European Aspergillus PCR Initiative Research Articles

The Emergence of COVID-19 Associated Mucormycosis: Analysis of Cases From 18 Countries

Coronavirus disease 2019 (COVID-19)–associated mucormycosis (CAM) has recently been increasingly reported, particularly among patients with uncontrolled diabetes. Patients with diabetes and hyperglycemia often display an inflammatory state that may be potentiated by the activation of antiviral immunity to SARS-CoV-2, and thus may favor secondary infections. We analyze 80 published and unpublished cases of CAM, with a predominance (42/80) of cases from India. Uncontrolled diabetes mellitus as well as systemic corticosteroid treatment represented major comorbid predisposing factors and rhino-orbital cerebral mucormycosis was the most frequent presentation of disease. Mortality was high at 49%, driven particularly by those with pulmonary or disseminated mucormycosis and those with cerebral involvement. Furthermore, a significant proportion of surviving patients suffered life-changing morbidities (loss of vision in 46% of survivors). Our review indicates that CAM may be a relevant complication of severe COVID-19, particularly in those with uncontrolled diabetes. Funding: Martin Hoenigl received funding from Astellas for two investigator initiated studies (ISR005824 and ISR005838), and was supported by the National Institutes of Health, Grant UL1TR001442. Agostinho Carvalho was supported by the Fundação para a Ciência e a Tecnologia (FCT) (UIDB/50026/2020 and UIDP/50026/2020), the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000039), the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 847507, and the “la Caixa” Foundation (ID 100010434) and FCT under the agreement LCF/PR/HR17/52190003.Declaration of Interest: MH received research funding from Gilead, Pfizer, Astellas, Scynexis and NIH. JPG received speaker and expert advice fees from Pfizer and Gilead. NK has received research grants or honoraria as a speaker or advisor from Astellas, Gilead, MSD, and Pfizer, outside the submitted work. KL received consultancy fees from SMB Laboratoires Brussels, MSD and Gilead, travel support from Pfizer, speaker fees from FUJIFILM WAKO, Pfizer and Gilead, a service fee from Thermo fisher Scientific. OAC is supported by the German Federal Ministry of Research and Education, is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – CECAD, EXC 2030 – 390661388 and has received research grants from, is an advisor to, or received lecture honoraria from Actelion, Allecra Therapeutics, Al-Jazeera Pharmaceuticals, Amplyx, Astellas, Basilea, Biosys, Cidara, Da Volterra, Entasis, F2G, Gilead, Grupo Biotoscana, Immunic, IQVIA, Janssen, Matinas, Medicines Company, MedPace, Melinta Therapeutics, Menarini, Merck/MSD, Mylan, Nabriva, Noxxon, Octapharma, Paratek, Pfizer, PSI, Roche Diagnostics, Scynexis, and Shionogi. PLW performed diagnostic evaluations and received meeting sponsorship from Bruker, Dynamiker, and Launch Diagnostics; Speakers fees, expert advice fees and meeting sponsorship from Gilead; and speaker and expert advice fees 489 from F2G and speaker fees MSD and Pfizer. Is a founding member of the European Aspergillus PCR Initiative. ACh received funding support from educational grant of Pfizer, MSD Pharmaceutical Ltd, and Gilead. All other authors no conflicts.Ethical Approval: MISSING

Genus- and Species-Specific PCR Detection Methods.

PCR-based detection of fungal pathogens offers a sensitive and specific tool for the diagnosis of invasive fungal infections. A large variety of different clinical specimen types can be used as original material. However, certain precautions, in addition to the published MIQE guidelines [1], need to be taken to prevent contaminations from airborne fungal spores and PCR reagents. In addition, the European Aspergillus PCR Initiative (EAPCRI) recently defined standards for Aspergillus PCR [2, 3], following these recommendations leads to superior sensitivity. The combination of fungal PCR with the galactomannan ELISA assay increases the sensitivity for the detection of Aspergillus DNA from blood, compared to a single assay only [4, 5].

Comparison of Performance Characteristics of Aspergillus PCR in Testing a Range of Blood-Based Samples in Accordance with International Methodological Recommendations.

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.

