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PubMed2006

Dry reagent dipstick test combined with 23S rRNA PCR for molecular diagnosis of bacterial infection in arthroplasty.

Periprosthetic joint infections present a challenging problem in orthopaedics. Conventional methods for detection of arthroplasty infections rely on bacterial culture of synovial fluid aspirates. During recent years, however, molecular tests that are based on DNA amplification by the polymerase chain reaction (PCR), followed by electrophoretic analysis of the products, have been introduced. We report a simple and inexpensive assay that allows visual detection and confirmation of the PCR-amplified sequences by hybridization within minutes. The assay is performed in a dry reagent dipstick format (strip) and does not require special instrumentation. Universal primers are used for PCR of the 23S ribosomal RNA (rRNA) gene. The biotinylated amplification product is hybridized with dA-tailed probes that are specific for six pathogens commonly involved in periprosthetic joint infections. The mixture is applied to the strip, which is then immersed in the appropriate buffer. The buffer migrates along the strip by capillary action and rehydrates gold nanoparticles with oligo(dT) strands attached to their surface. The nanoparticles bind to the target DNA through hybridization, and the hybrids are captured by immobilized streptavidin at the test zone of the strip, producing a characteristic red line. Unbound nanoparticles are captured by immobilized oligo(dT) strands at the control zone of the strip, generating a second line. The dipstick test was applied to the detection of Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Enterococcus faesium, and Haemophilus influenza. Twelve samples of synovial fluids from patients were analyzed for the detection and identification of the infection caused by the six pathogens. The results were compared with bacterial cultures.

Arthroplasty↗
PubMed1996

Dynamic mutations pose unique challenges for the molecular diagnostics laboratory.

Routine clinical molecular testing of diseases associated with unstable or dynamic trinucleotide repeat syndromes poses unique technical, medical, and ethical challenges to the laboratory. Although the pathophysiology of these disorders is to date still largely undefined, the uniformity of their genetics has led to the development of highly informative diagnostic tests. In general, amplification techniques, such as the polymerase chain reaction (PCR), are used to determine the size of alleles within the genes linked to these disorders. Technically, these assays require empirical optimization so that the PCR reactions are both robust and reproducible, and occasionally other methods must be used to confirm diagnoses. Beyond execution of the test, however, the molecular diagnostics laboratory needs also to be fundamentally involved in the process of interpreting these tests in the correct clinical context and in setting policy as to how these data are presented to patients.

Chemistry, Clinical↗
PubMed2006

Introduction to molecular cystic fibrosis testing.

Technology improvements are rapidly bringing molecular diagnostics into routine laboratories. Recent recommendations for cystic fibrosis carrier testing by the American College of Medical Genetics (ACMG) have led to commercial test kit development and increased testing volumes. Molecular testing of genetic diseases presents a variety of challenges and situations that may be unfamiliar to laboratories with limited molecular genetic experience. We will briefly review the disease and discuss mutation testing indications, methodologies, quality assurance, and reporting issues associated with cystic fibrosis testing.

Cystic Fibrosis↗
PubMed2002

Laboratory diagnosis of metabolic myopathies.

The metabolic myopathies are a heterogeneous group of disorders inherited by a variety of modes that include gene defects in both the nuclear and mitochondrial genomes. Many factors impact on the expression of the pathogenic mutations that cause these disorders including genetic background, environmental factors, and coexisting disorders. Molecular technology has greatly improved the ability to make definitive diagnoses in many of the metabolic myopathies in the last decade and particularly has demonstrated that the wide diversity in the severity of mutations contributes to understanding genotype-phenotype correlations. In some cases, molecular testing obviates the necessity to perform an invasive muscle biopsy. However, it is also clear that the diagnostic yield from molecular testing is incomplete and particularly low among the mitochondrial myopathies as a group, ranging from approximately 6% to 19% in well-classified high-risk groups. Therefore, it is often essential to combine clinical, biochemical, histopathologic, and molecular data for each patient in order to arrive at a definitive diagnosis. The approach to the laboratory diagnosis of metabolic myopathies is described emphasizing both noninvasive and invasive testing, highlighting the molecular methodologies with the benefits and disadvantages of each technology, and documenting how to determine whether patients have coexisting disorders.

Clinical Laboratory Techniques↗
PubMed2003

[Hereditary neuropathies].

