A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

No evidence of enteric viral involvement in the new neonatal porcine diarrhoea syndrome in Danish pigs.

BACKGROUND: The aim of this study was to investigate whether the syndrome New Neonatal Porcine Diarrhoea Syndrome (NNPDS) is associated with a viral aetiology. Four well-managed herds experiencing neonatal diarrhoea and suspected to be affected by NNPDS were included in a case-control set up. A total of 989 piglets were clinically examined on a daily basis. Samples from diarrhoeic and non-diarrhoeic piglets at the age of three to seven days were selected for extensive virological examination using specific real time polymerase chain reactions (qPCRs) and general virus detection methods. RESULTS: A total of 91.7% of the animals tested positive by reverse transcription qPCR (RT-qPCR) for porcine kobuvirus 1 (PKV-1) while 9% and 3% were found to be positive for rotavirus A and porcine teschovirus (PTV), respectively. The overall prevalence of porcine astrovirus (PAstV) was 75% with 69.8% of the PAstV positive pigs infected with PAstV type 3. No animals tested positive for rotavirus C, coronavirus (TGEV, PEDV and PRCV), sapovirus, enterovirus, parechovirus, saffoldvirus, cosavirus, klassevirus or porcine circovirus type 2 (PCV2). Microarray analyses performed on a total of 18 animals were all negative, as were eight animals examined by Transmission Electron Microscopy (TEM). Using Next Generation de novo sequencing (de novo NGS) on pools of samples from case animals within all herds, PKV-1 was detected in four herds and rotavirus A, rotavirus C and PTV were detected in one herd each. CONCLUSIONS: Our detailed analyses of piglets from NNPDS-affected herds demonstrated that viruses did not pose a significant contribution to NNPDS. However, further investigations are needed to investigate if a systemic virus infection plays a role in the pathogenesis of NNPDS.

A case-control study of risk factors for rotavirus infections in adults, Denmark, 2005-2009.

Rotavirus (RV) infections affect young children, but can also occur in adults. We sought to identify risk factors for RV infections in adults aged ⩾18 years in Denmark, and to describe illness and genotyping characteristics. From March 2005 to February 2009, we recruited consecutive cases of laboratory-confirmed RV infection and compared them with healthy controls matched by age, gender and municipality of residence. We collected information on illness characteristics and exposures using postal questionnaires. We calculated univariable and multivariable matched odds ratios (mOR) with conditional logistic regression. The study comprised 65 cases and 246 controls. Illness exceeded 10 days in 31% of cases; 22% were hospitalized. Cases were more likely than controls to suffer serious underlying health conditions [mOR 5·6, 95% confidence interval (CI) 1·7-18], and to report having had close contact with persons with gastrointestinal symptoms (mOR 9·4, 95% CI 3·6-24), in particular young children aged 18 years. Close contact with young children or adults with gastrointestinal symptoms is the main risk factor for RV infection in adults in Denmark. RV vaccination assessments should consider that RV vaccination in children may indirectly reduce the burden of disease in adults.

Emergence of enterovirus D68 in Denmark, June 2014 to February 2015.

From June 2014 through February 2015, respiratory samples from 130 Danish patients were screened for enterovirus D68 (EV-D68). Fourteen EV-D68 cases were detected, of which 12 presented with respiratory symptoms, and eight had known underlying disease. The median age of EV-D68 cases was three years (interquartile range: 0­30 years). Acute flaccid paralysis (AFP) was not detected although Danish EV-D68 strains showed > 98% nt identity with EV-D68-strains from AFP cases from the United States and France.

Emergence of enterovirus 71 C4a in Denmark, 2009 to 2013.

