A Comprehensive Triple-Repeat Primed PCR and a Long-Range PCR Agarose-Based Assay for Improved Genotyping of Guanine-Adenine-Adenine Repeats in Friedreich Ataxia.

Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide [guanine-adenine-adenine (GAA)] repeat in intron 1 of the FXN gene. It is common in the White population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease.

Detection of autoantibodies to 3-hydroxy-3-methylglutaryl-coenzyme a reductase by ELISA in a reference laboratory setting.

Background: We investigated the performance of an ELISA for the detection of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) IgG antibodies in immune-mediated necrotizing myopathies (IMNM).

Methods: Patients positive for HMGCR antibodies (n=61) or negative (n=78) by protein immunoprecipitation (IP), and healthy controls (n=100) were used to evaluate the ELISA. Unique consecutive serum samples (n=155) received at ARUP Laboratories for HMGCR IgG testing by ELISA were also investigated and analysed for serum muscle enzymes (aldolase, creatine kinase, and myoglobin).

Phenotypic characterization of Sodalis praecaptivus sp. nov., a close non-insect-associated member of the Sodalis-allied lineage of insect endosymbionts.

A Gram-stain-negative bacterium, isolated from a human wound was previously found to share an unprecedentedly close relationship with Sodalis glossinidius and other members of the Sodalis-allied clade of insect symbionts. This relationship was inferred from sequence analysis of the 16S rRNA gene and genomic comparisons and suggested the strain belonged to a novel species. Biochemical and genetic analyses supported this suggestion and demonstrated that the organism has a wide repertoire of metabolic properties, which is consistent with the presence of a relatively large gene inventory.

Laboratory evaluation of anti-phospholipid syndrome: a preliminary prospective study of phosphatidylserine/prothrombin antibodies in an at-risk patient cohort.

Immunoglobulin (Ig)G/IgM autoantibodies to phosphatidylserine/prothrombin (aPS/PT) were evaluated individually and in combination with criteria anti-phospholipid (aPL) tests in a prospectively ascertained cohort of patients at risk for anti-phospholipid syndrome (APS). One hundred and sixty (160) consecutive requests for lupus anti-coagulant (LAC) from the University of Utah Health Sciences Center were identified during 8 weeks. Of these, 104 unique patients had additional requests for cardiolipin (aCL) and/or beta2 glycoprotein I (aβ2 GPI) IgG and/or IgM; samples were retained and analysed for aPS/PT, aCL and/or aβ2 GPI IgG and IgM antibodies.

Antiphospholipid syndrome and the relevance of antibodies to negatively charged phospholipids in diagnostic evaluation.

The presence of lupus anticoagulant, moderate-to-high levels of IgG and/or IgM antibodies to beta-2 glycoprotein I or cardiolipin in association with at least one of the two major clinical manifestations (thrombosis and/or pregnancy-related morbidity) are required for a diagnosis of definite antiphospholipid syndrome (APS). The realization that certain negatively charged phospholipid (PL) autoantibodies broadly react with antibodies directed against cardiolipin and may be more reliable and specific markers for APS has led to the search for diagnostic assays with greater predictability for disease evaluation and management. This review focuses on the state-of-the-art analytical and clinical performance of IgG and IgM antibodies directed against negatively charged PLs, specifically phosphatidic acid (aPA), phosphatidylinositol (aPI), and phosphatidylserine (aPS), as well as the APhL assay, which contains a proprietary mixture of phospholipid antigens.

Severely depressed interleukin-17 production by human neonatal mononuclear cells.

Background: The role of T-helper 17 cells (Th17) in neonatal host defense remains to be fully elucidated. Interleukin (IL)-17 plays an important role in the immune response to bacterial and fungal pathogens by promoting inflammation.

Methods: We examined neonatal production of IL-17 in mixed mononuclear cells (MMCs) isolated from umbilical cord blood for comparison with adult peripheral blood mononuclear cell controls.

14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends.

Current classification criteria for definite Antiphospholipid Syndrome (APS) require the use of three laboratory assays to detect antiphospholipid antibodies (aCL, anti-β2GPI and LA) in the presence of at least one of the two major clinical manifestations (i.e. thrombosis or pregnancy morbidity) of the syndrome.

Neisseria oralis sp. nov., isolated from healthy gingival plaque and clinical samples.

A polyphasic analysis was undertaken of seven independent isolates of gram-negative cocci collected from pathological clinical samples from New York, Louisiana, Florida and Illinois and healthy subgingival plaque from a patient in Virginia, USA. The 16S rRNA gene sequence similarity among these isolates was 99.7-100 %, and the closest species with a validly published name was Neisseria lactamica (96.