BACKGROUND: Hereditary neuropathies constitute a heterogeneous group of diseases that make up a significant proportion of peripheral nerve disease cases. MATERIAL AND METHODS: The paper is based on a review of recent literature, including searches on Medline, and our own clinical and research experience. RESULTS: Charcot-Marie-Tooth disease, itself a heterogeneous disease, is the most common hereditary neuropathy. Several variants of neuropathy are associated with hereditary metabolic disorders. Diagnostic methods include nerve conduction velocity studies and electromyography, quantitative sensory testing, molecular genetic diagnostic testing, and in selected cases processing of nerve biopsies and skin biopsies for determination of epidermal nerve fibre densities. INTERPRETATION: A thorough family history of neuropathic symptoms and signs, and preferably clinical and electrophysiological examination of relatives is essential for the diagnosis of hereditary neuropathy. Molecular genetic analysis is promising for accurate classification of these diseases. Nerve biopsy is only helpful in selected cases.

Charcot-Marie-Tooth Disease↗
PubMed2000

Men's attitudes regarding genetic testing for hereditary prostate cancer risk.

OBJECTIVES: Little is known about the attitudes of men unselected for a family history for prostate cancer concerning genetic testing for prostate cancer risk or genetic testing for inherited cancer predisposition. To explore this, we examined the interest in molecular testing for hereditary prostate cancer (HPC) predisposition among a self-selected cohort of 342 men presenting for prostate cancer screening. METHODS: Participants were surveyed concerning their attitudes about DNA testing for HPC predisposition and knowledge of prostate cancer-associated risk factors, including heredity. RESULTS: Of the participants completing the survey, 92% expressed interest in learning about DNA testing, and 89% stated that they would undergo DNA analysis for HPC predisposition, if available. Twenty-eight percent of respondents failed to demonstrate an adequate understanding of the concept of "inherited tendency." The demonstrated level of understanding of this concept did not differ by the respondent's family history, although it varied by race. An interest in learning about or undergoing testing did not vary by race, family history, or demonstrated understanding of the concept of inherited risk. CONCLUSIONS: Among men presenting for routine prostate cancer screening, interest in learning about testing for HPC predisposition and in having such testing performed may be high. The data also provide evidence that, in a population of men unselected for family history, interest in molecular testing for this common, male-specific cancer may parallel the high interest level demonstrated among women in DNA testing for inherited breast and ovarian cancer risk.

Adult↗
PubMed2004

Phenotypic and genotypic characterization of Enterococcus spp. of different origins.

Sixty-four strains of Enterococcus spp. of different origin were identified using traditional phenotypic, biochemical and cultural tests, together with molecular tests. API 20 STREP tests identified the species at a preliminary level, only one strain remaining unidentified. Strains belonging to the genus Enterococcus were tested with genus-specific primers, while species-level identification was carried out with the 16S-23S rDNA intergenic region (ITS) and species-specific primers for E. faecium and E. faecalis. Those strains found to be negative with the species-specific primers were subjected to 16S rDNA partial sequencing.

Animals↗
PubMed2005

Rapid and/or high-throughput genotyping for human red blood cell, platelet and leukocyte antigens, and forensic applications.

BACKGROUND: Traditionally, transfusion medicine, platelet and human leukocyte antigen (HLA) typing, and forensic medicine relied on serologic studies. METHODS: In recent years, molecular testing on nucleic acids has been increasingly applied to these areas. Although conventional molecular diagnostic methods such as PCR-sequence-specific priming, PCR-restriction fragment-length polymorphism, PCR-single-strand conformation polymorphism, sequence-based typing, and DNA fingerprinting have been shown to perform well, their use is limited by long turnaround times, high cost, labor-intensiveness, the need for special technical skills, and/or the high risk of amplicon contamination. With advance of fast and/or high-throughput methods and platforms that often combine amplification and detection, a new era of molecular genotyping is emerging in these fields dominated by serology for a century. As new targets, short tandem repeats, mitochondrial DNA and Y-chromosome sequences were introduced for forensic applications. This article reviews the current status of the application of rapid and/or high-throughput genotyping methods to these areas. RESULTS: The results are already promising with real-time PCR, pyrosequencing, microarrays, and mass spectrometry and show high concordance rates with classic serologic and earlier manual molecular diagnostic methods. Exploration of other emerging methodologies will likely further enhance the diagnostic utility of molecular testing in these areas.

Blood Platelets↗
PubMed2005

How unexpected are unexpected findings in prenatal cytogenetic diagnosis? A literature review.

