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With the proposal to include Aspergillus PCR in the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) definitions for fungal disease, commercially manufactured assays may be required to provide standardization and accessibility. The PathoNostics AsperGenius assay represents one such test that has the ability to detect a range of Aspergillus species as well as azole resistance in Aspergillus fumigatus . Its performance has been validated on bronchoalveolar lavage (BAL) fluid and serum specimens, but recent evidence suggests that testing of plasma may have enhanced sensitivity over that with serum. We decided to evaluate the analytical and clinical performances of the PathoNostics AsperGenius assay for testing of plasma. For the analytical evaluations, plasma was spiked with various concentrations of Aspergillus genomic DNA before extraction following international recommendations, using two automated platforms. For the clinical study, 211 samples from 10 proven/probable invasive aspergillosis (IA) and 2 possible IA cases and 27 controls were tested. The limits of detection for testing of DNA extracted using the bioMérieux EasyMag and Qiagen EZ1 extractors were 5 and 10 genomes/0.5-ml sample, respectively. In the clinical study, true positivity was significantly greater than false positivity ( P < 0.0001). The sensitivity and specificity obtained using a single positive result as significant were 80% and 77.8%, respectively. If multiple samples were required to be positive, specificity was increased to 100%, albeit sensitivity was reduced to 50%. The AsperGenius assay provided good clinical performance, but the predicted improvement of testing with plasma was not seen, possibly as a result of target degradation attributed to sample storage. Prospective testing is required to determine the clinical utility of this assay, particularly for the diagnosis of azole-resistant disease.

Quantitative real-time PCR (qPCR) is increasingly used to detect Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia (PCP), but there are differences in the nucleic acids targeted, DNA only versus whole nucleic acid (WNA), and also the target genes for amplification. Through the Fungal qPCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicentre and monocentre evaluation of PCP qPCR assays was performed. For the multicentre study, 16 reference laboratories from eight different countries, performing 20 assays analysed a panel consisting of two negative and three PCP positive samples. Aliquots were prepared by pooling residual material from 20 negative or positive- P. jirovecii bronchoalveolar lavage fluids (BALFs). The positive pool was diluted to obtain three concentrations (pure 1:1; 1:100; and 1:1000 to mimic high, medium, and low fungal loads respectively). The monocentre study compared five in-house and five commercial qPCR assays testing 19 individual BALFs on the same amplification platform. Across both evaluations and for all fungal loads, targeting WNA and the mitochondrial small sub-unit (mtSSU) provided the earliest Cq values, compared to only targeting DNA and the mitochondrial large subunit, the major surface glycoprotein or the beta-tubulin genes. Thus, Reverse Transcriptase-qPCR targeting the mtSSU gene could serve as a basis for standardizing the P. jirovecii load, which is essential if qPCR is to be incorporated into clinical care pathways as the reference method, accepting that additional parameters such as amplification platforms still need evaluation

Invasive fungal disease (IFD) can be caused by a range of pathogens. Conventional diagnosis has the capacity to detect most causes of IFD, but poor performance limits impact. The introduction of non-culture diagnostics, including the detection of (1-3)-β-D-Glucan (BDG), has shown promising performance for the detection of IFD in variety of clinical settings. Recently, the Dynamiker® Fungus (1-3)-β-D-Glucan assay (D-BDG) was released as an IFD diagnostic test. This article describes an evaluation of the D-BDG assay for the diagnosis of invasive aspergillosis (IA), invasive candidiasis (IC) and Pneumocystis pneumonia (PCP) across several high-risk patient cohorts and provides comparative data with the Associates of Cape Cod Fungitell® and BioRad Platelia™ Aspergillus Ag (GM) assays. There were 163 serum samples from 121 patients tested, from 21 probable IA cases, 28 proven IC cases, six probable PCP cases, one probable IFD case, 14 possible IFD cases and 64 control patients. For proven/probable IFD the mean BDG concentration was 209pg/ml, significantly greater than the control population (73pg/ml; P

