Molecular diagnostics in cancer patients with suspected respiratory mold infections.

INTRODUCTION

Pneumonia caused by molds is a serious complication in patients with severe underlying diseases including hematologic malignancies and the critically ill ( 1 ). In these patients, aspergillosis is the most prevalent invasive mold infection diagnosed ( 2 ). Other mold infections, including mucormycosis, may not be differentiated from aspergillosis by clinical presentation but require unique management strategies due to antifungal resistance and rapid disease progression. In addition, viral infections are increasingly recognized as risk factors for respiratory mold infections and may be a target for specific intervention to improve patient outcome ( 3 ).

While Aspergillus detection by molecular tests or antigen detection identifies these fungi with a higher sensitivity as compared to culture and microscopy in bronchoalveolar lavage fluid (BALF), optimal diagnostic strategies to identify a broader spectrum of molds remain undefined. Besides mycological culture, specific qPCR assays, broad-range qPCR with amplicon sequencing, and fluorescence in situ hybridization (FISH) targeting ribosomal RNA (rRNA) represent sensitive molecular detection methods. As FISH targeting rRNA does not require previous amplification of the molecular target due to a high number of ribosomes in replicating fungi, it has the potential to be a sensitive, specific, and rapid diagnostic tool, but it has not been studied for the diagnosis of mold infections using BALF.

We compared different molecular strategies, that is, broad-range fungal qPCR with amplicon sequencing, specific real-time qPCR assays targeting Aspergillus and Mucorales, and FISH detection of Aspergillus and Mucorales with fungal cultivation using diverse media from BALF to identify the etiology of invasive pulmonary mold infections in cancer patients to improve patient care. In subgroups of patients, fungal DNA was amplified by qPCR from serum to compare results with BALF testing.

MATERIALS AND METHODS

Study population and ethics approval

We conducted a prospective multicenter study at 10 clinical sites and 2 central diagnostic laboratories performing qPCR (Wü4i, University of Würzburg), mycological culture, fungal FISH, and viral diagnostics (Robert Koch-Institut). The study was approved by the ethics committee of the University Hospital Würzburg (#270/15).

After informed consent, adult cancer patients with new or worsening pulmonary infiltrates unresponsive to antibacterial therapy, suspicious for an invasive mold infection, were included. Clinical data (underlying disease, risk factors for mold infections, radiologic findings, antimicrobial therapy) were documented at participating centers in a web-based database at the time of inclusion and around day 90 ( ClinicalSurveys.net ).

BALF samples were collected according to local standards and sent to both central laboratories for diagnostic testing in addition to local center-specific diagnostic assessment procedures.

In total, 234 episodes in 222 patients were included between May 2016 and May 2018. More than one episode per patient was allowed after resolution of clinical signs from the previous episode at the investigator’s discretion. Ten episodes from 10 patients were excluded from the analysis as BALF was not received in both central laboratories, and 14 samples from 14 patients were excluded because baseline clinical data were not reported ( Fig. 1 ). In a subgroup of patients ( n = 175), serum was collected between 3 days each prior and after BALF sampling, stored frozen, and later analyzed by fungal qPCR.

Fungal diseases were classified according to the EORTC/MSG criteria as published in 2008 ( 4 ). Microbiologic criteria included cultivation of a mold pathogen, microscopy demonstrating mold hyphae or positive galactomannan from BALF.

Laboratory procedures

For cultivation of fungi, BALF was centrifuged (1,600 g for 10 min), and cultivation of the pellet was performed on Sabouraud-Dextrose agar, Candida ID agar, and inhibitory mold agar (IMA) (all from Becton-Dickinson, Heidelberg, Germany); Birdseed agar for identification of melanin-producing Cryptococcus ; Scedosporium select agar containing benomyl; and Sabouraud-Dextrose broth (all manufactured at RKI), using 50 µL for each medium (300 µL total volume used for cultivation), and cultures were incubated at 37°C and 26°C (Birdseed agar only). Plates were checked daily for growth for 14 days. Yeast isolates were identified by matrix-assisted laser desorption/ionization time-of-flight and molds by phenotype and sequencing of the ITS region, except for Aspergillus , which was identified by sequencing of a part of the beta-tubulin gene. Cultivated Aspergillus fumigatus was genotyped by cell surface protein typing ( 5 ).

