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Immunocompromised patients are highly susceptible to invasive aspergillosis. Herein, we identified a homozygous deletion mutation (507 del C) resulting in a frameshift (N170I) and early stop codon in the fungal binding Dectin-2 receptor, in an immunocompromised patient. The mutated form of Dectin-2 was weakly expressed, did not form clusters at/near the cell surface and was functionally defective. Peripheral blood mononuclear cells from this patient were unable to mount a cytokine (tumor necrosis factor, interleukin 6) response to Aspergillus fumigatus, and this first identified Dectin-2–deficient patient died of complications of invasive aspergillosis., We identified a Dectin-2 N170I mutation in an immunocompromised patient who died of complications of invasive aspergillosis. This mutation results in an early stop codon and poor receptor expression and renders Dectin-2 functionally defective.

Background While the serological detection of (1→3)-β-D-glucan (BDG) can indicate invasive fungal disease (IFD), false positivity occurs. Nevertheless, the presence of BDG can still be recognized by the host’s innate immune system and persistent BDG antigenemia, in the absence of IFD, can result in deleterious proinflammatory immune responses. Methods During the XXX (INTENSE) study into the preemptive use of micafungin to prevent invasive candidiasis (IC) after abdominal surgery, the serum burden of BDG was determined to aid diagnosis of IC. Data from the INTENSE study were analyzed to determine whether BDG was associated with organ failure and patient mortality, while accounting for the influences of IC and antifungal therapy. Results A BDG concentration >100 pg/mL was associated with a significantly increased Sequential Organ Failure Assessment score (≤100 pg/mL: 2 vs >100 pg/mL: 5; P < .0001) and increased rates of mortality (≤100 pg/mL: 13.7% vs >100 pg/mL: 39.0%; P = .0002). Multiple (≥2) positive results >100 pg/mL or a BDG concentration increasing >100 pg/mL increased mortality (48.1%). The mortality rate in patients with IC and a BDG concentration >100 pg/mL and ≤100 pg/mL was 42.3% and 25.0%, respectively. The mortality rate in patients without IC but a BDG concentration >100 pg/mL was 37.3%. The use of micafungin did not affect the findings. Conclusions The presence of persistent or increasing BDG in the patient’s circulation is associated with significant morbidity and mortality after abdominal surgery, irrespective of IC. The potential lack of a specific therapeutic focus has consequences when trying to manage these patients, and when designing clinical trials involving patients where host-associated BDG concentrations may be elevated. Clinical Trials Registration NCT01122368.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77–100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Background Neutropenic sepsis remains a common treatment complication for patients receiving systemic anti-cancer treatment. The UK National Institute for Health and Care Excellence have not recommended switching from empirical intravenous antibiotics to oral antibiotics within 48 h for patients assessed as low risk for septic complications because of uncertainty about whether this would achieve comparable outcomes to using intravenous antibiotics for longer. The UK National Institute for Health Research funded the EASI-SWITCH trial to tackle this uncertainty. Methods The trial is a pragmatic, randomised, non-inferiority trial that aims to establish the clinical and cost-effectiveness of early switching from intravenous to oral antibiotics in cancer patients with low-risk neutropenic sepsis. Patients ≥ 16 years, receiving systemic anti-cancer treatment (acute leukaemics/stem cell transplants excluded), with a temperature of > 38 °C, neutrophil count ≤ 1.0 × 109/L, MASCC (Multinational Association of Supportive Care in Cancer) score ≥ 21 and receiving IV piperacillin/tazobactam or meropenem for less than 24 h are eligible to participate. Patients are randomised 1:1 either (i) to switch to oral ciprofloxacin and co-amoxiclav within 12–24 h of commencing intravenous antibiotics, completing at least 5 days total antibiotics (intervention), or (ii) to continue intravenous antibiotics for at least 48 h, with ongoing antibiotics being continued at the physician’s discretion (control). Patients are discharged home when their physician deems it appropriate. The primary outcome measure is a composite of treatment failures as assessed at day 14. The criteria for treatment failure include fever persistence or recurrence 72 h after starting intravenous antibiotics, escalation from protocolised antibiotics, hospital readmission related to infection/antibiotics, critical care support or death. Based on a 15% treatment failure rate in the control group and a 15% non-inferiority margin, the recruitment target is 230 patients. Discussion If the trial demonstrates non-inferiority of early switching to oral antibiotics, with potential benefits for patient quality of life and resource savings, this finding will have significant implications for the routine clinical management of those with low-risk neutropenic sepsis. Trial registration ISRCTN: 84288963. Registered on the 1 July 2015. 10.1186/ISRCTN84288963. EudraCT: 2015-002830-35.

Standardization of Aspergillus polymerase chain reaction (PCR) protocols has progressed, and analytical validity of blood-based assays has been formally established. It remains necessary to consider how the tests can be used in practice to maximize clinical utility. To determine the optimal diagnostic strategies and influence on patient management, several factors require consideration, including the patient population, incidence of invasive aspergillosis (and other fungal disease), and the local antifungal prescribing policy. Technical issues such as specimen type, ease of sampling, frequency of testing, access to testing centers, and time to reporting will also influence the use of PCR in clinical practice. Interpretation of all diagnostic tests is dependent on the clinical context and molecular assays are no exception, but with the proposal to incorporate Aspergillus PCR into the second revision of the consensus guidelines for defining invasive fungal disease the acceptance and understanding of molecular tests should improve.

Contains fulltext : 226024.pdf (Publisher’s version ) (Closed access) 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 PCR Initiative, a working group of the International Society for Human and Animal Mycology, a multicenter and monocenter evaluation of PCP qPCR assays was performed. For the multicenter study, 16 reference laboratories from eight different countries, performing 20 assays analyzed 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 monocenter 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.

Despite the availability of four different classes of antifungal agents, invasive fungal infections, in particular mould diseases, continue to have a high crude mortality rate in adult haematology patients, especially when diagnosed late. Early diagnosis, resulting in prompt and adequate antifungal intervention, is of great importance when trying to improve the overall outcome of these infections, but depends on the availability of rapid and sensitive diagnostic tools. The medical community has developed and continues to evaluate a continuum of antifungal strategies (starting with prophylaxis followed by empirical therapy and more recently a diagnostic-driven or pre-emptive approach) to better tackle these life-threatening diseases. While the empirical approach seems to have lost some of its popularity, the jury is still out about the pros and cons of universal antifungal prophylaxis in at-risk adult haematology patients compared with an approach that uses radiological and mycological diagnostic methods with good to excellent negative predictive values (also erroneously called pre-emptive), trying to exclude the presence of an invasive fungal disease. Whilst awaiting the results of comparative clinical studies, believers and non-believers around the globe continue to argue about the advantages and shortcomings of both strategies. The debate presented here provides a rationale for both prophylaxis for 'high-risk' haematology patients as well as for a more targeted approach based on the appropriate use of mycological, radiological, immunological (and genetic) methods for the diagnosis of invasive fungal diseases.

