This is a prospective study of threshold-driven and continuous response variables from independent control and experimental groups of subjects. The intensive care units in the five hospitals include 63 beds and will provide 2292 potentially suitable critically ill subjects annually during a 3-year prospective study.

In patients with suspected IPA, all types of samples will be collected twice a week for analysis from the date of diagnosis until at least 2 consecutive negative results are obtained, or the patient will be discharged from an intensive care unit. If available, stored aliquots of samples taken 14 - 1 days before the day of diagnosis (mainly the serum and BALF/ETA (bronchoalveolar lavage fluid/endotracheal aspirate) used for galactomannan screening) will be used for Pentraxin 3 (PTX3), Aspergillus qPCR, and HPLC-ESI-FTICR analyses. Results will be reported to clinicians in variable time frames depending on the centre.

The control and experimental group sizes are expected to be approximately equal. Based on the previous experimental and published data, the expected AUC value is 0.8 for the power of study limiting laboratory assay. For Ptx3 and qPCR Aspergillus, a sample size of 13 in each group will be sufficient to detect a significant difference, assuming a power of 80%, and a two-sided significance level of 0.05. The second assay used in the study would require only 7 subjects in each group based on a mean difference of 4303 and an estimated standard deviation of 2500 to detect significant differences assuming the power of 80%, a two-sided significance level of 0.05, and the t-test. When an appropriate threshold is applied to the latter assay, the expected AUC value is 0.99, thus rendering the minimum required number of subjects to 3 for each study group. The investigators expect to enrol more subjects (up to a total of 90) to allow for a possible variation of the values in the groups. Statistical analysis for clinical samples will be performed in R 4.0.2 using standard libraries for exploratory data analysis. Descriptive statistics will be reported in the form of mean, median, standard deviation, standard error of the mean, and coefficient of variation. Differences in male proportion and age will be evaluated using the Wilcoxon rank-sum test for equal variance. Sample frequency will be compared with Fisher's exact test. The normal distribution of the data (age and gender) will be tested using the Shapiro-Wilk normality test. If the data do not meet the normality requirement, the nonparametric Kruskal-Wallis test will be followed by the post hoc pairwise Wilcoxon signed-rank test for further data analysis; for all above-mentioned tests, p \<0.05 will be considered statistically significant. R library cutpointr estimates the optimal cut-off value, and the ROC characteristics, specificity, and sensitivity will be determined. Maximize spline, gam, and loss estimates for metrics accuracy and a sum of sensitivity and specificity, and Kernel estimate for Youden index methods will be used for cut-off value estimates. False-negative and false-positive rates will be calculated as controls based on probable IPA and non-IPA.

Patients with suspected IPA defined by the presence of host factors, suggestive imaging, and clinical symptoms, according to the 2008 consensus definitions from the European Organization for the Research and Treatment of Cancer / Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) revised in 2019, will be prospectively recruited in five hospital centres in the Czech Republic from 1st June 2023 to 31st December 2025 (Cohort 1). Mycological and clinical investigations will be performed according to the usual procedures in each centre (imaging, mycological culture, identification using microscopy, and histopathological examination). Molecular detection of hyphae recognized in tissue samples, BALF, and/or ETA will be performed using Aspergillus quantitative PCR (qPCR). IPA-specific acute phase biomarker PTX3 elevation will be analysed in BALF and/or ETA and serum commercial PTX3 immunoassays. Mass spectrometry identification of specific Aspergillus siderophores TafC, TafB, Fc, and secondary metabolite Gtx in the tissue biopsies (if available), urine, BC (intubated patient), BALF, and/or ETA will be performed using HPLC/ESI-FTICR (co-PI 1). All other investigations and treatments, including treatment for aspergillosis, will be based on the standards of each co-applicant team in accordance with the procedures defined in each centre. Population characteristics, clinical and biological data, underlying conditions, medical history, biology, imaging, time to diagnosis, and treatment will be collected for each patient after enrolment and recorded in each centre using an online Castor EDC (Castor's Decentralized Clinical Trials Platform, www.castoredc.com). Routine detection of galactomannan antigen, 1,3-β-D-glucan, PTX3, and Aspergillus qPCR assay will be performed in accordance with the procedures defined in co-participant Public Health Institute Ostrava and Faculty of Medicine, University of Ostrava. The HPLC/ESI-FTICR analysis will be performed by the Institute of Microbiology, Academy of Sciences of the Czech Republic in Prague. TafC enrichment by affinity purification column will be performed by co-applicant University Hospital Olomouc, and MS analyses (HPLC/ESI-FTICR, ESI-FTICR, and MALDI-TOF) will be performed by co-applicant Institute of Microbiology, Academy of Sciences, Czech Republic in Prague. The day of sampling of the first histological or positive mycological specimen will be defined as day 0 (D0). Patients will be classified at month 6 as having a possible, probable, or proven IPA according to the 2019 EORTC/MSGERC criteria.