The objective of this review was to gain understanding about unexpected findings in prenatal cytogenetic diagnosis. This category of results might be excluded from prenatal testing when new molecular tests such as I-FISH and QF-PCR will be applied in a future scenario of targeted testing. The literature was systematically searched for publications wherein the term unexpected or a synonym refers to testing results with specific problems. On the selected articles a qualitative analysis was performed, using the methods of cross-case analysis and within-case analysis. Sixteen articles published between 1979 and 2003 were selected. Analysis led to the classification of four problems of unexpected findings: I. unexpected for professionals; II. unexpected for patients; III. uncertainty; IV. other difficult counselling issues. We conclude that currently the problems of unexpected findings relate only slightly to their unexpected character. Instead, the main problems of unexpected findings relate to uncertainty and other aspects which create difficult counselling issues. As such, unexpected findings can be distinguished only gradually from standard results. Before targeted testing can be applied it is necessary to establish exact criteria in order to discern unexpected findings from standard testing results.

Amniocentesis↗
PubMedDate not supplied

[2 cases of Lowe syndrome].

Löwe syndrome is a form of X-linked mental retardation with short stature, cataracts, renal tubular dysfunction and hypotonia. We present 2 cases to illustrate this rare entity, but also to discuss the suggestive aspect of the face and to underline the importance of molecular tests for genetic counselling. Both cases associate ocular, cerebral and renal defects. Molecular tests changed the recurrence risk in the first family.

Facies↗
PubMed2004

Toward self-assembled surface-mounted prismatic altitudinal rotors. A test case: molecular rectangle.

[structure: see text] A self-assembly path toward prismatic molecular rotors based on transversely reactive terminally metalated molecular rods and pyridine-terminated star connectors is outlined. The concept is tested on the assembly of the biphenyl rod [Ph(2)P(CH(2))(3)PPh(2)Pt(+)-C(6)H(4)-C(6)H(4)-Pt(+)[Ph(2)P(CH(2))(3)PPh(2)] and 4,4'-bipyridyl into a molecular rectangle, fully characterized by NMR and MS, including diffusion-ordered NMR and collision-induced dissociation MS.

Journal Article↗
PubMed1995

Molecular and clinical findings in a family with dentatorubral-pallidoluysian atrophy.

Herein we describe the molecular and clinical findings in a North American Caucasian family with dentatorubral-pallidoluysian atrophy (DRPLA). These patients all presented with an autosomal dominant neurodegenerative disorder characterized by a variable combination of clinical symptoms including seizures, ataxia, dementia, choreiform movements, mental retardation, and psychiatric disease. Neuroradiologic findings in the index case revealed deep subcortical white matter changes on magnetic resonance imaging. Prior to referral, the family carried a diagnosis of Huntington's disease (HD). Subsequent direct molecular testing for HD failed to identify the HD expansion mutation in affected individuals. Molecular testing for DRPLA, however, demonstrated the presence of the recently characterized DRPLA expansion mutation in all affected individuals. The size of the expansion correlated with the age of onset of clinical symptoms. As DRPLA has rarely been reported in North American and European populations, the molecular confirmation of DRPLA in this family provides support for the hypothesis that DRPLA may not be as geographically restricted as once thought.

Adolescent↗
PubMed2001

Diagnosis of hepatitis C virus (HCV) infection and laboratory monitoring of its therapy.

BACKGROUND: Just after the identification and characterization of hepatitis C virus (HCV) in 1989, tests for the detection of HCV antibodies or HCV RNA in serum were developed. The enzyme-linked immunosorbent assays (ELISAs) and confirmatory/supplemental analytical antibody tests were improved in sensitivity and specificity with the development of further generations of these assays. Application of molecular tests for detecting, quantifying, and characterization of the infecting virus became very important in management of HCV infection. OBJECTIVE AND DESIGN: This review summarizes the assays developed for the diagnosis and management of HCV infection. Strategies for the diagnosis and monitoring with the advantages and disadvantages of the assays based on the setting and goal are discussed according to data in the literature and our experience. RESULTS: Specific laboratory diagnostic tests for hepatitis C virus infection may be discussed under two titles: (i) Serological antibody tests which detect anti-HCV in serum or plasma; (ii) Molecular tests which detect HCV RNA genome, investigate viral load, and determine the characteristics of the genome. Strategies in different laboratory settings which screen populations with different HCV prevalences vary. CONCLUSIONS: Anti-HCV positive result in a low-risk setting such as blood banks should be confirmed with an analytical antibody test. Then a HCV RNA test should be performed on serum of the person with a positive or indeterminate confirmatory test result. On the contrary, anti-HCV positive test result in high-risk population or a situation where HCV infection is suspected, it is likely to be true positive and confirmation with HCV RNA test will be significant. Quantitative HCV RNA test and genotyping should be performed if therapy is considered.

Hepacivirus↗
PubMed2004

Molecular genetic testing for malignant hyperthermia susceptibility.

