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Background: This meta-analysis examines the comparative diagnostic performance of polymerase chain reaction (PCR) for the diagnosis of Pneumocystis pneumonia (PCP) on different respiratory tract samples, in both human immunodeficiency virus (HIV) and non-HIV populations., Methods: A total of 55 articles met inclusion criteria, including 11 434 PCR assays on respiratory specimens from 7835 patients at risk of PCP. QUADAS-2 tool indicated low risk of bias across all studies. Using a bivariate and random-effects meta-regression analysis, the diagnostic performance of PCR against the European Organisation for Research and Treatment of Cancer-Mycoses Study Group definition of proven PCP was examined., Results: Quantitative PCR (qPCR) on bronchoalveolar lavage fluid provided the highest pooled sensitivity of 98.7% (95% confidence interval [CI], 96.8%-99.5%), adequate specificity of 89.3% (95% CI, 84.4%-92.7%), negative likelihood ratio (LR-) of 0.014, and positive likelihood ratio (LR+) of 9.19. qPCR on induced sputum provided similarly high sensitivity of 99.0% (95% CI, 94.4%-99.3%) but a reduced specificity of 81.5% (95% CI, 72.1%-88.3%), LR- of 0.024, and LR+ of 5.30. qPCR on upper respiratory tract samples provided lower sensitivity of 89.2% (95% CI, 71.0%-96.5%), high specificity of 90.5% (95% CI, 80.9%-95.5%), LR- of 0.120, and LR+ of 9.34. There was no significant difference in sensitivity and specificity of PCR according to HIV status of patients., Conclusions: On deeper respiratory tract specimens, PCR negativity can be used to confidently exclude PCP, but PCR positivity will likely require clinical interpretation to distinguish between colonization and active infection, partially dependent on the strength of the PCR signal (indicative of fungal burden), the specimen type, and patient population tested., Competing Interests: Potential conflicts of interest. A. A. has received honoraria from Gilead Sciences. S. C.-A. A. has received united educational grants from F2G Ltd and consulted for MSD Australia. T. R. R. has received grants from Pfizer and the Irish Department of Agriculture, Food and the Marine and honoraria from Gilead and Menarini Pharma. P. L. W. has performed diagnostic evaluations and received meeting sponsorship from Associates of Cape Cod, Bruker, Dynamiker, and Launch Diagnostics; speaker fees, expert advice fees, and meeting sponsorship from Gilead; and speaker and expert advice fees from F2G. J. P. D. has received personal fees from F2G, Gilead Sciences, and Pfizer. W. J. H. has received lecture honoraria from AbbVie, Amgen, AstraZeneca, Celgene/BMS, Gilead Sciences, Janssen, MSD, and Pfizer; has received support to attend meetings and travel support from AbbVie, Alexion, Astellas, Janssen, Lilly, MSD, Novartis, and Pfizer; and has participated on advisory boards for AbbVie, Amgen, AstraZeneca, Basilea, Celgene/BMS, Gilead Sciences, Janssen, and Sanofi-Aventis. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Objectives: To determine whether early switch to oral antibiotic treatment in adults with neutropenic sepsis at low risk of complications is non-inferior to switching later., Methods: This non-inferiority, parallel-group, randomized, open-label clinical trial enrolled UK adults hospitalized with neutropenic sepsis. Participants were randomly assigned to either switch to oral ciprofloxacin plus co-amoxiclav within 12-24 hours or to continue intravenous treatment for at least 48 hours. The primary outcome was a composite measure of treatment failure, 14 days after randomization. The non-inferiority margin was 15%., Results: There were 129 participants from 16 centres and 125 were assessed for the primary outcome. Of these, 113 patients completed protocolized treatment and comprised the per-protocol population. In total, 9 (14.1%) of 64 patients in the standard care arm met the primary end point, compared with 15 (24.6%) of 61 in the early switch arm, giving a risk difference of 10.5% (1-sided 95% CI, -∞% to 22%; p 0.14). In the per-protocol population, 8 (13.3%) of the 60 patients in the standard care arm met the primary end point, compared with 9 (17%) of 53 in the intervention arm giving a risk difference of 3.7% (one-sided 95% CI, -∞% to 14.8%; p 0.59). Duration of hospital stay was shorter in the intervention arm (median 2 [inter-quartile range (IQR) 2-3] vs. 3 days [IQR 2-4]; p 0.002)., Discussion: Although non-inferiority of early oral switch was found in the per-protocol population, the intervention was not non-inferior in the intent-to-treat population., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This overview of reviews (i.e., an umbrella review) is designed to reappraise the validity of systematic reviews (SRs) and meta-analyses related to the performance of Aspergillus PCR tests for the diagnosis of invasive aspergillosis in immunocompromised patients. The methodological quality of the SRs was assessed using the AMSTAR-2 checklist; the quality of the evidence (QOE) within each SR was appraised following the GRADE approach. Eight out of 12 SRs were evaluated for qualitative and quantitative assessment. Five SRs evaluated Aspergillus PCR on bronchoalveolar lavage fluid (BAL) and three on blood specimens. The eight SRs included 167 overlapping reports (59 evaluating PCR in blood specimens, and 108 in BAL), based on 107 individual primary studies (98 trials with a cohort design, and 19 with a case-control design). In BAL specimens, the mean sensitivity and specificity ranged from 0.57 to 0.91, and from 0.92 to 0.97, respectively (QOE: very low to low). In blood specimens (whole blood or serum), the mean sensitivity ranged from 0.57 to 0.84, and the mean specificity from 0.58 to 0.95 (QOE: low to moderate). Across studies, only a low proportion of AMSTAR-2 critical domains were unmet (1.8%), demonstrating a high quality of methodological assessment. Conclusions. Based on the overall methodological assessment of the reviews included, on average we can have high confidence in the quality of results generated by the SRs.