DNA extraction for fungal PCR testing was performed by combining BALF supernatant and pellet fraction using bead-beating and a commercial kit as described previously ( 6 ). Briefly, both BALF fractions were separated after centrifugation. The pellet fraction only was bead-beaten to free cell-associated DNA into the liquid phase. Both fractions were re-united, and cell-free DNA was extracted using a commercial kit. DNA from serum was extracted as described before ( 7 ). Specific qPCR assays were used to detect DNA of Aspergillus with melt curve analysis for species identification ( 8 , 9 ) and Mucorales with species identification using Sanger sequencing ( 10 , 11 ). A broad-range assay targeting a region of the 28S rRNA gene using primers 12f and 13r was performed in the initial samples ( n = 42) but stopped due to a low diagnostic yield ( 12 ). Human DNA was quantified as described before by targeting the human ribosomal RNA gene ( 6 , 13 ). DNA extraction from serum and qPCR specific for Aspergillus were compliant with the recommendation of the fungal PCR initiative (FPCRI) ( 14 ). Appropriate controls were included to document efficient DNA extraction, absence of PCR inhibition, and contaminating fungal DNA ( 7 ). Amplicons were identified by Sanger sequencing (Mucorales and broad-range PCR) ( 10 ).

GM was quantified from BALF using ELISA (Platelia Antigen ELISA, BioRad, Feldkirchen, Germany) according to manufacturer’s instructions. Positive samples were defined by optical density index of ≥0.5 (BALF) and ≥0.5 (serum).

FISH was performed from BALF pellets after incubation of 1 mL with 2 mL of paraformaldehyde (PFA) for 30 min at room temperature. Fixed BALF (300 µL) was spotted on two microscopy slides using a cytospin centrifuge. The first slide was incubated with hybridization buffer containing a specific probe targeting Aspergillus with the Cy3 dye, two probes targeting common agents of mucormycosis with the Cy5 dye, and the unspecific probe EUK 516 targeting eukaryotic cells with the dye Alexa Fluor 488 ( 15 , 16 ). The second slide was incubated with the non-sense probe EUB coupled with Cy3, Cy5, and Alexa Fluor 488 to control for autofluorescence and unspecific hybridization as a negative control. Hybridization, post-hybridization washes, and slight preparation for microscopy were performed as reported previously ( 16 ). Epifluorescence microscopy was performed as described previously ( 17 ). Images were taken by a technician blinded for clinical data, culture, and PCR results. Positive hybridization was assumed when fungal elements showed a probe signal in the absence of evidence for unspecific hybridization or autofluorescence in the negative controls with non-sense probes. Control fungal elements ( Candida parapsilosis yeast cells, A. fumigatus conidia and hyphae, conidia and hyphae of Lichtheimia corymbifera ) grown overnight in liquid broth and fixed with PFA were included in each experiment as positive control and documentation of specific hybridization.

Additional methods including testing for viruses, in vitro susceptibility testing, and typing of A. fumigatus are presented in the Supplemental file .

Statistical analysis

Statistical analyses were performed in GraphPad Prism 9.1.0 for Windows (GraphPad Software, San Diego, USA) and Stata 17 (Stata Corp., College Station, Texas, USA).

RESULTS

Patient characteristics

The final analysis included 210 pneumonia episodes in 198 patients ( Fig. 1 ). Two patients provided three samples, eight patients provided two samples from subsequent pneumonia episodes.

Patients’ underlying diseases were mostly hematological malignancies, predominantly acute leukemias with chemotherapy-associated neutropenia or stem cell transplantation ( Table 1 ). In 13 episodes, medical history included a mold infection (aspergillosis: 12, mucormycosis: 1) that had resolved before inclusion into the study. A probable/proven fungal infection was subsequently diagnosed in 8/13 (62%) vs 67/197 (34%) ( P = 0.05). Antibacterial agents during the 72 h before bronchoscopy were administered in 79%, antivirals in 31%, and non-mold active antifungals in 6% of all episodes. Mold active antifungals were administered in 58% of patients for a median of 18 (range: 1–351) days ( Table 1 ).

Mycologic testing from BALF

Microbiologic laboratory results are summarized in Table 2 for all samples ( n = 210) and for the subgroup from proven/probable invasive fungal infection (IFI) episodes ( n = 75) ( Fig. 2 ). For a subgroup of episodes ( n = 187), Aspergillus GM antigen was tested from BALF ( Table 2 ).

Culture was less likely to demonstrate growth of Aspergillus than specific Aspergillus qPCR from BALF in all episodes (15 [7%] vs 45 [21%]; P = 0.0001) and in probable/proven episodes (14 [19%] vs 35 [47%]; P = 0.07). BALF with cultivation of Aspergillus demonstrated lower sample quantification cycles (Cqs) in qPCR ( n = 14; mean = 40.9; 95% CI = 35.6–46.2) than in culture-negative ( n = 35; mean = 43.6; 95% CI = 41.7–45.6) samples. A. fumigatus was cultivated on IMA in 9/15 (60%) cases and Sabouraud-Dextrose agar in 7/15 (47%) culture-positive samples. Sequencing of cultivated Aspergillus confirmed A. fumigatus sensu stricto in all cases.

