, cubitus valgus, short neck, widely separated nipples, and pigmented nevi were obtained from the patients' medical records. The aim of the present study was to evaluate the screening of a group of short-stature girls for TS based on the number of CAG repeats of the androgen receptor gene analyzed by GeneScan software. Patient samples with two alleles (heterozygous) were 49/64 (76.5%) and with one allele (homozygous) were 15/64 (23.5%). A karyotype was determined in 30 patients, 9 homozygous and 21 heterozygous. In the homozygous group, 6/9 were 45,X and 3/9 were 46,XX. In the heterozygous group, 17/21 were 46,XX, and 4/21 were TS patients with mosaicism (45,X/46,XX; 45,X/46XiXq; 46XdelXp). The pattern obtained by GeneScan in two patients with mosaicism in the karyotype was an imbalance between the peak heights of the two alleles, suggesting that this imbalance could be present when there is a mosaicism. The frequency of TS abnormalities (18.7%) did not differ between TS and 46,XX girls. Thus, it is important to accurately assess the incidence of TS in growth-retarded girls, even in the absence of other dysmorphisms. In this study, we diagnosed 6 cases of TS 45,X (9.4%) by molecular analysis, with a 100% sensitivity and 85% specificity. This molecular analysis was able to

  10. Accurate molecular dynamics and nuclear quantum effects at low cost by multiple steps in real and imaginary time: Using density functional theory to accelerate wavefunction methods.

    PubMed

    Kapil, V; VandeVondele, J; Ceriotti, M

    2016-02-01

    The development and implementation of increasingly accurate methods for electronic structure calculations mean that, for many atomistic simulation problems, treating light nuclei as classical particles is now one of the most serious approximations. Even though recent developments have significantly reduced the overhead for modeling the quantum nature of the nuclei, the cost is still prohibitive when combined with advanced electronic structure methods. Here we present how multiple time step integrators can be combined with ring-polymer contraction techniques (effectively, multiple time stepping in imaginary time) to reduce virtually to zero the overhead of modelling nuclear quantum effects, while describing inter-atomic forces at high levels of electronic structure theory. This is demonstrated for a combination of MP2 and semi-local DFT applied to the Zundel cation. The approach can be seamlessly combined with other methods to reduce the computational cost of path integral calculations, such as high-order factorizations of the Boltzmann operator or generalized Langevin equation thermostats.

  11. Accurate molecular dynamics and nuclear quantum effects at low cost by multiple steps in real and imaginary time: Using density functional theory to accelerate wavefunction methods

    NASA Astrophysics Data System (ADS)

    Kapil, V.; VandeVondele, J.; Ceriotti, M.

    2016-02-01

    The development and implementation of increasingly accurate methods for electronic structure calculations mean that, for many atomistic simulation problems, treating light nuclei as classical particles is now one of the most serious approximations. Even though recent developments have significantly reduced the overhead for modeling the quantum nature of the nuclei, the cost is still prohibitive when combined with advanced electronic structure methods. Here we present how multiple time step integrators can be combined with ring-polymer contraction techniques (effectively, multiple time stepping in imaginary time) to reduce virtually to zero the overhead of modelling nuclear quantum effects, while describing inter-atomic forces at high levels of electronic structure theory. This is demonstrated for a combination of MP2 and semi-local DFT applied to the Zundel cation. The approach can be seamlessly combined with other methods to reduce the computational cost of path integral calculations, such as high-order factorizations of the Boltzmann operator or generalized Langevin equation thermostats.

  12. Molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: an update of new CYP21A2 mutations.

    PubMed

    Concolino, Paola; Mello, Enrica; Zuppi, Cecilia; Capoluongo, Ettore

    2010-08-01

    Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia, an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Approximately 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. A list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http:www.imm.Ki.se/CYPalleles/cyp21.htm). Unfortunately, the last update of this database was in 2006. However, over the last 4 years many other novel CYP21A2 mutations have been reported in PubMed. The aim of this review is to provide a focus on the molecular and genetic aspects of the diagnosis of 21-hydroxylase deficiency. In addition, an updated list of the last new CYP21A2 mutations is included.

  13. The Clinical, Serological and Molecular Diagnosis of Emerging Dengue Infection at a Tertiary Care Institute in Southern, India

    PubMed Central

    Neeraja, Mamidi; Lakshmi, Vemu; Dash, P.K.; Parida, M.M.; Rao, P.V.L.

    2013-01-01

    Introduction: Dengue is an acute viral infection which presents as uneventful pyrexia to a fatal complication. This infection is increasingly being recognized as the world’s major emerging tropical disease and an important public health problem. This article highlights the clinical manifestations of Dengue virus infection and the various molecular tests that were used for its laboratory diagnosis. Methods: Serum samples from 713 suspected cases of Dengue were collected between August and December 2007. The clinical profiles of 123 hospitalized patients were analyzed. Serology, RT- PCR, virus isolation and sequencing were done. Results: The most common clinical symptoms were fever, thrombocytopenia, rash and elevated liver enzymes. The demonstration of the Dengue RNA in 5.16% samples, the detection of Dengue specific IgM antibodies in 18% samples and the isolation of the DENV-4 and the DENV-3 viruses from the clinical samples confirmed this Dengue outbreak. A co -infection with Chikungunya was observed in 2.06% of the cases. The phylogenetic analysis revealed that the Indian Dengue-4 isolates from this outbreak belonged to the genotype I. This study clearly indicated the sudden dominance of DENV-4 in an Indian Dengue outbreak. Conclusion: The surveillance of the Dengue viruses needs to be closely monitored for the emergence of newer serotype(s) in hitherto unknown areas. PMID:23634396

  14. Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.

    PubMed

    Ferguson, Tanya M; Weigel, Kris M; Lakey Becker, Annie; Ontengco, Delia; Narita, Masahiro; Tolstorukov, Ilya; Doebler, Robert; Cangelosi, Gerard A; Niemz, Angelika

    2016-01-01

    Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse(®) mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >10(4) fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse(®) method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 10(4) and 10(5) colony forming units (cfu)/mL M.tb. At 10(3) cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse(®) method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with 1 mL input volume (N = 41). The semi-automated PureLyse(®) method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented, and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings. PMID:26785769

  15. Proxy Molecular Diagnosis from Whole-Exome Sequencing Reveals Papillon-Lefevre Syndrome Caused by a Missense Mutation in CTSC

    PubMed Central

    Erzurumluoglu, A. Mesut; Alsaadi, Muslim M.; Rodriguez, Santiago; Alotaibi, Tahani S.; Guthrie, Philip A. I.; Lewis, Sian; Ginwalla, Aasiya; Gaunt, Tom R.; Alharbi, Khalid K.; Alsaif, Fahad M.; Alsaadi, Basma M.; Day, Ian N. M.

    2015-01-01

    Papillon-Lefevre syndrome (PLS) is an autosomal recessive disorder characterised by severe early onset periodontitis and palmoplantar hyperkeratosis. A previously reported missense mutation in the CTSC gene (NM_001814.4:c.899G>A:p.(G300D)) was identified in a homozygous state in two siblings diagnosed with PLS in a consanguineous family of Arabic ancestry. The variant was initially identified in a heterozygous state in a PLS unaffected sibling whose whole exome had been sequenced as part of a previous Primary ciliary dyskinesia study. Using this information, a proxy molecular diagnosis was made on the PLS affected siblings after consent was given to study this second disorder found to be segregating within the family. The prevalence of the mutation was then assayed in the local population using a representative sample of 256 unrelated individuals. The variant was absent in all subjects indicating that the variant is rare in Saudi Arabia. This family study illustrates how whole-exome sequencing can generate findings and inferences beyond its primary goal. PMID:25799584

  16. [Modern algorithms of diagnosis of benign tumors of the mammary gland: the role of molecular-genetic methods].

    PubMed

    Dubinina, V G; Chetverikov, S G; Zavoloka, A V; Moroziuk, O N

    2014-01-01

    Experience of treatment in 2010-2012 yrs of the patients, suffering mammarial gland tumors, in The Center of Reconstructive and Restoration Medicine (The University Clinic) of The Odessa National Medical University was analyzed. There were examined 143 women with morphologically confirmed mammarial gland cancer (MGT), 56--benign mammary gland tumors and 50 healthy women. Molecular-genetic investigation was performed in the patients-women: there were determined the gene C634G polymorphism of VEGF and of the gene G308A of TNF--a with subsequent estimation of correlation of the mutations quantity and the mammarial gland diseases rate. Algorithm of differential diagnosis of benign tumors must include estimation of polymorphism of the VEGF gene C634G. While revealing of the heterozygous or homozygous bearers of mutation with the gene C634G polymorphism of VEGF the risk of the MGC occurrence is enhancing, what may serve as additional criterion for expedience for conduction of operative treatment in such patients.

  17. Molecular Identification of Q Fever in Patients with a Suspected Diagnosis of Dengue in Brazil in 2013-2014.

    PubMed

    Mares-Guia, Maria Angélica M M; Rozental, Tatiana; Guterres, Alexandro; Ferreira, Michelle Dos Santos; Botticini, Renato De Gasperis; Terra, Ana Kely Carolina; Marraschi, Sandro; Bochner, Rosany; Lemos, Elba R S

    2016-05-01

    Q fever is an important cause of undifferentiated fever that is rarely recognized or reported in Brazil. The objective of this study was to look for the presence of Coxiella burnetii during a dengue fever outbreak in the municipality of Itaboraí, Rio de Janeiro, Brazil, where this bacterium had previously infected humans and domesticated animals. Blood samples from clinically suspected dengue fever patients were tested by polymerase chain reaction (PCR) for C. burnetii; the DNA was detected in nine (3.3%) of 272 patients. One was coinfected with dengue virus, which was also detected in another 166 (61.3%) patients. The nucleotide sequence of PCR amplification and DNA sequencing of the IS1111 transposase elements in the genome of C. burnetii exhibited 99% identity with the sequence in GenBank. The detection of C. burnetii in patients suspected of dengue fever indicates that awareness and knowledge of Q fever should be strengthened and that this bacterium is present in Brazil. Finally, because a negative molecular result does not completely rule out the diagnosis of Q fever and the serological assay based on seroconversion was not available, the actual number of this zoonosis is likely to be much higher than that reported in this study. PMID:26928831

  18. [Diagnosis of early gastric marginal zone lymphoma (MALT lymphoma) in endoscopic biopsies. Report of a case that demonstrates the utility of immunohistochemistry and the molecular analysis.].

    PubMed

    Piña-Oviedo, S; Fend, F; Kramer, M; Fournier, F; Farca, A; Ortiz-Hidalgo, C

    2008-01-01

    MALT lymphomas are a subtype of low grade lymphomas that represent 7-8% of all B-cell lymphomas originated in extranodal sites. Nearly 50% of the cases present as primary gastric lymphomas.They arise from the acquired MALT developed during the course of H. pylori chronic infection. Microscopically,the presence of lymphoid follicles with follicular colonization, marginal zone ("centrocytoid")cells and lymphoepithelial lesions is characteristic.Histopathological diagnosis may result difficult in superficial endoscopic biopsies showing a typical lymphoid infiltrates. Using Wotherspoon criteria along with molecular analyses is useful in borderline lesions. We present the case of a 62 years old female with chronic gastritis. A small endoscopic biopsy was performed and the presence of a heterogeneous lymphoid infiltrate in the lamina propia was observed. Immuno histochemical profile showed focal co expression of CD20 andCD43 and kappa light chain restriction. The diagnosis of an atypical lymphoid infiltrate highly suspicious of MALT lymphoma was established. PCR molecular analysis corroborated the monoclonal pattern of the lymphoid cells and the diagnosis of lymphoma. Immunohistochemical and molecular analyses in cases of lymphoid infiltrates suspicious of malignancy (Wotherspoon 3 and 4) will aid in the adequate diagnosis between chronic gastritis and MALT lymphoma, which is crucial for prognosis,treatment and patient's outcome.Key words: Chronic reactive gastritis, marginal zone lymphoma, MALT lymphoma, gastriclym.

  19. Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer

    PubMed Central

    Charanya, Tauseef; York, Timothy; Bloch, Sharon; Sudlow, Gail; Liang, Kexian; Garcia, Missael; Akers, Walter J.; Rubin, Deborah; Gruev, Viktor; Achilefu, Samuel

    2014-01-01

    Abstract. Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6±1.65) and flat lesions (12.1±1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.41). Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy. PMID:25473883

  20. Trimodal color-fluorescence-polarization endoscopy aided by a tumor selective molecular probe accurately detects flat lesions in colitis-associated cancer

    NASA Astrophysics Data System (ADS)

    Charanya, Tauseef; York, Timothy; Bloch, Sharon; Sudlow, Gail; Liang, Kexian; Garcia, Missael; Akers, Walter J.; Rubin, Deborah; Gruev, Viktor; Achilefu, Samuel

    2014-12-01

    Colitis-associated cancer (CAC) arises from premalignant flat lesions of the colon, which are difficult to detect with current endoscopic screening approaches. We have developed a complementary fluorescence and polarization reporting strategy that combines the unique biochemical and physical properties of dysplasia and cancer for real-time detection of these lesions. Using azoxymethane-dextran sodium sulfate (AOM-DSS) treated mice, which recapitulates human CAC and dysplasia, we show that an octapeptide labeled with a near-infrared (NIR) fluorescent dye selectively identified all precancerous and cancerous lesions. A new thermoresponsive sol-gel formulation allowed topical application of the molecular probe during endoscopy. This method yielded high contrast-to-noise ratios (CNR) between adenomatous tumors (20.6±1.65) and flat lesions (12.1±1.03) and surrounding uninvolved colon tissue versus CNR of inflamed tissues (1.62±0.41). Incorporation of nanowire-filtered polarization imaging into NIR fluorescence endoscopy shows a high depolarization contrast in both adenomatous tumors and flat lesions in CAC, reflecting compromised structural integrity of these tissues. Together, the real-time polarization imaging provides real-time validation of suspicious colon tissue highlighted by molecular fluorescence endoscopy.

  1. More parasitic myositis cases in humans in Australia, and the definition of genetic markers for the causative agents as a basis for molecular diagnosis.

    PubMed

    Koehler, Anson V; Spratt, David M; Norton, Robert; Warren, Sanchia; McEwan, Belinda; Urkude, Ravindra; Murthy, Suresh; Robertson, Thomas; McCallum, Naomi; Parsonson, Fiona; Bradbury, Richard S; Gasser, Robin B

    2016-10-01

    Since 1998, there have been six reported human cases of myositis in Australia, attributable to infection with the nematode Haycocknema perplexum. However, an unequivocal diagnosis of H. perplexum infection and associated disease has been seriously compromised by a lack of molecular markers for this nematode. Here, we report new cases of disseminated myositis in two male patients from the states of Queensland and Tasmania in Australia, respectively; genetically characterize the causative agent from each case; and, also establish a PCR-based sequencing approach as a tool to support the diagnosis of future cases and to underpin epidemiological studies.