Introduction: Fungal PCR has undergone considerable standardization and, together with the availability of commercial assays, external quality assessment schemes, and extensive performance validation data, is ready for widespread use for the screening and diagnosis of invasive fungal disease (IFD)., Areas Covered: Drawing on the experience and knowledge of the leads of the various working parties of the Fungal PCR initiative, this review will address general considerations concerning the use of molecular tests for the diagnosis of IFD, before focusing specifically on the technical and clinical aspects of molecular testing for the main causes of IFD and recent technological developments., Expert Opinion: For infections caused by Aspergillus, Candida , and Pneumocystis jirovecii , PCR testing is recommended, and combination with serological testing will likely enhance the diagnosis. For other IFD (e.g. mucormycosis), molecular diagnostics represent the only non-classical mycological approach toward diagnoses, and continued performance validation and standardization have improved confidence in such testing. The emergence of antifungal resistance can be diagnosed, in part, through molecular testing. Next-generation sequencing has the potential to significantly improve our understanding of fungal phylogeny, epidemiology, pathogenesis, mycobiome/microbiome, and interactions with the host, while identifying novel and existing mechanisms of antifungal resistance and novel diagnostic/therapeutic targets.

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., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

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 × 10 9 /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. https://doi.org/10.1186/ISRCTN84288963. EudraCT: 2015-002830-35.

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.

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.

Personalized medicine provides a strategic approach to the management of IA. The incidence of IA in high-risk hematology populations is relatively low (<10%), despite unavoidable 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., (Copyright © 2017 American Society for Microbiology.)

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: <.0001). The sensitivity, specificity, and diagnostic odds ratio for proven/probable IFD was 81.4%, 78.1%, and 15.5, respectively. Significant BDG false positivity (9/13) was associated post abdominal surgery. D-BDG showed fair and good agreement with the Fungitell®, and GM assays, respectively. In conclusion, the D-BDG provides a useful adjunct test to aid the diagnosis of IFD, with technical flexibility that will assist laboratories processing low sample numbers. Further, large scale, prospective evaluation is required to confirm the clinical validity and determine clinical utility., (© The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2017 American Society for Microbiology.)

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., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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. 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. 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.