Enterovirus (EV) 71 has emerged as a primary cause of severe neurologic enterovirus infection in the aftermath of the global polio eradication effort. Eleven subgenotypes of EV71 exist, the C4 subgenotype being associated with large outbreaks in Asia with high mortality rates. This subgenotype has rarely been reported in Europe. In the period between 1 January 2009 and 31 December 2013 a total of 1,447 EV positive samples from 1,143 individuals were sent to the Statens Serum Institute (SSI), and 938 samples from 913 patients were genotyped at the Danish National World Health Organization Reference laboratory for Poliovirus at SSI. Echovirus 6 (E06) (n=141 patients), echovirus 30 (E30) (n=114), coxsackievirus A6 (CA06) (n=96) and EV71 (n=63) were the most prevalent genotypes. We observed a shift in circulating EV71 subgenotypes during the study period, with subgenotype C4 dominating in 2012. A total of 34 EV71 patients were found to be infected with strains of the C4 subgenotype, and phylogenetic analysis revealed that they belonged to the C4a lineage. In our study, the proportions of cases with cerebral and/or sepsis-like symptoms were similar in those affected by C4a (19/34) and those with C1 and C2 (15/35). The majority (n=30) of the 34 EV71 C4 cases were children≤5 years of age, and males (n=22) were over-represented. Continued EV surveillance is required to monitor the spread of EV71 C4 in Denmark and the rest of Europe.

Suspected zoonotic transmission of rotavirus group A in Danish adults.

Group A rotaviruses infect humans and a variety of animals. In July 2006 a rare rotavirus strain with G8P[14] specificity was identified in the stool samples of two adult patients with diarrheoa, who lived in the same geographical area in Denmark. Nucleotide sequences of the VP7, VP4, VP6, and NSP4 genes of the identified strains were identical. Phylogenetic analyses showed that both Danish G8P[14] strains clustered with rotaviruses of animal, mainly, bovine and caprine, origin. The high genetic relatedness to animal rotaviruses and the atypical epidemiological features suggest that these human G8P[14] strains were acquired through direct zoonotic transmission events.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

A method of dosimetry for synchrotron microbeam radiation therapy using radiochromic films of different sensitivity.

This paper describes a method of film dosimetry used to measure the peak-to-valley dose ratios for synchrotron microbeam radiation therapy (MRT). Two types of radiochromic film (manufactured by International Specialty Products, NJ, USA) were irradiated in a phantom and also flush against a microbeam collimator (beam width 25 microm, centre-to-centre spacing 200 microm) on beamline BL28 B2 at the SPring-8 synchrotron. Four experiments are reported: (1) the HD-810 and EBT varieties of radiochromic film were used to record 'peak' dose and 'valley' (regions in between peaks) dose, respectively; (2) a stack of HD-810 film sheets was microbeam-irradiated and analysed to investigate a possible dose build-up effect; (3) a very high MRT dose was delivered to HD-810 film to elicit a measurable valley dose to compare with the result obtained using broad beam radiation; (4) the half value layer of the beam with and without the microbeam collimator was measured to investigate the effect of the collimator on the beam quality. The valley dose obtained for films placed flush against the collimator was approximately 0.2% of the peak dose. Within the water phantom, the valley dose had increased to between 0.7 and 1.8% of the peak dose, depending on the depth in the phantom. We also demonstrated, experimentally and by Monte Carlo simulation, that the dose is not maximal on the surface and that there is a dose build-up effect. The microbeam collimator did not make an appreciable difference to the beam quality. The values of the peak-to-valley ratio reported in this paper are higher than those predicted by previously published Monte Carlo simulation papers.

Angular resolution in magnification radiography and the observation of x-ray wave interaction signatures.