Introduction: Pneumocystis jirovecii is a ubiquitous fungus, which causes pneumonia in humans. Diagnosis was hampered by the inability to culture the organism, and based on microscopic examination of respiratory samples or clinical presentation. New assays can assist in the diagnosis and even aid with the emergence of resistant infections.\ud \ud Areas covered: This manuscript will provide background information on Pneumocystis pneumonia (PcP). Diagnosis, from radiological to non-microbiological (e.g. Lactate dehydrogenase) and microbiological investigations (Microscopy, PCR, β-D-Glucan) will be discussed. Recommendations on prophylactic and therapeutic management will be covered.\ud \ud Expert commentary: PcP diagnosis using microscopy is far from optimal and false negatives will occur. With an incidence of 1% or less, the pre-test probability of not having PcP is 99% and testing is suited to excluding disease. Microscopy provides a high degree of diagnostic confidence but it is not infallible, and its lower sensitivity limits its application. Newer diagnostics (PCR, β-D-Glucan) can aid management and improve performance when testing less invasive specimens, such as upper respiratory samples or blood, alleviating clinical pressure. Combination testing may allow PcP to be both diagnosed and excluded, and molecular testing can assist in the detection of emerging resistant PcP.

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

Purpose In mechanically ventilated patients, the endotracheal tube is an essential interface between the patient and ventilator, but inadvertently, it also facilitates the development of ventilator-associated pneumonia (VAP) by subverting pulmonary host defenses. A number of investigations suggest that bacteria colonizing the oral cavity may be important in the etiology of VAP. The present study evaluated microbial changes that occurred in dental plaque and lower airways of 107 critically ill mechanically ventilated patients. Materials and Methods Dental plaque and lower airways fluid was collected during the course of mechanical ventilation, with additional samples of dental plaque obtained during the entirety of patients' hospital stay. Results A “microbial shift” occurred in dental plaque, with colonization by potential VAP pathogens, namely, Staphylococcus aureus and Pseudomonas aeruginosa in 35 patients. Post-extubation analyses revealed that 70% and 55% of patients whose dental plaque included S aureus and P aeruginosa, respectively, reverted back to having a predominantly normal oral microbiota. Respiratory pathogens were also isolated from the lower airways and within the endotracheal tube biofilms. Conclusions To the best of our knowledge, this is the largest study to date exploring oral microbial changes during both mechanical ventilation and after recovery from critical illness. Based on these findings, it was apparent that during mechanical ventilation, dental plaque represents a source of potential VAP pathogens.

Standardized methodologies for the molecular detection of invasive aspergillosis (IA) have been established by the European Aspergillus PCR Initiative for the testing of whole blood, serum, and plasma. While some comparison of the performance of Aspergillus PCR when testing these different sample types has been performed, no single study has evaluated all three using the recommended protocols. Standardized Aspergillus PCR was performed on 423 whole-blood pellets (WBP), 583 plasma samples, and 419 serum samples obtained from hematology patients according to the recommendations. This analysis formed a bicenter retrospective anonymous case-control study, with diagnosis according to 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) consensus definitions (11 probable cases and 36 controls). Values for clinical performance using individual and combined samples were calculated. For all samples, PCR positivity was significantly associated with cases of IA (for plasma, P = 0.0019; for serum, P = 0.0049; and for WBP, P = 0.0089). Plasma PCR generated the highest sensitivity (91%); the sensitivities for serum and WBP PCR were 80% and 55%, respectively. The highest specificity was achieved when testing WBP (96%), which was significantly superior to the specificities achieved when testing serum (69%, P = 0.0238) and plasma (53%, P = 0.0002). No cases were PCR negative in all specimen types, and no controls were PCR positive in all specimens. This study confirms that Aspergillus PCR testing of plasma provides robust performance while utilizing commercial automated DNA extraction processes. Combining PCR testing of different blood fractions allows IA to be both confidently diagnosed and excluded. A requirement for multiple PCR-positive plasma samples provides similar diagnostic utility and is technically less demanding. Time to diagnosis may be enhanced by testing multiple contemporaneously obtained sample types.

There is sufficient evidence that is at least equivalent to that used to include galactomannan enzyme immunoassay and β-d-glucan testing for polymerase chain reaction to be included in the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group definitions., Background. Aspergillus polymerase chain reaction (PCR) was excluded from the European Organisation for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) definitions of invasive fungal disease because of limited standardization and validation. The definitions are being revised. Methods. A systematic literature review was performed to identify analytical and clinical information available on inclusion of galactomannan enzyme immunoassay (GM-EIA) (2002) and β-d-glucan (2008), providing a minimal threshold when considering PCR. Categorical parameters and statistical performance were compared. Results. When incorporated, GM-EIA and β-d-glucan sensitivities and specificities for diagnosing invasive aspergillosis were 81.6% and 91.6%, and 76.9% and 89.4%, respectively. Aspergillus PCR has similar sensitivity and specificity (76.8%–88.0% and 75.0%–94.5%, respectively) and comparable utility. Methodological recommendations and commercial PCR assays assist standardization. Although all tests have limitations, currently, PCR is the only test with independent quality control. Conclusions. We propose that there is sufficient evidence that is at least equivalent to that used to include GM-EIA and β-d-glucan testing, and that PCR is now mature enough for inclusion in the EORTC/MSG definitions.