Culture was less likely to demonstrate growth of Mucorales than specific qPCR from BALF in all episodes (1 [0.5%] vs 10 [5%]; P = 0.048) and in probable/proven episodes (1 [1%] vs 7 [9%]; P = 0.09). Mucorales DNA was detected at Cq 29.95 in a case with cultivation of Rhizomucor pusillus on IMA only. Culture-negative samples demonstrated different amounts of DNA ( n = 9; mean = 36.49; 95% CI = 30.25–42.73). Of note, Mucorales DNA was detected in 6 of 7 BALF (86%) of patients with probable/proven infection together with Aspergillus DNA suggesting coinfection.

Broad-range fungal PCR was performed in a subgroup of BALF samples ( n = 42) and was positive in 37 (88%). In 10 (27%) of these samples, specific qPCRs identified DNA of a mold pathogen ( Aspergillus 9; Mucorales 2). In only one of these samples, sequencing of the broad-range amplicon identified Aspergillus as the specific qPCR detected. Here, Aspergillus was also cultivated from BAL, and Aspergillus DNA was also amplified from blood. In contrast, sequencing identified yeasts that were also cultivated from BAL in six samples. In three samples, the amplicon of the broad-range qPCR could not be identified due to mixed sequence reads. This suggests a high rate of detection of colonizing yeasts by broad-range qPCR rather than amplification of mold DNA. The frequent occurrence of yeasts in BALF was confirmed by culture (67/210; 32%) and FISH (71/210; 34%).

Inhibition of the PCR reaction was detected in 5 of 210 (2%) BALF samples and in 5 of 117 (4.3%) serum samples. One Aspergillus qPCR in BALF was positive in the context of detected PCR inhibition. No sample showed growth of a mold and negative mold qPCR in the context of PCR inhibition.

The mean human DNA amount in BALF was 7.1 ng/mL (140 ng/mL to 1,043 µg/mL). The median human DNA content was higher in non-neutropenic episodes ( n = 64) than in neutropenic episodes ( n = 146) (13 µg/mL vs 5.2 µg/mL BALF; P = 0.001). For patients with neutropenia, a human DNA content >14 µg/mL BALF was associated with a higher likelihood of positive Aspergillus qPCR (30% vs 15%; P = 0.09).

Culture was less likely to grow Aspergillus than FISH to identify Aspergillus by hybridization of fungal structures with the Aspergillus probe (15 [7%] vs 26 [13%]; P = 0.0001) in all episodes but not in probable/proven episodes (14 [19%] vs 15 [20%]; n.s.). FISH targeting Aspergillus detected conidia ( n = 21), hyphae ( n = 1), or both ( n = 4) by hybridization ( Fig. 3 ). Culture was less likely to grow Mucorales than FISH to identify Mucorales by hybridization of fungal structures with the Mucorales probe (1 [0.5%] vs 6 [3%]; n.s.) in all episodes and probable/proven episodes (1 [2%] vs 1 [2%]; n.s.). Culture was as likely to grow yeasts as FISH to detect yeast cells by positive hybridization of yeasts with the unspecific probe (67 [32%] vs 71 [34%]; n.s.) in all episodes and in proven/probable episodes (30 [40%] vs 32 [43%]). Detection of Aspergillus and Candida by FISH was predictors of subsequent cultivation of Aspergillus (9/15 [60%] vs 5/55 [9%]; P = 0.0001) and Candida (18/32 [56%] vs 12/31 [39%]; P = 0.01).

Among Aspergillus qPCR-positive samples ( n = 45), FISH detected Aspergillus in 11/45 (24%) samples as compared to 15/165 (7%) Aspergillus qPCR-negative BALF ( P = 0.006). Aspergillus qPCR Cq values were not different between Aspergillus FISH positive ( n = 11) and negative samples ( n = 34) (data not shown). Among Mucorales qPCR-positive samples ( n = 10), FISH detected conidia with hybridization of the Mucorales probe in only one sample (10%) as compared to 5/200 (2.5%) (n.s.).

Detection of Aspergillus by culture, specific qPCR, or FISH was not significantly impaired by the presence of systemic mold-active antifungal prophylaxis (data not shown).

Additional results on BALF testing for respiratory viruses and CMV are presented in the supplemental file.