High resolution x-ray imaging studies have demonstrated significant radiographic contrast enhancements that are attributed to wave interactions within the sample. This paper reviews diffraction and refraction in the context of medical radiography, describing signatures produced by each process and the necessary experimental conditions for observing them. The concept of angular resolution is introduced and applied to current x-ray source and detector configurations, testing their ability to record these features. It is difficult to record interference patterns arising from refractive phase shifts because their formation requires a mono-energetic beam. The refraction of x-rays across boundaries, as described by Snell's law, produces strong contrast enhancements when they are struck at close to the glancing incidence. Deflections are proportional to the change in electron density (at energies above the K-edge) and square root of the wavelength, so they can be observed with a poly-energetic beam. Diffraction can also be observed with white radiation, but produce fringes with far narrower separation under the same irradiation conditions. In both cases, the observation of wave interaction signatures requires a propagation distance between the sample and detector, and selection of an appropriate geometric magnification, which can be estimated using a simple model presented here.

Materials analysis using x-ray linear attenuation coefficient measurements at four photon energies.

The analytical properties of an accurate parameterization scheme for the x-ray linear attenuation coefficient are examined. The parameterization utilizes an additive combination of N compositional- and energy-dependent coefficients. The former were derived from a parameterization of elemental cross-sections using a polynomial in atomic number. The compositional-dependent coefficients are referred to as the mixture parameters, representing the electron density and higher order statistical moments describing elemental distribution. Additivity is an important property of the parameterization, allowing measured x-ray linear attenuation coefficients to be written as linear simultaneous equations, and then solved for the unknown coefficients. The energy-dependent coefficients can be determined by calibration from measurements with materials of known composition. The inverse problem may be utilized for materials analysis, whereby the simultaneous equations represent multi-energy linear attenuation coefficient measurements, and are solved for the mixture parameters. For in vivo studies, the choice of measurement energies is restricted to the diagnostic region (approximately 20 keV to 150 keV), where the parameterization requires N >or= 4 energies. We identify a mathematical pathology that must be overcome in order to solve the inverse problem in this energy regime. An iterative inversion strategy is presented for materials analysis using four or more measurements, and then tested against real data obtained at energies 32 keV to 66 keV. The results demonstrate that it is possible to recover the electron density to within +/-4% and fourth mixture parameter. It is also a key finding that the second and third mixture parameters cannot be recovered, as they are of minor importance in the parameterization at diagnostic x-ray energies.

Multimodality monitoring as a guide to treatment of intracranial hypertension after severe brain injury.

Transcranial doppler measurements of blood flow velocity in the middle cerebral artery were made during treatment of raised intracranial pressure (ICP) in 22 patients with severe brain injury. Twenty patients also had continuous measurement of arterial and jugular bulb venous oxygen saturation (SJO2). The transcranial Doppler parameters studied included both mean flow velocity and pulsatility index (PI). Successful treatment was defined as a reduction of ICP to less than 20 mm Hg with improvement or preservation of cerebral perfusion pressure (CPP) above 60 mm Hg. Successful therapy was associated with a significant rise in SJO2 and reduction of cerebral arteriovenous oxygen content difference (AVDO2) and PI only when the pretreatment CPP was less than 60 mm Hg. An increase in CPP beyond 70 mm Hg did not further improve cerebral oxygen delivery and PI, suggesting that autoregulation became a factor above this CPP threshold. Treatment failure during administration of hypnotic drugs resulted in a reduction in arterial pressure, CPP, SJO2, and mean velocity and in an increase in PI and AVDO2, despite a decrease in ICP. CPP is the most important parameter to monitor during ICP therapy. It should be maintained above 70 mm Hg in patients with severe brain injury.

The effect of changes in cerebral perfusion pressure upon middle cerebral artery blood flow velocity and jugular bulb venous oxygen saturation after severe brain injury.

