The current state of laboratory mycology and access to antifungal treatment in Europe: a European Confederation of Medical Mycology survey

Access to the appropriate tools is crucial for early diagnosis and clinical management of invasive fungal infections. This Review aims to describe the invasive fungal infection diagnostic capacity of Europe to better understand the status and the most pressing aspects that need improvement. To our knowledge, this is the ﬁrst time that the mycological diagnostic capability and access to antifungal treatments of institutions has been evaluated at a pan-European level. Between Nov 1, 2021, and Jan 31, 2022, 388 institutions in Europe self-assessed their invasive fungal infection management capability. Of the 388 participating institutions from 45 countries, 383 (99%) had access to cultures, 375 (97%) to microscopy, 363 (94%) to antigen-detection assays, 329 (85%) to molecular tests (mostly PCR), and 324 (84%) to antibody tests for diagnosis and management. With the exception of microscopy, there were considerable diﬀerences in access to techniques among countries according to their gross domestic product. At least one triazole was available in 363 (94%) of the institutions, one echinocandin in 346 (89%), and liposomal amphotericin B in 301 (78%), with country gross domestic product-based diﬀerences. Diﬀerences were also observed in the access to therapeutic drug monitoring. Although Europe is well prepared to manage invasive fungal infections, some institutions do not have access to certain diagnostic tools and antifungal drugs, despite most being considered essential by WHO. These limitations need to be overcome to ensure that all patients receive the best diagnostic and therapeutic management.


Introduction
The prevalence of invasive fungal infections continues to increase in Europe and worldwide. 1Europe is home to large populations at risk for invasive fungal infections, including haematological and oncological patients, patients requiring intensive medical care, recipients of solid organ transplants, and older populations. 2The prevalence of invasive fungal infection is increasing in the intensive care unit (ICU) population, including people with respiratory viral infections, particularly influenza and COVID-19.][7][8] With almost 800 million inhabitants 9 and located in the northern hemisphere, Europe presents a wide diversity in terms of environmental climates, 10 access to health care, 11 and citizen income. 12Four European countries are located within the top ten of the highest-income countries in the International Monetary Fund list of 2021, with an average gross domestic product (GDP) of greater than US$62 000. 13However, there are also countries within Europe with lower average GDP, closer to those of countries in Africa or Asia.These discrepancies might jeopardise the access to appropriate mycological diagnoses and treatments and, therefore, result in increased death.Moreover, cases of invasive fungal infections due to strains with intrinsic or acquired resistance to available antifungals have been described. 8,14,15This resistance increases the need to make specialised diagnostic tools more widely available for better management of such infections.
Hence, as part of a continued effort from the European Confederation of Medical Mycology (ECMM), [16][17][18] this Review aims to describe the invasive fungal infection diagnostic capacity of Europe to better understand the current situation and the most pressing issues that need improvement.Similar studies have been performed before, although restricted to national experiences. 19,20To the best of our knowledge, this is the first time that the mycological diagnostic capability and access to antifungal treatments of institutions has been evaluated at a pan-European level.

Procedure
Data were collected via an online electronic case report form between November, 2021, and January, 2022.Before analysis, the answers from each participant were validated to ensure data coherence and completeness.The queries covered different categories; namely, institution profile, perceptions on invasive fungal infections in the respective institution, microscopy, culture and fungal identification, serology, antigendetection, molecular assays, and therapeutic drug monitoring.In most categories, participants had to reply dichotomously to whether or not the respective technique was available in their places of work.Participants could specify availability onsite or through an outsourced institution for serology, antigen-detection molecular and specifically of mucormycosis, could be answered with a Lickert scale, ranging from 1 (very low) to 5 (very high; appendix p 2).
Institutions in European sovereign states, de facto independent countries, and self-governing dependencies and regions were contacted by email and asked to participate. 21Mass emailing was targeted not only to close collaborators of the authors, but also members of scientific societies, such as the International Society of Human and Animal Mycology and the ECMM.Online scientific repositories (ClinicalTrials.gov,EU Clinical Trials Register, Google Scholar, PubMed, and ScienceDirect) and journals in mycology were screened for a larger list of potential participants.Additionally, online advertisements were launched in the social networks LinkedIn and Twitter.
Participating institutions were classified according to their country GDP per capita to analyse whether there were statistically significant differences between European countries in the availability of antifungals and diagnostic tests.Three cutoffs were established, dividing the continent in countries and regions with a GDP greater than $45 000, GDP between $30 000 and $45 000, and GDP less than $30 000, according to the International Monetary Fund for 2021. 13rthermore, participating institutions were assessed to place them in one of the ECMM excellence categories: blue, silver, gold, or diamond (appendix p 6). 22 The sole intention of this classification was to determine which accreditation levels these institutions could achieve in case of application.
Data are presented as frequencies and percentages.Proportions were laid out in contingency tables and compared with Fisher's Exact test (variables with at least one cell with expected value <5) and χ² test (variables with all cells with expected value >5), as appropriate.P values less than 0•05 were considered statistically significant.SPSS, version 27.0, was used for statistical analyses.

