Comparison of QIAsymphony automated and QIAamp manual DNA extraction systems for measuring Epstein-Barr virus DNA load in whole blood using real-time PCR.

Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively).

Endoplasmic reticulum stress causes EBV lytic replication.

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication.

Comparative analysis and supragenome modeling of twelve Moraxella catarrhalis clinical isolates.

Background: M. catarrhalis is a gram-negative, gamma-proteobacterium and an opportunistic human pathogen associated with otitis media (OM) and exacerbations of chronic obstructive pulmonary disease (COPD). With direct and indirect costs for treating these conditions annually exceeding $33 billion in the United States alone, and nearly ubiquitous resistance to beta-lactam antibiotics among M.

A one-step, real-time PCR assay for rapid detection of rhinovirus.

One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons.

Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli.

We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E.

Mycoplasma pneumoniae and atypical Stevens-Johnson syndrome: a case series.

Mycoplasma pneumoniae is a common cause of community-acquired respiratory illness in the adolescent population. Stevens-Johnson syndrome is an extrapulmonary manifestation that has been associated with M. pneumoniae infections.

Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus.

Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients.

Diagnosis of human metapneumovirus infection in immunosuppressed lung transplant recipients and children evaluated for pertussis.

Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is known to cause respiratory tract infections in children and immunocompromised individuals. Given the difficulties of identifying hMPV by conventional culture, molecular techniques could improve the detection of this virus in clinical specimens. In this study, we developed a real-time reverse transcription-PCR (RT-PCR) assay designed to detect the four genetic lineages of hMPV.

Extensive genomic plasticity in Pseudomonas aeruginosa revealed by identification and distribution studies of novel genes among clinical isolates.

The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen.

Identification, distribution, and expression of novel genes in 10 clinical isolates of nontypeable Haemophilus influenzae.

We hypothesize that Haemophilus influenzae, as a species, possesses a much greater number of genes than that found in any single H. influenzae genome. This supragenome is distributed throughout naturally occurring infectious populations, and new strains arise through autocompetence and autotransformation systems.

Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by TaqMan PCR and in peripheral blood lymphocytes by competitive PCR.

Epstein-Barr virus (EBV) DNA load values were measured in samples of whole blood (n = 60) and plasma (n = 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n = 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r(2) > 0.

Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

Background: Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples.

Evaluation of Chlamydia pneumoniae and Mycoplasma pneumoniae as etiologic agents of persistent cough in adolescents and adults.

Chlamydia pneumoniae and Mycoplasma pneumoniae were evaluated as agents of persistent cough in adolescents and adults (n = 491). Tests of 473 respiratory specimens by culture or PCR or both identified four episodes (0.8%) of M.