  2. Novel molecular beacon probe-based real-time RT-PCR assay for diagnosis of Crimean-Congo hemorrhagic fever encountered in India.

    PubMed

    Kamboj, Aman; Pateriya, Atul Kumar; Mishra, Anamika; Ranaware, Pradip; Kulkarni, Diwakar D; Raut, Ashwin Ashok

    2014-01-01

    Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

  3. Molecular dynamics investigations of ozone on an ab initio potential energy surface with the utilization of pattern-recognition neural network for accurate determination of product formation.

    PubMed

    Le, Hung M; Dinh, Thach S; Le, Hieu V

    2011-10-13

    The singlet-triplet transformation and molecular dissociation of ozone (O(3)) gas is investigated by performing quasi-classical molecular dynamics (MD) simulations on an ab initio potential energy surface (PES) with visible and near-infrared excitations. MP4(SDQ) level of theory with the 6-311g(2d,2p) basis set is executed for three different electronic spin states (singlet, triplet, and quintet). In order to simplify the potential energy function, an approximation is adopted by ignoring the spin-orbit coupling and allowing the molecule to switch favorably and instantaneously to the spin state that is more energetically stable (lowest in energy among the three spin states). This assumption has previously been utilized to study the SiO(2) system as reported by Agrawal et al. (J. Chem. Phys. 2006, 124 (13), 134306). The use of such assumption in this study probably makes the upper limits of computed rate coefficients the true rate coefficients. The global PES for ozone is constructed by fitting 5906 ab initio data points using a 60-neuron two-layer feed-forward neural network. The mean-absolute error and root-mean-squared error of this fit are 0.0446 eV (1.03 kcal/mol) and 0.0756 eV (1.74 kcal/mol), respectively, which reveal very good fitting accuracy. The parameter coefficients of the global PES are reported in this paper. In order to identify the spin state with high confidence, we propose the use of a pattern-recognition neural network, which is trained to predict the spin state of a given configuration (with a prediction accuracy being 95.6% on a set of testing data points). To enhance the prediction effectiveness, a buffer series of five points are validated to confirm the spin state during the MD process to gain better confidence. Quasi-classical MD simulations from 1.2 to 2.4 eV of total internal energy (including zero-point energy) result in rate coefficients of singlet-triplet transformation in the range of 0.027 ps(-1) to 1.21 ps(-1). Also, we find very

  4. Molecular diagnosis of Prader-Willi syndrome: Parent-of-origin dependent methylation sites and non-isotopic detection of (CA){sub n} dinucleotide repeat polymorphisms

    SciTech Connect

    Lerer, I.; Meiner, V.; Pashut-Lavon, I.; Abeliovich, D.

    1994-08-01

    We describe our experience in the molecular diagnosis of 22 patients suspected of Prader-Willi syndrome (PWS) using a DNA probe PW71 (D15S63) which detects a parent-of-origin specific methylated site in the PWS critical region. The cause of the syndrome was determined as deletion or uniparental disomy according to the segregation of (CA){sub n} dinucleotide repeat polymorphisms of the PWS/AS region and more distal markers of chromosome 15. In 10 patients the clinical diagnosis was confirmed by the segregation of (CA){sub n}, probably due to paternal microdeletion in the PWs critical region which did not include the loci D15S97, D15S113, GABRB3, and GABRA5. This case demonstrates the advantage of the DNA probe PW71 in the diagnosis of PWS. 31 refs., 2 figs., 3 tabs.

  5. Implementation of next-generation sequencing for molecular diagnosis of hereditary breast and ovarian cancer highlights its genetic heterogeneity.

    PubMed

    Pinto, Pedro; Paulo, Paula; Santos, Catarina; Rocha, Patrícia; Pinto, Carla; Veiga, Isabel; Pinheiro, Manuela; Peixoto, Ana; Teixeira, Manuel R

    2016-09-01

    Molecular diagnosis of hereditary breast and ovarian cancer (HBOC) by standard methodologies has been limited to the BRCA1 and BRCA2 genes. With the recent development of new sequencing methodologies, the speed and efficiency of DNA testing have dramatically improved. The aim of this work was to validate the use of next-generation sequencing (NGS) for the detection of BRCA1/BRCA2 point mutations in a diagnostic setting and to study the role of other genes associated with HBOC in Portuguese families. A cohort of 94 high-risk families was included in the study, and they were initially screened for the two common founder mutations with variant-specific methods. Fourteen index patients were shown to carry the Portuguese founder mutation BRCA2 c.156_157insAlu, and the remaining 80 were analyzed in parallel by Sanger sequencing for the BRCA1/BRCA2 genes and by NGS for a panel of 17 genes that have been described as involved in predisposition to breast and/or ovarian cancer. A total of 506 variants in the BRCA1/BRCA2 genes were detected by both methodologies, with a 100 % concordance between them. This strategy allowed the detection of a total of 39 deleterious mutations in the 94 index patients, namely 10 in BRCA1 (25.6 %), 21 in BRCA2 (53.8 %), four in PALB2 (10.3 %), two in ATM (5.1 %), one in CHEK2 (2.6 %), and one in TP53 (2.6 %), with 20.5 % of the deleterious mutations being found in genes other than BRCA1/BRCA2. These results demonstrate the efficiency of NGS for the detection of BRCA1/BRCA2 point mutations and highlight the genetic heterogeneity of HBOC. PMID:27553368

  6. Evaluation of a gp63–PCR Based Assay as a Molecular Diagnosis Tool in Canine Leishmaniasis in Tunisia

    PubMed Central

    Guerbouj, Souheila; Djilani, Fattouma; Bettaieb, Jihene; Lambson, Bronwen; Diouani, Mohamed Fethi; Ben Salah, Afif; Ben Ismail, Riadh; Guizani, Ikram

    2014-01-01

    A gp63PCR method was evaluated for the detection and characterization of Leishmania (Leishmania) (L.) parasites in canine lymph node aspirates. This tool was tested and compared to other PCRs based on the amplification of 18S ribosomal genes, a L. infantum specific repetitive sequence and kinetoplastic DNA minicircles, and to classical parasitological (smear examination and/or culture) or serological (IFAT) techniques on a sample of 40 dogs, originating from different L. infantum endemic regions in Tunisia. Sensitivity and specificity of all the PCR assays were evaluated on parasitologically confirmed dogs within this sample (N = 18) and control dogs (N = 45) originating from non–endemic countries in northern Europe and Australia. The gp63 PCR had 83.5% sensitivity and 100% specificity, a performance comparable to the kinetoplast PCR assay and better than the other assays. These assays had comparable results when the gels were southern transferred and hybridized with a radioactive probe. As different infection rates were found according to the technique, concordance of the results was estimated by (κ) test. Best concordance values were between the gp63PCR and parasitological methods (74.6%, 95% confidence intervals CI: 58.8–95.4%) or serology IFAT technique (47.4%, 95% CI: 23.5–71.3%). However, taken together Gp63 and Rib assays covered most of the samples found positive making of them a good alternative for determination of infection rates. Potential of the gp63PCR-RFLP assay for analysis of parasite genetic diversity within samples was also evaluated using 5 restriction enzymes. RFLP analysis confirmed assignment of the parasites infecting the dogs to L. infantum species and illustrated occurrence of multiple variants in the different endemic foci. Gp63 PCR assay thus constitutes a useful tool in molecular diagnosis of L. infantum infections in dogs in Tunisia. PMID:25153833

  7. Korean Society for Laboratory Medicine Practice Guidelines for the Molecular Diagnosis of Middle East Respiratory Syndrome During an Outbreak in Korea in 2015

    PubMed Central

    Ki, Chang-Seok; Lee, Hyukmin; Sung, Heungsup; Kim, Sinyoung; Seong, Moon-Woo; Yong, Dongeun; Kim, Jae-Seok; Lee, Mi-Kyung; Choi, Jong-Rak; Kim, Jeong-Ho

    2016-01-01

    For two months between May and July 2015, a nationwide outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) occurred in Korea. On June 3, 2015, the Korean Society for Laboratory Medicine (KSLM) launched a MERS-CoV Laboratory Response Task Force (LR-TF) to facilitate clinical laboratories to set up the diagnosis of MERS-CoV infection. Based on the WHO interim recommendations, the Centers for Disease Control and Prevention of United States guidelines for MERS-CoV laboratory testing, and other available resources, the KSLM MERS-CoV LR-TF provided the first version of the laboratory practice guidelines for the molecular diagnosis of MERS-CoV to the clinical laboratories on June 12, 2015. The guidelines described here are an updated version that includes case definition, indications for testing, specimen type and protocols for specimen collection, specimen packing and transport, specimen handling and nucleic acid extraction, molecular detection of MERS-CoV, interpretation of results and reporting, and laboratory safety. The KSLM guidelines mainly focus on the molecular diagnosis of MERS-CoV, reflecting the unique situation in Korea and the state of knowledge at the time of publication. PMID:26915607

  8. Korean Society for Laboratory Medicine Practice Guidelines for the Molecular Diagnosis of Middle East Respiratory Syndrome During an Outbreak in Korea in 2015.

    PubMed

    Ki, Chang Seok; Lee, Hyukmin; Sung, Heungsup; Kim, Sinyoung; Seong, Moon Woo; Yong, Dongeun; Kim, Jae Seok; Lee, Mi Kyung; Kim, Mi Na; Choi, Jong Rak; Kim, Jeong Ho

    2016-05-01

    For two months between May and July 2015, a nationwide outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) occurred in Korea. On June 3, 2015, the Korean Society for Laboratory Medicine (KSLM) launched a MERS-CoV Laboratory Response Task Force (LR-TF) to facilitate clinical laboratories to set up the diagnosis of MERS-CoV infection. Based on the WHO interim recommendations, the Centers for Disease Control and Prevention of United States guidelines for MERS-CoV laboratory testing, and other available resources, the KSLM MERS-CoV LR-TF provided the first version of the laboratory practice guidelines for the molecular diagnosis of MERS-CoV to the clinical laboratories on June 12, 2015. The guidelines described here are an updated version that includes case definition, indications for testing, specimen type and protocols for specimen collection, specimen packing and transport, specimen handling and nucleic acid extraction, molecular detection of MERS-CoV, interpretation of results and reporting, and laboratory safety. The KSLM guidelines mainly focus on the molecular diagnosis of MERS-CoV, reflecting the unique situation in Korea and the state of knowledge at the time of publication.

  9. Novel, Precise, Accurate Ion-Pairing Method to Determine the Related Substances of the Fondaparinux Sodium Drug Substance: Low-Molecular-Weight Heparin

    PubMed Central

    Deshpande, Amol A.; Madhavan, P.; Deshpande, Girish R.; Chandel, Ravi Kumar; Yarbagi, Kaviraj M.; Joshi, Alok R.; Moses Babu, J.; Murali Krishna, R.; Rao, I. M.

    2016-01-01

    Fondaparinux sodium is a synthetic low-molecular-weight heparin (LMWH). This medication is an anticoagulant or a blood thinner, prescribed for the treatment of pulmonary embolism and prevention and treatment of deep vein thrombosis. Its determination in the presence of related impurities was studied and validated by a novel ion-pair HPLC method. The separation of the drug and its degradation products was achieved with the polymer-based PLRPs column (250 mm × 4.6 mm; 5 μm) in gradient elution mode. The mixture of 100 mM n-hexylamine and 100 mM acetic acid in water was used as buffer solution. Mobile phase A and mobile phase B were prepared by mixing the buffer and acetonitrile in the ratio of 90:10 (v/v) and 20:80 (v/v), respectively. Mobile phases were delivered in isocratic mode (2% B for 0–5 min) followed by gradient mode (2–85% B in 5–60 min). An Evaporative Light Scattering Detector (ELSD) was connected to the LC system to detect the responses of chromatographic separation. Further, the drug was subjected to stress studies for acidic, basic, oxidative, photolytic, and thermal degradations as per ICH guidelines and the drug was found to be labile in acid, base hydrolysis, and oxidation, while stable in neutral, thermal, and photolytic degradation conditions. The method provided linear responses over the concentration range of the LOQ to 0.30% for each impurity with respect to the analyte concentration of 12.5 mg/mL, and regression analysis showed a correlation coefficient value (r2) of more than 0.99 for all the impurities. The LOD and LOQ were found to be 1.4 µg/mL and 4.1 µg/mL, respectively, for fondaparinux. The developed ion-pair method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness. PMID:27110496

  10. Novel, Precise, Accurate Ion-Pairing Method to Determine the Related Substances of the Fondaparinux Sodium Drug Substance: Low-Molecular-Weight Heparin.

    PubMed

    Deshpande, Amol A; Madhavan, P; Deshpande, Girish R; Chandel, Ravi Kumar; Yarbagi, Kaviraj M; Joshi, Alok R; Moses Babu, J; Murali Krishna, R; Rao, I M

    2016-01-01

    Fondaparinux sodium is a synthetic low-molecular-weight heparin (LMWH). This medication is an anticoagulant or a blood thinner, prescribed for the treatment of pulmonary embolism and prevention and treatment of deep vein thrombosis. Its determination in the presence of related impurities was studied and validated by a novel ion-pair HPLC method. The separation of the drug and its degradation products was achieved with the polymer-based PLRPs column (250 mm × 4.6 mm; 5 μm) in gradient elution mode. The mixture of 100 mM n-hexylamine and 100 mM acetic acid in water was used as buffer solution. Mobile phase A and mobile phase B were prepared by mixing the buffer and acetonitrile in the ratio of 90:10 (v/v) and 20:80 (v/v), respectively. Mobile phases were delivered in isocratic mode (2% B for 0-5 min) followed by gradient mode (2-85% B in 5-60 min). An Evaporative Light Scattering Detector (ELSD) was connected to the LC system to detect the responses of chromatographic separation. Further, the drug was subjected to stress studies for acidic, basic, oxidative, photolytic, and thermal degradations as per ICH guidelines and the drug was found to be labile in acid, base hydrolysis, and oxidation, while stable in neutral, thermal, and photolytic degradation conditions. The method provided linear responses over the concentration range of the LOQ to 0.30% for each impurity with respect to the analyte concentration of 12.5 mg/mL, and regression analysis showed a correlation coefficient value (r(2)) of more than 0.99 for all the impurities. The LOD and LOQ were found to be 1.4 µg/mL and 4.1 µg/mL, respectively, for fondaparinux. The developed ion-pair method was validated as per ICH guidelines with respect to accuracy, selectivity, precision, linearity, and robustness. PMID:27110496

  11. Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy.