Invasive fungal diseases are an important cause of morbidity and mortality in a wide range of patients, and early diagnosis and management are a challenge. We therefore did a review of the scientific literature to generate a series of key recommendations for the appropriate use of microbiological, histological, and radiological diagnostic methods for diagnosis of invasive fungal diseases. The recommendations emphasise the role of microscopy in rapid diagnosis and identification of clinically significant isolates to species level, and the need for susceptibility testing of all Aspergillus spp, if treatment is to be given. In this Review, we provide information to improve understanding of the importance of antigen detection for cryptococcal disease and invasive aspergillosis, the use of molecular (PCR) diagnostics for aspergillosis, and the crucial role of antibody detection for chronic and allergic aspergillosis. Furthermore, we consider the importance of histopathology reporting with a panel of special stains, and emphasise the need for urgent (

Contains fulltext : 193204.pdf (Publisher’s version ) (Closed access) The European Society for Clinical Microbiology and Infectious Diseases, the European Confederation of Medical Mycology and the European Respiratory Society Joint Clinical Guidelines focus on diagnosis and management of aspergillosis. Of the numerous recommendations, a few are summarized here. Chest computed tomography as well as bronchoscopy with bronchoalveolar lavage (BAL) in patients with suspicion of pulmonary invasive aspergillosis (IA) are strongly recommended. For diagnosis, direct microscopy, preferably using optical brighteners, histopathology and culture are strongly recommended. Serum and BAL galactomannan measures are recommended as markers for the diagnosis of IA. PCR should be considered in conjunction with other diagnostic tests. Pathogen identification to species complex level is strongly recommended for all clinically relevant Aspergillus isolates; antifungal susceptibility testing should be performed in patients with invasive disease in regions with resistance found in contemporary surveillance programmes. Isavuconazole and voriconazole are the preferred agents for first-line treatment of pulmonary IA, whereas liposomal amphotericin B is moderately supported. Combinations of antifungals as primary treatment options are not recommended. Therapeutic drug monitoring is strongly recommended for patients receiving posaconazole suspension or any form of voriconazole for IA treatment, and in refractory disease, where a personalized approach considering reversal of predisposing factors, switching drug class and surgical intervention is also strongly recommended. Primary prophylaxis with posaconazole is strongly recommended in patients with acute myelogenous leukaemia or myelodysplastic syndrome receiving induction chemotherapy. Secondary prophylaxis is strongly recommended in high-risk patients. We strongly recommend treatment duration based on clinical improvement, degree of immunosuppression and response on imaging.

Personalized medicine provides a strategic approach to the management of IA. The incidence of IA in high-risk hematology populations is relatively low (Aspergillus exposure in patients with a potentially similar clinical risk. Nonclinical variables, including genetic mutations that increase susceptibility to IA, could explain why only certain patients develop the disease. This study screened for mutations in 322 hematology patients classified according to IA status and developed a predictive model based on genetic risk, established clinical risk factors, and diagnostic biomarkers. Genetic markers were determined by real-time PCR and, with clinical risk factors and Aspergillus PCR results, subjected to multilogistic regression analysis to identify a best-fit model for predicting IA. The probability of IA was calculated, and an optimal threshold was determined. Mutations in dectin-1 (rs7309123) and DC-SIGN (rs11465384 and rs7248637), allogeneic stem cell transplantation, respiratory virus infection, and Aspergillus PCR positivity were all significant risk factors for developing IA and were combined in a predictive model. An optimal threshold requiring three positive factors generated a mean sensitivity/specificity of 70.4%/89.2% and a probability of developing IA of 56.7%. In patients with no risk factors, the probability of developing IA was 2.4%, compared to >79.1% in patients with four or more factors. Using a risk threshold of 50%, preemptive therapy would have been prescribed for 8.4% of the population. This pilot study shows that patients can be stratified according to risk of IA, providing personalized medicine based on strategic evidence for the management of IA. Further studies are required to confirm this approach.

The Dynamiker® Fungus (1-3)-β-D-Glucan Assay (D-BDG) has recently become available in the Western Hemisphere for the diagnosis of invasive fungal disease (IFD). Evaluations of its performance for Pneumocystis pneumonia (PcP) are limited. A retrospective evaluation of D-BDG diagnosis of PcP was performed (23 PcP cases and 23 controls). Sensitivity and specificity were 87% and 70%, respectively, reducing the positivity threshold to 45 pg/ml increased sensitivity (96%), whereas a threshold of 300 pg/ml increased specificity (96%). The performance of D-BDG for the detection of PcP is comparable to other IFD, but sensitivity is below that required to confidently exclude PcP.

Galactomannan enzyme immune assay (GM EIA) is a nonculture test for detecting invasive aspergillosis (IA) forming a key part of diagnosis and management. Recent reports have questioned the reproducibility of indices after sample storage. To investigate this, 198 serum samples (72 from cases and 126 from controls) and 61 plasma samples (24 from cases and 37 from controls), initially tested between 2010 and 2013, were retested to determine any change in index. Data were also collected on circulatory protein levels for false-positive serum samples. Serum indices significantly declined on retesting (median: initial, 0.50, retest, 0.23; P < 0.0001). This was shown to be diagnosis dependent as the decline was apparent on retesting of control samples (median: initial 0.50, retest 0.12; P < 0.0001), but was not evident with case samples (median: initial, 0.80, retest, 0.80; P = 0.724). Plasma samples showed little change on reanalysis after long-term storage at 4°C. Retesting after freezing showed a decrease in index values for controls (median: initial 0.40, retest 0.26; P = 0.0505), but no significant change in cases. Circulatory proteins showed a correlation between serum albumin concentration and difference in index value on retesting. Overall, this study suggests that a lack of reproducibility in GM EIA positivity is only significant when disease is absent. Retesting after freezing helps to differentiate false-positive GM EIA results and, with consecutive positivity, could help to improve accuracy in predicting disease status. The freezing of samples prior to testing could potentially reduce false-positivity rates and the need to retest.

The burden of human disease related to medically important fungal pathogens is substantial. An improved understanding of antifungal pharmacology and antifungal pharmacokinetics–pharmacodynamics has resulted in therapeutic drug monitoring (TDM) becoming a valuable adjunct to the routine administration of some antifungal agents. TDM may increase the probability of a successful outcome, prevent drug-related toxicity and potentially prevent the emergence of antifungal drug resistance. Much of the evidence that supports TDM is circumstantial. This document reviews the available literature and provides a series of recommendations for TDM of antifungal agents.

Directed therapy aims to target antifungal drugs to patients most likely to have fungal disease and to reduce the use of empirical therapy. The term embraces both pre-emptive therapy, where biomarkers are used to detect early signs of infection before there is clinical disease, and diagnostically driven approaches, which rely on radiological signs to detect early manifestations of clinical disease. Better targeting of drugs can potentially reduce antifungal expenditure, limit the development of resistance and improve outcomes for patients. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy

Samples from patients at high risk for invasive aspergillosis (IA) were prospectively collected and analyzed for the presence of molecular markers of fungal infection. Serum specimens were screened for galactomannan and Aspergillus DNA, and whole-blood specimens were screened only for Aspergillus DNA. Fungal infections were categorized according to the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Forty-seven cases (proven and probable IA) and 31 controls (no evidence of IA) were selected retrospectively for this case-control study, comprising 803 samples, in order to determine the performance of whole-blood PCR, serum PCR, and serum galactomannan testing. Although no single assay was able to detect every case of IA, a combination of different assays provided the best performance. There was no significant difference between the use of whole-blood and serum specimens for PCR-based diagnosis of IA, but there was a trend for whole blood to be more sensitive (85% versus 79%) and to yield an earlier positive result (36 days versus 15 days) than for serum. However, DNA extraction from serum specimens is easier and faster than that from whole-blood specimens, and it allows the same specimen to be used for both galactomannan and PCR assays. In conclusion, the appropriate sample type for DNA extraction should be determined by the local requirements and the technical platforms available at each individual center. A combination of biomarker tests offered the best diagnostic utility for detecting IA.