Timing of Candida invasive fungal disease relative to critical care unit admission (Nâ =â 359). (DOC 27 kb)

Area under the operating curve for clinical decision rules. (DOC 110 kb)

Therapies received, fungal colonisation, mortality and length of stay by invasive fungal disease subgroup. (DOC 61 kb)

Description of patients included at each time point in each of the development and validation samples. (DOC 37 kb)

Site of Candida invasive fungal disease (Nâ =â 359). (DOC 31 kb)

Final risk models at admission, 24Â h and end of calendar day 3. (DOC 44 kb)

Modern wind turbine blades are large composite structures with complex geometry, subjected to many different loading cases and structural constraints. As a consequence, design and analysis of wind turbine blades is laborious and computational resource requirements are high. In this thesis, an improved design methodology for composite wind turbine blades has been developed that allows a larger scope and greater flexibility than the traditional design process, whilst maintaining moderate computational resource requirements. The methodology incorporates a highly flexible parametric model into a structural optimisation procedure, and uses data mining techniques to reduce the computational resource requirements without sacrificing flexibility or accuracy. The method is demonstrated by application to two wind turbine blade design problems: a standard blade for use in high wind speed areas and a specialised blade designed for use in areas with low wind speeds. The specific requirements of each design problem were determined with a quantitative comparison of high and low wind speed blade structures. Common simplifications that are used in wind turbine blade design were assessed, namely material placement and internal structural geometry along with critical load assumptions and the loading approximation method. The results indicated that the problem formulation should include additional materials placement variables, a wider range of constraints, and additional loading cases using accurate loading approximation methods. The consequence of incorporating the additional variables, loading cases and constraints is that the complexity of the optimisation problem is increased. In order to remedy this, data mining techniques were used to systematically simplify the problem formulations. Correlations between pairs of structural responses were identified, which allowed the number of loading case evaluations and constraints to be reduced, and the most significant variables were identified, and the remainder eliminated. The simplified problem formulations were solved using a genetic algorithm to determine optimum high and low wind speed blade designs. The resulting designs have significantly reduced mass and materials cost compared to the traditionally-designed baseline blades, particularly for the low wind speed blade design, which features a spar cap that is predominately CFRP, and a greater proportion of material in the aerodynamic shell than the traditional design.

The European Aspergillus PCR Initiative (EAPCRI) has provided recommendations for the PCR testing of whole blood (WB) and serum/plasma. It is important to test these recommended protocols on nonsimulated “ in vivo ” specimens before full clinical evaluation. The testing of an animal model of invasive aspergillosis (IA) overcomes the low incidence of disease and provides experimental design and control that is not possible in the clinical setting. Inadequate performance of the recommended protocols at this stage would require reassessment of methods before clinical trials are performed and utility assessed. The manuscript describes the performance of EAPCRI protocols in an animal model of invasive aspergillosis. Blood samples taken from a guinea pig model of IA were used for WB and serum PCR. Galactomannan and β- d -glucan detection were evaluated, with particular focus on the timing of positivity and on the interpretation of combination testing. The overall sensitivities for WB PCR, serum PCR, galactomannan, and β- d -glucan were 73%, 65%, 68%, and 46%, respectively. The corresponding specificities were 92%, 79%, 80%, and 100%, respectively. PCR provided the earliest indicator of IA, and increasing galactomannan and β- d -glucan values were indicators of disease progression. The combination of WB PCR with galactomannan and β- d -glucan proved optimal (area under the curve [AUC], 0.95), and IA was confidently diagnosed or excluded. The EAPRCI-recommended PCR protocols provide performance comparable to commercial antigen tests, and clinical trials are warranted. By combining multiple tests, IA can be excluded or confirmed, highlighting the need for a combined diagnostic strategy. However, this approach must be balanced against the practicality and cost of using multiple tests.

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