    PubMed

    Xiao, Heng; Chen, Yuqi; Yuan, Erfeng; Li, Wei; Jiang, Zhuoran; Wei, Lai; Su, Haomiao; Zeng, Weiwu; Gan, Yunjiu; Wang, Zijing; Yuan, Bifeng; Qin, Shanshan; Leng, Xiaohua; Zhou, Xin; Liu, Songmei; Zhou, Xiang

    2016-09-14

    Early cancer diagnosis is of great significance to relative cancer prevention and clinical therapy, and it is crucial to efficiently recognize cancerous tumor sites at the molecular level. Herein, we proposed a versatile and efficient strategy based on aptamer recognition and photoactivation imaging for cancer diagnosis. This is the first time that a visible light-controlled photoactivatable aptamer-based platform has been applied for cancer diagnosis. The photoactivatable aptamer-based strategy can accurately detect nucleolin-overexpressed tumor cells and can be used for highly selective cancer cell screening and tissue imaging. This strategy is available for both formalin-fixed paraffin-embedded tissue specimens and frozen sections. Moreover, the photoactivation techniques showed great progress in more accurate and persistent imaging to the use of traditional fluorophores. Significantly, the application of this strategy can produce the same accurate results in tissue specimen analysis as with classical hematoxylin-eosin staining and immunohistochemical technology.

  12. Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy.

    PubMed

    Xiao, Heng; Chen, Yuqi; Yuan, Erfeng; Li, Wei; Jiang, Zhuoran; Wei, Lai; Su, Haomiao; Zeng, Weiwu; Gan, Yunjiu; Wang, Zijing; Yuan, Bifeng; Qin, Shanshan; Leng, Xiaohua; Zhou, Xin; Liu, Songmei; Zhou, Xiang

    2016-09-14

    Early cancer diagnosis is of great significance to relative cancer prevention and clinical therapy, and it is crucial to efficiently recognize cancerous tumor sites at the molecular level. Herein, we proposed a versatile and efficient strategy based on aptamer recognition and photoactivation imaging for cancer diagnosis. This is the first time that a visible light-controlled photoactivatable aptamer-based platform has been applied for cancer diagnosis. The photoactivatable aptamer-based strategy can accurately detect nucleolin-overexpressed tumor cells and can be used for highly selective cancer cell screening and tissue imaging. This strategy is available for both formalin-fixed paraffin-embedded tissue specimens and frozen sections. Moreover, the photoactivation techniques showed great progress in more accurate and persistent imaging to the use of traditional fluorophores. Significantly, the application of this strategy can produce the same accurate results in tissue specimen analysis as with classical hematoxylin-eosin staining and immunohistochemical technology. PMID:27550088

  13. Communication: Rate coefficients of the H + CH{sub 4} → H{sub 2} + CH{sub 3} reaction from ring polymer molecular dynamics on a highly accurate potential energy surface

    SciTech Connect

    Meng, Qingyong Chen, Jun Zhang, Dong H.

    2015-09-14

    The ring polymer molecular dynamics (RPMD) calculations are performed to calculate rate constants for the title reaction on the recently constructed potential energy surface based on permutation invariant polynomial (PIP) neural-network (NN) fitting [J. Li et al., J. Chem. Phys. 142, 204302 (2015)]. By inspecting convergence, 16 beads are used in computing free-energy barriers at 300 K ≤ T ≤ 1000 K, while different numbers of beads are used for transmission coefficients. The present RPMD rates are in excellent agreement with quantum rates computed on the same potential energy surface, as well as with the experimental measurements, demonstrating further that the RPMD is capable of producing accurate rates for polyatomic chemical reactions even at rather low temperatures.

  14. Laboratory Diagnosis of Amebiasis

    PubMed Central

    Tanyuksel, Mehmet; Petri, William A.

    2003-01-01

    The detection of Entamoeba histolytica, the causative agent of amebiasis, is an important goal of the clinical microbiology laboratory. To assess the scope of E. histolytica infection, it is necessary to utilize accurate diagnostic tools. As more is discovered about the molecular and cell biology of E. histolytica, there is great potential for further understanding the pathogenesis of amebiasis. Molecular biology-based diagnosis may become the technique of choice in the future because establishment of these protozoa in culture is still not a routine clinical laboratory process. In all cases, combination of serologic tests with detection of the parasite (by antigen detection or PCR) offers the best approach to diagnosis, while PCR techniques remain impractical in many developing country settings. The detection of amebic markers in serum in patients with amebic colitis and liver abscess appears promising but is still only a research tool. On the other hand, stool antigen detection tests offer a practical, sensitive, and specific way for the clinical laboratory to detect intestinal E. histolytica. All the current tests suffer from the fact that the antigens detected are denatured by fixation of the stool specimen, limiting testing to fresh or frozen samples. PMID:14557296

  15. Rapid detection of live methicillin-resistant Staphylococcus aureus by using an integrated microfluidic system capable of ethidium monoazide pre-treatment and molecular diagnosis

    PubMed Central

    Liu, Yu-Hsin; Wang, Chih-Hung; Wu, Jiunn-Jong; Lee, Gwo-Bin

    2012-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium resistant to all existing penicillin and lactam-based antimicrobial drugs and, therefore, has become one of the most prevalent antibiotic-resistant pathogens found in hospitals. The multi-drug resistant characteristics of MRSA make it challenging to clinically treat infected patients. Therefore, early diagnosis of MRSA has become a public-health priority worldwide. Conventionally, cell-culture based methodology and microscopic identification are commonly used for MRSA detection. However, they are relatively time-consuming and labor-intensive. Recently, molecular diagnosis based on nucleic acid amplification techniques, such as polymerase chain reaction (PCR), has been widely investigated for the rapid detection of MRSA. However, genomic DNA of both live and dead pathogens can be distinguished by conventional PCR. These results thus could not provide sufficient confirmation of an active infection for clinicians. In this study, live MRSA was rapidly detected by using a new integrated microfluidic system. The microfluidic system has been demonstrated to have 100% specificity to detect live MRSA with S. aureus and other pathogens commonly found in hospitals. The experimental results showed that the limit of detection for live MRSA from biosamples was approximately 102 CFU/μl. In addition, the entire diagnostic protocol, from sample pre-treatment to fluorescence observation, can be automatically completed within 2.5 h. Consequently, this microfluidic system may be a powerful tool for the rapid molecular diagnosis of live MRSA. PMID:24019858

  16. Accurate Molecular Dimensions from Stearic Acid Monolayers.

    ERIC Educational Resources Information Center

    Lane, Charles A.; And Others

    1984-01-01

    Discusses modifications in the fatty acid monolayer experiment to reduce the inaccurate moleculary data students usually obtain. Copies of the experimental procedure used and a Pascal computer program to work up the data are available from the authors. (JN)

  17. An International MDS/MPN Working Group’s perspective and recommendations on molecular pathogenesis, diagnosis and clinical characterization of myelodysplastic/myeloproliferative neoplasms

    PubMed Central

    Mughal, Tariq I.; Cross, Nicholas C.P.; Padron, Eric; Tiu, Ramon V.; Savona, Michael; Malcovati, Luca; Tibes, Raoul; Komrokji, Rami S.; Kiladjian, Jean-Jacques; Garcia-Manero, Guillermo; Orazi, Attilio; Mesa, Ruben; Maciejewski, Jaroslaw P.; Fenaux, Pierre; Itzykson, Raphael; Mufti, Ghulam; Solary, Eric; List, Alan F.

    2015-01-01

    In the 2008 WHO classification, chronic myeloid malignancies that share both myelodysplastic and myeloproliferative features define the myelodysplastic/myeloproliferative group, which includes chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, atypical chronic myeloid leukemia, refractory anemia with ring sideroblasts and thrombocytosis, and myelodysplastic/myeloproliferative unclassified. With the notable exception of refractory anemia with ring sideroblasts and thrombocytosis, there is much overlap among the various subtypes at the molecular and clinical levels, and a better definition of these entities, an understanding of their biology and an identification of subtype-specific molecular or cellular markers are needed. To address some of these challenges, a panel comprised of laboratory and clinical experts in myelodysplastic/myeloproliferative was established, and four independent academic MDS/MPN workshops were held on: 9th March 2013, in Miami, Florida, USA; 6th December 2013, in New Orleans, Louisiana, USA; 13th June 2014 in Milan, Italy; and 5th December 2014 in San Francisco, USA. During these meetings, the current understanding of these malignancies and matters of biology, diagnosis and management were discussed. This perspective and the recommendations on molecular pathogenesis, diagnosis and clinical characterization for adult onset myelodysplastic/myeloproliferative is the result of a collaborative project endorsed and supported by the MDS Foundation. PMID:26341525

  18. Value of the oral swab for the molecular diagnosis of dogs in different stages of infection with Leishmania infantum.

    PubMed

    Aschar, Mariana; de Oliveira, Eveline Tozzi Braga; Laurenti, Marcia Dalastra; Marcondes, Mary; Tolezano, Jose Eduardo; Hiramoto, Roberto Mitsuyoshi; Corbett, Carlos Eduardo P; da Matta, Vania Lucia Ribeiro

    2016-07-30

    OS and CS combination based on qPCR notably improves the detection of both AD and SD dogs. In conclusion, OS proved to be a suitable sample for the molecular diagnosis of infected dogs with clinical signs of VL, but not for dogs with inapparent infection. For these, we recommend the combination of OS results with CS and/or serology in order to reach relevant positivity for L. infantum. Finally, another advantage of using OS or both noninvasive samples is the increased likelihood of diagnosing seronegative dogs. PMID:27369584

  19. Current Proceedings in the Molecular Dissection of Hepatocellular Adenomas: Review and Hands-on Guide for Diagnosis.

    PubMed

    Goltz, Diane; Fischer, Hans-Peter

    2015-01-01

    Molecular dissection of hepatocellular adenomas has brought forward a diversity of well-defined entities. Their distinction is important for routine practice, since prognosis is tightly related to the individual subgroup. Very recent activity has generated new details on the molecular background of hepatocellular adenoma, which this article aims to integrate into the current concepts of taxonomy. PMID:26404250

  20. Overview of food allergy diagnosis

    PubMed Central

    MANEA, IRENA; AILENEI, ELENA; DELEANU, DIANA

    2016-01-01

    Food allergy is a condition with significant social and economic impact and a topic of intense concern for scientists and clinicians alike. Worldwide, over 220 million people suffer from some form of food allergy, but the number reported is just the tip of the iceberg. Recent years have brought new perspectives in diagnosing food allergy. Elucidating incriminated immunological mechanisms, along with drawing the clinical phenotype of food hypersensitivity reactions ensures an accurate diagnosis of food allergy. Moreover, molecular based allergy diagnosis, which is increasingly used in routine care, is a stepping-stone to improved management of food allergy patients. The aim of this review is to summarize the topic of IgE-mediated food allergy from the perspective of current diagnostic methods. PMID:27004019

  1. Coinfection with "Rickettsia sibirica subsp. mongolotimonae" and Rickettsia conorii in a Human Patient: a Challenge for Molecular Diagnosis Tools.

    PubMed

    Nogueras, María Mercedes; Roson, Beatriz; Lario, Sergio; Sanfeliu, Isabel; Pons, Immaculada; Anton, Esperança; Casanovas, Aurora; Segura, Ferran

    2015-09-01

    Rickettsioses are zoonoses transmitted by vectors. More than one agent can coexist in vectors. Although vectors may transmit more than one microorganism to humans, information on dual infections is scarce. We present a case of a patient with an atypical rickettsiosis diagnosis in whom two species of Rickettsia were detected.

  2. Next-generation sequencing (NGS) as a fast molecular diagnosis tool for left ventricular noncompaction in an infant with compound mutations in the MYBPC3 gene.

    PubMed

    Schaefer, Elise; Helms, Pauline; Marcellin, Luc; Desprez, Philippe; Billaud, Philippe; Chanavat, Valérie; Rousson, Robert; Millat, Gilles

    2014-03-01

    Left ventricular noncompaction (LVNC) is a clinically heterogeneous disorder characterized by a trabecular meshwork and deep intertrabecular myocardial recesses that communicate with the left ventricular cavity. LVNC is classified as a rare genetic cardiomyopathy. Molecular diagnosis is a challenge for the medical community as the condition shares morphologic features of hypertrophic and dilated cardiomyopathies. Several genetic causes of LVNC have been reported, with variable modes of inheritance, including autosomal dominant and X-linked inheritance, but relatively few responsible genes have been identified. In this report, we describe a case of a severe form of LVNC leading to death at 6 months of life. NGS sequencing using a custom design for hypertrophic cardiomyopathy panel allowed us to identify compound heterozygosity in the MYBPC3 gene (p.Lys505del, p.Pro955fs) in 3 days, confirming NGS sequencing as a fast molecular diagnosis tool. Other studies have reported neonatal presentation of cardiomyopathies associated with compound heterozygous or homozygous MYBPC3 mutations. In this family and in families in which parental truncating MYBPC3 mutations are identified, preimplantation or prenatal genetic screening should be considered as these genotypes leads to neonatal mortality and morbidity.

  3. Prenatal diagnosis of a partial trisomy 13q (q14-->qter): phenotype, cytogenetics and molecular characterization by spectral karyotyping and array comparative genomic hybridization.

    PubMed

    Machado, I N; Heinrich, J K; Campanhol, C; Rodrigues-Peres, R M; Oliveira, F M; Barini, R

    2010-03-16

    Partial trisomy 13q is an uncommon chromosomal abnormality with variable phenotypic expression. We report prenatal diagnosis of partial trisomy 13q in a fetus with partial agenesis of the cerebellar vermis, partial agenesis of the corpus callosum, hydrops and polyhydramnios. G-banding karyotyping, spectral karyotyping and array comparative genomic hybridization (aCGH) analysis of fetal blood were performed. Cytogenetic analysis of fetal blood displayed 46,XX,add(4)(q28). The parental karyotypes were normal. A girl was delivered at 34 weeks gestation; she died within 2 h. Autopsy confirmed all the prenatal findings and also showed agenesis of the diaphragm. Spectral karyotyping identified the additional material's origin as chromosome 13. aCGH was carried out and showed amplification of distal regions of the long arm of chromosome 13 from region 13q14 to qter. This is the first report of a fetus with molecular characterization of a partial trisomy 13q (q14-->qter), present as a de novo unbalanced translocation at chromosome 4q. This case demonstrates the usefulness of molecular characterization of malformed fetuses for prenatal diagnosis and counseling.

  4. Prenatal diagnosis of a partial trisomy 13q (q14-->qter): phenotype, cytogenetics and molecular characterization by spectral karyotyping and array comparative genomic hybridization.

    PubMed

    Machado, I N; Heinrich, J K; Campanhol, C; Rodrigues-Peres, R M; Oliveira, F M; Barini, R

    2010-01-01

    Partial trisomy 13q is an uncommon chromosomal abnormality with variable phenotypic expression. We report prenatal diagnosis of partial trisomy 13q in a fetus with partial agenesis of the cerebellar vermis, partial agenesis of the corpus callosum, hydrops and polyhydramnios. G-banding karyotyping, spectral karyotyping and array comparative genomic hybridization (aCGH) analysis of fetal blood were performed. Cytogenetic analysis of fetal blood displayed 46,XX,add(4)(q28). The parental karyotypes were normal. A girl was delivered at 34 weeks gestation; she died within 2 h. Autopsy confirmed all the prenatal findings and also showed agenesis of the diaphragm. Spectral karyotyping identified the additional material's origin as chromosome 13. aCGH was carried out and showed amplification of distal regions of the long arm of chromosome 13 from region 13q14 to qter. This is the first report of a fetus with molecular characterization of a partial trisomy 13q (q14-->qter), present as a de novo unbalanced translocation at chromosome 4q. This case demonstrates the usefulness of molecular characterization of malformed fetuses for prenatal diagnosis and counseling. PMID:20391329

  5. RNA and DNA bacteriophages as molecular diagnosis controls in clinical virology: a comprehensive study of more than 45,000 routine PCR tests.