Middle cerebral artery blood flow velocity and jugular bulb venous oxygen saturation (SJO2) were measured by transcranial Doppler (TCD) ultrasonography and continuous venous oximetry, respectively, in 41 severely brain-injured patients. The purpose of the study was to examine the relationships between TCD flow velocity, SJO2, and alterations in blood pressure (BP), intracranial pressure (ICP), and cerebral perfusion pressure (CPP). In these patients, CPP was reduced either by rising ICP or by falling BP. Both forms of reduction of CPP resulted in a greater fall in diastolic flow velocity than other flow parameters. As CPP decreased below a critical value of 70 mm Hg, a progressive increase in TCD pulsatility index (PI) was observed (r = -0.942, p less than 0.0001), accompanied by a fall in SJO2 (r = 0.78, p less than 0.0001). At pressures above 70 mm Hg, there was no correlation of either PI or SJO2 with CPP. The relationship between PI and CPP held true in patients with both focal and diffuse pathologies and was the same whether changes in CPP resulted from alterations in ICP or BP. The PI and SJO2 correlated better with CPP than with ICP or BP. Transcranial Doppler ultrasonography can identify states of reduced CPP. Decreases in SJO2 with falling CPP suggested progressive failure of cerebral blood flow to meet metabolic demands. Monitoring of TCD and SJO2 may be used to define the optimum CPP level for management of severely brain-injured patients.

Role of intracranial pressure monitoring in severely head-injured patients without signs of intracranial hypertension on initial computerized tomography.

Previous studies have suggested that only a small proportion (< 15%) of comatose head-injured patients whose initial computerized tomography (CT) scan was normal or did not show a mass lesion, midline shift, or abnormal basal cisterns develop intracranial hypertension. The aim of the present study was to re-examine this finding against a background of more intensive monitoring and data acquisition. Eight severely head-injured patients with a Glasgow Coma Scale score of 8 or less, whose admission CT scan did not show a mass lesion, midline shift, or effaced basal cisterns, underwent minute-to-minute recordings of arterial blood pressure, intracranial pressure (ICP), and cerebral perfusion pressure (CPP) derived from blood pressure minus ICP. Intracranial hypertension (ICP > or = 20 mm Hg lasting longer than 5 minutes) was recorded in seven of the eight patients; in five cases the rise was pronounced in terms of both magnitude (ICP > or = 30 mm Hg) and duration. Reduced CPP (< or = 60 mm Hg lasting longer than 5 minutes) was recorded in five patients. Severely head-injured (comatose) patients whose initial CT scan is normal or does not show a mass lesion, midline shift, or abnormal cisterns nevertheless remain at substantial risk of developing significant secondary cerebral insults due to elevated ICP and reduced CPP. The authors recommend continuous ICP and blood pressure monitoring with derivation of CPP in all comatose head-injured patients.

A parameterization scheme for the x-ray linear attenuation coefficient and energy absorption coefficient.

A novel parameterization of x-ray interaction cross-sections is developed, and employed to describe the x-ray linear attenuation coefficient and mass energy absorption coefficient for both elements and mixtures. The new parameterization scheme addresses the Z-dependence of elemental cross-sections (per electron) using a simple function of atomic number, Z. This obviates the need for a complicated mathematical formalism. Energy dependent coefficients describe the Z-direction curvature of the cross-sections. The composition dependent quantities are the electron density and statistical moments describing the elemental distribution. We show that it is possible to describe elemental cross-sections for the entire periodic table and at energies above the K-edge (from 6 keV to 125 MeV), with an accuracy of better than 2% using a parameterization containing not more than five coefficients. For the biologically important elements 1 < or = Z < or = 20, and the energy range 30-150 keV, the parameterization utilizes four coefficients. At higher energies, the parameterization uses fewer coefficients with only two coefficients needed at megavoltage energies.

A feasibility study for megavoltage cone beam CT using a commercial EPID.