Discussion
Our Review evaluates the diagnostic and therapeutic capacity for the management of invasive fungal infections at a pan-European level.The study succeeded in collecting data from at least one institution from every European sovereign state with more than 100 000 inhabitants, except for Luxembourg.
When asked about the self-perception of most relevant fungal pathogens in their respective institutions, there was consensus among the participants.Candida spp was the most relevant fungal pathogen, followed by Aspergillus

Review
spp, Mucorales, Cryptococcus spp, and Fusarium spp.6][27][28][29][30][31][32][33][34][35][36] However, in other continents, there was major concern for Cryptococcus spp (Africa [55•0%] and Latin America and the Caribbean [67•0%]) 16,17 and Histoplasma spp (Africa [12•5%], Latin America and the Caribbean [48•0%], and Europe [4•1%]), 16,17 probably because of regional endemicity 37 or a larger number of uncontrolled HIV infections. 31lthough the ruling of Europeans under the same institutions that encourages integration to a common government (ie, European Union and the Council of Europe) has been promoted for years, 38 GDP per capita differs substantially between countries. 13In this survey, we describe how the availability of individual assays and thus, the invasive fungal infection management capacity correlates with GDP, limiting the compliance with available guidelines, and therefore affecting patient outcome. 27,29,30,32,33hen managing invasive fungal infection, access to appropriate diagnostics is a prerequisite for achieving favourable outcomes.Since 2018, WHO has developed and updated a list of essential in vitro diagnostics, although for invasive fungal infection this list is still insufficient. 39In this survey, regarding microscopy,