    PubMed

    Ninove, Laetitia; Nougairede, Antoine; Gazin, Celine; Thirion, Laurence; Delogu, Ilenia; Zandotti, Christine; Charrel, Remi N; De Lamballerie, Xavier

    2011-02-09

    Real-time PCR techniques are now commonly used for the detection of viral genomes in various human specimens and require for validation both external and internal controls (ECs and ICs). In particular, ICs added to clinical samples are necessary to monitor the extraction, reverse transcription, and amplification steps in order to detect false-negative results resulting from PCR-inhibition or errors in the technical procedure. Here, we performed a large scale evaluation of the use of bacteriophages as ICs in routine molecular diagnosis. This allowed to propose simple standardized procedures (i) to design specific ECs for both DNA and RNA viruses and (ii) to use T4 (DNA) or MS2 (RNA) phages as ICs in routine diagnosis. Various technical formats for using phages as ICs were optimised and validated. Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. The frequency of inefficient detection of ICs was analyzed according to the nature of the sample. Inhibitors of enzymatic reactions were detected at high frequency in specific sample types such as heparinized blood and bone marrow (>70%), broncho-alveolar liquid (41%) and stools (36%). The use of T4 and MS2 phages as ICs proved to be cost-effective, flexible and adaptable to various technical procedures of real-time PCR detection in virology. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids.

  6. Molecular diagnosis of Prader-Willi syndrome: Comparison of cytogenetic and molecular genetic data including parent of origin dependent methylation DNA patterns

    SciTech Connect

    Butler, M.G.

    1996-01-11

    This letter to the editor discusses a recent study concerning the parent-of-origin methylation site at D15S63 and its application in molecular diagnostics confirming Prader-Willi syndrome (PWS). It concludes that more research is needed to clear up questions which remain regarding the causation of PWS. 20 refs., 1 tab.

  7. [Molecular diagnosis of HFE mutations in routine laboratories. Results of a survey from reference laboratories in France].

    PubMed

    Jouanolle, Anne-Marie; Gérolami, Victoria; Ged, Cécile; Grandchamp, Bernard; Le Gac, Gérald; Pissard, Serge; Rochette, Jacques; Aguilar-Martinez, Patricia

    2012-01-01

    HFE-related hemochromatosis (HFE hemochromatosis) or type 1 hemochromatosis is an autosomal recessive disease characterized by progressive iron overload usually expressed in adulthood. The HFE gene, located on the short arm of chromosome 6 (6p21.3), encodes a protein that plays a crucial role in iron metabolism by modulating hepcidin synthesis in the liver. Homozygosity for the p.Cys282Tyr mutation accounts for nearly 80% of cases of hemochromatosis in France. Genetic testing is the key investigation to confirm the diagnosis of HFE hemochromatosis. A survey on routine practices was carried out among the eight reference laboratories of the French national network on genetic iron disorders. The main findings from this survey are as follows: 1) the p.Cys282Tyr mutation must be searched for as an initial step to establish the diagnosis of HFE hemochromatosis. This is in agreement with the recommendations of the French Health Authority (HAS) published in 2005. In these recommendations, homozygosity for the p.Cys282Tyr mutation with at least elevated transferrin saturation, is considered the only genotype that confirms of the diagnosis of HFE hemochromatosis; 2) in combination with the p.Cys282Tyr mutation (compound heterozygous genotypes), the p.Ser65Cys and the p.His63Asp variants may contribute to the occurrence of mild iron overload; 3) family screening is mandatory following the detection of homozygous individuals for the p.Cys282Tyr mutation.

  8. A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy

    PubMed Central

    Ben Rekaya, Mariem; Laroussi, Nadia; Messaoud, Olfa; Jones, Mariem; Jerbi, Manel; Bouyacoub, Yosra; Chargui, Mariem; Kefi, Rym; Fazaa, Becima; Boubaker, Mohamed Samir; Boussen, Hamouda; Mokni, Mourad; Abdelhak, Sonia; Zghal, Mohamed; Khaled, Aida; Yacoub-Youssef, Houda

    2014-01-01

    Xeroderma pigmentosum Variant (XP-V) form is characterized by a late onset of skin symptoms. Our aim is the clinical and genetic investigations of XP-V Tunisian patients in order to develop a simple tool for early diagnosis. We investigated 16 suspected XP patients belonging to ten consanguineous families. Analysis of the POLH gene was performed by linkage analysis, long range PCR, and sequencing. Genetic analysis showed linkage to the POLH gene with a founder haplotype in all affected patients. Long range PCR of exon 9 to exon 11 showed a 3926 bp deletion compared to control individuals. Sequence analysis demonstrates that this deletion has occurred between two Alu-Sq2 repetitive sequences in the same orientation, respectively, in introns 9 and 10. We suggest that this mutation POLH NG_009252.1: g.36847_40771del3925 is caused by an equal crossover event that occurred between two homologous chromosomes at meiosis. These results allowed us to develop a simple test based on a simple PCR in order to screen suspected XP-V patients. In Tunisia, the prevalence of XP-V group seems to be underestimated and clinical diagnosis is usually later. Cascade screening of this founder mutation by PCR in regions with high frequency of XP provides a rapid and cost-effective tool for early diagnosis of XP-V in Tunisia and North Africa. PMID:24877075

  9. The Simplexa™ Group A Strep Direct Assay: a sample-to-answer molecular assay for the diagnosis of group A streptococcal pharyngitis.

    PubMed

    Tabb, Michelle M; Batterman, Hollis J

    2016-01-01

    The Simplexa™ Group A Strep Direct assay is intended for use on the Integrated Cycler for detection of Group A Streptococcus (GAS) directly from throat swabs that have not undergone nucleic acid extraction. A prospective study of 1352 samples in 4 geographically diverse sites showed an overall prevalence of GAS of 15.4%. The assay demonstrated 97.4% sensitivity and 95.2% specificity versus culture. The positive predictive value compared to culture was 72.7%. However, 46 out of 57 discrepant samples were Group A Strep positive when tested using a bi-directional sequencing method illustrating the increased sensitivity of the assay compared to culture for detection of GAS. Rapid and accurate diagnosis of GAS allows for timely treatment to decrease complications of this prevalent organism that continues to cause substantial morbidity and mortality worldwide. PMID:26679835

  10. Cervical cancer: Biomarkers for diagnosis and treatment.

    PubMed

    Dasari, Subramanyam; Wudayagiri, Rajendra; Valluru, Lokanatha

    2015-05-20

    Cervical cancer is a major gynecological cancer which involves uncontrolled cell division and tissue invasiveness of the female uterine cervix. With the availability of new technologies researchers have increased their efforts to develop novel biomarkers for early diagnosis, and evaluation and monitoring of therapeutic treatments. This approach will help in the development of early diagnosis and in increasing treatment efficacy with decreased recurrence. The present review explains the currently available biomarkers for cervical cancer diagnosis and prognosis. Apart from the currently available biomarkers the review also explains strategies for the development of biomarkers based on cellular and molecular approaches such as DNA, protein and other metabolic markers with suitable clinical examples. The investigations of specific proteins, enzymes and metabolites will establish more useful biomarkers for accurate detection and management of gynecological cancers especially cervical cancer.

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

    PubMed Central

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

    2015-01-01

    Context: 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. Objective: This study aimed to evaluate a novel diagnostic algorithm combining mutation detection and miRNA expression to improve the diagnostic yield of molecular cytology. Setting: 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. Design: 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. Results: 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%. Conclusions: 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

  12. Improving molecular diagnosis of Chinese patients with Charcot-Marie-Tooth by targeted next-generation sequencing and functional analysis

    PubMed Central

    Liu, Zhi-Jun; Ni, Wang; Li, Hong-Fu; Xiao, Bao-Guo; Wu, Zhi-Ying

    2016-01-01

    Charcot-Marie-Tooth (CMT) disease is the most common hereditary peripheral neuropathy. More than 50 causative genes have been identified. The lack of genotype-phenotype correlations in many CMT patients make it difficult to decide which genes are affected. Recently, targeted next-generation sequencing (NGS) has been introduced as an alternative approach for diagnosis of genetic disorders. Here, we applied targeted NGS in combination with PMP22 duplication/deletion analysis to screen causative genes in 22 Chinese CMT families. The novel variants detected by targeted NGS were then further studied in cultured cells. Of the 22 unrelated patients, 8 had PMP22 duplication. The targeted NGS revealed 10 possible pathogenic variants in 11 patients, including 7 previously reported variants and 3 novel heterozygous variants (GJB1: p.Y157H; MFN2: p.G127S; YARS: p.V293M). Further classification of the novel variants according to American College of Medical Genetics and Genomics (ACMG) standards and guidelines and functional analysis in cultured cells indicated that p.Y157H in GJB1 was pathogenic, p.G127S in MFN2 was likely pathogenic, while p.V293M in YARS was likely benign. Our results suggest the potential for targeted NGS to make a more rapid and precise diagnosis in CMT patients. Moreover, the functional analysis is required when the novel variants are indistinct. PMID:27027447

  13. The Use of Next-Generation Sequencing in Molecular Diagnosis of Neurofibromatosis Type 1: A Validation Study

    PubMed Central

    Maruoka, Ryo; Takenouchi, Toshiki; Torii, Chiharu; Shimizu, Atsushi; Misu, Kumiko; Higasa, Koichiro; Matsuda, Fumihiko; Ota, Arihito; Tanito, Katsumi; Kuramochi, Akira; Arima, Yoshimi; Otsuka, Fujio; Yoshida, Yuichi; Moriyama, Keiji; Niimura, Michihito; Saya, Hideyuki

    2014-01-01

    Aims: We assessed the validity of a next-generation sequencing protocol using in-solution hybridization-based enrichment to identify NF1 mutations for the diagnosis of 86 patients with a prototypic genetic syndrome, neurofibromatosis type 1. In addition, other causative genes for classic genetic syndromes were set as the target genes for coverage analysis. Results: The protocol identified 30 nonsense, 19 frameshift, and 8 splice-site mutations, together with 10 nucleotide substitutions that were previously reported to be pathogenic. In the remaining 19 samples, 10 had single-exon or multiple-exon deletions detected by a multiplex ligation-dependent probe amplification method and 3 had missense mutations that were not observed in the normal Japanese SNP database and were predicted to be pathogenic. Coverage analysis of the genes other than the NF1 gene included on the same diagnostic panel indicated that the mean coverage was 115-fold, a sufficient depth for mutation detection. Conclusions: The overall mutation detection rate using the currently reported method in 86 patients who met the clinical diagnostic criteria was 92.1% (70/76) when 10 patients with large deletions were excluded. The results validate the clinical utility of this next-generation sequencing-based method for the diagnosis of neurofibromatosis type 1. Comparable detection rates can be expected for other genetic syndromes, based on the results of the coverage analysis. PMID:25325900

  14. European guidance for the molecular diagnosis of pseudohypoparathyroidism not caused by point genetic variants at GNAS: an EQA study

    PubMed Central

    Garin, Intza; Mantovani, Giovanna; Aguirre, Urko; Barlier, Anne; Brix, Bettina; Elli, Francesca M; Freson, Kathleen; Grybek, Virginie; Izzi, Benedetta; Linglart, Agnès; Perez de Nanclares, Guiomar; Silve, Caroline; Thiele, Susanne; Werner, Ralf

    2015-01-01

    Pseudohypoparathyroidism is a rare endocrine disorder that can be caused by genetic (mainly maternally inherited inactivating point mutations, although intragenic and gross deletions have rarely been reported) or epigenetic alterations at GNAS locus. Clinical and molecular characterization of this disease is not that easy because of phenotypic, biochemical and molecular overlapping features between both subtypes of the disease. The European Consortium for the study of PHP (EuroPHP) designed the present work with the intention of generating the standards of diagnostic clinical molecular (epi)genetic testing in PHP patients. With this aim, DNA samples of eight independent PHP patients carrying GNAS genetic and/or epigenetic defects (three patients with GNAS deletions, two with 20q uniparental disomy and three with a methylation defect of unknown origin) without GNAS point mutations were anonymized and sent to the five participant laboratories for their routine genetic analysis (methylation-specific (MS)-MLPA, pyrosequencing and EpiTYPER) and interpretations. All laboratories were able to detect methylation defects and, after the data analysis, the Consortium compared the results to define technical advantages and disadvantages of different techniques. To conclude, we propose as first-level investigation in PHP patients copy number and methylation analysis by MS-MLPA. Then, in patients with partial methylation defect, the result should be confirmed by single CpG bisulphite-based methods (ie pyrosequencing), whereas in case of a complete methylation defect without detectable deletion, microsatellites or SNP genotyping should be performed to exclude uniparental disomy 20. PMID:25005735

  15. In vivo molecular imaging using nanomaterials: general in vivo characteristics of nano-sized reagents and applications for cancer diagnosis.

    PubMed

    Rosenblum, Lauren T; Kosaka, Nobuyuki; Mitsunaga, Makoto; Choyke, Peter L; Kobayashi, Hisataka

    2010-10-01

    Nanoparticles present a new collection of contrast agents for the field of in vivo molecular imaging. This review focuses on promising molecular imaging probes for optical and magnetic resonance imaging based on four representative nanomaterial(s) platforms: quantum dots, upconversion phosphors, superparamagnetic iron oxides, and dendrimer-based agents. Quantum dots are extremely efficient fluorescent nanoparticles with size-tunable emission properties, enabling high sensitivity and greater depth penetration. Their heavy metal composition and long retention in the body, however, pose concerns for clinical translational applications. Upconversion phosphors generate excellent signal-to-background contrast because they emit light with higher energy than the excitation photons and autofluorescence signals. For MRI, iron oxide particles also generate excellent signal and have been used in liver imaging and for cell tracking studies. As they are metabolized through endogenous iron salvage pathways, they have already been introduced as clinical contrast agents. Lastly, dendrimers, a 'soft' nanoparticle, can be used as a structural basis for the attachment of small molecule imaging agents and/or targeting groups. This array of nanoparticles should offer insights into the uses and potentials of nanoparticles for the molecular imaging.

  16. Improvement of the management of infants, children and adults with a molecular diagnosis of Enterovirus meningitis during two observational study periods.

