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.

A method for estimating radiation interaction coefficients for tissues from single energy CT.

A parametric model for the x-ray linear attenuation coefficient is used to describe the compositional dependence of Hounsfield numbers measured by medical CT scanners. Measurements with materials of known density and composition, that span and evenly sample the compositional range of tissues, are written as linear simultaneous equations and solved for model coefficients. An algorithm is identified for this purpose. Results are expressed as atomic cross-sections in units of barn per electron divided by the attenuation coefficient for water. With the CT scanner characterised, a virtual CT scan can be simulated to predict HN for tissues based upon their known density and composition. Similar calculations using the tabulations and mixture rule deliver attenuation coefficients and mass energy absorption coefficients for mono-energetic radiation 10 keV to 20 MeV. Results are presented for measurements with a radiotherapy CT simulator, the RMI-467 phantom with tissue substitute materials, plus common polymer materials and silicon. Published measurements with earlier generations of the phantom and tissue substitutes using different CT scanners are also considered. Measured atomic cross-sections differ from expectations for mono-energetic radiation due to the use of a filtered spectrum and energy integrating detection system. The cross-sections for different CT scanners are similar, without large variations with kVp. Results are presented showing the relationship between predicted HN for tissues, electron density and photon interaction coefficients for healthy tissues and mono-energetic radiation. A strategy is suggested for accommodating strongly attenuating materials such as calculi and metallic implants.

Capture and analysis of radiation dose reports for radiology.

Radiographic imaging systems can produce records of exposure and dose parameters for each patient. A variety of file formats are in use including plain text, bit map images showing pictures of written text and radiation dose structured reports as text or extended markup language files. Whilst some of this information is available with image data on the hospital picture archive and communication system, access is restricted to individual patient records, thereby making it difficult to locate multiple records for the same scan protocol. This study considers the exposure records and dose reports from four modalities. Exposure records for mammography and general radiography are utilized for repeat analysis. Dose reports for fluoroscopy and computed tomography (CT) are utilized to study the distribution of patient doses for each protocol. Results for dosimetric quantities measured by General Radiography, Fluoroscopy and CT equipment are summarised and presented in the Appendix. Projection imaging uses the dose (in air) area product and derived quantities including the dose to the reference point as a measure of the air kerma reaching the skin, ignoring movement of the beam for fluoroscopy. CT uses the dose indices CTDIvol and dose length product as a measure of the dose per axial slice, and to the scanned volume. Suitable conversion factors are identified and used to estimate the effective dose to an average size patient (for CT and fluoroscopy) and the entrance skin dose for fluoroscopy.

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.

Human group A rotavirus infections in children in Denmark: detection of reassortant G9 strains and zoonotic P[14] strains.

One of the leading causes of severe childhood gastroenteritis are group A rotaviruses, and they have been found to be associated with ∼40% of the annual gastroenteritis-associated hospitalizations in young Danish children <5years of age (Fischer et al., 2011). In this study, we investigated the diversity of rotavirus strains circulating among young children <5years of age, presenting with gastroenteritis disease either at the general practitioner or in the hospital, during the period 2009-2013. A total of 831 rotavirus positive stool samples were genotyped in the study period, and the majority of samples (74%) were from hospitalized children. G and P genotypes were successfully determined for 826 of samples, with G1P[8] being the most commonly detected genotype. Detection of G1 showed a decreasing trend over time, and an inverse trend was seen for the emerging G9P. The common human genotypes (G1/G3/G4/G9P[8] and G2P[4]) were detected in the majority of samples (n=733, 88.2%). Rare genotype combinations such as G6P[14] were detected in <1% of samples. Rare genotype strains and strains which failed to amplify in genotyping RT-PCR were subjected to genetic characterization by sequencing one or all of the following genes; VP7, VP4, VP6 and NSP4. Sequences of sufficient length and quality were available for all 4 genes for 28 strains. Phylogenetic analysis revealed that reassortant G9P[4] strains circulated with 3 different genotype combinations. As rotavirus vaccines are not widely used in Denmark or its neighboring countries, the diversity of rotavirus strains identified in this study most likely reflects naturally occurring selection pressures and viral evolution.

Feasibility study for DEXA using synchrotron CT at 20-35 keV.

A nonlinear model for the x-ray linear attenuation coefficient µ is employed for dual energy x-ray analysis (DEXA). Nonlinear simultaneous equations formed by µ and energy dependent model parameters are solved for the electron density N(e) and fourth compositional ratio R(4) which has the same 'units' as the atomic number. Computed tomography data was acquired at 20-35 keV using bending magnet synchrotron radiation, a double crystal monochromator, a rotation stage and an area detector. Test objects contained liquid samples as mixtures of ethanol, water and salt solutions with known density and composition. Various noise sources are identified and give µ uncertainties of 1-2%. A fan beam geometry allowed the detection of forward scattered radiation with measured µ being 6% lower than expectations for a narrow beam. Energy dependent model parameters were obtained by solving linear simultaneous equations formed by µ and material parameters based upon N(e) and R(4). DEXA accuracy was studied as a function of photon energy and sample composition. Propagation of errors analysis identifies the importance of the fractional compositional cross-products whose difference at the two beam energies should exceed 0.1, requiring 10 keV or more separation. For a reasonable approximation for the adjustable model parameters, the mean difference between the DEXA solution and true values (ΔN(e), ΔR(4)) are (1.0%, 0.5%) for soft tissue and (1.5%, 0.8%) for bone like samples.