Diagnosis of invasive aspergillosis (IA) remains challenging. With a relatively low incidence of disease, the use of expensive empirical antifungal therapy exposes many patients to unnecessary toxicity. Diagnosis places emphasis on specific but temporal radiological evidence. Circulating biomarker diagnosis has shown potential, but assays show variable performance, take several hours to perform, and require a degree of technical ability. A novel and simple lateral-flow device (LFD) using monoclonal antibody JF5, which targets an extracellular glycoprotein, has been developed and potentially removes any technical requirements, reducing processing time considerably. In this study, we evaluate the performance of this LFD compared to real-time PCR (targeting the 28S rRNA gene) and galactomannan (GM) detection when testing serum from a European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group, National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG)-defined hematological population. In a proven/probable-IA population versus a no-IA population, the LFD performance was comparable to that of both PCR and galactomannan enzyme immunoassay. Specificity (98.0%) was similar to that of PCR (96.6%) and slightly superior to that of GM (91.5%). Sensitivity (81.8%) was inferior to that of PCR (95.5%) but better than that of GM (77.3%). In combination with PCR, it provided both 100% sensitivity and 100% specificity. The LFD permits rapid testing of easily obtainable specimens, to be used as an adjunct test, before confirmation by other investigations. Its simplicity provides centers without specialist diagnostics with a test with clinical performance superior to that of classical microbiological approaches and results that can be used to direct antifungal management. In summary, microbiological diagnosis of IA is difficult and options are limited, with variable performance. An LFD assay targeting a novel specific biomarker has been developed, one which is methodologically simple and provides good clinical performance, particularly if combined with PCR.

Contains fulltext : 175113.pdf (Publisher’s version ) (Closed access) A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

BACKGROUND: Given the predominance of invasive fungal disease (IFD) amongst the non-immunocompromised adult critically ill population, the potential benefit of antifungal prophylaxis and the lack of generalisable tools to identify high risk patients, the aim of the current study was to describe the epidemiology of IFD in UK critical care units, and to develop and validate a clinical risk prediction tool to identify non-neutropenic, critically ill adult patients at high risk of IFD who would benefit from antifungal prophylaxis. METHODS: Data on risk factors for, and outcomes from, IFD were collected for consecutive admissions to adult, general critical care units in the UK participating in the Fungal Infection Risk Evaluation (FIRE) Study. Three risk prediction models were developed to model the risk of subsequent Candida IFD based on information available at three time points: admission to the critical care unit, at the end of 24 h and at the end of calendar day 3 of the critical care unit stay. The final model at each time point was evaluated in the three external validation samples. RESULTS: Between July 2009 and April 2011, 60,778 admissions from 96 critical care units were recruited. In total, 359 admissions (0.6 %) were admitted with, or developed, Candida IFD (66 % Candida albicans). At the rate of candidaemia of 3.3 per 1000 admissions, blood was the most common Candida IFD infection site. Of the initial 46 potential variables, the final admission model and the 24-h model both contained seven variables while the end of calendar day 3 model contained five variables. The end of calendar day 3 model performed the best with a c index of 0.709 in the full validation sample. CONCLUSIONS: Incidence of Candida IFD in UK critical care units in this study was consistent with reports from other European epidemiological studies, but lower than that suggested by previous hospital-wide surveillance in the UK during the 1990s. Risk modeling using classical statistical methods produced relatively simple risk models, and associated clinical decision rules, that provided acceptable discrimination for identifying patients at 'high risk' of Candida IFD. TRIAL REGISTRATION: The FIRE Study was reviewed and approved by the Bolton NHS Research Ethics Committee (reference: 08/H1009/85), the Scotland A Research Ethics Committee (reference: 09/MRE00/76) and the National Information Governance Board (approval number: PIAG 2-10(f)/2005).

Objective The objective of this study was to audit the compliance and implementation of the British Society for Medical Mycology standards of care for patients with invasive infections in UK hospitals. Methods A multidisciplinary audit questionnaire regarding the processing of microbiology and histopathology specimens, radiology imaging and clinical management of patients with invasive fungal infections was distributed to UK hospitals. Results The study has shown that speciation of Candida and Aspergillus isolates from sterile sites was performed in 42–98% of hospitals. Microscopy of bronchoscopy specimens was not undertaken in 13 of 62 (21%) laboratories. Cryptococcal culture and antigen were undertaken routinely in abnormal CSF in 40–75% and 31–83% of at-risk patients but only in 12% of abnormal CSFs in patients without risk factors. Detailed fungal morphology was provided by

There are conflicting reports on the agreement between the Clinical and Laboratory Standards Institute disk diffusion (M44-A) and reference broth microdilution (M27-A) methods for determination of antifungal susceptibility of yeasts. The antifungal susceptibility of 541 yeasts, the majority of which were from the oral cavity, was determined using these two methods and the accuracy of the disk diffusion method assessed for clinical testing of various Candida species. Of the strains tested, Candida albicans predominated (390 out of 541). The classification of susceptibility determined by the disk diffusion method was largely in concordance with that obtained using the broth dilution method, regardless of species within Candida genus. The overall observed agreement between these two methods was 94.7% for fluconazole and 96.7% for voriconazole was with a 'very major' discrepancy level of 1.5% and 1.7% respectively. This study demonstrates a strong agreement of the simple disk diffusion method with the more labour intensive 'gold standard' broth microdilution method. These findings would support the use of the disk diffusion method in a routine mycology service.