    PubMed

    Archimbaud, Christine; Ouchchane, Lemlih; Mirand, Audrey; Chambon, Martine; Demeocq, François; Labbé, André; Laurichesse, Henri; Schmidt, Jeannot; Clavelou, Pierre; Aumaître, Olivier; Regagnon, Christel; Bailly, Jean-Luc; Henquell, Cécile; Peigue-Lafeuille, Hélène

    2013-01-01

    Enteroviruses (EVs) are a major cause of aseptic meningitis, and RNA detection using molecular assay is the gold standard diagnostic test. The aim of this study was to assess the impact of an EV positive diagnosis on the clinical management of patients admitted for meningitis over the course of two observational study periods (2005 and 2008-09) in the same clinical departments. We further investigated in multivariate analysis various factors possibly associated with hospital length of stay (LOS) in all age groups (infants, children, and adults). The results showed an overall improvement in the management of patients (n = 142) between the study periods, resulting in a significantly shorter hospital LOS for adults and children, and a shorter duration of antibiotic use for adults and infants. In multivariate analysis, we observed that the time from molecular test results to discharge of patients and the median duration of antibiotic treatment were associated with an increase in LOS in all age groups. In addition, among adults, the turnaround time of the molecular assay was significantly correlated with LOS. The use of CT scan in children and hospital admission outside the peak of EV prevalence in infants tended to increase LOS. In conclusion, the shorter length of stay of patients with meningitis in this study was due to various factors including the rapidity of the EV molecular test (particularly in adults), greater physician responsiveness after a positive result (in adults and children), and greater experience on the part of physicians in handling EV meningitis, as evidenced by the shorter duration of antibiotic use in adults and infants.

  17. A Molecular Score by Quantitative PCR as a New Prognostic Tool at Diagnosis for Chronic Lymphocytic Leukemia Patients

    PubMed Central

    Stamatopoulos, Basile; Meuleman, Nathalie; De Bruyn, Cécile; Pieters, Karlien; Anthoine, Géraldine; Mineur, Philippe; Bron, Dominique; Lagneaux, Laurence

    2010-01-01

    Background Several markers have been proposed to predict the outcome of chronic lymphocytic leukemia (CLL) patients. However, discordances exist between the current prognostic factors, indicating that none of these factors are totally perfect. Methodology/Principal Findings Here, we compared the prognostic power of new RNA-based markers in order to construct a quantitative PCR (qPCR) score composed of the most powerful factors. ZAP70, LPL, CLLU1, microRNA-29c and microRNA-223 were measured by real time PCR in a cohort of 170 patients with a median follow-up of 64 months (range3-330). For each patient, cells were obtained at diagnosis and RNA was extracted from purified CD19 cells. The best markers were included in a qPCR score, which was thereafter compared to each individual factor. Statistical analysis showed that all five RNA-based markers can predict treatment-free survival (TFS), but only ZAP70, LPL and microRNA-29c could significantly predict overall survival (OS). These three markers were thus included in a simple qPCR score that was able to significantly predict TFS and OS by dividing patients into three groups (0/3, 1-2/3 and 3/3). Median TFS were >210, 61 and 24 months (P<0.0001) and median OS were >330, 242 and 137 months (P<0.0001), respectively. Interestingly, TFS results were also confirmed in Binet stage A patients (P<0.0001). When compared to other classical factors, this score displays the highest univariate Cox hazard ratio (TFS: HR = 9.45 and OS: HR = 13.88) but also provides additional prognostic information. Conclusions In our hands, this score is the most powerful tool for CLL risk stratification at the time of diagnosis. PMID:20862275

  18. Characterization of feline T cell receptor gamma (TCRG) variable region genes for the molecular diagnosis of feline intestinal T cell lymphoma.

    PubMed

    Moore, Peter F; Woo, Jennifer C; Vernau, William; Kosten, Sandra; Graham, Petra S

    2005-07-15

    A diagnosis of intestinal lymphoma is currently made on the basis of clinical and morphologic criteria. This can prove problematic for many reasons that include inadequate sample size, the coexistence of lymphoma and inflammation, and the inability to assess architectural integrity of all tissue compartments in biopsy specimens obtained endoscopically. The detection of a clonal population of cells in a lymphoproliferative lesion represents an important criterion for the diagnosis of neoplasia, but this has not been assessed in feline intestinal lymphoma. T cell receptor gamma (TCRG) gene rearrangement analysis using polymerase chain reaction (PCR) is a methodology that can be used to detect clonality in T cell populations. The basis of this assay depends on the assessment of the junctional diversity that results from rearrangement of TCRG V (variable) and J (joining) gene segments. Feline TCRG transcripts from normal small intestine and spleen were obtained using a rapid amplification of cDNA ends (5'RACE) method. Limited diversity of TCRG V and J gene segments was observed. The high degree of sequence homology in the TCRG V and J gene segments was exploited to develop a PCR test for the assessment of TCRG V--J junctional diversity and hence clonality determination of T cell populations in cats. Molecular clonality determination was applied to feline intestinal lymphoplasmacytic inflammatory bowel disease (IBD) (9 cats), and transmural and mucosal T cell lymphoma (28 cats). Clonal rearrangement of the TCRG V--J junction was detected in 22 of 28 intestinal T cell lymphomas, and oligoclonality was detected in 3 intestinal T cell lymphomas. This contrasted with the detection of polyclonal rearrangement in normal intestinal tissues (3 cats) and in lymphoplasmacytic IBD (9 cats). It is proposed that assessment of TCRG V--J junctional diversity for the detection of clonality represents an important adjunctive tool for the diagnosis of T cell lymphoma in the cat.

  19. Molecular diagnosis of Fragile X syndrome in subjects with intellectual disability of unknown origin: implications of its prevalence in regional Pakistan.

    PubMed

    Kanwal, Madiha; Alyas, Saadia; Afzal, Muhammad; Mansoor, Atika; Abbasi, Rashda; Tassone, Flora; Malik, Sajid; Mazhar, Kehkashan

    2015-01-01

    Fragile-X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and affects 0.7-3.0% of intellectually compromised population of unknown etiology worldwide. It is mostly caused by repeat expansion mutations in the FMR1 at chromosome Xq27.3. The present study aimed to develop molecular diagnostic tools for a better detection of FXS, to assess implementation of diagnostic protocols in a developing country and to estimate the prevalence of FXS in a cohort of intellectually disabled subjects from Pakistan. From a large pool of individuals with below normal IQ range, 395 subjects with intellectual disability of unknown etiology belonging to different regions of the country were recruited. Conventional-PCR, modified-PCR and Southern blot analysis methods were employed for the detection of CGG repeat polymorphisms in the FMR1 gene. Initial screening with conventional-PCR identified 13 suspected patients. Subsequent investigations through modified PCR and Southern blot analyses confirmed the presence of the FMR1 mutation, suggesting a prevalence of 3.5% and 2.8% (mean 3.3%) among the male and female ID patients, respectively. These diagnostic methods were further customized with the in-house conditions to offer robust screening of referral patients/families for diagnostics and genetic counseling. Prescreening and early diagnosis are crucial for designing a prudent strategy for the management of subjects with ID. Outcome of the study recommends health practitioners for implementation of molecular based FXS diagnosis in routine clinical practice to give a better care for patients similar to the ones included in the study. PMID:25875842

  20. Usefulness of conventional transbronchial needle aspiration in the diagnosis, staging and molecular characterization of pulmonary neoplasias by thin-prep based cytology: experience of a single oncological institute

    PubMed Central

    Ramieri, Maria Teresa; Marandino, Ferdinando; Visca, Paolo; Salvitti, Tommaso; Gallo, Enzo; Casini, Beatrice; Giordano, Francesca Romana; Frigieri, Claudia; Caterino, Mauro; Carlini, Sandro; Rinaldi, Massimo; Ceribelli, Anna; Pennetti, Annarita; Alò, Pier Luigi; Pescarmona, Edoardo; Filippetti, Massimo

    2016-01-01

    Background Conventional transbronchial needle aspiration (c-TBNA) contributed to improve the bronchoscopic examination, allowing to sample lesions located even outside the tracheo-bronchial tree and in the hilo-mediastinal district, both for diagnostic and staging purposes. Methods We have evaluated the sensitivity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the c-TBNA performed during the 2005–2015 period for suspicious lung neoplasia and/or hilar and mediastinal lymphadenopathy at the Thoracic endoscopy of the Thoracic Surgery Department of the Regina Elena National Cancer Institute, Rome. Data from 273 consecutive patients (205 males and 68 females) were analyzed. Results Among 158 (58%) adequate specimens, 112 (41%) were neoplastic or contained atypical cells, 46 (17%) were negative or not diagnostic. We considered in the analysis first the overall period; then we compared the findings of the first [2005–2011] and second period [2012–2015] and, finally, only those of adequate specimens. During the overall period, sensibility and accuracy values were respectively of 53% and 63%, in the first period they reached 41% and 53% respectively; in the second period sensibility and accuracy reached 60% and 68%. Considering only the adequate specimens, sensibility and accuracy during the overall period were respectively of 80% and 82%; the values obtained for the first period were 68% and 72%. Finally, in the second period, sensibility reached 86% and accuracy 89%. Carcinoma-subtyping was possible in 112 cases, adenocarcinomas being diagnosed in 50 cases; further, in 30 cases molecular predictive data could be obtained. Conclusions The c-TBNA proved to be an efficient method for the diagnosis/staging of lung neoplasms and for the diagnosis of mediastinal lymphadenopathy. Endoscopist’s skill and technical development, associated to thin-prep cytology and to a rapid on site examination (ROSE), were able to provide by c-TBNA a

  1. Usefulness of conventional transbronchial needle aspiration in the diagnosis, staging and molecular characterization of pulmonary neoplasias by thin-prep based cytology: experience of a single oncological institute

    PubMed Central

    Ramieri, Maria Teresa; Marandino, Ferdinando; Visca, Paolo; Salvitti, Tommaso; Gallo, Enzo; Casini, Beatrice; Giordano, Francesca Romana; Frigieri, Claudia; Caterino, Mauro; Carlini, Sandro; Rinaldi, Massimo; Ceribelli, Anna; Pennetti, Annarita; Alò, Pier Luigi; Pescarmona, Edoardo; Filippetti, Massimo

    2016-01-01

    Background Conventional transbronchial needle aspiration (c-TBNA) contributed to improve the bronchoscopic examination, allowing to sample lesions located even outside the tracheo-bronchial tree and in the hilo-mediastinal district, both for diagnostic and staging purposes. Methods We have evaluated the sensitivity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the c-TBNA performed during the 2005–2015 period for suspicious lung neoplasia and/or hilar and mediastinal lymphadenopathy at the Thoracic endoscopy of the Thoracic Surgery Department of the Regina Elena National Cancer Institute, Rome. Data from 273 consecutive patients (205 males and 68 females) were analyzed. Results Among 158 (58%) adequate specimens, 112 (41%) were neoplastic or contained atypical cells, 46 (17%) were negative or not diagnostic. We considered in the analysis first the overall period; then we compared the findings of the first [2005–2011] and second period [2012–2015] and, finally, only those of adequate specimens. During the overall period, sensibility and accuracy values were respectively of 53% and 63%, in the first period they reached 41% and 53% respectively; in the second period sensibility and accuracy reached 60% and 68%. Considering only the adequate specimens, sensibility and accuracy during the overall period were respectively of 80% and 82%; the values obtained for the first period were 68% and 72%. Finally, in the second period, sensibility reached 86% and accuracy 89%. Carcinoma-subtyping was possible in 112 cases, adenocarcinomas being diagnosed in 50 cases; further, in 30 cases molecular predictive data could be obtained. Conclusions The c-TBNA proved to be an efficient method for the diagnosis/staging of lung neoplasms and for the diagnosis of mediastinal lymphadenopathy. Endoscopist’s skill and technical development, associated to thin-prep cytology and to a rapid on site examination (ROSE), were able to provide by c-TBNA a

  2. Neurofibromatosis type 1 molecular diagnosis: what can NGS do for you when you have a large gene with loss of function mutations?

    PubMed

    Pasmant, Eric; Parfait, Béatrice; Luscan, Armelle; Goussard, Philippe; Briand-Suleau, Audrey; Laurendeau, Ingrid; Fouveaut, Corinne; Leroy, Chrystel; Montadert, Annelore; Wolkenstein, Pierre; Vidaud, Michel; Vidaud, Dominique

    2015-05-01

    Molecular diagnosis of neurofibromatosis type 1 (NF1) is challenging owing to the large size of the tumour suppressor gene NF1, and the lack of mutation hotspots. A somatic alteration of the wild-type NF1 allele is observed in NF1-associated tumours. Genetic heterogeneity in NF1 was confirmed in patients with SPRED1 mutations. Here, we present a targeted next-generation sequencing (NGS) of NF1 and SPRED1 using a multiplex PCR approach (230 amplicons of ∼150 bp) on a PGM sequencer. The chip capacity allowed mixing 48 bar-coded samples in a 4-day workflow. We validated the NGS approach by retrospectively testing 30 NF1-mutated samples, and then prospectively analysed 279 patients in routine diagnosis. On average, 98.5% of all targeted bases were covered by at least 20X and 96% by at least 100X. An NF1 or SPRED1 alteration was found in 246/279 (88%) and 10/279 (4%) patients, respectively. Genotyping throughput was increased over 10 times, as compared with Sanger, with ∼90[euro ] for consumables per sample. Interestingly, our targeted NGS approach also provided quantitative information based on sequencing depth allowing identification of multiexons deletion or duplication. We then addressed the NF1 somatic mutation detection sensitivity in mosaic NF1 patients and tumours.

  3. Application of the NucliSENS easyMAG system for nucleic acid extraction: optimization of DNA extraction for molecular diagnosis of parasitic and fungal diseases.

    PubMed

    Jeddi, Fakhri; Piarroux, Renaud; Mary, Charles

    2013-01-01

    During the last 20 years, molecular biology techniques have propelled the diagnosis of parasitic diseases into a new era, as regards assay speed, sensitivity, and parasite characterization. However, DNA extraction remains a critical step and should be adapted for diagnostic and epidemiological studies. The aim of this report was to document the constraints associated with DNA extraction for the diagnosis of parasitic diseases and illustrate the adaptation of an automated extraction system, NucliSENS easyMAG, to these constraints, with a critical analysis of system performance. Proteinase K digestion of samples is unnecessary with the exception of solid tissue preparation. Mechanically grinding samples prior to cell lysis enhances the DNA extraction rate of fungal cells. The effect of host-derived nucleic acids on the extraction efficiency of parasite DNA varies with sample host cell density. The optimal cell number for precise parasite quantification ranges from 10 to 100,000 cells. Using the NucliSENS easyMAG technique, the co-extraction of inhibitors is reduced, with an exception for whole blood, which requires supplementary extraction steps to eliminate inhibitors.

  4. Accurate monotone cubic interpolation

    NASA Technical Reports Server (NTRS)

    Huynh, Hung T.

    1991-01-01

    Monotone piecewise cubic interpolants are simple and effective. They are generally third-order accurate, except near strict local extrema where accuracy degenerates to second-order due to the monotonicity constraint. Algorithms for piecewise cubic interpolants, which preserve monotonicity as well as uniform third and fourth-order accuracy are presented. The gain of accuracy is obtained by relaxing the monotonicity constraint in a geometric framework in which the median function plays a crucial role.