Simultaneous genotyping of rs12979860 and rs8099917 variants near the IL28B locus associated with HCV clearance and treatment response.

Recent genome-wide association studies have identified two host single-nucleotide polymorphisms (SNPs) near the IL28B gene (rs12979860 C/T and rs8099917 T/G) that are associated with sustained virological response in patients infected with the hepatitis C virus. Herein, we describe a rapid multiplexed dual-color fluorescence resonance energy transfer (FRET) probe assay that accurately genotypes for both SNPs simultaneously. A single-nucleotide extension assay was also developed for verification of genotypes.

Diagnostic assays for anti-PM/Scl IgG antibodies: heterogeneity in antibody response or lack of standardization?

Background: The aim of this study was to compare the correlation between new diagnostic methodologies for detecting anti-polymyositis/scleroderma (anti-PM/Scl) IgG antibodies associated with myositis and/or systemic scleroderma assays with existing platforms.

Methods: Sera from 164 samples previously tested for anti-PM/Scl IgG antibody by immunodiffusion, ID; 171 sera screened for anti-PM/Scl IgG by immunoprecipitation, IP; an additional group of 215 sera tested by ID and 46 healthy blood donor sera were retrospectively evaluated. Anti-PM/Scl IgG antibodies were measured using three PM/Scl-100 specific enzyme immunoassays (EIAs), PM1-alpha (PM1-α) EIA and a line immunoblot assay (LIA) for anti-PM/Scl-75 and -100 IgG antibodies.

Significant differences in the analytic concordance between anti-dsDNA IgG antibody assays for the diagnosis of systemic lupus erythematosus--implications for inter-laboratory testing.

Background: Anti-double stranded DNA (anti-dsDNA) autoantibodies are considered hallmark of systemic lupus erythematosus (SLE).

Methods: To determine concordance between assays for the detection of this marker, we analyzed 100 antinuclear antibody (ANA) positive sera with a homogeneous pattern and titers≥1:160 by indirect immunofluorescence assay (IFA) on HEp-2 cells, 100 consecutive anti-dsDNA IgG ELISA-negative as well as 100 healthy control samples using six commercial ELISAs and the Crithidia luciliae immunofluorescence test (CLIFT).

Results: The positivity rates for the ELISAs in the ANA positive group ranged from 55.

Isolation and characterization of "Pseudomonas andersonii" from four cases of pulmonary granulomas and emended species description.

"Pseudomonas andersonii" is a Gram-negative bacillus initially isolated from a granulomatous lung lesion. Novel species status has not been validated for this single strain. We report four additional cases of pulmonary granuloma involving P.

Nucleotide extension genotyping by high-resolution melting.

One limitation of small amplicon melting is the inability to genotype certain nearest-neighbor symmetric variations without manipulating the sample. We have developed a method for these exceptions: a high-resolution melting single nucleotide extension assay. Single nucleotide extension was performed in a new instrument, the LightScanner 32 (LS32), which uses capillary reaction tubes and is capable of real-time PCR and sequential high-resolution melting of 32 samples.

Simultaneous sequence analysis of the 16S rRNA and rpoB genes by use of RipSeq software to identify Mycobacterium species.

The 16S rRNA gene is commonly used to identify Mycobacterium spp., but alternative DNA targets can provide better resolution to the species level. We evaluated a novel system that enables simultaneous amplification, sequencing, and analysis of two different DNA targets in a single tube to identify clinical isolates of Mycobacterium spp.

Gastrointestinal Stromal Tumors: A Guide to the Diagnosis.

Gastrointestinal stromal tumors (GISTs) have emerged from being a poorly understood and therapeutically refractory sarcoma to a tumor whose biology has not only provided insight into a mechanism of oncogenesis but has also led to a rational basis for therapy. Most GISTs are characterized by KIT protein (CD117) expression and constitutive activating mutations in either the c-kit or platelet-derived growth factor receptor α genes. This information can now be obtained from routine formalin-fixed and paraffin-embedded tissue.

A comparison of Brucella IgG and IgM ELISA assays with agglutination methodology.

Despite brucellosis having a low incidence rate in developed nations, it still remains the leading zoonotic disease in the world. Culturing of Brucella spp. provides good specificity but in cases where the fever is intermittent, sensitivity is problematic.

Novel screening assay performance in pediatric celiac disease and adult dermatitis herpetiformis.

Objectives: : Several serologic assays are commercially available to aid in the diagnosis of gluten-sensitive enteropathy (GSE). Our objective in this study was to assess the performance of a novel combined antigen-screening assay for GSE.

Patients And Methods: : Deidentified sera from 111 pediatric patients suspected of having celiac disease (CD), 130 adults diagnosed with dermatitis herpetiformis (DH), and 77 pediatric and 49 adult normal controls were included in the study.