The commercially developed PathoNostics AsperGenius species assay is a multiplex real-time PCR capable of detecting aspergillosis and genetic markers associated with azole resistance. The assay is validated for testing bronchoalveolar lavage fluids, replacing the requirement for culture and benefiting patient management. Application of this assay to less invasive, easily obtainable samples (e.g., serum) might be advantageous. The aim of this study was to determine the analytical and clinical performance of the AsperGenius species and resistance assays for testing serum samples. For the analytical evaluations, serum samples were spiked with various concentrations of Aspergillus genomic DNA for extraction, following international recommendations. For the clinical study, 124 DNA extracts from 14 proven/probable invasive aspergillosis (IA) cases, 2 possible IA cases, and 33 controls were tested. The resistance assay was performed on Aspergillus fumigatus PCR-positive samples when a sufficient fungal burden was evident. The limits of detection of the species and resistance assays for A. fumigatus DNA were 10 and ≥75 genomes/sample, respectively. Nonreproducible detection at lower burdens was achievable for all markers. With a positivity threshold of 39 cycles, the sensitivity and specificity of the species assay were 78.6% and 100%, respectively. For 7 IA cases, at least one genetic region potentially associated with azole resistance was successfully amplified, although no resistance markers were detected in this small cohort. The AsperGenius assay provides good clinical performance with the added ability to detect azole resistance directly from noninvasive samples. While the available burden will limit application, it remains a significant advancement in the diagnosis and management of aspergillosis.

The accepted limitations associated with classic culture techniques for the diagnosis of invasive fungal infections have lead to the emergence of many non-culture-based methods. With superior sensitivities and quicker turnaround times, non-culture-based methods may aid the diagnosis of invasive fungal infections. In this review of the diagnostic service, we assessed the performances of two antigen detection techniques (enzyme-linked immunosorbent assay [ELISA] and latex agglutination) with a molecular method for the detection of invasive Candida infection and invasive aspergillosis. The specificities for all three assays were high (≥97%), although the Candida PCR method had enhanced sensitivity over both ELISA and latex agglutination with values of 95%, 75%, and 25%, respectively. However, calculating significant sensitivity values for the Aspergillus detection methods was not feasible due to a low number of proven/probable cases. Despite enhanced sensitivity, the PCR method failed to detect nucleic acid in a probable case of invasive Candida infection that was detected by ELISA. In conclusion, both PCR and ELISA techniques should be used in unison to aid the detection of invasive fungal infections.

Diagnostic galactomannan (GM) enzyme immunoassay (EIA) testing is formally validated only for serum, though in practice, plasma is occasionally tested. It is assumed, but not confirmed, that results will be comparable to those for serum. GM EIA when testing plasma was evaluated, providing sensitivity (85.7%) and specificity (85.4%) comparable to those for serum. Plasma index values were higher than those for serum; if plasma GM EIA were used to define probable cases, four additional cases would have been diagnosed.

The increasing importance of invasive fungal infection as a cause of severe morbidity and mortality in immunocompromised patients has fuelled the development of new antifungal agents, including new triazole agents and echinocandins. Failure to diagnose fungal infection adequately has hampered the evaluation of these drugs in clinical trials. Consequently, the evidence base behind many prophylactic and empirical treatment strategies has been poor. Recent advances have improved diagnostic criteria and opened the way for more rational usage of these expensive preparations. The proven efficacy and improved safety profile of newer agents is a major advantage. Furthermore, better understanding of the immune response may allow novel strategies of adjunctive therapy and immunomodulation.

Endocarditis secondary to Aspergillus niger has not been described in a leukaemic patient. We describe a case of A. niger endocarditis in a patient with acute myeloid leukaemia and refractory fever. The microbiological cause of his endocarditis was initially misdiagnosed because he fulfilled the Duke criteria for enterococcal endocarditis. A polymerase chain reaction test utilizing pan-fungal primers detected a product from an Aspergillus sp. The DNA was subsequently sequenced and was found to have 100% homology with A. niger. A postmortem revealed fungal endocarditis secondary to disseminated aspergillosis, without evidence of bacterial endocarditis. The patient was found to have a lung aspergilloma that was possibly occupationally acquired, and may have been long standing.

Outcomes for invasive fungal infections have greatly improved in the past decade, and several new antifungal drugs have been or will be licensed in the next few years. Early accurate diagnosis and appropriate treatment have major impact on survival. In a 1995 survey of laboratory practice in the UK for mycology, major disparities were seen, with many laboratories not undertaking even simple diagnostic procedures. Delays in processing and inadequate procedures for handling samples, incomplete or delayed reporting of results, or a combination of these, compromise the care of patients. In randomised trials of antifungal chemotherapy, optimum treatments and good alternatives for others have been defined for some infections. High-quality care requires a multidisciplinary approach to diagnosis and management. In this review, we propose microbiology, histopathology, radiology, and clinical auditing standards, with the evidence base for each reviewed. The standards are absolutes, and, therefore, provide a straightforward basis for improving services to patients if they are all implemented.

We welcome the efforts of the Antimicrobial Resistance Funders' Forum, led by the UK Medical Research Council, to coordinate approaches for research on antimicrobial resistance, as reported by Geoff Watts (Aug 2, p 391).1 However, we wish to voice our concerns at the apparent restriction of these efforts to antibacterial resistance. Although undoubtedly an area of major global concern, bacteria are not the only microbes for which resistance is a growing threat to human health and wellbeing. In particular, drug resistance in fungal pathogens needs urgent research attention.

Invasive fungal disease is a significant cause of morbidity and mortality in the immunocompromised patient. Early and accurate diagnosis is essential to improve outcome. This article explores non-culture based methods as aids to diagnosis as well as a means to risk stratify patients in order to determine the need for targeted therapy.

Invasive aspergillosis is a significant cause of morbidity and mortality within at-risk groups. Directed antifungal chemotherapy, guided by effective screening algorithms that incorporate reliable and validated molecular assays, reduces the morbidity associated with empirical administration and allows earlier diagnosis. The efficient extraction of nucleic acid from Aspergillus fumigatus is the main limiting factor for successful Aspergillus PCR from clinical specimens. With the integration of automated extraction platforms, assessment of the suitability of these platforms for specific targets is of paramount importance. In this study, four extraction robots (Applied Biosystems MagMAX, bioMérieux easyMAG, Qiagen EZ1 and Roche MagNA Pure LC) were evaluated for their ability to extract clinically significant levels of A. fumigatus from blood. All of the platforms could detect 101 c.f.u. ml−1 from EDTA whole blood, although only the easyMAG, EZ1 and MagNA Pure had 100 % reproducibility at this level. Despite good analytical sensitivity, contamination associated with the easyMAG platform excluded its use for diagnostic Aspergillus PCR. The EZ1 and MagNA Pure platforms demonstrated equivalent high sensitivity and negative predictive values (97.4–100 %), essential for screening assays.