  5. Accurate Finite Difference Algorithms

    NASA Technical Reports Server (NTRS)

    Goodrich, John W.

    1996-01-01

    Two families of finite difference algorithms for computational aeroacoustics are presented and compared. All of the algorithms are single step explicit methods, they have the same order of accuracy in both space and time, with examples up to eleventh order, and they have multidimensional extensions. One of the algorithm families has spectral like high resolution. Propagation with high order and high resolution algorithms can produce accurate results after O(10(exp 6)) periods of propagation with eight grid points per wavelength.

  6. SPLASH: Accurate OH maser positions

    NASA Astrophysics Data System (ADS)

    Walsh, Andrew; Gomez, Jose F.; Jones, Paul; Cunningham, Maria; Green, James; Dawson, Joanne; Ellingsen, Simon; Breen, Shari; Imai, Hiroshi; Lowe, Vicki; Jones, Courtney

    2013-10-01

    The hydroxyl (OH) 18 cm lines are powerful and versatile probes of diffuse molecular gas, that may trace a largely unstudied component of the Galactic ISM. SPLASH (the Southern Parkes Large Area Survey in Hydroxyl) is a large, unbiased and fully-sampled survey of OH emission, absorption and masers in the Galactic Plane that will achieve sensitivities an order of magnitude better than previous work. In this proposal, we request ATCA time to follow up OH maser candidates. This will give us accurate (~10") positions of the masers, which can be compared to other maser positions from HOPS, MMB and MALT-45 and will provide full polarisation measurements towards a sample of OH masers that have not been observed in MAGMO.

  7. Diagnosis of lubricating oil by evaluating cyanide and carbon molecular emission lines in laser induced breakdown spectra

    NASA Astrophysics Data System (ADS)

    Elnasharty, I. Y.; Kassem, A. K.; Sabsabi, M.; Harith, M. A.

    2011-08-01

    To prevent engine failure it is essential to change lubricating oil regularly before it loses its protective properties. It is also necessary to monitor the physical and chemical conditions of the oil to reliably determine the optimum oil-change intervals. The present work focuses on studying evolution of the cyanide (CN) and carbon (C 2) molecular spectral emission lines in the laser induced breakdown spectra of lubricating oil as a function of its consumption. The intensities of these molecular bands have been taken as indicator of engine oil degradation at certain mileage. Furthermore, the percentage of decay of CN and C 2 integral intensity values at the corresponding mileage was calculated in order to relate it to the degree of consumption of the motor oil. Such percentage decay of the CN and C 2 integral intensities have been found to increase gradually with increasing mileage which is accompanied with increasing depletion of engine oil. The results of using LIBS technique in the present measurements proved that it is possible to have a direct, straightforward and easy method for prediction of lubricating oil degree of consumption. This may facilitate scheduling the proper time and/or mileage intervals for changing the oil to avoid any possibility of engine failure.

  8. Diagnosis of Familial Wolf-Hirschhorn Syndrome due to a Paternal Cryptic Chromosomal Rearrangement by Conventional and Molecular Cytogenetic Techniques

    PubMed Central

    Venegas-Vega, Carlos A.; Zepeda, Luis M.; Garduño-Zarazúa, Luz M.; Berumen, Jaime; Kofman, Susana; Cervantes, Alicia

    2013-01-01

    The use of conventional cytogenetic techniques in combination with fluorescent in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) microarrays is necessary for the identification of cryptic rearrangements in the diagnosis of chromosomal syndromes. We report two siblings, a boy of 9 years and 9 months of age and his 7-years- and 5-month-old sister, with the classic Wolf-Hirschhorn syndrome (WHS) phenotype. Using high-resolution GTG- and NOR-banding karyotypes, as well as FISH analysis, we characterized a pure 4p deletion in both sibs and a balanced rearrangement in their father, consisting in an insertion of 4p material within a nucleolar organizing region of chromosome 15. Copy number variant (CNV) analysis using SNP arrays showed that both siblings have a similar size of 4p deletion (~6.5 Mb). Our results strongly support the need for conventional cytogenetic and FISH analysis, as well as high-density microarray mapping for the optimal characterization of the genetic imbalance in patients with WHS; parents must always be studied for recognizing cryptic balanced chromosomal rearrangements for an adequate genetic counseling. PMID:23484094

  9. Jacobsen Syndrome: Surgical Complications due to Unsuspected Diagnosis, the Importance of Molecular Studies in Patients with Craniosynostosis

    PubMed Central

    Linares Chávez, Etzalli P.; Toral López, Jaime; Valdés Miranda, Juan M.; González Huerta, Luz M.; Perez Cabrera, Adrian; del Refugio Rivera Vega, María; Messina Baas, Olga M.; Cuevas-Covarrubias, Sergio A.

    2016-01-01

    Jacobsen syndrome (JBS) is an uncommon contiguous gene syndrome. About 85-92% of cases have a de novo origin. Clinical variability and severity probably depend on the size of the affected region. The typical clinical features in JBS include intellectual disability, growth retardation, craniofacial dysmorphism as well as craniosynostosis, congenital heart disease, and platelet abnormalities. The proband was a 1 year/3-month-old Mexican male. Oligonucleotide-SNP array analysis using the GeneChip Human Cytoscan HD was carried out for the patient from genomic DNA. The SNP array showed a 14.2-Mb deletion in chromosome 11q23.3q25 (120,706-134,938 Mb), which involved 163 RefSeq genes in the database of genomic variation. We report a novel deletion in JBS that increases the knowledge of the variability in the mutation sites in this region and expands the spectrum of molecular and clinical defects in this syndrome. PMID:26997943

  10. Pathological, immunological and molecular diagnosis of rabies in clinically suspected animals of different species using four detection techniques in Jordan.

    PubMed

    Faizee, N; Hailat, N Q; Ababneh, M M K; Hananeh, W M; Muhaidat, A

    2012-04-01

    A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty-seven brain tissue samples from various animal species - dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) - were provided by the Vaccine and Sera Department/Al-Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at -20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24h only, and 21 (72.42%) were positive using RT-PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non-suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT-PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT-PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT-PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.

  11. Molecular history of Richter syndrome: origin from a cell already present at the time of chronic lymphocytic leukemia diagnosis.

    PubMed

    Rossi, Davide; Spina, Valeria; Forconi, Francesco; Capello, Daniela; Fangazio, Marco; Rasi, Silvia; Martini, Maurizio; Gattei, Valter; Ramponi, Antonio; Larocca, Luigi M; Bertoni, Francesco; Gaidano, Gianluca

    2012-06-15

    Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation.

  12. Molecular history of Richter syndrome: origin from a cell already present at the time of chronic lymphocytic leukemia diagnosis.

    PubMed

    Rossi, Davide; Spina, Valeria; Forconi, Francesco; Capello, Daniela; Fangazio, Marco; Rasi, Silvia; Martini, Maurizio; Gattei, Valter; Ramponi, Antonio; Larocca, Luigi M; Bertoni, Francesco; Gaidano, Gianluca

    2012-06-15

    Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation. PMID:21796624

  13. Molecular diagnosis of Theileria and Babesia species infecting cattle in Northern Spain using reverse line blot macroarrays

    PubMed Central

    García-Sanmartín, Josune; Nagore, Daniel; García-Pérez, Ana L; Juste, Ramón A; Hurtado, Ana

    2006-01-01

    Background Piroplasmosis in cattle is caused by tick-borne haemoprotozoan parasites of the genera Theileria and Babesia. Molecular detection techniques offer higher sensitivity and specificity than microscopy examination methods and serological tests. A reverse line blot (RLB) macroarray that included generic and species-specific probes for Theileria annulata, Theileria buffeli, Babesia bovis, Babesia bigemina, Babesia divergens and Babesia major was used to study the presence and identity of the piroplasm species infecting 263 bovine blood samples from 79 farms, most of them in Northern Spain. Microscopy examination of blood smears and haematology were also performed whenever possible to identify animals with parasitaemia. Results RLB hybridisation identified infection in 54.0% of the samples, whereas only 28.8% were positive by microscopy examination. The most frequently found species was T. buffeli, present in 42.6% of the samples. T. annulata was found in 22 samples (8.4%) from 12 farms, including 9 farms (14 samples) located in Northern Spain where presence of the vector is not very common. Babesia infections were less frequently detected: B. major was found in 3.0% of the samples, B. bigemina in 2.7%, B. bovis in 2.3% and B. divergens in 1.1%. Mixed infections were detected in 14 samples, accounting for six different combinations of species. Conclusion This is the first report in which B. major and B. divergens have been detected in Spain using molecular identification techniques and the first time that B. bovis has been detected in Northern Spain. The detection of T. annulata in Northern Spain suggests that the distribution of Mediterranean theileriosis might be changing. Samples with positive RLB hybridisation but negative microscopy had haematology values within the normal ranges suggesting that they corresponded to chronic carriers that may serve as reservoirs of the infection. In this sense, sensitive and specific laboratorial tests like RLB that clearly

  14. Redescription and molecular diagnosis of Hepatozoon theileri (Laveran, 1905) (Apicomplexa: Adeleorina: Hepatozoidae), infecting Amietia quecketti (Anura: Pyxicephalidae).

    PubMed

    Netherlands, Edward C; Cook, Courtney A; Smit, Nico J; du Preez, Louis H

    2014-08-01

    Blood smears prepared from the peripheral blood of 20 wild caught Amietia quecketti (Boulenger) from the North-West University Botanical Gardens, North West Province, South Africa, were examined for the presence of haemogregarines. A haemogregarine species comparative in morphology, host and geographical locality to that of Haemogregarina theileri Laveran, 1905 was detected. The original description of H. theileri was based solely on frog peripheral blood gamont stages. Later, further parasite stages, including trophozoites and merogonic liver stages, were recorded in a related Amietia sp. from equatorial Africa. This species was originally classified as a member of the genus Haemogregarina Danilewsky, 1885, but due to the close life cycle and morphological resemblance to those of Hepatozoon species, H. theileri was later transferred from Haemogregarina to Hepatozoon Miller, 1908. In the present study, meront and merozoite stages not described before, along with previously observed trophozoite, immature and mature gamont stages, are described from the peripheral blood of hosts. In addition, comparative phylogenetic analysis of the partial 18S rDNA sequence of Hepatozoon theileri to those of other haemogregarine species, including those of species of Hepatozoon and a Haemogregarina, support the taxonomic transfer of H. theileri to Hepatozoon, nesting H. theileri within a clade comprising species parasitising other amphibians. This is the first molecular and phylogenetic analysis of an African anuran species of Hepatozoon.

  15. Photodynamic molecular beacon triggered by fibroblast activation protein on cancer-associated fibroblasts for diagnosis and treatment of epithelial cancers.

    PubMed

    Lo, Pui-Chi; Chen, Juan; Stefflova, Klara; Warren, Michael S; Navab, Roya; Bandarchi, Bizhan; Mullins, Stefanie; Tsao, Ming; Cheng, Jonathan D; Zheng, Gang

    2009-01-22

    Fibroblast activation protein (FAP) is a cell-surface serine protease highly expressed on cancer-associated fibroblasts of human epithelial carcinomas but not on normal fibroblasts, normal tissues, and cancer cells. We report herein a novel FAP-triggered photodynamic molecular beacon (FAP-PPB) comprising a fluorescent photosensitizer and a black hole quencher 3 linked by a peptide sequence (TSGPNQEQK) specific to FAP. FAP-PPB was effectively cleaved by both human FAP and murine FAP. By use of the HEK293 transfected cells (HEK-mFAP, FAP(+); HEK-vector, FAP(-)), systematic in vitro and in vivo experiments validated the FAP-specific activation of FAP-PPB in cancer cells and mouse xenografts, respectively. FAP-PPB was cleaved by FAP, allowing fluorescence restoration in FAP-expressing cells while leaving non-expressing FAP cells undetectable. Moreover, FAP-PPB showed FAP-specific photocytotoxicity toward HEK-mFAP cells whereas it was non-cytotoxic toward HEK-Vector cells. This study suggests that the FAP-PPB is a potentially useful tool for epithelial cancer detection and treatment.

  16. Pyridoxine dependent epilepsy and antiquitin deficiency: clinical and molecular characteristics and recommendations for diagnosis, treatment and follow-up.

    PubMed

    Stockler, Sylvia; Plecko, Barbara; Gospe, Sidney M; Coulter-Mackie, Marion; Connolly, Mary; van Karnebeek, Clara; Mercimek-Mahmutoglu, Saadet; Hartmann, Hans; Scharer, Gunter; Struijs, Eduard; Tein, Ingrid; Jakobs, Cornelis; Clayton, Peter; Van Hove, Johan L K

    2011-01-01

    Antiquitin (ATQ) deficiency is the main cause of pyridoxine dependent epilepsy characterized by early onset epileptic encephalopathy responsive to large dosages of pyridoxine. Despite seizure control most patients have intellectual disability. Folinic acid responsive seizures (FARS) are genetically identical to ATQ deficiency. ATQ functions as an aldehyde dehydrogenase (ALDH7A1) in the lysine degradation pathway. Its deficiency results in accumulation of α-aminoadipic semialdehyde (AASA), piperideine-6-carboxylate (P6C) and pipecolic acid, which serve as diagnostic markers in urine, plasma, and CSF. To interrupt seizures a dose of 100 mg of pyridoxine-HCl is given intravenously, or orally/enterally with 30 mg/kg/day. First administration may result in respiratory arrest in responders, and thus treatment should be performed with support of respiratory management. To make sure that late and masked response is not missed, treatment with oral/enteral pyridoxine should be continued until ATQ deficiency is excluded by negative biochemical or genetic testing. Long-term treatment dosages vary between 15 and 30 mg/kg/day in infants or up to 200 mg/day in neonates, and 500 mg/day in adults. Oral or enteral pyridoxal phosphate (PLP), up to 30 mg/kg/day can be given alternatively. Prenatal treatment with maternal pyridoxine supplementation possibly improves outcome. PDE is an organic aciduria caused by a deficiency in the catabolic breakdown of lysine. A lysine restricted diet might address the potential toxicity of accumulating αAASA, P6C and pipecolic acid. A multicenter study on long term outcomes is needed to document potential benefits of this additional treatment. The differential diagnosis of pyridoxine or PLP responsive seizure disorders includes PLP-responsive epileptic encephalopathy due to PNPO deficiency, neonatal/infantile hypophosphatasia (TNSALP deficiency), familial hyperphosphatasia (PIGV deficiency), as well as yet unidentified conditions and nutritional

  17. Gene expression fingerprint of uterine serous papillary carcinoma: identification of novel molecular markers for uterine serous cancer diagnosis and therapy

    PubMed Central

    Santin, A D; Zhan, F; Cane', S; Bellone, S; Palmieri, M; Thomas, M; Burnett, A; Roman, J J; Cannon, M J; Shaughnessy, J; Pecorelli, S

    2005-01-01

    Uterine serous papillary cancer (USPC) represents a rare but highly aggressive variant of endometrial cancer, the most common gynecologic tumour in women. We used oligonucleotide microarrays that interrogate the expression of some 10 000 known genes to profile 10 highly purified primary USPC cultures and five normal endometrial cells (NEC). We report that unsupervised analysis of mRNA fingerprints readily distinguished USPC from normal endometrial epithelial cells and identified 139 and 390 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary USPC when compared to NEC. Many of the genes upregulated in USPC were found to represent adhesion molecules, secreted proteins and oncogenes, such as L1 cell adhesion molecule, claudin-3 and claudin-4, kallikrein 6 (protease M) and kallikrein 10 (NES1), interleukin-6 and c-erbB2. Downregulated genes in USPC included SEMACAP3, ras homolog gene family, member I (ARHI), and differentially downregulated in ovarian carcinoma gene 1. Quantitative RT–PCR was used to validate differences in gene expression between USPC and NEC for several of these genes. Owing to its potential as a novel therapeutic marker, expression of the high-affinity epithelial receptor for Clostridium perfringens enterotoxin (CPE) claudin-4 was further validated through immunohistochemical analysis of formalin-fixed paraffin-embedded specimens from which the primary USPC cultures were obtained, as well as an independent set of archival USPC specimens. Finally, the sensitivity of primary USPC to the administration of scalar doses of CPE in vitro was also demonstrated. Our results highlight the novel molecular features of USPC and provide a foundation for the development of new type-specific therapies against this highly aggressive variant of endometrial cancer. PMID:15785748

  18. Testing the burden of rare variation in arrhythmia-susceptibility genes provides new insights into molecular diagnosis for Brugada syndrome.

