Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.     

Back to BAC: the use of infectious clone technologies for viral mutagenesis

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate the propagation and manipulation of large DNA fragments in molecular biology studies for uses such as genome sequencing projects and genetic disease models. To facilitate these studies, methodologies have been developed to introduce specific mutations that can be directly applied to the mutagenesis of infectious clones (icBAC) using BAC technologies. This has resulted in rapid identification of gene function and expression at unprecedented rates. Here we review the major developments in BAC mutagenesis in vitro. This review summarises the technologies used to construct and introduce mutations into herpesvirus icBAC. It also explores developing technologies likely to provide the next leap in understanding these important viruses.     

Bifidobacterium infantis 35624 modulates host inflammatory processes beyond the gut

Certain therapeutic microbes, including Bifidobacteria infantis (B. infantis) 35624 exert beneficial immunoregulatory effects by mimicking commensal-immune interactions; however, the value of these effects in patients with non-gastrointestinal inflammatory conditions remains unclear. In this study, we assessed the impact of oral administration of B. infantis 35624, for 6‒8 weeks on inflammatory biomarker and plasma cytokine levels in patients with ulcerative colitis (UC) (n = 22), chronic fatigue syndrome (CFS) (n = 48) and psoriasis (n = 26) in three separate randomized, double-blind, placebo-controlled interventions. Additionally, the effect of B. infantis 35624 on immunological biomarkers in healthy subjects (n = 22) was assessed. At baseline, both gastrointestinal (UC) and non-gastrointestinal (CFS and psoriasis) patients had significantly increased plasma levels of C-reactive protein (CRP) and the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) compared with healthy volunteers. B. infantis 35624 feeding resulted in reduced plasma CRP levels in all three inflammatory disorders compared with placebo. Interestingly, plasma TNF-α was reduced in CFS and psoriasis while IL-6 was reduced in UC and CFS. Furthermore, in healthy subjects, LPS-stimulated TNF-α and IL-6 secretion by peripheral blood mononuclear cells (PBMCs) was significantly reduced in the B. infantis 35624-treated groups compared with placebo following eight weeks of feeding. These results demonstrate the ability of this microbe to reduce systemic pro-inflammatory biomarkers in both gastrointestinal and non-gastrointestinal conditions. In conclusion, these data show that the immunomodulatory effects of the microbiota in humans are not limited to the mucosal immune system but extend to the systemic immune system.     

Abnormal pregnancy: early diagnosis by US and serum chorionic gonadotropin levels

Simultaneous sonography and quantitative serum human chorionic gonadotropin (HCG) levels from 126 women with threatened abortion were compared. Of 56 women with normal outcome, 39 (70%) had a gestation sac greater than or equal to 5 mm in mean sac diameter, and in each case the HCG level was 1,800 milli-international units (mIU/ml) or greater. The serum HCG levels strongly correlated with the gestation sac sizes to a mean sac diameter of 25 mm. Of 70 abnormal pregnancies, 31 demonstrated a gestation sac. Of these, 20 women (65%) had disproportionately low HCG levels relative to sac size, including 12 in whom the HCG level was less than 1,800 mIU/ml. One woman with an early molar pregnancy had a disproportionately elevated HCG level. Correlation of sonograms with a simultaneous measurement of serum HCG level is a useful method for evaluating threatened spontaneous abortion. A disproportionately low HCG level relative to gestation sac size is evidence for an abnormal pregnancy.     

Diagnostic value of detecting JC virus DNA in cerebrospinal fluid of patients with progressive multifocal leukoencephalopathy.

JC virus DNA was detected by PCR in the cerebrospinal fluid of 17 of 23 (73.9%) patients with confirmed cases of progressive multifocal leukoencephalopathy and 2 of 48 (4.2%) controls without progressive multifocal leukoencephalopathy. The sensitivity and specificity of this PCR were 74 and 95.8%, respectively, while the positive and negative predictive values were 89.5 and 88.5%, respectively.     

Effect of time elapsed since previous voiding on the detection ofChlamydia trachomatis antigens in urine

To determine if the time elapsed since previous voiding affects the sensitivity of an enzyme immunoassay (EIA) to detectChlamydia trachomatis in urine, 882 women and 428 men were tested for chlamydial infection in urethral specimens by isolation in cell culture (women and men) and EIA with blocking confirmation (women only). Of the 36 women (4.1 %) and 38 men (8.9 %) who were positive forChlamydia trachomatis in the urethra, 55.5 % (20/36) and 81.6 % (31/38) respectively were positive in the first void urine (FVU) sediment by confirmed EIA. In women the sensitivity of the EIA performed on FVU was 67.8 % (19/28) if the urine had been in the bladder <3 hours and decreased to 12.5 % (1/8) if longer times had elapsed (odds ratio 13.7; 95 % confidence interval 1.4 to 700.0) with no obvious confounding. In men a weaker association was present (odds ratio 2.7; 95 % confidence interval 0.4 to 22.3). These findings should enable investigators to optimize the analysis of urine to diagnose chlamydial infections.     

Predicting competency in automated machine use in an acquired brain injury population using neuropsychological measures.

The purpose of the current study was to explore whether performance on standardised neuropsychological measures could predict functional ability with automated machines and services among people with an acquired brain injury (ABI). Participants were 45 individuals who met the criteria for mild, moderate or severe ABI and 15 control participants matched on demographic variables including age- and education. Each participant was required to complete a battery of neuropsychological tests, as well as performing three automated service delivery tasks: a transport automated ticketing machine, an automated teller machine (ATM) and an automated telephone service. The results showed consistently high relationship between the neuropsychological measures, both as single predictors and in combination, and level of competency with the automated machines. Automated machines are part of a relatively new phenomena in service delivery and offer an ecologically valid functional measure of performance that represents a true indication of functional disability.     

Renal function in unilateral nephrectomy subjects.

Renal function in 32 subjects who had undergone unilateral nephrectomy (17 transplant donors and 15 subjects with unilateral renal disease) was compared with that of 22 normal subjects. The age-adjusted glomerular filtration rate was lower in transplant donors (79 +/- 15% of normal) than in those whose nephrectomy was performed for unilateral renal disease (90 +/- 12% of normal). The donors were also significantly older at nephrectomy (48 +/- 10 years versus 24 +/- 13 years, p less than 0.001). This finding may represent less capacity for compensatory hypertrophy. Proximal tubular and medullary function as assessed by 15-minute phenolsulfonphthalein excretion, maximum urinary concentration in response to water deprivation plus exogenous vasopressin, and urinary acidification in response to an oral acid load were all within normal limits for glomerular filtration rate. Overall renal function was well preserved after nephrectomy. A small number of patients did have increased cast excretion, which may signify the presence of mild renal disease in these subjects.     

Septa of Bertin: a sonographic pseudotumor

The sonographic features of six renal "pseudotumors" representing septa of Bertin are described. Septa of Bertin are isoechoic with renal parenchyma and demonstrate a characteristic splaying of the central sinus echoes. CT performed in all six cases revealed the "mass" to be normally enhancing renal cortex. The characteristic radiographic features of septa of Bertin are reviewed and the split sinus sign is discussed.