BACKGROUND: For more than 30 yr, the in vitro contracture test (IVCT) was the only appropriate diagnostic tool for malignant hyperthermia (MH). After the introduction of molecular genetics into MH research, guidelines for molecular genetic diagnosis of MH susceptibility were published. The aim of this study was to establish applicability of the guidelines, sensitivity, and specificity of genetic testing in MH and advantages for studied patients. METHODS: The IVCT was performed following the guidelines of the European MH Group. Mutation analyses were performed by amplification of genomic DNA by polymerase chain reaction and restriction enzyme digestion. RESULTS: Two hundred eight individuals underwent MH testing between January 2001 and April 2003. In 32 of 67 initially genetic-tested patients, the familial mutation was identified, and they were diagnosed as MH susceptible. The IVCT followed negative genetic test results in 20 patients, and all but one had negative IVCT results. Three patients were scheduled to undergo elective surgery, and IVCT and genetic testing were performed simultaneously. All three had positive IVCT results and were carriers of their familial mutation. CONCLUSIONS: In families with known MH mutations, there is a 50% chance of reliably confirming MH susceptibility by noninvasive testing. The authors found the negative predictive value of genetic testing to be 0.95 (95% confidence interval, 0.75-0.99), but for patient safety, they still recommend following the guidelines for genetic testing in MH and therefore performing an IVCT in case of negative genetic results.

Anesthesia, Conduction↗
PubMed2003

Papanicolaou tests and molecular analyses using new fluid-based specimen collection technology in 3000 Japanese women.

A fluid-based Papanicolaou test has been established to improve sample collection and preparation. This study was the first large-scale investigation in Japan to examine the feasibility of using fluid-based Papanicolaou specimens to detect human papillomavirus (HPV) using Hybrid Capture II and polymerase chain reaction (PCR). Three thousand patients who visited Keio University Hospital between October 2000 and February 2001 were enrolled in the study. The results of the fluid-based Papanicolaou tests corresponded well with those of conventional Papanicolaou smears (96.8% concordance). The sensitivities of cervical neoplasia detection using the fluid-based Papanicolaou test (73.9%) and Hybrid Capture II (76.3%, P=0.55) were not significantly different. Among the cervical intraepithelial neoplasia 3 and squamous cell carcinoma specimens, HPV 16 and HPV 52 were predominantly detected using the PCR method. Although some DNA samples extracted from the fluid-based specimens were degradaded, PCR and direct sequencing could be performed without difficulty even after 1 year of specimen storage. We conclude that fluid-based Papanicolaou specimens can be applied to investigate HPV infection.

DNA, Viral↗
PubMed2005

Multiplex detection of Ehrlichia and Anaplasma species pathogens in peripheral blood by real-time reverse transcriptase-polymerase chain reaction.

Tick-borne infections are responsible for many emerging diseases in humans and several vertebrates. These include human infections with Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Because single or co-infections can result from tick bites, the availability of a rapid, multiplex molecular test will be valuable for timely diagnosis and treatment. Here, we describe a multiplex molecular test that can detect single or co-infections with up to five Ehrlichia and Anaplasma species. The test protocol includes the magnetic capture-based purification of 16S ribosomal RNA, its enrichment, and specific-pathogen(s) detection by real-time reverse transcriptase-polymerase chain reaction. We also report a unique cloning strategy to develop positive controls in the absence of a pathogen's genomic DNA. The test was assessed by examining blood samples from dogs suspected to be positive for ehrlichiosis. The dog was chosen as the model system because it is susceptible to acquire infections with up to five pathogens of the genera Ehrlichia and Anaplasma. The test identified single infections in the canine host with E. chaffeensis, E. canis, E. ewingii, A. phagocytophilum, and A. platys and co-infection with E. canis and A. platys. The multipathogen detection and novel positive control development procedures described here will be valuable in monitoring infections in people, other vertebrates, and ticks.

Anaplasma↗
PubMed2026

Cost-Effectiveness and the Economics of Genomic Testing and Molecularly Matched Therapies.

Cost-effectiveness analysis of precision oncology can help guide value-driven care. Next-generation sequencing is increasingly cost-efficient over single gene testing because diagnostic algorithms require multiple individual gene tests to determine biomarker status. Matched targeted therapy is often not cost-effective due to the high cost associated with drug treatment. However, genomic profiling can promote cost-effective care by identifying patients who are unlikely to benefit from therapy. Additional applications of genomic profiling such as universal testing for hereditary cancer syndromes and germline testing in patients with cancer may represent cost-effective approaches compared with traditional history-based diagnostic methods.

Humans↗