Multicenter Clinical Evaluation of the Automated Aries Bordetella Assay.

Molecular methods offer superior sensitivity and specificity and reduce testing turnaround time from days to hours for detection of Bordetella pertussis and Bordetella parapertussis In this study, we evaluated the performance of the automated PCR-based Aries Bordetella Assay, which detects both B. pertussis and B. parapertussis directly from nasopharyngeal swab specimens. The limits of detection (LoDs) were 1,800 CFU·ml-1 for B. pertussis and 213 CFU·ml-1 for B. parapertussis The assay detected 16/18 unique B. pertussis/B. parapertussis strains. Of 71 potentially cross-reacting organisms, 5 generated false positives in 1/6 replicates; none of 6 additional Bordetella spp. were erroneously detected. Specimens were stable at 20 to 25°C for at least 10 h, at 4 to 8°C for 10 days, and at temperatures not exceeding -70°C for 6 months. Of 1,052 nasopharyngeal specimens from patients with suspected pertussis, 3.0% (n = 32) were B. pertussis positive and 0.2% (n = 2) were B. parapertussis positive. Combining these data with Aries Bordetella Assay data from 57 nasopharyngeal samples with previously confirmed B. pertussis or B. parapertussis data and with data from 50 contrived B. parapertussis samples, the proportions of positive and negative agreement of the respective Aries assays with the reference assays were 97.1% and 99.0% for B. pertussis and 100% and 99.7% for B. parapertussis The Aries Bordetella Assay provides accurate detection and distinction of B. pertussis and B. parapertussis infections within 2 h. (This study has been registered at ClinicalTrials.gov under registration no. NCT02862262.).

Multicenter Evaluation of the Solana Group A Streptococcus Assay: Comparison with Culture.

We compared group A Streptococcus (GAS) culture with a rapid helicase-dependent amplification (HDA) method using 1,082 throat swab specimens. The HDA method demonstrated 98.2% sensitivity and 97.2% specificity. GAS prevalence by culture was 20.7%, and it was 22.6% using the HDA method. In 35 min, the HDA method provided rapid, sensitive GAS detection, making culture confirmation unnecessary.

Human Infection with Ehrlichia muris-like Pathogen, United States, 2007-2013(1).

An Ehrlichia muris-like (EML) pathogen was detected among 4 patients in Minnesota and Wisconsin during 2009. We characterized additional cases clinically and epidemiologically. During 2004-2013, blood samples from 75,077 patients from all 50 United States were tested by PCR from the groEL gene for Ehrlichia spp. and Anaplasma phagocytophilum. During 2007-2013, samples from 69 (0.1%) patients were positive for the EML pathogen; patients were from 5 states: Indiana (1), Michigan (1), Minnesota (33), North Dakota (3), and Wisconsin (31). Most (64%) patients were male; median age was 63 (range 15-94) years; and all 69 patients reported likely tick exposure in Minnesota or Wisconsin. Fever, malaise, thrombocytopenia, and lymphopenia were the most common symptoms. Sixteen (23%) patients were hospitalized (median 4 days); all recovered, and 96% received doxycycline. Infection with the EML pathogen should be considered for persons reporting tick exposure in Minnesota or Wisconsin.

Detection of Group A Streptococcus in Pharyngeal Swab Specimens by Use of the AmpliVue GAS Isothermal Helicase-Dependent Amplification Assay.

We evaluated the clinical performance (sensitivity and specificity) of the AmpliVue group A Streptococcus (GAS) isothermal helicase-dependent amplification assay using 1,192 pharyngeal swab specimens. AmpliVue GAS assay results were compared to the results of routine throat cultures on selective streptococcal blood agar plates. The sensitivity and specificity of the AmpliVue GAS assay were 98.3% (95% confidence interval [CI], 95 to 100%) and 93.2% (95% CI, 91 to 95%), respectively.

Clinical evaluation of a real-time PCR assay for identification of Salmonella, Shigella, Campylobacter (Campylobacter jejuni and C. coli), and shiga toxin-producing Escherichia coli isolates in stool specimens.