    PubMed

    Le Scouarnec, Solena; Karakachoff, Matilde; Gourraud, Jean-Baptiste; Lindenbaum, Pierre; Bonnaud, Stéphanie; Portero, Vincent; Duboscq-Bidot, Laëtitia; Daumy, Xavier; Simonet, Floriane; Teusan, Raluca; Baron, Estelle; Violleau, Jade; Persyn, Elodie; Bellanger, Lise; Barc, Julien; Chatel, Stéphanie; Martins, Raphaël; Mabo, Philippe; Sacher, Frédéric; Haïssaguerre, Michel; Kyndt, Florence; Schmitt, Sébastien; Bézieau, Stéphane; Le Marec, Hervé; Dina, Christian; Schott, Jean-Jacques; Probst, Vincent; Redon, Richard

    2015-05-15

    The Brugada syndrome (BrS) is a rare heritable cardiac arrhythmia disorder associated with ventricular fibrillation and sudden cardiac death. Mutations in the SCN5A gene have been causally related to BrS in 20-30% of cases. Twenty other genes have been described as involved in BrS, but their overall contribution to disease prevalence is still unclear. This study aims to estimate the burden of rare coding variation in arrhythmia-susceptibility genes among a large group of patients with BrS. We have developed a custom kit to capture and sequence the coding regions of 45 previously reported arrhythmia-susceptibility genes and applied this kit to 167 index cases presenting with a Brugada pattern on the electrocardiogram as well as 167 individuals aged over 65-year old and showing no history of cardiac arrhythmia. By applying burden tests, a significant enrichment in rare coding variation (with a minor allele frequency below 0.1%) was observed only for SCN5A, with rare coding variants carried by 20.4% of cases with BrS versus 2.4% of control individuals (P = 1.4 × 10(-7)). No significant enrichment was observed for any other arrhythmia-susceptibility gene, including SCN10A and CACNA1C. These results indicate that, except for SCN5A, rare coding variation in previously reported arrhythmia-susceptibility genes do not contribute significantly to the occurrence of BrS in a population with European ancestry. Extreme caution should thus be taken when interpreting genetic variation in molecular diagnostic setting, since rare coding variants were observed in a similar extent among cases versus controls, for most previously reported BrS-susceptibility genes.

  19. Morphological and Molecular Diagnosis of Anisakid Nematode Larvae from Cutlassfish (Trichiurus lepturus) off the Coast of Rio de Janeiro, Brazil

    PubMed Central

    Borges, Juliana Novo; Cunha, Luiz Felipe Gullo; Santos, Helena Lúcia Carneiro; Monteiro-Neto, Cassiano; Santos, Cláudia Portes

    2012-01-01

    Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank. PMID:22792329

  20. Morphological and molecular diagnosis of anisakid nematode larvae from cutlassfish (Trichiurus lepturus) off the coast of Rio de Janeiro, Brazil.

    PubMed

    Borges, Juliana Novo; Cunha, Luiz Felipe Gullo; Santos, Helena Lúcia Carneiro; Monteiro-Neto, Cassiano; Portes Santos, Cláudia

    2012-01-01

    Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank. PMID:22792329

  1. Cell-free DNA testing: an aid to prenatal sonographic diagnosis.

    PubMed

    Chitty, Lyn S

    2014-04-01

    Sonographic diagnosis of fetal abnormalities is based on the recognition of sonographic patterns associated with structural abnormalities. Although diagnosis in some situations, such as neural tube defects, gastroschisis, and omphalocoele, can be straightforward, in many situations, the constellation of fetal abnormalities suggest an underlying chromosomal or genetic cause. In these situations, invasive testing is needed to provide the information required to make a definitive diagnosis, and thus accurately counsel parents. Since the identification of cell-free fetal DNA in maternal plasma, the potential for non-invasive prenatal diagnosis is increasingly becoming possible. In this chapter, the current role and future potential of non-invasive prenatal diagnosis, combined with new molecular techniques as an aid to sonographic diagnosis, will be discussed.

  2. Evaluation of the Broad-Range PCR/ESI-MS Technology in Blood Specimens for the Molecular Diagnosis of Bloodstream Infections

    PubMed Central

    Jordana-Lluch, Elena; Giménez, Montserrat; Quesada, Mª Dolores; Rivaya, Belén; Marcó, Clara; Domínguez, Mª Jesús; Arméstar, Fernando; Martró, Elisa; Ausina, Vicente

    2015-01-01

    Background Rapid identification of the etiological agent in bloodstream infections is of vital importance for the early administration of the most appropriate antibiotic therapy. Molecular methods may offer an advantage to current culture-based microbiological diagnosis. The goal of this study was to evaluate the performance of IRIDICA, a platform based on universal genetic amplification followed by mass spectrometry (PCR/ESI-MS) for the molecular diagnosis of sepsis-related pathogens directly from the patient’s blood. Methods A total of 410 whole blood specimens from patients admitted to Emergency Room (ER) and Intensive Care Unit (ICU) with clinical suspicion of sepsis were tested with the IRIDICA BAC BSI Assay (broad identification of bacteria and Candida spp.). Microorganisms grown in culture and detected by IRIDICA were compared considering blood culture as gold standard. When discrepancies were found, clinical records and results from other cultures were taken into consideration (clinical infection criterion). Results The overall positive and negative agreement of IRIDICA with blood culture in the analysis by specimen was 74.8% and 78.6%, respectively, rising to 76.9% and 87.2% respectively, when compared with the clinical infection criterion. Interestingly, IRIDICA detected 41 clinically significant microorganisms missed by culture, most of them from patients under antimicrobial treatment. Of special interest were the detections of one Mycoplasma hominis and two Mycobacterium simiae in immunocompromised patients. When ICU patients were analyzed separately, sensitivity, specificity, positive and negative predictive values compared with blood culture were 83.3%, 78.6%, 33.9% and 97.3% respectively, and 90.5%, 87.2%, 64.4% and 97.3% respectively, in comparison with the clinical infection criterion. Conclusions IRIDICA is a promising technology that offers an early and reliable identification of a wide variety of pathogens directly from the patient’s blood

  3. Utility of immunohistochemical investigation of SDHB and molecular genetic analysis of SDH genes in the differential diagnosis of mesenchymal tumors of GIT.

    PubMed

    Dubova, M; Sedivcova, M; Michal, M; Kokoskova, B; Ryska, A; Smid, D; Daum, O

    2015-02-01

    Loss of expression of beta subunit of succinate dehydrogenase (SDHB) was proved to be present in a subgroup of KIT/PDGFRA wt gastrointestinal stromal tumors (GISTs). To evaluate possible diagnostic utility of SDHB immunohistochemistry in the differential diagnostics of mesenchymal tumors of gastrointestinal tract (GIT), 11 cases of KIT/PDGFRA wt GISTs, 12 gastric schwannomas (GSs), 20 solitary fibrous tumors (SFTs), 4 leiomyomas (LMs), 16 leiomyosarcomas (LMSs), 5 synovial sarcomas (SSs), 3 endometrioid stromal sarcomas (ESSs), and 1 ileal inflammatory myofibroblastic tumor (IMT) were investigated for SDHB immunoexpression together with molecular genetic analysis of genes encoding succinate dehydrogenase (SDH). Three recent cases of KIT/PDGFRA mutant GISTs were used as controls. Among the 11 KIT/PDGFRA wt GISTs, 6 expressed SDHB, 1 of them harboring a sequence change of SDHD. All SDHB-negative cases were SDHB-D wt. In 1 of the control GIST cases molecular genetic analysis revealed an SDHD sequence change in addition to a mutation in KIT exon 11. No SFT was truly SDHB-negative, but in 2 of them the staining was impossible to analyze. Furthermore, 1 SFT carried an SDHB and another 1 SDHD sequence change. All GSs, LMs, LMSs, SSs, ESSs, and IMT were SDHB-positive or non-analyzable, and SDHB-D wt. Additional factors may play a role in regulating expression of SDHB. Furthermore, SDHB immunohistochemistry alone may be misleading in excluding tumors other than GIST (especially SFT) in the differential diagnosis of KIT/PDGFRA wt mesenchymal tumors of GIT.

  4. [Diagnosis: synovial fluid analysis].

    PubMed

    Gallo Vallejo, Francisco Javier; Giner Ruiz, Vicente

    2014-01-01

    Synovial fluid analysis in rheumatological diseases allows a more accurate diagnosis in some entities, mainly infectious and microcrystalline arthritis. Examination of synovial fluid in patients with osteoarthritis is useful if a differential diagnosis will be performed with other processes and to distinguish between inflammatory and non-inflammatory forms. Joint aspiration is a diagnostic and sometimes therapeutic procedure that is available to primary care physicians.

  5. Accurate Optical Reference Catalogs

    NASA Astrophysics Data System (ADS)

    Zacharias, N.

    2006-08-01

    Current and near future all-sky astrometric catalogs on the ICRF are reviewed with the emphasis on reference star data at optical wavelengths for user applications. The standard error of a Hipparcos Catalogue star position is now about 15 mas per coordinate. For the Tycho-2 data it is typically 20 to 100 mas, depending on magnitude. The USNO CCD Astrograph Catalog (UCAC) observing program was completed in 2004 and reductions toward the final UCAC3 release are in progress. This all-sky reference catalogue will have positional errors of 15 to 70 mas for stars in the 10 to 16 mag range, with a high degree of completeness. Proper motions for the about 60 million UCAC stars will be derived by combining UCAC astrometry with available early epoch data, including yet unpublished scans of the complete set of AGK2, Hamburg Zone astrograph and USNO Black Birch programs. Accurate positional and proper motion data are combined in the Naval Observatory Merged Astrometric Dataset (NOMAD) which includes Hipparcos, Tycho-2, UCAC2, USNO-B1, NPM+SPM plate scan data for astrometry, and is supplemented by multi-band optical photometry as well as 2MASS near infrared photometry. The Milli-Arcsecond Pathfinder Survey (MAPS) mission is currently being planned at USNO. This is a micro-satellite to obtain 1 mas positions, parallaxes, and 1 mas/yr proper motions for all bright stars down to about 15th magnitude. This program will be supplemented by a ground-based program to reach 18th magnitude on the 5 mas level.

  6. Molecular diagnosis of Papaya meleira virus (PMeV) from leaf samples of Carica papaya L. using conventional and real-time RT-PCR.

    PubMed

    Abreu, Paolla M V; Piccin, João G; Rodrigues, Silas P; Buss, David S; Ventura, José A; Fernandes, Patricia M B

    2012-03-01

    Papaya meleira virus (PMeV) is the causal agent of papaya sticky disease. This study describes two methods for molecular diagnosis of PMeV using conventional and real-time PCR. These methods were shown to be more efficient than current methods of viral detection using extraction of PMeV dsRNA and observation of symptoms in the field. The methods described here were used to evaluate the effect of inoculation of papaya plants with purified PMeV dsRNA on the progress of PMeV infection. A single inoculation with PMeV dsRNA was observed to delay the progress of the virus infection by several weeks. The possibility of vertical transmission of PMeV was also investigated. No evidence was found for PMeV transmission through seeds collected from diseased fruit. The implications of these results for the epidemiology of PMeV and the management of papaya sticky disease are discussed.

  7. Guidelines for splicing analysis in molecular diagnosis derived from a set of 327 combined in silico/in vitro studies on BRCA1 and BRCA2 variants.

    PubMed

    Houdayer, Claude; Caux-Moncoutier, Virginie; Krieger, Sophie; Barrois, Michel; Bonnet, Françoise; Bourdon, Violaine; Bronner, Myriam; Buisson, Monique; Coulet, Florence; Gaildrat, Pascaline; Lefol, Cédrick; Léone, Mélanie; Mazoyer, Sylvie; Muller, Danielle; Remenieras, Audrey; Révillion, Françoise; Rouleau, Etienne; Sokolowska, Joanna; Vert, Jean-Philippe; Lidereau, Rosette; Soubrier, Florent; Sobol, Hagay; Sevenet, Nicolas; Bressac-de Paillerets, Brigitte; Hardouin, Agnès; Tosi, Mario; Sinilnikova, Olga M; Stoppa-Lyonnet, Dominique

    2012-08-01

    Assessing the impact of variants of unknown significance (VUS) on splicing is a key issue in molecular diagnosis. This impact can be predicted by in silico tools, but proper evaluation and user guidelines are lacking. To fill this gap, we embarked upon the largest BRCA1 and BRCA2 splice study to date by testing 272 VUSs (327 analyses) within the BRCA splice network of Unicancer. All these VUSs were analyzed by using six tools (splice site prediction by neural network, splice site finder (SSF), MaxEntScan (MES), ESE finder, relative enhancer and silencer classification by unanimous enrichment, and human splicing finder) and the predictions obtained were compared with transcript analysis results. Combining MES and SSF gave 96% sensitivity and 83% specificity for VUSs occurring in the vicinity of consensus splice sites, that is, the surrounding 11 and 14 bases for the 5' and 3' sites, respectively. This study was also an opportunity to define guidelines for transcript analysis along with a tentative classification of splice variants. The guidelines drawn from this large series should be useful for the whole community, particularly in the context of growing sequencing capacities that require robust pipelines for variant interpretation.

  8. Vaginal tampons as specimen collection device for the molecular diagnosis of non-ulcerative sexually transmitted infections in antenatal clinic attendees.