Many countries have observed an increase in the incidence of invasive fungal infections (IFIs) over the past two decades with emergence of new risk factors and isolation of new fungal pathogens. Early diagnosis and appropriate antifungal treatment remain the cornerstones of successful outcomes. However, due to non-specific clinical presentations and limited availability of rapid diagnostic tests, in more than half of cases antifungal treatment is inappropriate. As a result, the emergence of antifungal resistance both in yeasts and mycelial fungi is becoming increasingly common. The Delhi Chapter of the Indian Association of Medical Microbiologists (IAMM-DC) organized a 1 day workshop in collaboration with BSAC on 10 December 2015 in New Delhi to design a road map towards the development of a robust antifungal stewardship programme in the context of conditions in India. The workshop aimed at developing a road map for optimizing better outcomes in patients with IFIs while minimizing unintended consequences of antifungal use, ultimately leading to reduced healthcare costs and prevention development of resistance to antifungals. The workshop was a conclave of all stakeholders, eminent experts from India and the UK, including clinical microbiologists, critical care specialists and infectious disease physicians. Various issues in managing IFIs were discussed, including epidemiology, diagnostic and therapeutic algorithms in different healthcare settings. At the end of the deliberations, a consensus opinion and key messages were formulated, outlining a step-by-step approach to tackling the growing incidence of IFIs and antifungal resistance, particularly in the Indian scenario., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

PCR can aid in the diagnosis of invasive fungal disease (IFD). While the large number of "in-house" methodologies drives technological diversity, providing robustness, they make it difficult to identify optimal strategies, limiting standardization, and widespread acceptance. No matter how efficient, PCR utility will be limited by the quality of extracted nucleic acid. This chapter highlights benefits and limitations affecting the nucleic acid extraction process, before focusing on recent recommendations that through multicenter evaluation have provided optimal and standardized methodology.

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).

The application of molecular technologies to aid diagnosis and management of infectious diseases has had a major impact and many assays are in routine use. Diagnosis of aspergillosis has lagged behind. Lack of standardization and limited commercial interest have meant that PCR was not included in consensus diagnostic criteria for invasive fungal disease. In the last ten years careful evaluation and validation by the Aspergillus European PCR initiative with the development of standardized extraction, amplification and detection protocols for various specimen types, has provided the opportunity for clinical utility to be investigated. PCR has the potential to not only exclude a diagnosis of invasive aspergillosis but in combination with antigen testing may offer an approach for the early diagnosis and treatment of invasive aspergillosis in high-risk populations, with the added benefit of detection of genetic markers associated with antifungal resistance.

The EuropeanAspergillusPCR 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., (Copyright © 2016 White et al.)

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., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

The use of serum or plasma for Aspergillus PCR testing facilitates automated and standardized technology. Recommendations for serum testing are available, and while serum and plasma are regularly considered interchangeable for use in fungal diagnostics, differences in galactomannan enzyme immunoassay (GM-EIA) performance have been reported and are attributed to clot formation. Therefore, it is important to assess plasma PCR testing to determine if previous recommendations for serum are applicable and also to compare analytical performance with that of serum PCR. Molecular methods testing serum and plasma were compared through multicenter distribution of quality control panels, with additional studies to investigate the effect of clot formation and blood fractionation on DNA availability. Analytical sensitivity and time to positivity (TTP) were compared, and a regression analysis was performed to identify variables that enhanced plasma PCR performance. When testing plasma, sample volume, preextraction-to-postextraction volume ratio, PCR volume, duplicate testing, and the use of an internal control for PCR were positively associated with performance. When whole-blood samples were spiked and then fractionated, the analytical sensitivity and TTP were superior when testing plasma. Centrifugation had no effect on DNA availability, whereas the presence of clot material significantly lowered the concentration (P = 0.028). Technically, there are no major differences in the molecular processing of serum and plasma, but the formation of clot material potentially reduces available DNA in serum. During disease, Aspergillus DNA burdens in blood are often at the limits of PCR performance. Using plasma might improve performance while maintaining the methodological simplicity of serum testing., (Copyright © 2015 Loeffler et al.)

Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity., (Copyright © 2015 White et al.)

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., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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 (<48 hours) and optimised imaging for patients with suspected invasive fungal infection. All 43 recommendations are auditable and should be used to ensure best diagnostic practice and improved outcomes for patients., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nonculture-based tests are gaining popularity in the diagnosis of invasive fungal disease (IFD), but PCR is excluded from disease-defining criteria because of limited standardization and a lack of commercial assays. Commercial PCR assays may have a standardized methodology while providing quality assurance. The detection of PCR products by a surface-enhanced Raman scattering (SERS) assay potentially provides superior analytical sensitivity and multiplexing capacity compared to that of real-time PCR. Using this approach, the RenDx Fungiplex assay was developed to detect Candida and Aspergillus. Analytical and clinical evaluations of the assay were undertaken using extraction methods according to European Aspergillus PCR Initiative (EAPCRI) recommendations. A total of 195 previously extracted samples (133 plasma, 49 serum, and 13 whole blood) from 112 patients (29 with proven/probable IFD) were tested. The 95% limit of detection of Candida and Aspergillus was 200 copies per reaction, with an overall reproducibility of 92.1% for detecting 20 input copies per PCR, and 89.8% for the nucleic acid extraction-PCR-SERS process for detecting fungal burdens of <20 genome equivalents per sample. A clinical evaluation showed that assay positivity significantly correlated with IFD (P < 0.0001). The sensitivity of the assay was 82.8% and was similar for both Candida (80.0%) and Aspergillus (85.7%). The specificity was 87.5% and was increased (97.5%) by using a multiple (≥ 2 samples) PCR-positive threshold. In summary, the RenDx Fungiplex assay is a PCR-SERS assay for diagnosing IFD and demonstrates promising clinical performance on a variety of samples. This was a retrospective clinical evaluation, and performance is likely to be enhanced through a prospective analysis of clinical validity and by determining clinical utility., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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 10(1) 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., (© 2014 The Authors.)

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 Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 range of opportunist pathogens in cancer and transplant patients continues to increase. New treatment modalities and forms of immunosuppression following transplantation have improved survival from the underlying disease but can lead to prolonged immunosuppression and increased risk of infection. NICE guidelines for the management of neutropenic sepsis are now available but have aroused some controversy, particularly over the recommendation for quinolone prophylaxis in high-risk patient groups. In addition to neutropenia, long-term defects in cell-mediated immunity are exposing patients to risk of chronic, viral, protozoal and fungal infection. Advances in diagnostic techniques have the potential to improve management and limit unnecessary empirical treatment, allowing a move towards a diagnosis-driven strategy. However, interpreting the clinical validity and utility of some of these assays can be difficult, particularly for low-prevalence infection where the positive predictive value of any diagnostic test is likely to be low and prompt empirical antibacterial therapy is still indicated in neutropenic patients., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

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.

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.

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.

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

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).

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.

PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as "compliant" or "noncompliant," according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 microl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (>or=3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 microl.

Human protothecosis is a rare infection caused by a member of the genus Prototheca, an ubiquitous alga. Traumatic inoculation can cause infections in the immunocompetent host while disseminated infection is mainly seen in patients with compromised immunity. We report here an unusual case of disseminated infection in a cancer patient. Traumatic removal of a Hickman catheter in this patient, led to the development of a severe skin and subcutaneous tissue infection with algaemia. These infections are often indolent and difficult to treat, with paucity of information available as to guidelines for diagnosis and effective therapeutic options.

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, <500 ng/mL) was significantly more common in patients with aspergillosis than control subjects (62% vs 32%; P < .001). Frequency of MBL deficiency was similar among patients with aspergillosis irrespective of response to antifungal therapy (P = .10)., Conclusions: This study is the first, to our knowledge, to show an association between MBL deficiency and acute invasive aspergillosis in humans. Further study is required to investigate the causal nature of this association and to define whether diagnosis of MBL deficiency may identify immunocompromised patients at increased risk of invasive aspergillosis.