Review
which was generally available at most institutions (97%), we observed restrictions in the access to calcofluor white stain, which was more accessible in countries with a GDP of greater than $45 000, probably related to its high cost. 40This could be especially relevant for the diagnosis of aspergillosis or mucormycosis, for which this fluorescent dye is strongly recommended. 26,35[28][29][30][32][33][34][35][36] Most of the European institutions (99%) could process isolates, which is 10 percentage points more than Asian institutions (89•2%), 41 and 20% more compared with Latin American and Caribbean institutions (78•0%). 17Within Europe, we observed that the availability of tests for specific identification varied according to country GDP.This uneven distribution was especially relevant for access to MALDI-TOF MS.In regions with a GDP of less than $30 000, only 31% of the institutions had access to this technique, compared with 73% in countries with a GDP of between $30 000 and $45 000, and 89% when the GDP was greater than $45 000.Nevertheless, the availability of MALDI-TOF MS was reported to be much lower in African (17•5%), 16 Asian (12•3%), 41 and Latin American and Caribbean (20•0%) institutes. 17][43] The access to susceptibility testing is much higher in Europe (94%) as compared with surveys in Africa (62•5%), 16 Asia (58•9%), 41 or Latin America and the Caribbean (61•0%). 17Considering the increasing number of reports of cases of invasive fungal infection due to strains resistant to various antifungals, either intrinsically or acquired, and to the continuous discovery of new pathogenic fungal species, 8,14,15 this puts Europe in a much better situation in the fight against antifungal resistance compared with other continents.However, it is still not an ideal situation because antifungal susceptibility testing for yeast is more frequently available than for moulds and there are still several European institutions that do not perform tests routinely.
The WHO list of essential systemic antifungal drugs comprises amphotericin B deoxycholate and liposomal formulation, anidulafungin, caspofungin, fluconazole, flucytosine, itraconazole, micafungin, and voriconazole. 44here were statistically significant differences in the access to liposomal amphotericin B, itraconazole, voriconazole, and flucytosine.In all cases, there was a clear gradient between the availability of the drug and the GDP of the country of the respective institution.Limited availability also applied to other antifungals not included in the WHO list until 2021, such as all echinocandins, 45 or to the broad spectrum triazoles isavuconazole and posaconazole, and terbinafine, all of which are still not in the list.Isavuconazole and voriconazole are the recommended antifungals for invasive aspergillosis; 27,35 however, they were only available in 78% and 20% of institutions with a GDP of less than $30 000, respectively.Echinocandins, which are strongly recommended for the treatment of candidemia, 36 were available in only 72% of countries with the lowest GDP.Liposomal amphotericin B, isavuconazole, and posaconazole are the preferred options for mucormycosis; 26,29 however, in countries with a GDP of less than $30 000 these were available in 20-50% of the reporting institutions.These results show how access to important antifungals is associated with GDP.
Access to therapeutic drug monitoring also varied across Europe.Particularly for voriconazole, therapeutic drug monitoring is essential to provide an adequate antifungal dose and reduce drug-related adverse events. 27,29,30,32,33herapeutic drug monitoring availability was closely related to the GDP of the country in which each of the institutions was located.
This study has several limitations.First, there were no replies from the least populated countries and regions, which might be associated with several factors.It was more difficult to contact institutions from these countries and regions due to reduced research activity or a lack of international collaborations.Conversely, the lower number of inhabitants in these regions might suggest the scarcity of specialised health institutions within their borders or the automatic transfer of patients to other neighbouring countries with health-system agreements for severe diseases such as invasive fungal infections. 46,47he second limitation could be associated with the number of institutions per country, which might be closely related to the traditional collaboration partnerships in the research environment.Third, the data for this

Review
survey were collected during a pandemic, in which laboratory professionals, microbiologists, and infectious disease specialists might have had time restrictions to complete the survey.Fourth, the data from institutions with greater experience and capacity for invasive fungal infection diagnosis and treatment might not be extrapolated to non-major institutions.Last, further analysis of the specific problems of each of the countries is needed to better understand and make policies focused on specific needs.
Overall, we conclude that the general status of invasive fungal infection diagnostic capacity of Europe is at an acceptable level in many countries, but there are substantial differences based on GDP that need to be overcome so that every patient in Europe receives the best diagnostic and therapeutic management and, thus, the best possible outcome of invasive fungal infections.Contributors MH, J-PG, ES, and OAC contributed to study design.JS-G, MH, J-PG, ES, and OAC conceived the study idea.JS-G collected and validated the data, performed the statistical plan and analysis, and drafted the first version of the manuscript.All authors contributed to data interpretation, manuscript writing, and review of the manuscript.

Declaration of interests
JS-G received payments or honoraria for lectures, presentations, speakers' bureaus, manuscript writing, or educational events from Gilead and Pfizer, outside of the submitted work.MH received grants or contracts from Gilead, Pfizer, Astellas, Euroimmune, MSD, Pulmocide, Scynexis, and F2G, outside of the submitted work.J-PG received grants or contracts from Pfizer, and consulting fees from Gilead and Pfizer, outside of the submitted work.AA-I received payments or honoraria for lectures, presentations, speakers' bureaus, manuscript writing, or educational events from Gilead and Pfizer; received support for attending meetings and travel from Gilead; participated on a data safety monitoring board or advisory board for JPI-AMR; and had a leadership or fiduciary role in board, society, committee or advocacy groups that were either paid or unpaid, from WHO, European Society of Clinical Microbiology and Infectious Diseases, Fungal Infection Study Group, and Global Action For Fungal Infections, outside of the submitted work.KL received grants or contracts from Thermo Fisher Scientific and TECOmedical; consulting fees from Gilead, MSD, and MRM Health; and payment or honoraria for lectures, presentations, speakers' bureaus, manuscript writing, or educational events from Pfizer, Gilead, and