Mycologic testing from serum

For a subgroup of episodes ( n = 175), serum samples were investigated by fungal qPCRs, which detected Aspergillus ( n = 15 [9%]) or Mucorales ( n = 8 [5%]). Among episodes with available qPCRs from serum and BALF ( n = 117), Aspergillus qPCR in serum was more likely in BALF-positive samples (11/33 [33%] vs 2/84 [2%]; P = 0.0001). Mucorales qPCR in serum was more likely in BALF-positive samples (5/8 [63%] vs 3/109 [3%]; P = 0.0001). In episodes with BALF and serum positive by Aspergillus qPCR ( n = 11), qPCR from BALF became positive before serum (40.66 ± 5.58 vs 43.25 ± 5.35; P = 0.12). In episodes positive by Mucorales qPCR from BALF and serum ( n = 5), qPCR from BALF became positive 2.4 Cq before serum (data not shown).

In proven/probable IFI episodes, detection of Aspergillus DNA was more likely in BALF than in serum (35/75 [47%] vs 11/66 [17%]; P = 0.0002) in contrast to Mucorales DNA (7/75 [9%] vs 5/66 [8%]; P = 0.73).

Follow-up after BALF testing

Patients were followed for 0–328 (median 88) days after BALF sampling. At day 90, 120 (66%) patients survived, 28 (13%) patients were lost, and 62 episodes (34%) were reported as lethal. A fatal outcome was more likely among proven/probable mold infection (34/75; 45%), in Aspergillus or Mucorales BALF qPCR-positive episodes (23/49; 47%), among serum Aspergillus or Mucorales qPCR-positive episodes (14/23; 61%), and in culture of Aspergillus -positive BALF (10/16; 63%). In contrast, episodes with FISH-positive BALF were reported as lethal in only 11/33 (33%), with CMV detection by PCR (2/6; 33%) and respiratory virus detection (12/33; 36%).

DISCUSSION

Respiratory mold infections are serious complications in cancer patients, especially those with treatment-associated neutropenia or after allogenic stem cell transplantation. Identification of molds is a prerequisite for optimal patient management as it allows for specific treatment. In the present study, we were able to demonstrate that specific qPCRs targeting Aspergillus and Mucorales on BALF are the most sensitive strategy to identify these pathogens in cancer patients with suspected mold infection. Specific qPCRs are superior to cultivation from BALF and qPCR from blood, which showed lower amounts of fungal DNA detected by specific fungal qPCRs, especially in aspergillosis. Broad-range qPCR from BALF rarely identified mold DNA but frequently detected yeast colonization as confirmed by culture and FISH. Mucorales qPCR detected DNA mostly in BALF that was also positive for Aspergillus DNA suggesting coinfections, indicating that a broad diagnostic spectrum including Mucorales may benefit patients with fungal pneumonia by a potential for treatment optimization.

Microbiologic laboratory tests including detection of hyphae or cultivation of mold pathogens are considered gold standards in the diagnosis of respiratory mold infections. Methods for cultivation of molds vary widely. Preanalytic issues including BAL sampling, sample handling before cultivation, cultured volume, and duration of cultivation may impact the yield of fungal cultures. Experts report sensitivities below 30% for cultivation of Aspergillus from patients with invasive pulmonary aspergillosis ( 18 , 19 ). Using additional media, we were not able to improve the culture yield. Selective media allow for more sensitive cultivation of slow-growing molds such as Scedosporium due to inhibition of Aspergillus by benomyl, which is also not active against frequent agents of mucormycosis ( 20 ). While important Mucorales are resistant to benomyl, a selective medium did not improve cultivation in our study despite frequent co-occurrence of Mucorales and Aspergillus as detected by PCR ( 21 ). In addition, IMA and niger seed agar to identify Cryptococcus did not improve cultivation of pathogenic fungi in this study with frequent antifungal use at the time of sampling ( 21 ).

The detection of mold hyphae by microscopy is thought to be indicative of invasive mold infection in cancer patients. A rapid differentiation between hyphal morphologies such as septate and non-septate hyphae helps guide patient management and therefore is indicated for many samples including BALF when fungal etiology needs to be considered ( 22 ). However, as species resolution by microscopy is limited and small particles such as conidia are difficult to identify in standard fungal staining, we evaluated FISH targeting fungal rRNA to identify fungal elements in BALF. We regularly detected fungal elements, including yeast cells, conidia of mold pathogens, but only rarely mold hyphae. While mold hyphae visualized by FISH may actually represent lower respiratory tract infection, the detection of spores of facultative pathogenic and fungi such as A. fumigatus may reflect recent exposure to this ubiquitous mold and may not be an indicator of invasive diseases.