Bone marrow transplantation and stem cell transplantation have become standard therapies offering potential cures for a number of hematologic malignancies and immunologic disorders. Severe infection remains a life threatening complication after transplantation, contributes significantly to morbidity, and may necessitate admission to the ICU. It is estimated that between 20 and 40% of patients receiving bone marrow transplant will require ICU admission in the initial posttransplantation phase. Historically, survival rates after admission to the ICU are dismal, particularly if mechanical ventilation is required for respiratory failure. Other organ involvement worsens the prognosis still further and has led to proposals for rationing or restricting access to critical care units and supportive measures. Recent studies have reported small but significant improvements in outcome after critical illness. Whether this improvement is a result of changes in levels of supportive care or a more defined patient selection is uncertain. Moreover, risk factors identifying patients who will benefit most from intensive support are poorly defined. However, it is generally accepted that respiratory failure requiring invasive mechanical ventilation is associated with a poor prognosis in this patient group. Early involvement of intensivists in the management of critical illness in transplant recipients is likely to continue to improve survival in this group of patients.

Objectives: (1)To assess the diagnostic utility of quantitative cultures of non-bronchoscopic lung lavage (NBL) in ventilator-associated pneumonia and evaluate the role of the Bacterial Index; (2) To assess the predictive value of NBL surveillance quantitative cultures in ventilated patients; (3) To evaluate the Clinical Pulmonary Infection Score (CPIS) system in ventilated patients. Design: A prospective comparison of NBL with bronchoscopic bronchoalveolar lavage and protected specimen brush. Setting: Three intensive care units in academic tertiary care centres. Patients: 145 adults ventilated for 72 h, with and without clinical signs of pneumonia. Interventions: Sampling of lower airway secretions by NBL, bronchoalveolar lavage and protected specimen brush. Main outcome measures: Diagnostic reliability of quantitative cultures, Bacterial Index and CPIS. Results: 34 episodes of clinical ventilator-associated pneumonia were documented in 32 patients. 9 episodes were confirmed by concordant blood/pleural culture or post-mortem lung examination. Qualitative concordance of the predominant pathogen between sequential NBL: bronchoalveolar lavage and protected specimen brush was 83%. Sensitivity and specificity of non-directed bronchial lavage at a threshold of 104 CFU/ml were 68%and 70% respectively (p = 0.003) and were comparable with the bronchoscopic methods. However, the low positive predictive value of NBL indicates that quantitation in the absence of clinical signs is unlikely to be useful. Bacterial Index did not improve discriminatory power of quantitation compared with bacterial load of predominant organism. Mean CPIS for confirmed pneumonia cases was 8.4 - 1.01, significantly higher than for non-pneumonia observations (p = < 0.0001). Conclusion: NBL is a simple, safe, cheap, readily-available method of diagnosing ventilator-associated pneumonia with comparable diagnostic accuracy to bronchoscopic techniques. Quantitation of respiratory tract cultures can exclude pneumonia in patients with equivocal clinical signs. The diagnostic threshold should vary depending on the length of ventilation, likelihood of pneumonia and antibiotic administration. The Bacterial Index is a flawed mathematical device that has no contributory role in pneumonia diagnosis. The CPIS has some diagnostic role in selected cohorts of ventilated patients.

Preadmission administration of antibiotics to patients with suspected meningococcal infection has decreased the likelihood of obtaining an isolate and has stimulated development of rapid and reliable non-culture-based diagnostic methods. The sensitivity of the conventional test card latex agglutination test (TCLAT) for detection of capsular polysaccharide has been reported to be suboptimal. In the United Kingdom meningococcal DNA detection by PCR has become readily available and is now used as a first-line investigation. Recently, the performance of latex antigen detection has been markedly improved by ultrasound enhancement. Three tests for laboratory confirmation of meningococcal infection, (i) PCR assays, (ii) TCLAT, and (iii) ultrasound-enhanced latex agglutination test (USELAT), were compared in a retrospective study of 125 specimens (serum, plasma, and cerebrospinal fluid specimens) from 90 patients in whom meningococcal disease was suspected on clinical grounds. Samples were from patients with (i) culture-confirmed meningococcal disease, (ii) culture-negative but PCR-confirmed meningococcal disease, and (iii) clinically suspected but non-laboratory-confirmed meningococcal disease. USELAT was found to be nearly five times more sensitive than TCLAT. Serogroup characterization was obtained by both PCR and USELAT for 44 samples; all results were concordant and agreed with the serogroups determined for the isolates when the serogroups were available. For 12 samples negative by USELAT, the serogroup was determined by PCR; however, for 12 other specimens for which PCR had failed to indicate the serogroup, USELAT gave a result. USELAT is a rapid, low-cost method which can confirm a diagnosis, identify serogroups, and guide appropriate management of meningococcal disease contacts. A complementary non-culture-based confirmation strategy of USELAT for local use supported by a centralized PCR assay service for detection of meningococci would give the benefits of timely information and improved epidemiological data.

Objectives: The objective was to determine the current epidemiology of infective endocarditis. Patients and Methods: All microbiologically positive episodes of infective endocarditis treated at The University Hospital of Wales over a 9-year period from March 1987 to March 1996 was reviewed. Patients originated from the catchment area of The University Hospital of Wales or were referred from other hospitals in Wales. Data extraction was from records held in the Microbiology Department and, whenever possible, from patients' casenotes. The epidemiological parameters were: (1) age and sex of patients; (2) distribution of affected sites; (3) frequency of predisposing risk factors (cardiac and extracardiac); (4) incidence of early prosthetic valve endocarditis; and (5) mortality rates. Results: There were 128 microbiologically positive episodes of endocarditis in 125 patients. The mean age of the population was 53.1 years and the aortic valve was the most frequently involved site of infection (51.6%). A presumed source of infection was identified in 20% if episodes. The commonest predisposing cardiac risk factor in native valve episodes was bicuspid aortic valve (16.7%) but there was no identifiable cardiac risk factor in a much larger proportion (37.7%) of native valve episodes. There was a low incidence (0.6%) of culture positive early prosthetic valve episodes and low mortality rates for both native and prosthetic valve endocarditis (12.3% and 24.5%) in this study. Viridans streptococci were the predominant organisms. In prosthetic valve episodes with onset after the 60th postoperative day but within one postoperative year the identity of the isolate suggested, in most cases, perioperative valve contamination. Conclusions: The epidemiology of infective endocarditis has undergone significant change. Inability to detect clinically common predisposing lesions, and the frequent absence of any identifiable predisposing cardiac risk factor mean that initial diagnosis is often difficult and demands a high index of suspicion. There was a low incidence of culture positive early prosthetic valve episodes and there were low mortality rates for both native and prosthetic valve endocarditis; these figures suggest improvements in cardiac care. The microbiological evidence indicates that the duration of the postoperative time period used for classifying prosthetic valve endocarditis into ‘early’ and ‘late’ episodes should be extended from 60 days to 1 year.