Search strategy and selection criteria
ClinicalTrials.gov,EU Clinical Trials Register, Google Scholar, PubMed, and ScienceDirect were searched for registrations and publications from Jan 1, 2015, to Jan 1, 2022, without language or trial or publication restrictions.A combination of terms such as "antifungal", "capacity", "case", "diagnosis", "diagnostic", "Europe", "fungal", "IFD", "IFI", "invasive fungal disease", "invasive fungal infection", "fungal disease", "fungal infection", "laboratory", "microbiology", "mycology", "mycoses", "patient", "report", and "[name of each of the European countries and territories]" was included in the search string.Average gross domestic product lists from the International Monetary Fund were used to distribute and classify the results of the participating institutions in three countries, enabling a comparison on the basis of economic situation for each country.
FUJIFILM Wako, outside of the submitted work.SAA reports grants or contracts from Cidara; payment or honoraria for lectures, presentations, speakers' bureaus, manuscript writing, or educational events from Gilead; and support for attending meetings and travel from Astellas, outside of the submitted work.VÖ received grants or contracts from International Health Management Associates and Sentry, outside of the submitted work.OAC received grants or contracts from Amplyx, Basilea, Bundesministerium für Bildung und Forschung, Cidara, Deutsches Zentrum für Infektionsforschung, EU Directorate-General for Research and Innovation (grant: 101037867), F2G, Gilead, Matinas, MedPace, MSD, Mundipharma, Octapharma, Pfizer, and Scynexis; consulting fees from AbbVie, Amplyx, Biocon, Biosys, Cidara, Da Volterra, Gilead, Matinas, MedPace, Menarini, Molecular Partners, Mycoses Study Group Education and Research Consortium (MSG-ERC), Noxxon, Octapharma, Pardes, PSI, Scynexis, and Seres; payment or honoraria for lectures, presentations, speakers' bureaus, manuscript writing, or educational events from Abbott, Al-Jazeera Pharmaceuticals, Astellas, Grupo Biotoscana/United Medical/Knight, Hikma, MedScape, MedUpdate, Merck-MSD, Mylan, and Pfizer; payment for expert testimony from Cidara; patents planned, issued, or pending from the German Patent and Trademark Office; participation on a data safety monitoring board or advisory board for Actelion, Allecra, Cidara, Entasis, IQVIA, Janssen, MedPace, Paratek, PSI, Pulmocide, and Shionogi; and other financial or non-financial interests from Deutsche Gesellschaft für Hämatologie und Medizinische Onkologie, Deutsche Gesellschaft für Information und Wissen, European Confederation of Medical Mycology, International Society for Human and Animal Mycology, MSG-ERC, and Wiley, outside of the submitted work.ES and AV declare no competing interests.

Figure 2 :
Figure 2: Histogram of the access to therapeutic drug monitoring in analysed European institutions Currency is US$.χ² test used to obtain p value.

Table 1 : Baseline characteristics of participating institutions in Europe Review
GDP=gross domestic product.ICU=intensive care unit.(Table2continues on next page) 23SI=Clinical and Laboratory Standards Institute.ELISA=enzyme-linked immunosorbent assay.EUCAST=European Committee on Antimicrobial Susceptibility Testing.GDP=gross domestic product.LAT=latex agglutination test.LFA=lateral flow assay.LFD=lateral flow device.MALDI-TOF MS=matrix-assisted laser desorption/ionisationtime-of-flight mass spectrometry.*ComparedwithFisher'sExact test.†Compared with χ² test.‡Aspergillus-specificLFD is a tool used in clinical microbiology to detect extracellular mannoprotein antigen secretion, which is only active when there is Aspergillus growing, by using the JF5 monoclonal antibody.23§Aspergillus-specificLFAis a tool capable of detecting galactomannan and has a shorter turnaround time as compared with ELISA.23