When compared with the other detection methods, FISH results offer potential clues for improving diagnostic tests for respiratory mold infections. First, FISH targeting rRNA was less sensitive than PCR targeting fungal DNA. This may be caused by a lack of fluorescence signal used to define the presence of conidia. Aspergillus conidia may represent metabolically inactive structures that are not identified by FISH due to a low rRNA copy number and therefore low fluorescence signal intensity. They may become identifiable by FISH after germination due to sensing of nutrients after inhalation, a process that occurs asynchronously in different conidia, leading first to swelling of conidia and later polarized growth as germ tubes and later mycelia in germinating conidia. A preincubation with nutrients or germination inducers may improve FISH positivity and may improve cultivation ( 23 ). Alternatively, PCR may frequently detect free DNA released by antifungals or host defense. Therefore, optimization of DNA extraction should include testing of conidia, free DNA, and hyphae. Indeed, DNA yield can be different for extraction kits used for conidia vs hyphae ( 24 ). Interestingly, protocols that do not have any fungal lysis step allow for fungal DNA detection in BALF, indicating that free DNA released through immune cells or antifungal therapy might be amplified from BALF ( 25 ). In conclusion, while recommendation for the detection of Aspergillus DNA from blood has been published by the FPCRI based on extensive studies, our data suggest that there is a need for further standardization of BAL testing with a potential role of FISH in defining the target of fungal DNA extraction to generate evidence-based recommendations ( 26 ).

Although FISH has the potential for sensitive and specific detection of fungi in BALF, it has also technical limitations. First, the use of short oligonucleotides offers more limited species resolution than PCR, potentially yielding outside hits, that is, failure to discriminate between closely related fungi. Second, if fluorescence is used for detection, autofluorescence of fungal material requires adequate controls to rule out false positives.

Coinfections with Aspergillus and Mucorales have been described in diverse patient populations, including hematologic malignancy, organ transplantation, diabetes, and severe viral pneumonia, by detection of these fungi predominantly in respiratory samples (BALF, tissue biopsy) and blood ( 27 ). Such infections may pose significant therapeutic challenges. When microbiologic tests on BALF are based on Aspergillus antigen and even culture, mucormycosis may not be detected, potentially leading to inadequate patient management. Previously, it had been demonstrated that Mucorales can be detected by qPCR in 9.5% of patients with probable invasive aspergillosis by qPCR ( 28 , 29 ). Our finding that DNA load of Mucorales in BALF is not significantly higher than in serum confirms previous studies demonstrating the usefulness of qPCR from serum for the early diagnosis and assessment of treatment response in mucormycosis in patients with hematologic malignancy ( 30 ). However, as only five of eight patients with detection of Mucorales DNA in BALF and available serum were serum positive by Mucorales qPCR, BALF testing appears mandatory, in line with a sensitivity of 85% of serum Mucorales PCR published by Millon et al. ( 30 ).

Viral respiratory tract infections increase the risk of developing pulmonary aspergillosis, probably due to impairment of the epithelial barriers or inability to generate an effective immune response ( 3 ). While most research has been performed on CMV, influenza A virus, and recently SARS-CoV-2, other viruses have been reported mainly in transplant recipients. Among community-acquired respiratory viruses, rhinovirus has been previously shown to be the predominant virus cultivated from respiratory specimens in patients with hematologic malignancy, both with and without stem cell transplantation ( 31 ). A reliable detection enables patient isolation to prevent further transmission. However, in the absence of a viral pandemic during the study period, respiratory viruses and CMV were infrequently identified in our patients.

In conclusion, a combination of specific qPCRs from BALF is the most sensitive molecular test to identify Aspergillus and Mucorales in cancer patients with suspected mold pneumonia. The predominant detection of Mucorales DNA in the presence of Aspergillus DNA supports routine testing beyond Aspergillus in patients at high risk for fungal pneumonia. The use of FISH to visualize fungal structures in clinical samples may offer optimization of diagnostic tests.

ACKNOWLEDGMENTS

The authors thank Ilka McCormick Smith and Dunja Wilmes for excellent laboratory work.

Funding was obtained from the Sander Stiftung (2015.083.1) to V.R. and J.L.

V.R. and J.L. designed the overall study protocol. D.T., D.K., J.K., I.W., T.L., J. Steinmann, O.A.C., P.K., S.S., L.M., S.W.K., W.J.H., B.W., and G.M. were responsible for patient management and data collection. J. Springer, V.R., J.G., S.V., J.R., J.J.V., and T.E. performed and interpreted the analysis and collected the data. V.R. and J. Springer drafted the initial manuscript with input from D.T., J.L., J.H., and H.E. V.R. and J. Springer performed the statistical analysis. All authors helped to analyze or interpret data, contributed to the revision and correction of the final manuscript, approved the final version, and agreed to be accountable for all aspects of the work.