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.

A systematic review and meta-analysis was done on the use of PCR tests for the diagnosis of invasive aspergillosis. Data from more than 10000 blood, serum, or plasma samples obtained from 1618 patients at risk for invasive aspergillosis were retrieved from 16 studies. Overall, the mean diagnostic odds ratios (DORs) of PCR for proven and probable cases were similar whether two consecutive positive samples were required to define positivity (DOR 15.97 [95% CI 6.83-37.34]) or a single positive PCR test was required (DOR 16.41 [95% CI 6.43-41.88]). Sensitivity and specificity of PCR for two consecutive positive samples were 0.75 (95% CI 0.54-0.88) and 0.87 (95% CI 0.78-0.93), respectively, and if only a single positive sample was required, these values were 0.88 (95% CI 0.75-0.94) and 0.75 (95% CI 0.63-0.84), respectively. Whereas specificity based on a single positive test was significantly lower (p=0.027) than two positive tests, the sensitivity and DOR did not differ significantly. A single PCR-negative result is thus sufficient to exclude a diagnosis of proven or probable invasive aspergillosis. However, two positive tests are required to confirm the diagnosis because the specificity is higher than that attained from a single positive test. Populations at risk varied and there was a lack of homogeneity of the PCR methods used. Efforts are underway to devise a standard for Aspergillus sp PCR for screening, which will help enable formal validation of PCR and estimate its use in patients most likely to benefit.

Techniques for the diagnosis of invasive fungal infection, including antigen testing, nucleic acid detection and radiological imaging, have improved greatly in recent years. They have the potential to impact on patient management through replacing empirical antifungal strategies with targeted and pre-emptive therapy. Factors that influence performance of these diagnostic tests include underlying disease, the prevalence of fungal infection in particular populations and prophylactic antifungal drug strategies. Understanding these factors is necessary for rational use of antifungal agents and optimal management and prevention of fungal infection in immunosuppressed patients.

PCR is a useful tool to aid in the diagnosis of invasive aspergillosis. However, it is essential that an optimal method be agreed to allow inclusion in future consensus diagnosis criteria. It should be used in conjunction with other methods (e.g., galatomannan (GM) ELISA and high resolution computed tomography (HRCT)) to enhance the opportunity for detection of this devastating infection. This manuscript will try to highlight the benefits but mainly the limitations occurring throughout the process of molecular testing. It will focus on real-time methods although many of the points will be relevant to block-based amplification.

Background: Amphotericin B is a widely used broad-spectrum antifungal agent, despite being associated with significant adverse events, including nephrotoxicity., Methods: The present prospective study collected data on outcomes for 418 adult patients treated consecutively with polyenes in hematology and oncology wards in 20 hospitals in Europe., Results: Patients initially received amphotericin B deoxycholate (62% of patients), liposomal amphotericin B (27%), or other lipid formulations of amphotericin B (11%). Of the patients initially treated with amphotericin B deoxycholate, 36% had therapy switched to lipid formulations of amphotericin B, primarily because of increased serum creatinine levels (in 45.7% of patients) or other amphotericin B-attributable adverse events (in 41.3% of patients). Nephrotoxicity, which was defined as a > or = 50% increase in the serum creatinine level, developed in 57% of patients with normal kidney function at baseline. Predictors of nephrotoxicity included formulation type and duration of treatment. Compared with patients without nephrotoxicity, patients with nephrotoxicity had a higher mortality rate (24%), and their mean length of stay in the hospital was prolonged by 8.6 days. Slight increases in the serum creatinine level (i.e., > or = 50%) were associated with a significantly longer stay in the hospital. Severe nephrotoxicity (i.e., a > or = 200% increase in the serum creatinine level) was a significant predictor of death, as were severe underlying medical conditions and documented fungal infection., Conclusion: This prospective study confirmed that, in European hospitals, amphotericin B formulations have a major influence on the length of stay in the hospital and nephrotoxicity-associated mortality.