Enteric illness affects millions of individuals annually in the United States and results in >50,000 hospitalizations. The rapid and accurate identification of bacterial pathogens associated with gastroenteritis can aid acute patient management decisions, including the use of antibiotic therapy and infection control. This study compared the ProGastro SSCS multiplex real-time PCR assay (Gen-Probe Prodesse, San Diego, CA) to culture for the identification of Campylobacter spp. (Campylobacter jejuni and Campylobacter coli), Salmonella spp., and Shigella spp. and to broth enrichment followed by an FDA-cleared enzyme immunoassay (EIA) for the identification of Shiga toxin-producing Escherichia coli (STEC) isolates in stool specimens. Stool samples submitted in preservatives for routine culture and EIA were prospectively enrolled and tested at four clinical centers. Discrepancies between the ProGastro SSCS assay and culture or EIA were resolved using bidirectional sequencing. The overall prevalence of the pathogens as detected by culture was 5.6% (1.8% Campylobacter, 1.8% Salmonella, 1.3% Shigella, and 0.8% STEC). When results based on the ProGastro SSCS assay and bidirectional sequencing were applied, the overall prevalence increased to 8.3% (2.3% Campylobacter, 2.6% Salmonella, 1.8% Shigella, and 1.6% STEC). Following resolution of the discrepant results, the sensitivity of the ProGastro SSCS assay was 100% for all pathogens, and the specificities ranged from 99.4% to 100%. The sensitivity of culture compared to sequence-confirmed ProGastro SSCS results ranged from 52.9% to 76.9%, with the specificities ranging from 99.9% to 100%. Overall, these results suggest that the ProGastro SSCS assay is highly sensitive and specific in a clinical setting.

How novel molecular diagnostic technologies and biomarkers are revolutionizing genetic testing and patient care.

Technological applications and novel biomarkers in the field of molecular diagnostics have never been evolving at a more rapid pace. These novel applications have the promise to change the face of clinical care as we move into the era of personalized medicine. While some of these technologies and biomarkers have been adopted by some clinical laboratories, most laboratories face a steep learning curve in bringing these dramatically new and different molecular diagnostic applications on board. Furthermore, interpreting the vast amounts and new types of data produced by these novel applications brings forth challenges for laboratorians and clinicians alike. In this article, we discuss how some of these emerging novel molecular diagnostic technologies and analytes, such as next-generation sequencing, chromosomal microarray, microRNAs and circulating fetal nucleic acids are revolutionizing patient care and personalized medicine.

Biological variation among african trypanosomes: I. Clonal expression of virulence is not linked to the variant surface glycoprotein or the variant surface glycoprotein gene telomeric expression site.

The potential association of variant surface glycoprotein (VSG) gene expression with clonal expression of virulence in African trypanosomes was addressed. Two populations of clonally related trypanosomes, which differ dramatically in virulence for the infected host, but display the same apparent VSG surface coat phenotype, were characterized with respect to the VSG genes expressed as well as the chromosome telomeric expression sites (ES) utilized for VSG gene transcription. The VSG gene sequences expressed by clones LouTat 1 and LouTat 1A of Trypanosoma brucei rhodesiense were identical, and gene expression in both clones occurred precisely by the same gene conversion events (duplication and transposition), which generated an expression-linked copy (ELC) of the VSG gene. The ELC was present on the same genomic restriction fragments in both populations and resided in the telomere of a 330-kb chromosome; a single basic copy of the LouTat 1/1A VSG gene, present in all variants of the LouTat 1 serodeme, was located at an internal site of a 1.5-Mb chromosome. Restriction endonuclease mapping of the ES telomere revealed that the VSG ELC of clones LouTat 1 and 1A resides in the same site. Therefore, these findings provide evidence that the VSG gene ES and, potentially, any cotranscribed ES-associated genes do not play a role in the clonal regulation of virulence because trypanosome clones LouTat 1 and 1A, which differ markedly in their virulence properties, both express identical VSG genes from the same chromosome telomeric ES.

Salivary gland-type lung carcinomas: an EGFR immunohistochemical, molecular genetic, and mutational analysis study.

Salivary gland-type lung carcinomas are uncommon neoplasms of the lung, the two most common being adenoid cystic carcinoma and mucoepidermoid carcinoma. Although they usually have an indolent behavior, adenoid cystic carcinomas can be more aggressive, with 5-year survival as low as 55%. Unfortunately, these tumors do not respond well to chemotherapy. In contrast to the most common subtypes of lung carcinomas, epidermal growth factor receptor studies have not been carried out in this group of tumors. Herein we report a series of 24 cases (12 adenoid cystic and 12 mucoepidermoid carcinomas) tested for epidermal growth factor receptor protein expression, epidermal growth factor receptor gene copy gains, and epidermal growth factor receptor gene mutational status, through immunohistochemistry, fluorescence in situ hybridization, and sequencing of the exons 18-21, respectively. Overall, 91 and 92% of the adenoid cystic carcinomas and mucoepidermoid carcinomas expressed epidermal growth factor receptor protein. Chromosome 7 polysomy occurred in 25% of the cases (four adenoid cystic carcinomas and two mucoepidermoid carcinomas). No epidermal growth factor receptor gene amplification was detected and no mutation was found in exons 18-21 of the epidermal growth factor receptor gene. Immunoexpression of epidermal growth factor receptor in salivary gland-type lung carcinomas is not related to epidermal growth factor receptor gene copy number or mutational status.