O.A.C. reports grants or contracts from iMi, iHi, DFG, BMBF, Cidara, DZIF, EU-DG RTD, F2G, Gilead, MedPace, MSD, Mundipharma, Octapharma, Pfizer, Scynexis; consulting fees from Abbvie, AiCuris, Basilea, Biocon, Boston Strategic Partners, Cidara, Elion Therapeutics, Gilead, GSK, IQVIA, Janssen, Matinas, MedPace, Menarini, Melinta, Molecular Partners, MSG-ERC, Mundipharma, Noxxon, Octapharma, Pardes, Partner Therapeutics, Pfizer, PSI, Scynexis, Seres, Seqirus, Shionogi, The Prime Meridian Group; speaker and lecture honoraria from Abbott, Abbvie, Akademie für Infektionsmedizin, Al-Jazeera Pharmaceuticals/Hikma, amedes, AstraZeneca, Deutscher Ärzteverlag, Gilead, GSK, Grupo Biotoscana/United Medical/Knight, Ipsen Pharma, Medscape/WebMD, MedUpdate, MSD, Moderna, Mundipharma, Noscendo, Paul-Martini-Stiftung, Pfizer, Sandoz, Seqirus, Shionogi, streamedup!, Touch Independent, Vitis; payment for expert testimony Cidara; participation on a DRC, DSMB, DMC, Advisory Board for AstraZeneca, Cidara, IQVIA, Janssen, MedPace, Melinta, PSI, Pulmocide, Vedanta Biosciences. P.K. reports grants or contracts from German Federal Ministry of Research and Education (BMBF) B-FAST (Bundesweites Forschungsnetz Angewandte Surveillance und Testung) and NAPKON (Nationales Pandemie Kohorten Netz, German National Pandemic Cohort Network) of the Network University Medicine (NUM), the State of North Rhine-Westphalia, and the Dr. Heinz Lux-Stiftung; consulting fees from Ambu GmbH, Gilead Sciences, infill healthcare communication GmbH, Mundipharma Research Limited, Noxxon N.V. and Pfizer Pharma; honoraria for lectures from Akademie für Infektionsmedizin e.V., Ambu GmbH, Astellas Pharma, BioRad Laboratories Inc., Datamed GmbH, European Confederation of Medical Mycology, Gilead Sciences, GPR Academy Ruesselsheim, HELIOS Kliniken GmbH, Jazz Pharmaceuticals Germany GmbH, Lahn-Dill-Kliniken GmbH, medupdate GmbH, MedMedia GmbH, MSD Sharp & Dohme GmbH, Pfizer Pharma GmbH, Scilink Comunicación Científica SC, streamedup! GmbH, University Hospital and LMU Munich and VITIS GmbH; participation on an Advisory Board from Ambu GmbH, Gilead Sciences, Mundipharma Research Limited and Pfizer Pharma; a filed patent at the German Patent and Trade Mark Office (DE 10 2021 113 007.7); other non-financial interests from Elsevier, Wiley and Taylor & Francis online outside the submitted work. G.M. reports honoraria from Gilead Sciences.

Contributor Information

V. Rickerts, Email: rickertsv@RKI.de.

Kimberly E. Hanson, University of Utah, Salt Lake City, Utah, USA

DATA AVAILABILITY

Data are available upon request from the authors.

ETHICS APPROVAL

The study was approved by the ethics committee of the University hospital Würzburg (#270/15)

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.01201-25 .

jcm.01201-25-s0001.docx

Additional methods on viral diagnostics, in vitro resistance, and molecular typing of Aspergillus fumigatus .

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

REFERENCES

1. Thompson GR III, Young J-AH. 2021. Aspergillus infections. N Engl J Med 385:1496–1509. doi: 10.1056/NEJMra2027424

2. Koehler P, Hamprecht A, Bader O, Bekeredjian-Ding I, Buchheidt D, Doelken G, Elias J, Haase G, Hahn-Ast C, Karthaus M, et al. 2017. Epidemiology of invasive aspergillosis and azole resistance in patients with acute leukaemia: the SEPIA study. Int J Antimicrob Agents 49:218–223. doi: 10.1016/j.ijantimicag.2016.10.019

3. Salazar F, Bignell E, Brown GD, Cook PC, Warris A. 2022. Pathogenesis of respiratory viral and fungal coinfections. Clin Microbiol Rev 35:e0009421. doi: 10.1128/CMR.00094-21

4. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T, Pappas PG, Maertens J, Lortholary O, Kauffman CA, et al. 2008. Revised definitions of invasive fungal disease from the 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 group. Clin Infect Dis 46:1813–1821. doi: 10.1086/588660

5. Gao LJ, Sun Y, Wan Z, Li RY, Yu J. 2013. CSP typing of Chinese Aspergillus fumigatus isolates: identification of additional CSP types. Med Mycol 51:683–687. doi: 10.3109/13693786.2013.770609

6. Springer Jan, White PL, Kessel J, Wieters I, Teschner D, Korczynski D, Liebregts T, Cornely OA, Schwartz S, Elgeti T, Meintker L, Krause SW, Posso RB, Heinz WJ, Fuhrmann S, Vehreschild JJ, Einsele H, Rickerts V, Loeffler J. 2018. A comparison of Aspergillus and Mucorales PCR testing of different bronchoalveolar lavage fluid fractions from patients with suspected invasive pulmonary fungal disease. J Clin Microbiol 56:e01655-17. doi: 10.1128/JCM.01655-17

7. Springer J, Lackner M, Nachbaur D, Girschikofsky M, Risslegger B, Mutschlechner W, Fritz J, Heinz WJ, Einsele H, Ullmann AJ, Löffler J, Lass-Flörl C. 2016. Prospective multicentre PCR-based Aspergillus DNA screening in high-risk patients with and without primary antifungal mould prophylaxis. Clin Microbiol Infect 22:80–86. doi: 10.1016/j.cmi.2015.09.009

8. Springer J, Loeffler J, Heinz W, Schlossnagel H, Lehmann M, Morton O, Rogers TR, Schmitt C, Frosch M, Einsele H, Kurzai O. 2011. Pathogen-specific DNA enrichment does not increase sensitivity of PCR for diagnosis of invasive aspergillosis in neutropenic patients. J Clin Microbiol 49:1267–1273. doi: 10.1128/JCM.01679-10

9. Springer J, Schloßnagel H, Heinz W, Doedt T, Soeller R, Einsele H, Loeffler J. 2012. A novel extraction method combining plasma with a whole-blood fraction shows excellent sensitivity and reproducibility for patients at high risk for invasive aspergillosis. J Clin Microbiol 50:2585–2591. doi: 10.1128/JCM.00523-12

10. Springer J, Goldenberger D, Schmidt F, Weisser M, Wehrle-Wieland E, Einsele H, Frei R, Löffler J. 2016. Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species. J Med Microbiol 65:227–234. doi: 10.1099/jmm.0.000218

11. Springer J, Lackner M, Ensinger C, Risslegger B, Morton CO, Nachbaur D, Lass-Flörl C, Einsele H, Heinz WJ, Loeffler J. 2016. Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples. J Med Microbiol 65:1414–1421. doi: 10.1099/jmm.0.000375

12. Khot PD, Ko DL, Fredricks DN. 2009. Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol 75:1559–1565. doi: 10.1128/AEM.02383-08

13. Khot PD, Ko DL, Hackman RC, Fredricks DN. 2008. Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BMC Infect Dis 8:73. doi: 10.1186/1471-2334-8-73

14. White PL, Mengoli C, Bretagne S, Cuenca-Estrella M, Finnstrom N, Klingspor L, Melchers WJG, McCulloch E, Barnes RA, Donnelly JP, Loeffler J. 2011. Evaluation of Aspergillus PCR protocols for testing serum specimens. J Clin Microbiol 49:3842–3848. doi: 10.1128/JCM.05316-11

15. Rickerts V, Khot PD, Myerson D, Ko DL, Lambrecht E, Fredricks DN. 2011. Comparison of quantitative real time PCR with sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens. BMC Infect Dis 11:202. doi: 10.1186/1471-2334-11-202

16. Dalimot JJ, Smith IMC, Gerkrath J, Hartmann S, Cornely OA, Lee SC, Heitman J, Rickerts V. 2022. Identification of mucormycosis by fluorescence in situ hybridization targeting ribosomal RNA in tissue samples. J Fungi (Basel) 8:289. doi: 10.3390/jof8030289

17. Kessel J, Hogardt M, Aspacher L, Wichelhaus TA, Gerkrath J, Rosenow E, Springer J, Rickerts V. 2022. Exclusion of Mucorales co-infection in a patient with Aspergillus flavus sinusitis by fluorescence in situ hybridization (FISH). J Fungi (Basel) 8:306. doi: 10.3390/jof8030306