Prospective multicentre PCR-based Aspergillus DNA screening in high-risk patients with and without primary antifungal mould prophylaxis

Invasive aspergillosis (IA) is associated with significant morbidity and mortality, and, among other factors, this is due to a delay in diagnosis performed with conventional techniques. A prospective, multicentre study was conducted to evaluate the efficacy of Aspergillus DNA screening in the early diagnosis of IA. Patients undergoing haematopoietic stem cell transplantation or chemotherapy for acute leukaemia were enrolled for biomarker screening. Three centres applied the same protocol for in-house PCR, which was compliant with the European Aspergillus PCR Initiative recommendations, to guarantee the highest diagnostic standards. Two thousand one hundred and twenty-eight sera from 213 patients were investigated and stratified according to the revised European Organization for the Research and Treatment of Cancer/Mycoses Study Group criteria for invasive fungal disease. The incidence rates of probable and possible IA were 18% and 38%, respectively. The sensitivity, specificity and positive predictive value (PPV) of PCR were superior in antifungal drug-naive patients, being 71.4%, 92.3%, and 62.5%, respectively. The last of these key performance indicators (PPV) was moderate in patients receiving primary prophylaxis, at 5.4%. Negative predictive values for both strategies applied were 100% with and 98.3% without antifungal mould prophylaxis. PCR has the potential to play a decisive role in the diagnosis and management of Aspergillus infections in centres not applying primary antifungal mould prophylaxis.

Polymerase Chain Reaction in the Diagnosis of Invasive Aspergillosis in Hematopoietic Stem Cell Transplant Recipients: A Meta-Analysis of Diagnostic Performance

Background: Invasive aspergillosis (IA) is associated with significant morbidity and mortality among hematopoietic-stem cell transplant (HSCT) recipients. Timely and accurate diagnosis of an active infection is needed in order to initiate targeted antifungal therapy. Although previous studies have assessed the performance of Aspergillusspp. PCR with promising findings, PCR has not yet been incorporated in the diagnostic algorithms by the guideline issuing organizations.Aim: Our goal was to evaluate PCR as a screening or a confirmatory tool for the diagnosis of IA among HSCT patients and to determine the different parameters that could contribute to contradictory reports from the literature regarding PCR performance. Thus, we performed a meta-analysis of clinical trials that evaluated the accuracy of PCR for IA performed on serum and whole blood of HSCT patients.Methods: We performed a bivariate meta-analysis of diagnostic data for Aspergillus spp. PCR on blood specimens across HSCT patients. We included all studies involving human subjects that assessed the performance of any PCR assay for IA on whole blood or serum and that used as reference standard the EORTC/MSG criteria. We performed 2 separate analyses either including or excluding the cases that were defined as “possible”. When possible cases where included they were classified as negative. Out of 37 total studies, 25 met strict quality criteria and were included.Results: 25 studies with 2,595 patients were analyzed. Six studies performed PCR on serum (23%) and the remaining used whole blood assays (77%). The pooled diagnostic performance of whole blood and serum PCR was moderate, with a sensitivity of 84% (95% CI 75%-91%). Specificity was 76% (95% CI 65%-84%) when possible cases were included and 85% (95% CI 74%-91%) when excluded, suggesting that a positive or negative result is unable on its own to confirm or exclude a suspected infection. In subgroup analysis, the performance of PCR on serum was not significantly different from whole blood when possible cases were included. Interestingly, exclusion of possible cases revealed higher specificity of serum PCR (94% vs. 80%, meta-regression p-value=0.05) over whole blood without altering the sensitivity (79% vs. 85%, meta-regression p-value=0.4) and high positive likelihood ratio (LR+=12.6) suggesting that serum PCR could be used as a confirmatory test. No methodological parameter significantly affected the performance of PCR. Importantly, PCR specificity was greatly increased when 2 positive PCR results were used to define positivity (73% vs. 95%, respectively), giving a high positive likelihood ratio of 12.8, whereas sensitivity decreased (85% vs 64%, respectively). Importantly, the European AspergillusPCR Initiative (EAPCRI) recommendations improved even further the performance of PCR when at least two positive specimens were used to define ‘PCR positivity’.Conclusions: Two positive PCR results should be considered highly indicative of an active Aspergillus spp. infection in HSCT patients. Use of the EAPCRI recommendations by clinical laboratories can further enhance PCR performance. DisclosuresNo relevant conflicts of interest to declare.