Fungal infection in critically ill patients is an increasingly prevalent problem. Candida spp. cause the majority of these infections in ICU. They occur most commonly in patients with severe underlying illness, multiple courses of antibiotics and intravascular catheters. Clinical diagnosis is difficult due to nonspecific signs and the frequent occurrence of widespread superficial colonization with Candida spp. in ventilated patients. Most patients are diagnosed using inferential evidence of infection, such as persistent pyrexia despite antibiotics, raised serum C-reactive protein and the presence of individual risk factors. Amphotericin B and fluconazole are the most commonly used anti-fungals dependent on the identity of the fungus. Most of these infections are endogenous; however, a proportion may be caused via the hands of healthcare staff or contaminated medical equipment.

The treatment of deep fungal infection in haematological malignancy remains controversial due to the limited number of antifungal agents available and problems over their spectrum and dose-limiting side-effects. Difficulties in diagnosis mean that most treatments are begun empirically; amphotericin B remains the drug of choice. Emerging resistance may limit the usefulness of fluconazole and other azoles in some areas. Lipid preparations of amphotericin B have reduced the toxicity of this agent, but some issues of dosage and efficacy remain. Adjunctive treatments aimed at augmenting the host response to infection may have a role to play in deep fungal infection.

The standard test card agglutination of antibody-coated latex by Neisseria meningitidis. Streptococcus group B, Haemophilus influenzae type b and Streptococcus pneumoniae antigens has been compared with a technique involving local concentration of the coated latex in an ultrasonic standing wave. The detection of positive control antigen was enhanced, compared with the test-card procedure, over a 16 to 64 fold range on exposure to ultrasound. Sample filtration eliminated non-specific agglutination on ultrasonic exposure of latex in control serum, urine or concentrated urine. Tests of meningitis patient body fluids showed increased detection of antigen with ultrasound for CSF (11/14 > 7/14) serum (8/13 > 3/13) and concentrated urine (8/17 > 2/17) compared to test card assays. The ultrasound detection of antigen in serum or concentrated urine was comparable to that achieved with CSF on test cards. Serum dilution experiments showed that ultrasound could detect antigen in serum over a 1000 fold concentration range.

This paper describes a simple and rapid method for the differentiation of Candida albicans from other yeast species in primary cultures based on colonial morphology following incubation in carbon dioxide. The technique has superior sensitivity to the traditional germ-tube method and requires no additional laboratory tests. In a busy laboratory, this can result in significant savings in cost and time, as well as improvements in patient care.

BackgroundThere is increasing evidence that invasive fungal disease (IFD) is more likely to occur in non-neutropenic patients in critical care units. A number of randomised controlled trials (RCTs) have evaluated antifungal prophylaxis in non-neutropenic, critically ill patients, demonstrating a reduction in the risk of proven IFD and suggesting a reduction in mortality. It is necessary to establish a method to identify and target antifungal prophylaxis at those patients at highest risk of IFD, who stand to benefit most from any antifungal prophylaxis strategy.ObjectivesTo develop and validate risk models to identify non-neutropenic, critically ill adult patients at high risk of invasiveCandidainfection, who would benefit from antifungal prophylaxis, and to assess the cost-effectiveness of targeting antifungal prophylaxis to high-risk patients based on these models.DesignSystematic review, prospective data collection, statistical modelling, economic decision modelling and value of information analysis.SettingNinety-six UK adult general critical care units.ParticipantsConsecutive admissions to participating critical care units.InterventionsNone.Main outcome measuresInvasive fungal disease, defined as a blood culture or sample from a normally sterile site showing yeast/mould cells in a microbiological or histopathological report. For statistical and economic modelling, the primary outcome was invasiveCandidainfection, defined as IFD-positive forCandidaspecies.ResultsSystematic review: Thirteen articles exploring risk factors, risk models or clinical decision rules for IFD in critically ill adult patients were identified. Risk factors reported to be significantly associated with IFD were included in the final data set for the prospective data collection.Data collection: Data were collected on 60,778 admissions between July 2009 and March 2011. Overall, 383 patients (0.6%) were admitted with or developed IFD. The majority of IFD patients (94%) were positive forCandidaspecies. The most common site of infection was blood (55%). The incidence of IFD identified in unit was 4.7 cases per 1000 admissions, and for unit-acquired IFD was 3.2 cases per 1000 admissions.Statistical modelling: Risk models were developed at admission to the critical care unit, 24 hours and the end of calendar day 3. The risk model at admission had fair discrimination (c-index 0.705). Discrimination improved at 24 hours (c-index 0.823) and this was maintained at the end of calendar day 3 (c-index 0.835). There was a drop in model performance in the validation sample.Economic decision model: Irrespective of risk threshold, incremental quality-adjusted life-years of prophylaxis strategies compared with current practice were positive but small. Incremental costs of the prophylaxis strategies compared with current practice were positive for most strategies, although a few strategies were cost saving. Incremental net benefits of each prophylaxis strategy compared with current practice were positive for most, but not all, of the strategies. Cost-effectiveness acceptability curves showed that risk assessment and prophylaxis at the end of calendar day 3 was the strategy most likely to be cost-effective when the risk threshold was 1% or 2%. At a lower risk threshold (0.5%) it was most cost-effective to assess risk at each time point; this led to a relatively high proportion of patients receiving antifungal prophylaxis (30%), which may lead to additional burden from increased resistance. The estimates of cost-effectiveness were highly uncertain and the value of further research for the whole population of interest is high relative to the research costs.ConclusionsThe results of the Fungal Infection Risk Evaluation (FIRE) Study, derived from a highly representative sample of adult general critical care units across the UK, indicated a low incidence of IFD among non-neutropenic, critically ill adult patients. IFD was associated with substantially higher mortality, more intensive organ support and longer length of stay. Risk modelling produced simple risk models that provided acceptable discrimination for identifying patients at ‘high risk’ of invasiveCandidainfection. Results of the economic model suggested that the current most cost-effective treatment strategy among non-neutropenic, critically ill adult patients admitted to NHS adult general critical care units is a strategy of risk assessment and antifungal prophylaxis at the end of calendar day 3, but this finding is highly uncertain and future studies should consider the potential impact of increased resistance.FundingFunding for this study was provided by the Health Technology Assessment programme of the National Institute for Health Research.A previous version of this report was published in February 2013. The report was subsequently modified to reflect a substantial reduction in the unit cost of fluconazole that took place between the original analysis being conducted and the publication of the report.