Utility of microsatellite analysis in evaluation of pregnancies with placental mesenchymal dysplasia.

OBJECTIVE: To demonstrate a role for microsatellite analysis in the evaluation of prenatal cases with possible placental mesenchymal dysplasia (PMD). METHODS: We present a case report in which several molecular analyses of amniocytes and products of conception were used in combination with cytogenetic and ultrasound studies to evaluate a pregnancy with a clinical suspicion of PMD. A combination of Southern blotting analysis, methylation-sensitive polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) was utilized to evaluate methylation patterns in the Beckwith-Wiedemann syndrome (BWS) and Prader-Willi/Angelman syndrome (PW/AS) critical regions. A series of microsatellite markers were used to evaluate the possibility of an androgenetic cell line. RESULTS: Methylation studies performed for the BWS and PW/AS critical regions were abnormal and consistent with a molecular diagnosis of BWS and Angelman syndrome. Further studies of amniocytes using microsatellite markers identified androgenetic and biparental cell lines in approximately a 10:1 ratio, respectively. CONCLUSIONS: Prenatal ultrasonography, karyotyping and molecular genetic evaluation for BWS alone were not sufficient to identify the underlying etiology of PMD in this case. The androgenetic cell line was only identified after microsatellite analysis.

Mosaic ring 20 with no detectable deletion by FISH analysis: Characteristic seizure disorder and literature review.

Ring chromosome 20 is a rare chromosome disorder characterized by a typical seizure phenotype consisting of complex partial seizures, frequent progression to generalized tonic or tonic-clonic seizures, and nocturnal frontal lobe seizures with frequent episodes of non-convulsive status epilepticus. Development may be normal or mildly delayed, followed by cognitive and behavioral decline after seizure onset. Here, we describe a patient with a typical severe seizure phenotype and a mosaic ring chromosome 20 without loss of p or q subtelomere regions or telomeric sequences. The ring had a longer telomere length than either of the telomere ends of its homologous chromosome 20 by quantitative fluorescence in situ hybridization analysis, suggesting that it might be derived from telomere-telomere fusion. The phenotypic comparison of this patient and other chromosome 20 cases that had terminal deletions of 20qter (n = 1) and 20pter (n = 7), shows that the epilepsy phenotype and electroencephalographic abnormalities are characteristic in patients with ring chromosome 20. Several hypotheses have been proposed to address the elusive mechanisms underlying the seizure disorder in ring chromosome 20. These possibilities include haploinsufficiency of two epilepsy genes CHRNA4 and KCNQ2 located at 20qter, silencing of these genes by a telomere position effect, or microdeletions or rearrangements of genetic material during the ring formation.

A case of mosaic supernumerary ring chromosome 15 with two copies of the segment 15p11.1-q14.

Although supernumerary marker chromosomes derived from chromosome 15 (SMC(15)) are the most common marker chromosome in humans, ring SMC(15)s are rare. Here we report on a 16-month-old patient who has a ring SMC(15) with two copies of the segment 15p11.1-q14 region. She exhibits hypotonia, developmental delay, speech delay, microstomia, micrognathia, and other mild dysmorphic features. The ring was present in 22% of her peripheral blood lymphocyte cells. FISH study revealed that the ring was derived from chromosome 15, and had neither telomere sequence nor satellite III paracentromeric DNA. It had alpha satellite DNA, and two copies of the segment 15q11.2 to CTD 2125J1 (at 15q14, 2.2 Mbp telomeric of the common breakpoint 5). The ring-containing cells had four copies of 15p11.1-q14. The ring can be described as r(15)(::p11.1 --> q14::q14 --> p11.1::). Southern-blot analysis of the methylation pattern in the PW/AS critical region showed biparental inheritance, and the ring was maternally derived. This patient's phenotype was comparable to ring SMC(15) patients with three copies of the Prader-Willi/Angelman syndrome (PWS/AS) critical region.

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.