key: cord-0915914-io2v4mq7
authors: Kirchhoff, Lisa; Braun, Lukas Miles; Schmidt, Dirk; Dittmer, Silke; Dedy, Jutta; Herbstreit, Frank; Stauf, Raphael; Steckel, Nina Kristin; Buer, Jan; Rath, Peter‐Michael; Steinmann, Joerg; Verhasselt, Hedda Luise
title: COVID‐19‐associated pulmonary aspergillosis in ICU patients in a German reference centre: Phenotypic and molecular characterisation of Aspergillus fumigatus isolates
date: 2022-02-17
journal: Mycoses
DOI: 10.1111/myc.13430
sha: e857199b2e160be017f78ed22694e7cd5720f6f3
doc_id: 915914
cord_uid: io2v4mq7

BACKGROUND: COVID‐19‐associated invasive pulmonary aspergillosis (CAPA) is associated with increased mortality. Cases of CAPA caused by azole‐resistant Aspergillus fumigatus strains have been reported. OBJECTIVES: To analyse the twelve‐month CAPA prevalence in a German tertiary care hospital and to characterise clinical A. fumigatus isolates from two German hospitals by antifungal susceptibility testing and microsatellite genotyping. Patients/Methods. Retrospective observational study in critically ill adults from intensive care units with COVID‐19 from 17 February 2020 until 16 February 2021 and collection of A. fumigatus isolates from two German centres. EUCAST broth microdilution for four azole compounds and microsatellite PCR with nine markers were performed for each collected isolate (N = 27) and additional for three non‐COVID A. fumigatus isolates. RESULTS: welve‐month CAPA prevalence was 7.2% (30/414), and the rate of azole‐resistant A. fumigatus isolates from patients with CAPA was 3.7% with detection of one TR34/L98H mutation. The microsatellite analysis revealed no major clustering of the isolates. Sequential isolates mainly showed the same genotype over time. CONCLUSIONS: Our findings demonstrate similar CAPA prevalence to other reports and a low azole‐resistance rate. Genotyping of A. fumigatus showed polyclonal distribution except for sequential isolates.

Invasive pulmonary aspergillosis (IPA) is a severe fungal infection with a high mortality rate. 1 IPA usually occurs in severely immunocompromised patients with prolonged neutropenia 2 but also in patients on intensive care units (ICU) with viral pneumonia are more susceptible to fungal superinfections as seen in influenzaassociated pulmonary aspergillosis (IAPA). 3 Since the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 reports of COVID-19-associated pulmonary aspergillosis (CAPA) raised concerns about this superinfection as an additional contributing factor to mortality. 4 Diagnosis and management of patients with CAPA are challenging due to missing host factors and typical radiological signs; fungal diagnostic approaches are impaired by a reduced use of bronchoscopy and concurrent insufficient sensitivity of circulating galactomannan (GM) in serum [10] . Therefore, there is an urgent need to study the characteristics of this secondary mould infection. In the meantime, ECMM/ ISHAM consensus criteria for defining and managing CAPA have been published. 5 According to Prattes et al. 6 CAPA, occurring with a prevalence ranging between 1.7% and26.8%, is an independent and strong predictor of ICU mortality, leading to implications for antifungal therapy as well as emergence of azole-resistance. In IPA, patients from either the haematology ward or the ICU reveal a voriconazole-resistance rate of Aspergillus fumigatus of more than 16% in a Dutch monocentric study 7 whereas in a retrospective study in patients with CAPA, four azole-resistant A. fumigatus were detected (12.5%); three of which had the TR34/L98H resistance mutation in the cyp51A gene (9.4%). 8 Molecular typing methods enable strain differentiation of isolates from the same species to unveil the source of infection and potential transmission routes to characterise the epidemiology of infections. In previous studies, clonal relatedness of azole-resistant A. fumigatus strains in patients at high risk 7 and patients with cystic fibrosis 9 could not be verified. However, this has not been investigated so far in neither IAPA nor in CAPA. The aim of our study was to assess (i) CAPA twelve-month prevalence in critically ill ICU patients in a German tertiary care hospital, (ii) prevalence of azole-resistant 

All A. fumigatus isolates were further characterised using broth microdilution according to the EUCAST method for susceptibility testing of moulds. 

From isolates grown on Sabouraud agar (Thermo Scientific) for 24-48 h at 35°C, two 1 cm 2 agar blocks from opposing areas were punched out and lysed using Maxwell Tissue LEV Total RNA Purification Kit and Maxwell 16 instrument (both Promega Corp.).

Isolates with elevated MICs for at least one azole antifungal (itraconazole, voriconazole, posaconazole or isavuconazole) were further analysed for underlying mutations with the multiplex PCR AsperGeniusVR (PathoNostics). In a next step, the cyp51A gene was sequenced as described. 11 Sequences were then analysed using the FunResDB database 12 and matched with the non-mutated cyp51A sequence. Amino acid substitutions were correlated with published mutations and concomitant cross-resistance to azoles.