18. Latgé J-P, Chamilos G. 2019. Aspergillus fumigatus and aspergillosis in 2019. Clin Microbiol Rev 33:e00140-18. doi: 10.1128/CMR.00140-18

19. Danion F, Coste A, Le Hyaric C, Melenotte C, Lamoth F, Calandra T, Garcia-Hermoso D, Aimanianda V, Lanternier F, Lortholary O. 2023. What is new in pulmonary mucormycosis? J Fungi (Basel) 9:307. doi: 10.3390/jof9030307

20. Blyth CC, Harun A, Middleton PG, Sleiman S, Lee O, Sorrell TC, Meyer W, Chen SCA. 2010. Detection of occult Scedosporium species in respiratory tract specimens from patients with cystic fibrosis by use of selective media. J Clin Microbiol 48:314–316. doi: 10.1128/JCM.01470-09

21. Scognamiglio T, Zinchuk R, Gumpeni P, Larone DH. 2010. Comparison of inhibitory mold agar to sabouraud dextrose agar as a primary medium for isolation of fungi. J Clin Microbiol 48:1924–1925. doi: 10.1128/JCM.01814-09

22. Schelenz S, Barnes RA, Kibbler CC, Jones BL, Denning DW. 2009. Standards of care for patients with invasive fungal infections within the United Kingdom: a national audit. J Infect 58:145–153. doi: 10.1016/j.jinf.2008.12.006

23. Danion F, van Rhijn N van, Dufour AC, Legendre R, Sismeiro O, Varet H, Olivo-Marin J-C, Mouyna I, Chamilos G, Bromley M, Beauvais A, Latgé J-P. 2021. Aspergillus fumigatus, one uninucleate species with disparate offspring. J Fungi (Basel) 7:30. doi: 10.3390/jof7010030

24. Fredricks DN, Smith C, Meier A. 2005. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol 43:5122–5128. doi: 10.1128/JCM.43.10.5122-5128.2005

25. Springer J, Löffler J, Einsele H, White PL. 2020. The screening of blood by Aspergillus PCR and galactomannan ELISA precedes BAL detection in patients with proven and probable IA. Med Mycol 58:856–858. doi: 10.1093/mmy/myaa007

26. White PL, Bretagne S, Klingspor L, Melchers WJG, McCulloch E, Schulz B, Finnstrom N, Mengoli C, Barnes RA, Donnelly JP, Loeffler J. 2010. Aspergillus PCR: one step closer to standardization. J Clin Microbiol 48:1231–1240. doi: 10.1128/JCM.01767-09

27. Ling FX, Qu DM, Lu YQ, Li R, Zhao L. 2024. Successful treatment of mixed pulmonary Aspergillus and Mucor infection using intrabronchial amphotericin B infusion: a case report and literature review. BMC Pulm Med 24:436. doi: 10.1186/s12890-024-03234-z

28. Guegan H, Iriart X, Bougnoux ME, Berry A, Robert-Gangneux F, Gangneux JP. 2020. Evaluation of MucorGenius mucorales PCR assay for the diagnosis of pulmonary mucormycosis. J Infect 81:311–317. doi: 10.1016/j.jinf.2020.05.051

29. Wehrle-Wieland E, Affolter K, Goldenberger D, Tschudin Sutter S, Halter J, Passweg J, Tamm M, Khanna N, Stolz D. 2018. Diagnosis of invasive mold diseases in patients with hematological malignancies using Aspergillus, Mucorales, and panfungal PCR in BAL. Transpl Infect Dis 20:e12953. doi: 10.1111/tid.12953

30. Millon L, Caillot D, Berceanu A, Bretagne S, Lanternier F, Morio F, Letscher-Bru V, Dalle F, Denis B, Alanio A, Boutoille D, Bougnoux ME, Botterel F, Chouaki T, Charbonnier A, Ader F, Dupont D, Bellanger AP, Rocchi S, Scherer E, Gbaguidi-Haore H, Herbrecht R. 2022. Evaluation of serum mucorales polymerase chain reaction (PCR) for the diagnosis of mucormycoses: the MODIMUCOR prospective trial. Clin Infect Dis 75:777–785. doi: 10.1093/cid/ciab1066

31. Gabutti G, De Motoli F, Sandri F, Toffoletto MV, Stefanati A. 2020. Viral respiratory infections in hematological patients. Infect Dis Ther 9:495–510. doi: 10.1007/s40121-020-00313-6

Associated Data

Supplementary Materials

jcm.01201-25-s0001.docx

Additional methods on viral diagnostics, in vitro resistance, and molecular typing of Aspergillus fumigatus .

Data Availability Statement

Data are available upon request from the authors.