This study used a standard commercial electronic portal imaging device (EPID) area detector attached to an isocentric linear accelerator and the Feldkamp algorithm to produce cone beam tomographic reconstructions. The EPID has a active area of 32.5 x 32.5 cm2, and can record 12-bit images using two monitor units (MU), with a resolution of 2.1 x 2.0 mm2 FWHM. Since the EPID was not large enough to record the full patient projection at about 1.5 geometric magnification, it was necessary to offset the detector to collect half-cone projections. Corrections are required to convert pixel values into units of exit dose and to realign the projections to overcome the +/- 4 mm support arm sag. With a geometric magnification of 1.5 the sensitive volume is a cylinder of radius 21 cm and length 17 cm. Unfortunately, the patient couch contains metal bed support rails that lie just outside this cylinder, and produce streak artefacts in the reconstruction. Using 90 views the system delivers a central dose of 90 cGy, and has a density resolution of 4%.

What do general practitioners and community mental health teams talk about? Descriptive analysis of liaison meetings in general practice.

BACKGROUND: Liaison meetings between psychiatrists and general practitioners are now well established. Much has been written about their purpose and structure but little about their content. AIM: A study aimed to describe the clinical focus of meetings between a community mental health team and general practitioners and the nature of the professionals' interactions. METHOD: Audiotapes of six consecutive monthly meetings between a community mental health team and general practitioners in two general practices were analysed. RESULTS: Attendance rates among professionals were over 70%. Over 90% of discussion time was focused on patient-centred clinical matters. Almost two thirds of interactions were focused on patients receiving ongoing joint care; few interactions were devoted to new referrals or to patients who had not been assessed. Psychotic patients, although accounting for 15% of referrals, occupied 54% of patient-centred discussion time. Most interactions consisted of reciprocal information exchange between members of the community mental health team and general practitioners. CONCLUSION: The high attendance rates indicate that both general practitioners and community mental health team members considered these meetings as high priority. The steady move towards management of severely ill psychiatric patients in the community rather than in hospital requires close collaboration between primary and secondary care teams. The meetings described in this paper appear to be a simple, manageable and sustainable response to this need.

Measuring the burden of secondary insults in head-injured patients during intensive care.

Primary traumatic brain damage may be compounded by secondary pathophysiological insults that can occur soon after trauma, during transfer to hospital or subsequent treatment of the head-injured patient. The aim of this prospective study was to quantify the burden of a wide range of secondary insults occurring after head injury and to relate these to 12-month outcome. In 124 adult head-injured patients studied during intensive care using a computerized data collection system, < or = 14 clinically indicated physiological variables were measured minute-by-minute. Verified values falling outside threshold limits for > or = 5 min, as defined by the Edinburgh University Secondary Insult Grading scheme, were analysed by insult grade and duration. A greater incidence of secondary insults was detected than previous studies have indicated. Insults were found in 91% of patients and occurred in all severities of head trauma, at all ages, and at every level of Injury Severity Score (ISS). The cumulative durations were much greater than previously recorded although 85% of the total time was at the least severe grade. Short duration insults were common. In 71 patients, in whom 8 insults could be assessed (intracranial pressure, arterial hypo- and hypertension, cerebral perfusion pressure, hypoxemia, pyrexia, brady- and tachycardia), outcome at 12 months was analysed using logistic regression to determine the relative influence of age, admission Glasgow Coma Sumscore, ISS, pupil response on admission, and insult duration on both mortality and morbidity. The most significant predictors of mortality in this patient set were durations of hypotensive (p = .0064), pyrexic (p = .0137), and hypoxemic (p = .0244) insults. When good versus poor outcome was considered, hypotensive insults (p = .0118) and pupil response on admission (p = .0226) were significant.

Quantum dynamics under the influence of external magnetic fields.

In this paper, we describe a very versatile numerical model, which can be applied to study the quantum dynamics of an electron wave packet in the presence of external electric and magnetic fields. Detailed numerical analysis is carried out in the paper on the validity of such a model, including (1). comparison with analytical solution in the case of uniform magnetic field, and (2). stringent numerical tests in the case of both electric and magnetic fields. The algorithm presented here is found to be highly accurate, computationally efficient, and numerically stable. It also has the advantage of being able to accommodate arbitrarily complex magnetic field profiles as well as time-varying external fields.