Towards a standard for Aspergillus PCR - requirements, process and results

The diagnosis of invasive fungal disease remains challenging. The development of the European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria has helped standardise diagnosis, yet PCR is excluded, a result of limited standardisation and concurrent clinical validation. In 2006, the European Aspergillus PCR Initiative working party of International Society of Human and Animal Mycoses was formed with the aim of establishing a standard for PCR methodology for the diagnosis of invasive aspergillosis, to attain an accurate clinical utility of PCR and acceptance of PCR in future disease defining criteria. This manuscript will provide an overview of the standardisation process, before describing the potential samples available and the reasoning behind the choices made. It will summarise the key findings, provide the European Aspergillus PCR Initiative recommendations when testing whole blood and serum, and conclude with a synopsis of future European Aspergillus PCR Initiative perspectives and other key processes that will improve PCR based diagnosis.

Evaluation of Aspergillus PCR Protocols for Testing Serum Specimens

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.

Diagnostic Utility of DNA from Aspergillus in Whole Blood Specimens

For several years, PCR-based detection of Aspergillus DNA has been considered a promising tool for the diagnosis of invasive aspergillosis, but its broader implementation has been hampered by a lack of standardization and the uncertain diagnostic accuracy of the wide variety of published protocols. Recently, new attempts have been made to address this problem and define a quality standard for diagnostic Aspergillus PCR. This review summarizes technical aspects of up-to-date protocols, focusing on the recently proposed guidelines of the European Aspergillus PCR Initiative (EAPCRI) for whole blood PCR testing.

Standardization of Aspergillus PCR diagnosis

Blennow and Mattsson1 recently concluded in a reply to Meije et al.2 that standardization of Aspergillus PCR diagnosis is needed. We agree with this, which is why we set up a Working Group of the International Society of Human and Animal Mycology, known as the European Aspergillus PCR initiative (EAPCRI) (http://www.eapcri.eu). This group has forged ahead, developing the necessary framework for establishing standards.3

Standardization of Aspergillus PCR is needed

Meije et al. report in their letter ‘Silent and prolonged Aspergillus DNAemia in oncohematological patients receiving antifungal prophylaxis: a new phenomenon with clinical implications’ that Aspergillus PCR was a relevant and useful diagnostic tool in early detection of invasive aspergillosis (IA). This is in contrast to what we reported recently. Meije et al. report on six patients with IA for whom Aspergillus fumigatus PCR tests were persistently positive for several weeks before galactomannan Ag test turned positive and clinical symptoms developed. In our study, we followed patients undergoing reduced-intensity conditioning allogeneic hematopoietic SCT with weekly fungal PCR during the first 100 days post transplantation. We found that a single positive PCR test was not associated with IA. This is consistent with the findings of several other authors, including a study by Meije et al. that reported a low positive predictive value using a single positive PCR test. Regarding the association between multiple positive PCR tests and proven and probable IA that Meije et al. report in their letter, we could not draw any conclusion in our study because of low numbers. Our study differs from the experience of Meije et al. in that we did not find Aspergillus PCR to be useful in establishing the diagnosis of IA. One possible explanation for the differences between our study and the report by Meije et al. is that they may be due to the different fungal prophylaxes, fluconazole in our study vs itraconazole in the study by Meije et al. Another important reason for the differences in results between different centers is the lack of standardization of Aspergillus PCR. This is also indicated in a recent study evaluating Aspergillus PCR within the AmBiLoad trial. Using a nested Aspergillus PCR assay, the authors found that 43% of patients with proven and 39% of patients with probable invasive fungal infection had at least one positive test. However, when the authors analyzed the 43 positive samples with a quantitative PCR assay, only three turned out to be positive. Hopefully, the ongoing work of the European Aspergillus PCR initiative will eventually lead to a standardization enabling multicenter studies and comparison between different clinical centers.

Critical Stages of Extracting DNA from Aspergillus fumigatus in Whole-Blood Specimens

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.

Aspergillus PCR: One Step Closer to Standardization

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.