Microsatellite PCR was performed for all 32 A. fumigatus isolates as described previously by de Valk et al. 9 Hamming Distance was performed using PHYLOViZ 2.0 online software (https://www.phylo viz.net).

Statistical analysis was performed with Excel 2016 (Microsoft Corporation, Redmond, WA, USA) and GraphPad Prism 6.0 (GraphPad).

Overall, the total number of COVID-19 cases recorded on ICU within twelve months was 414 ( Table S1 .

In 16 (53%) cases, A. fumigatus could be isolated from respiratory specimen. Among the diagnostic procedures for CAPA definition, GM from respiratory specimen was positive in 77% followed by Aspergillus culture (57%) and PCR (30%). The GM median (IQR) was No mutation corresponding with azole-resistance was found in the A. fumigatus isolates derived from the centre in Nuremberg.

Next, to analyse molecular relationships, microsatellite typing was performed. The microsatellite derived clustering of all isolates from patients with and without COVID-19 is summarised in Figure 1 .

Assignment to the centre of origin or to CAPA classification by genotyping was not successful (data not shown). We also checked for clonal relatedness of isolates from patients with fatal outcome and 

In this study, we found a CAPA prevalence of 7.2% in 414 ICU COVID-19 patients from a German university hospital. Reports on CAPA prevalence around the world range from 3.8% 14 up to 40%. 15 In a recent multi-centre study, Prattes et al. 6 In comparison with a recent study from the Netherlands, 18 our data revealed lower rates for proven (0% vs. 2%) and probable CAPA (6.3% vs. 12%) but not for possible CAPA (1% vs. 1%).

Interestingly, in most of the CAPA cases, GM from respiratory specimen was positive (77%) followed by culture (57%) and PCR (30%). These findings correspond to the multi-national study of Janssen et al. 18 with 78% positivity for GM, 42% Aspergillus culture and only 17% PCR. Antifungal therapy or prophylaxis did not influence PCR results and therefore should not be the reason for the low positivity rate. 19 Another important aim of the study was to gain information about the relatedness of CAPA isolates. Therefore, we applied microsatellite genotyping, a well-established molecular approach with high discriminatory power. 13 Microsatellite typing showed polyclonality F I G U R E 1 Genetic relatedness of A. fumigatus isolates from patients with (blue) and without (green) underlying COVID-19 disease originating from the two centres Essen and Nuremberg based on microsatellite typing. The dendrogram was constructed based on UPGMA clustering of nine microsatellite markers

Non-COVID-19

of strains. It further revealed no major clustering neither regarding isolates from patients with or without COVID-19, nor the centre of origin, nor regarding CAPA classification. However, some, but not all sequential isolates seem to be related to each other. Why isolates 26

and 28 are not closely related as the other sequential isolates can only be assumed. In comparison with the other pairs of first and sequential isolates, pair 26/28 had the longest interval between sampling (five days). Possibly, the patient was infected with two distinct A. fumigatus genotypes.

To the best of our knowledge, no comparable genotyping data are available neither for IAPA, nor for CAPA. Steenwyk et al. 26 found that CAPA isolate genomes do not exhibit significant differences from the genome of a reference strain by using genome sequencing of four A. fumigatus strains. Additionally, all four CAPA isolates cluster together, which may be due to the fact they were all from the same geographic area. However, our findings, based on 27 isolates from patients with CAPA from two centres, showed no epidemiological association of isolates in terms of origin and underlying disease.

In the past, several approaches were used for genotyping A. fumigatus. With microsatellite and cyp51A sequence typing, Fuhren et al analysed the putative clonality of azole-resistant A. fumigatus strains from high-risk patients from either the haematology ward or the ICU and found no clonal spread of resistant strains [7] . Also more recently, a new genotyping method based on hypervariable tandem repeats within exons of surface protein-coding genes was established by Garcia-Rubio et al have been developed which is highly discriminatory and easy to perform [23] .

In summary, we found a CAPA twelve-month prevalence of

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Influenzaassociated aspergillosis in critically Ill patients

Clinical course and outcomes of critically ill patients with SARS-CoV-2 pneumonia in Wuhan, China: a singlecentered, retrospective, observational study

Defining and managing COVID-19-associated pulmonary aspergillosis: the 2020 ECMM/ ISHAM consensus criteria for research and clinical guidance

Risk factors and outcome of pulmonary aspergillosis in critically ill coronavirus disease 2019 patients-a multinational observational study by the European Confederation of Medical Mycology

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Prevalence and characterization of azole-resistant Aspergillus fumigatus in patients with cystic fibrosis: a prospective multicentre study in Germany

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Diagnosis and management of aspergillosis in the Netherlands: a national survey

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