Estimating photon interaction coefficients from single energy x-ray CT.

Single energy x-ray analysis is explored in the context of computed tomography (CT), whereby Hounsfield numbers (HN) are used to estimate electron density N(e) and parameters that describe composition. We examine measurements with tissue substitute materials and theoretical HN for a broad range of tissues. Results are combined with parametric models for the x-ray linear attenuation coefficient µ and energy absorption coefficient µ(en) to predict values at energies 10 keV to 20 MeV. At photon energies employed for CT, the fractional contribution to µ from composition is 0.1-0.4 for soft tissues to bone respectively, and is responsible for strong correlations between HN and N(e). The atomic density of tissues excluding lung is near constant allowing the models to be re-expressed as a function of N(e) alone. The transformed model is subjected to propagation of error analysis and results are presented as the ratio of uncertainties for µ or µ(en) to those for N(e). For soft tissues to bone the ratios are as follows: at photon energies 20-100 keV the ratio is 5.0-2.0, at intermediate energies it is unity and increases above 4 MeV to reach 1.5-2.0 at 20 MeV. Results are discussed in the context of attenuation correction and dosimetry calculations for the same range of photon energies.

Assessment of patient dose and image quality for cardiac CT with breast shields.

Breast shielding can reduce dose to the female breast, a radiosensitive organ receiving significant radiation during computed tomography (CT) chest examinations, particularly in cardiac CT, where Electrocardiogram dose modulation currently precludes the use of radial dose modulation to reduce breast dose. However, breast shields may produce artefacts affecting interpretation of coronary arteries. This study explores the dose savings and the effect of breast shields on image quality with torso and CT dose index body phantoms and an organ dose calculator. Change in dose calculated: 53-63 % (female breast), 82-85 % (lung), 79-84 % (oesophagus) and 76-80 % (effective dose) with larger dose reductions at lower kVp. Image quality is preserved when breast shields are placed after the scout no closer than 10 mm from the skin. Therefore, breast shields can be used in cardiac CT to reduce breast dose without compromising image quality. Revised conversion factors for dose length product to effective dose are suggested for cardiac CT without and with breast shields.

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.

Feasibility study for a novel method of dual energy x-ray analysis.

Dual energy x-ray analysis (DEXA) is investigated using a nonlinear model for the x-ray linear attenuation coefficient µ that is expressed as a function of electron density N(e) and the fourth compositional ratio R4. Nonlinear simultaneous equations are solved using a least-squares algorithm based upon the method of Levenberg and Marquardt. Measurements of µ for low atomic number materials (containing elements hydrogen to calcium) at energies 32-66 keV are used to study DEXA accuracy as a function of sample composition, photon energy and their separation ΔE. Results are presented for ΔE = 5-30 keV, for 2% measurement precision, and the doses involved are quantified. The model is subject to propagation of error analysis and results are presented for the relationship between measurement uncertainties and those for N(e) and R4. The analysis shows how DEXA accuracy is controlled by the fractional compositional cross-product, which represents the contribution of composition to µ, and how this can be optimized by careful selection of beam energies according to the compositional range of interest. Accurate DEXA is achieved over restricted energy and compositional ranges: soft tissues only at approximately 15-25 keV, all tissues at approximately 30-80 keV and, for situations where a higher dose can be tolerated, all tissues at approximately 4-8 MeV.

A model for multi-energy x-ray analysis.

Multi-energy x-ray analysis (MEXA) uses measurements of the x-ray linear attenuation coefficient µ, obtained at different photon energies to determine parameters that characterize the density and composition of materials. The key to achieving this goal is an accurate parameterization for µ, allowing measurements to be written as simultaneous equations and then solved. This author has reported such a model where mixtures are characterized by four or more statistical moments that describe the distribution of atomic number. These can be re-expressed as the product of the electron density N(e) and four or more compositional ratios R(k) with the same 'units' as atomic number (i.e. dimensionless). The model was turned to MEXA where it delivered reliable estimates for N(e) and R(4) and not the intermediate compositional ratios. This report studies the relationships between compositional ratios for tissues and tissue substitute materials. Correlations are identified leading to a new parameterization that is expressed as a nonlinear function of N(e), R(4) and other coefficients. The properties of the transformed parameterizations for µ and the energy absorption coefficient µ(en) are considered for low atomic number materials at energies 15-100 keV, and for a broader range of materials at energies 5 keV to 20 MeV. The interpretation of the parameters N(e) and R(4) is explored in terms of basis materials. The general case of three basis materials cannot be solved for all contributions, but the special case of just two basis materials can be fully solved.

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.