Development of a multiplex real-time RT-PCR assay for detection of influenza A, influenza B, RSV and typing of the 2009-H1N1 influenza virus.

A high-throughput real-time RT-PCR assay was developed to amplify and detect a conserved region of the hemagglutinin gene of the 2009-H1N1 influenza A virus using a minor groove binder-conjugated hybridization probe. The assay was paired with a separate triplex real-time assay that detects influenza A via the matrix gene, influenza B and RSV in a multiplex format and compared with the Centers for Disease Control and Prevention (CDC) rRT-PCR assay using 143 samples. The 2009-H1N1 portion of the multiplex assay had 100% correlation with the CDC assay, while the triplex assay had a 99% agreement.

Autoantibodies against phosphatidylserine, prothrombin and phosphatidylserine-prothrombin complex: identical or distinct diagnostic tools for antiphospholipid syndrome?

Background: To clarify the association and diagnostic utility of measuring antiphosphatidyl serine (aPS), antiprothrombin (aPT) and the antiphosphatidyl serine/prothrombin complex (aPS/PT) antibodies in patients with antiphospholipid syndrome (APS) and recurrent pregnancy loss (RPL).

Method: We measured IgG and IgM anti-aPS/PT (2 different kits) as well as the aPS and aPT autoantibodies by ELISA. All subjects were also tested for the presence of Lupus anticoagulant (LA), IgG and IgM anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (ass(2)GPI), and these served as the gold standard unless otherwise indicated.

Phylogenetic analysis of Mycobacterium aurum and Mycobacterium neoaurum with redescription of M. aurum culture collection strains.

We examined American Type Culture Collection (ATCC) strains of Mycobacterium aurum and Mycobacterium neoaurum by using multilocus DNA target sequencing. Apart from the type strain, all 10 ATCC M. aurum strains examined were classified incorrectly, with most being reclassified as belonging to the M.

An evaluation of two commercially available ELISAs and one in-house reference laboratory ELISA for the determination of human anti-rabies virus antibodies.

The envelope glycoprotein G of rabies virus in vaccines induces the production of neutralizing antibodies important in the protection against the disease. The measurement of anti-envelope glycoprotein antibodies is a good predictor of the degree of humoral immunity in people during anti-rabies treatment or after vaccination. Several assays exist for the serological determination of antibody protection against rabies virus infection.

Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to four Neisseria meningitidis serogroups.

Neisseria meningitidis is a gram-negative bacterium causing disease world wide with a fatality rate of 5-10%. Five serogroups, A, B, C, Y and W-135 are responsible for virtually all cases of the disease in humans. We have developed a multiplexed assay for the simultaneous quantitation of IgG antibody responses to the four most immunogenic (A, C, Y, and W-135) N.

Assessment of three commercially available serologic assays for detection of antibodies to Mycobacterium tuberculosis and identification of active tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a major world disease, with approximately 9 million new cases each year. Identification and treatment of active disease are essential for TB control. Serology may offer increased detection of active disease in patients with a positive tuberculin skin test (TST) or QuantiFERON-TB (QFT-G).

Clinical relevance of multiple antibody specificity testing in anti-phospholipid syndrome and recurrent pregnancy loss.

We wanted to evaluate whether testing for anti-phosholipid antibodies other than anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (abeta2GPI) immunoglobulin (Ig)G and IgM identifies patients with recurrent pregnancy loss (RPL) who may be positive for anti-phospholipid syndrome (APS). In a cross-sectional study comprising 62 patients with APS, 66 women with RPL, 50 healthy blood donors and 24 women with a history of successful pregnancies, we tested IgM and IgG antibodies to phosphatidic acid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol and phosphatidyl serine with and without beta-2 glycoprotein I (beta2GPI) from a single manufacturer as well as aCL and abeta2GPI antibodies. Diagnostic accuracies of individual and combined anti-phospholipid (aPL) assays were assessed by computing sensitivities, specificities, positive predictive values and negative predictive values together with their 95% confidence intervals.

Evaluation of a new commercial enzyme immunoassay for the detection of IgM antibodies to West Nile virus using a ratio method to eliminate nonspecific reactivity.

As West Nile virus (WNV) has become endemic in the United States, following the first reported cases in New York during the summer of 1999, the demand for specific serology has increased. Several IgM capture ELISA assays for the detection of WNV-specific IgM have been approved by the Food and Drug Administration for in vitro diagnostic testing, including kits from Focus Diagnostics and InBios International, Inc. The Focus Diagnostics IgM capture ELISA has a background subtraction protocol and the InBios IgM capture ELISA implements a ratio method to detect nonspecific reactivity due to rheumatoid factor, heterophile antibodies, and other interfering substances.