Immunotherapy can be defined as treatment directed at augmenting host immune defence mechanisms. Non-antimicrobial therapies and immunoprophylaxis in bone marrow transplantation (BMT) can be subdivided into three broad categories: passive immunotherapy with intravenous immunoglobulin (IVIG); cytokine therapy; and anti-endotoxin-directed treatments. Most studies using IVIG in BMT are prophylactic and suffer from variability in study design, type of IVIG and dosing regimens. Various effects on viral and bacterial infections and graft-versus-host disease (GVHD) have been reported but few if any have shown benefit in terms of improved patient survival. Moreover the immunomodulatory effect of immunoglobulin G preparations is frequently overlooked. With the exception of cytomegalovirus (CMV) pneumonitis, there is little evidence of benefit in the treatment of established infections and the relative benefits of hyperimmune preparations are poorly established. The development of haemopoietic growth factors has led to the widespread use of cytokines in BMT. The benefits of these agents both in the prevention of fever and infection and as adjuvants to standard antimicrobial therapy in established infection (e.g. invasive mycoses) are rapidly becoming apparent. Both human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and granulocyte colony-stimulating factor (rhG-CSF) have been shown to accelerate granulocyte recovery following BMT and reduce fever days, antibiotic usage and hospitalization. RhGM-CSF appears superior in these respects. The roles of interleukin 1 (IL1), IL3, IL6 and interferons are also under evaluation. As with the much publicised studies using anti-endotoxin antibodies as therapy in sepsis, there is little evidence of benefit in the few studies performed in BMT patients. Other strategies using prophylactic IVIG enriched for anti-endotoxin antibodies appear more promising. The concept of neutralizing the production of cytokines with monoclonal antibodies, receptor antagonists or other biological modifiers, is mainly directed at reducing tissue damage during the preparative regimen with the aim of reducing subsequent GVHD. Any effect this may have on infectious complications such as CMV disease has yet to be investigated. Cloned T cells cytotoxic against CMV have also been developed and studies using these preparations are underway.

Summary Objectives The aim of this study was to assess the clinical utility of enhanced diagnostics on the management of invasive fungal disease in high risk patients within an integrated care pathway and to audit compliance and efficacy of antifungal prophylaxis. Methods A cohort of 549 high risk haematology and stem-cell transplant recipients was followed over a 5 year period. The routine standard of care involved the use of antimould prophylaxis and a neutropenic care pathway utilizing twice weekly antigen and PCR testing. Results Prophylaxis with itraconazole was poorly tolerated and therapeutic levels could not be maintained. Antigen testing and PCR showed good clinical utility in the management of invasive aspergilosis with high sensitivity (98%) and negative predictive value (99.6%) when both tests were used together, allowing a diagnosis IA to be excluded and obviating the need for empirical antifungal agents. When used serially, multiple positive PCR and antigen test results enabled accurate diagnosis of IA with a specificity of 95% and a positive likelihood ratio of 11. Biomarkers preceded clinical signs in 85% of proven and probable invasive disease. Conclusion The combination of both tests showed optimum clinical utility for the diagnosis and management of IA in this high risk group.

Diagnosis of invasive aspergillosis remains a significant problem. PCR testing may aid diagnosis but is not yet included in disease-defining criteria due to a lack of standardization of assays and methodologies. This study investigated the analytical performance and the clinical sensitivity and specificity of the Myconostica MycAssay Aspergillus PCR (MAP) assay compared to those of a validated in-house Aspergillus PCR (IHP) test when testing serum specimens. Serum specimens spiked with Aspergillus genomic DNA had a limit of detection equivalent to 5 genomes and a linear dynamic range of 5 to >5 × 10 4 genomes for both assays. When testing clinical specimens ( n = 170), the MAP assay had a sensitivity of 60 to 70% and a specificity of 90.5 to 100%. The IHP assay had a sensitivity of 50 to 80% and a specificity of 100%. A commercially available Aspergillus PCR assay provides a methodology that is standardized and reagents that are quality controlled. This facilitates multicenter evaluation of the clinical utility of PCR diagnosis. The performance of the MAP assay is comparable to that of the IHP assay and to those in previously reported studies evaluating commercial tests (galactomannan enzyme-linked immunosorbent assay).

Methicillin-resistant Staphylococcus aureus septicemia is associated with significant morbidity and mortality and requires treatment with intravenous glycopeptides. For blood cultures positive for gram-positive cocci, 24 to 48 h is required for the detection of S. aureus bacteremia and the provision of antibiotic susceptibility testing results. We describe a molecular biology-based assay that requires 2 h from the time of initial positivity of blood cultures. The assay correctly detected 96% of the S. aureus isolates including all methicillin-resistant S. aureus isolates. Clinical data collected during the study suggest that 28% of patients with S. aureus bacteremia do not receive early and appropriate treatment and that 10% of patients may initially be receiving inappropriate glycopeptide treatment.

A standardized protocol for extracting DNA from Aspergillus fumigatus has been proposed by the European Aspergillus PCR Initiative (EAPCRI). Using meta-regression analysis, the EAPCRI showed certain stages of the process to be critical to providing a satisfactory analytical sensitivity. The study investigated each step of the EAPCRI protocol by elimination and monitored the influence on Aspergillus PCR performance.

Background. Invasive aspergillosis is a devastating infection with attributable mortality of 40% despite antifungal therapy. In animal models of aspergillosis, deficiency of mannose-binding lectin (MBL), a pattern recognition receptor that activates complement, is a susceptibility factor. MBL deficiency occurs in 20%-30% of the population. We hypothesized that MBL deficiency may be a susceptibility factor for invasive aspergillosis in humans. Methods. Serum MBL concentrations were measured by enzyme-linked immunosorbent assay in 65 patients with proven or probable acute invasive aspergillosis and 78 febrile immunocompromised control subjects. MBL concentrations and the frequency of MBL deficiency were compared. Results. The median serum MBL level was significantly lower in patients with aspergillosis than in control subjects (281 ng/mL vs 835 ng/mL; P=.007). MBL deficiency (MBL concentration

Despite improvements in medical technology, the definitive diagnosis of invasive fungal disease (IFD) is limited. The prevalence of disease is relatively low but many cases are undiagnosed. With the diagnosis of proven IFD dependent on histopathology or culture from a sterile site, clinicians have become more reliant on noninvasive nonculture diagnostic techniques. PCR technology has the capacity to overcome classical limitations but has its own drawbacks, resulting from an incomplete knowledge of the various disease processes and subsequent shortage of optimal specimens, leading to a lack of methodological standardization. This review will consider the general principles and limitations of fungal PCR before discussing genus-specific PCR applications. It is by no means a systematic review of the literature but is intended, where possible, to provide the reader with access to assays with proficient clinical performance.