    PubMed

    Sturm, P D J; Connolly, Cathy; Khan, Nasreen; Ebrahim, Safiya; Sturm, A Willem

    2004-02-01

    Self-inserted vaginal tampons for the molecular diagnosis of non-ulcerative STIs were evaluated. Cervical and vaginal swabs, tampons and urines were collected from 185 first-time antenatal clinic attendees. Cultures and nucleic acid amplification assays (NAA) were performed. The sensitivity of PCR on tampons for Trichomonas vaginalis was with 94% (CI 85-98%) significantly higher (P<0.001) than culture (50%, CI 38-62%) or urine (53%, CI 41-65%). Neisseria gonorrhoeae culture had a sensitivity of 64% (CI 36-86%), strand displacement assay (SDA) had a sensitivity of 79% (CI 49-94%) using tampon specimens, 57% (CI 30-81%) using endocervical swabs and 43% (CI 19-70%) using urines. There was no difference in sensitivity of SDA for Chlamydia trachomatis using tampon specimens, urine or endocervical swabs. The specificity approached 100% for all assays on all specimens. NAA on tampons for the detection of T. vaginalis, N. gonorrhoeae and C. trachomatis identified more infections than assays on swabs or urines. This reached statistical significance for T. vaginalis only.

  9. Hepatobiliary Tumors: Update on Diagnosis and Management

    PubMed Central

    Kabbach, Ghassan; Assi, Hussein A; Bolotin, George; Schuster, Michael; Lee, Hwa Jeong; Tadros, Micheal

    2015-01-01

    Tumors of the liver and biliary tree, mainly hepatocellular carcinoma and cholangiocarcinoma, are the second leading cause of cancer related death worldwide and the sixth leading cause of cancer related death among men in developed countries. Recent developments in biomarkers and imaging modalities have enhanced early detection and accurate diagnosis of these highly fatal malignancies. These advances include serological testing, micro-ribonucleic acids, fluorescence in situ hybridization, contrast-enhanced ultrasound, and hepatobiliary-phase magnetic resonance imaging. In addition, there have been major developments in the surgical and nonsurgical management of these tumors, including expansion of the liver transplantation criteria, new locoregional treatments, and molecularly targeted therapies. In this article, we review various types of hepatobiliary tumors and discuss new developments in their diagnosis and management. PMID:26623263

  10. Cloning, molecular characterization of a 13-kDa antigen from Schistosoma japonicum, Sj13, a putative salivary diagnosis candidate for Schistosomiasis japonica.

    PubMed

    Zhou, Yan-ping; Wu, Zhong-dao; Yang, Lin-lin; Sun, Xi; You, Xu; Yu, Xin-bing; Hu, Wei; Zheng, Huan-qin; Lv, Zhi-yue

    2009-10-01

    Saliva has been suggested as an easily accessible and a noninvasive diagnostic alternative for detection of antibodies. To identify and characterize Schistosoma japonicum (S. japonicum) antigens that are recognized by saliva of infected host, we have used a pool of saliva from infected patients to immunoscreen an egg cDNA library of S. japonicum. The open reading frame of the isolated two clones encodes same protein of 116 amino acids exhibiting 100% identity to an amino acid sequence (AY222893) of S. japonicum in NCBInr database. The protein encoded is inferred a secretory protein with a molecular mass of 13 kDa (Sj13) and shares no homology to any entries in the NCBInr database, demonstrating that Sj13 might be a schistosome-specific protein. Reverse transcriptase polymerase chain reaction, Western blotting, and immunolocalization analysis revealed Sj13 could be detected in cercaria, adult, and egg and was localized to forehead and tegument of cercaria, cell body ("cytons") of adult worm, egg shell, and epidermal plate of miracidium. Furthermore, Sj13 showed a good antigenicity when reacted with saliva or serum from schistosomiasis patients. The recombinant Sj13 (rSj13) expressed and purified from Escherichia coli was applied to detect its specific salivary antibody for schistosomiasis diagnosis by an indirect enzyme linked immunosorbent assay (ELISA). Preliminary laboratory test of 116 subjects, 40 with parasitologically proven S. japonicumm infection, 46 with other infectious diseases, and 30 negative controls exhibited 92.50% sensitivity with saliva/rSj13 and 95.00% with serum/SWAP (P > 0.05). The specificity of the ELISA using saliva/rSj13 was 92.11% versus 85.53% with serum/SWAP (P < 0.05). No direct correlations of anti-Sj13 IgG levels with egg counts in stool were observed in saliva detection. These results suggest that Sj13 specific salivary antibody detection may be useful as an antigen for the salivary diagnosis of schistosomiasis japonica and

  11. The molecular classification of hereditary endocrine diseases.

    PubMed

    Ye, Lei; Ning, Guang

    2015-12-01

    Hereditary endocrine diseases are an important group of diseases with great heterogeneity. The current classification for hereditary endocrine disease is mostly based upon anatomy, which is helpful for pathophysiological interpretation, but does not address the pathogenic variability associated with different underlying genetic causes. Identification of an endocrinopathy-associated genetic alteration provides evidence for differential diagnosis, discovery of non-classical disease, and the potential for earlier diagnosis and targeted therapy. Molecular diagnosis should be routinely applied when managing patients with suspicion of hereditary disease. To enhance the accurate diagnosis and treatment of patients with hereditary endocrine diseases, we propose categorization of endocrine diseases into three groups based upon the function of the mutant gene: cell differentiation, hormone synthesis and action, and tumorigenesis. Each category was further grouped according to the specific gene function. We believe that this format would facilitate practice of precision medicine in the field of hereditary endocrine diseases.

  12. Long-term clinical follow-up and molecular testing for diagnosis of the first Tunisian family with Alström syndrome.

    PubMed

    Chakroun, Amine; Ben Said, Mariem; Ennouri, Amine; Achour, Imen; Mnif, Mouna; Abid, Mohamed; Ghorbel, Abdelmonem; Marshall, Jan D; Naggert, Jürgen K; Masmoudi, Saber

    2016-09-01

    Alström syndrome is a clinically complex disorder characterized by progressive degeneration of sensory functions, resulting in visual and audiological impairment as well as metabolic disturbances. It is caused by recessively inherited mutations in the ALMS1 gene, which codes for a centrosomal/basal body protein. The purpose of this study was to investigate the genetic and clinical features of two Tunisian affected siblings with Alström syndrome. Detailed clinical examinations were performed including complete ophthalmic examination, serial audiograms and several biochemical and hormonal blood tests. For the molecular study, first genomic DNA was isolated using a standard protocol. Then, linkage analysis with microsatellite markers was performed and DNA array was used to detect known mutations. Subsequently, all ALMS1 exons were simultaneously sequenced for one affected patient with the TaGSCAN targeted sequencing panel. Finally, segregation of the causal variant was performed by Sanger sequencing. Both affected siblings had cone rod dystrophy with impaired visual acuity, sensorineural hearing loss and truncal obesity. One affected individual showed insulin resistance without diabetes mellitus. Other clinical features including cardiac and pulmonary dysfunction, hypothyroidism, hyperlipidemia, acanthosis nigricans, renal and hepatic dysfunction were absent. Genetic analysis showed the presence of a homozygous splice site mutation (c.10388-2A > G) in both affected siblings. Although Alström syndrome is relatively well characterized disease, this syndrome is probably misdiagnosed in Tunisia. Here, we describe the first report of Tunisian patients affected by this syndrome and carrying a homozygous ALMS1 mutation. The diagnosis was suspected after long-term clinical follow-up and confirmed by genetic testing. PMID:27523285

  13. Diagnosis of hepatitis B

    PubMed Central

    Song, Jeong Eun

    2016-01-01

    Hepatitis B virus (HBV) infection is a major global health problems leading to severe liver disease such as cirrhosis and hepatocellular carcinoma (HCC). HBV is a circular, partly double-stranded DNA virus with various serological markers: hepatitis B surface antigen (HBsAg) and anti-HBs, anti-HBc IgM and IgG, and hepatitis B e antigen (HBeAg) and anti-HBe. It is transmitted by sexual, parenteral and vertical route. One significant method to diminish the burden of this disease is timely diagnosis of acute, chronic and occult cases of HBV. First step of HBV diagnosis is achieved by using serological markers for detecting antigens and antibodies. In order to verify first step of diagnosis, to quantify viral load and to identify genotypes, quantitative or qualitative molecular tests are used. In this article, the serological and molecular tests for diagnosis of HBV infection will be reviewed. PMID:27761442

  14. Case Report: Whole exome sequencing helps in accurate molecular diagnosis in siblings with a rare co-occurrence of paternally inherited 22q12 duplication and autosomal recessive non-syndromic ichthyosis.

    PubMed Central

    Gupta, Aayush; Sharma, Yugal; Deo, Kirti; Vellarikkal, Shamsudheen; Jayarajan, Rijith; Dixit, Vishal; Verma, Ankit; Scaria, Vinod; Sivasubbu, Sridhar

    2015-01-01

    Lamellar ichthyosis (LI), considered an autosomal recessive monogenic genodermatosis, has an incidence of approximately 1 in 250,000. Usually associated with mutations in the transglutaminase gene ( TGM1), mutations in six other genes have, less frequently, been shown to be causative. Two siblings, born in a collodion membrane, presented with fish like scales all over the body. Karyotyping revealed duplication of the chromosome arm on 22q12+ in the father and two siblings. Whole exome sequencing revealed a homozygous p.Gly218Ser variation in TGM1; a variation reported earlier in an isolated Finnish population in association with autosomal recessive non-syndromic ichthyosis. This concurrence of a potentially benign 22q12+ duplication and LI, both rare individually, is reported here likely for the first time. PMID:26594337

  15. [RARE DISEASES DTC: DIAGNOSIS, TREATMENT AND CARE].

    PubMed

    Mendlovic, Joseph; Barash, Hila; Yardeni, Hadar; Banet-Levi, Yonit; Yonath, Hagith; Raas-Rothschild, Annick

    2016-04-01

    Rare diseases are chronic, progressive genetic disorders, which affect around 6-8% of the general population, mainly children. Therefore, in Israel approximately 500,000 people are probably affected by a rare disease. In this article, we review some of the issues pertaining to rare diseases, such as the need for accurate diagnosis which is necessary not only for specific care and treatment but also for informed family planning. In addition, we review the impact of the activities of patients' organizations on the awareness of rare diseases and their involvement in the creation of the Orphan Drug Act, which was the leading point on the way to drug development worldwide. During the last few years networks for reaching leading specialists' opinions on the way to proper diagnosis were created. Thereafter, the next generation genetic technologies, such as exome sequencing, have been a revolution in terms of options and hope for patients with rare undiagnosed diseases. Patients with rare diseases and their families are a challenge to the health care system, not only in terms of diagnosis and therapy, but also in terms of special needs. In addition, deciphering molecular pathways of rare diseases might be the key for understanding molecular events involved in common disorders. We emphasize the duty to ensure appropriate capacity and equal access to follow-up and clinical management of patients with rare diseases in Israel.

  16. [RARE DISEASES DTC: DIAGNOSIS, TREATMENT AND CARE].

    PubMed

    Mendlovic, Joseph; Barash, Hila; Yardeni, Hadar; Banet-Levi, Yonit; Yonath, Hagith; Raas-Rothschild, Annick

    2016-04-01

    Rare diseases are chronic, progressive genetic disorders, which affect around 6-8% of the general population, mainly children. Therefore, in Israel approximately 500,000 people are probably affected by a rare disease. In this article, we review some of the issues pertaining to rare diseases, such as the need for accurate diagnosis which is necessary not only for specific care and treatment but also for informed family planning. In addition, we review the impact of the activities of patients' organizations on the awareness of rare diseases and their involvement in the creation of the Orphan Drug Act, which was the leading point on the way to drug development worldwide. During the last few years networks for reaching leading specialists' opinions on the way to proper diagnosis were created. Thereafter, the next generation genetic technologies, such as exome sequencing, have been a revolution in terms of options and hope for patients with rare undiagnosed diseases. Patients with rare diseases and their families are a challenge to the health care system, not only in terms of diagnosis and therapy, but also in terms of special needs. In addition, deciphering molecular pathways of rare diseases might be the key for understanding molecular events involved in common disorders. We emphasize the duty to ensure appropriate capacity and equal access to follow-up and clinical management of patients with rare diseases in Israel. PMID:27323543

  17. Commercial Multiplex Technologies for the Microbiological Diagnosis of Sepsis

    PubMed Central

    Lebovitz, Evan E.; Burbelo, Peter D.

    2013-01-01

    In patients with suspected sepsis, rapid and accurate diagnosis of the causative infectious agent is critical. Although clinicians often use empiric antimicrobial therapy until the blood cultures are available to potentially adjust treatment, this approach is often not optimum for patient care. Recently, several commercial molecular multiplex technologies have shown promise for fast and comprehensive diagnosis of microorganisms and their antimicrobial resistance signatures. While one class of multiplex technologies is directed at improving the speed and diagnostic information obtained from positive blood cultures, the other identifies the causative microorganisms directly from clinical blood samples. The goal of this review is to provide an overview of these molecular technologies and describe their performance capabilities compared to standard blood cultures and in some cases to each other. We discuss the current clinical impact, limitations, and likely futures advances these multiplex technologies may have in guiding the management of patients with sepsis. PMID:23636778

  18. Diagnosis of toxoplasmosis and typing of Toxoplasma gondii.

    PubMed

    Liu, Quan; Wang, Ze-Dong; Huang, Si-Yang; Zhu, Xing-Quan

    2015-05-28

    Toxoplasmosis, caused by the obligate intracellular protozoan Toxoplasma gondii, is an important zoonosis with medical and veterinary importance worldwide. The disease is mainly contracted by ingesting undercooked or raw meat containing viable tissue cysts, or by ingesting food or water contaminated with oocysts. The diagnosis and genetic characterization of T. gondii infection is crucial for the surveillance, prevention and control of toxoplasmosis. Traditional approaches for the diagnosis of toxoplasmosis include etiological, immunological and imaging techniques. Diagnosis of toxoplasmosis has been improved by the emergence of molecular technologies to amplify parasite nucleic acids. Among these, polymerase chain reaction (PCR)-based molecular techniques have been useful for the genetic characterization of T. gondii. Serotyping methods based on polymorphic polypeptides have the potential to become the choice for typing T. gondii in humans and animals. In this review, we summarize conventional non-DNA-based diagnostic methods, and the DNA-based molecular techniques for the diagnosis and genetic characterization of T. gondii. These techniques have provided foundations for further development of more effective and accurate detection of T. gondii infection. These advances will contribute to an improved understanding of the epidemiology, prevention and control of toxoplasmosis.

  19. Accurate phase-shift velocimetry in rock.

    PubMed

    Shukla, Matsyendra Nath; Vallatos, Antoine; Phoenix, Vernon R; Holmes, William M

    2016-06-01

    Spatially resolved Pulsed Field Gradient (PFG) velocimetry techniques can provide precious information concerning flow through opaque systems, including rocks. This velocimetry data is used to enhance flow models in a wide range of systems, from oil behaviour in reservoir rocks to contaminant transport in aquifers. Phase-shift velocimetry is the fastest way to produce velocity