Adrien Caillet

Anne-Pauline Bellanger
email: apbellanger@chu-besancon.fr

Jean Christophe Navellou

Etienne Daguindau

Steffi Rocchi

Emeline Scherer

Ana Berceanu

Laurence Millon

Refractory invasive pulmonary aspergillosis due to Aspergillus flavus detected with the combination of two in-house Aspergillus qPCR

Keywords: Aspergillus flavus, invasive pulmonary aspergillosis, Aspergillus qPCR, galactomannan antigen, amphotericin B

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Invasive aspergillosis (IA) remains a clinically challenging opportunistic invasive fungal disease (IFD) in heavily immunocompromised patients. Aspergillus fumigatus is in cause of approximately 80% of IA; A. flavus being the second most frequent pathogenic species (approximately 15-20%) [START_REF] Krishnan | Aspergillus flavus : an emerging nonfumigatus Aspergillus species of significance[END_REF]. Allogenic hematopoietic stem cell transplantation (HSCT) is the only curative option for several hematological malignancies but is associated with prolonged periods of neutropenia. More than 1660 cases of IA were reported to the French National Referent Center of Invasive Fungal Disease from 2012 to 2018 [START_REF] Bretagne | Active Surveillance Program to Increase Awareness on Invasive Fungal Diseases: the French RESSIF Network (2012 to 2018)[END_REF]. The global mortality associated with IA remains high in France: 42.5% at 3 months reported by the French National Referent Center of Invasive Fungal Disease [START_REF] Bretagne | Active Surveillance Program to Increase Awareness on Invasive Fungal Diseases: the French RESSIF Network (2012 to 2018)[END_REF]. The early diagnosis of IA and the prompt initiation of antifungal therapy have been shown to reduce mortality in patients with IA [START_REF] Ullmann | Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline[END_REF].

For the past several years, our center has been applying a strategy of systematic IA screening combining the detection of galactomannan antigen (GM) detection and two in-house Aspergillus qPCRs [START_REF] Bellanger | Combining Aspergillus mitochondrial and ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients[END_REF][START_REF] Millon | Ribosomal and mitochondrial DNA target for real-time PCR diagnosis of invasive aspergillosis[END_REF]. The strategical choice of combining a mitochondrial and a ribosomal Aspergillus qPCR aimed at optimizing information on the Aspergillus species involved, while using a minimum quantity of DNA. Thus, instead of duplicating the same assay, we made the choice to perform two different complementary qPCRs [START_REF] Bellanger | Combining Aspergillus mitochondrial and ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients[END_REF][START_REF] Millon | Ribosomal and mitochondrial DNA target for real-time PCR diagnosis of invasive aspergillosis[END_REF][START_REF] Bretagne | Detection of Aspergillus Species DNA in bronchoalveolar lavage samples by competitive PCR[END_REF]. The ribosomal Aspergillus qPCR targets a 18S area specific of Aspergillus fumigatus, which is a very repeated target (estimated from 38 to 91 copies per genome [START_REF] Herrera | Strain-dependent variation of 18S ribosomal DNA copy number in Aspergillus fumigatus[END_REF]), thus, this qPCR is very sensitive. On the flipside, the mitochondrial Aspergillus qPCR targets a less repeated area (estimated at between 9 and 10 mitochondrial copies per single-copy gene [START_REF] Costa | Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum[END_REF]), but the targeted area is common to other Aspergillus thermophilic species (A. flavus, A. terreus, A. nidulans and A. niger), thus this qPCR is less sensitive but allow the detection of other thermophilic Aspergillus species. This strategy has proven useful for early detection of IA [START_REF] Bellanger | Combining Aspergillus mitochondrial and ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients[END_REF][START_REF] Millon | Ribosomal and mitochondrial DNA target for real-time PCR diagnosis of invasive aspergillosis[END_REF][START_REF] Bretagne | Detection of Aspergillus Species DNA in bronchoalveolar lavage samples by competitive PCR[END_REF].

Case report

A 58-year-old female patient was diagnosed with acute myeloid leukemia (AML) with complex karyotype and received an allogenic HSCT (D0) five months after the AML diagnosis. The last myelogram, after induction chemotherapy with idarubicin and aracytin, salvage and consolidation chemotherapy with amsacrine and etoposide, showed 6% of myeloblasts and the thoracic computed tomography (CT) scan before the allogenic HSCT was normal. The antifungal prevention was fluconazole from the day the conditioning regimen was begun.

On D14 the patient presented fever and thoracic pain. On D15, the chest CT showed lobar condensations and the galactomannan antigen (GM) was positive in serum (index 3.3). Both in-house Aspergillus qPCRs were negative. An antifungal treatment by isavuconazole was initiated, with 200 mg every 8 h during 2 days and then 200 mg per day (Figure 1). On D18, GM was still positive and the mitochondrial Aspergillus qPCR (mqPCR) turned positive (GM >3.5, Cycle quantification (Cq) 34; Figure 1). A broncholaveolar lavage fluid (BALF) was performed on D18: direct examination was negative, the fungal culture remained sterile, the Aspergillus qPCRs were negative and the galactomannane was positive (>3.5). The patient didn't have any fever at that time. The patient needed oxygen therapy from D20. Two tracheal aspiration were send to the laboratory for fungal culture on D22 and D24 but the cultures remained sterile.

On D24, the antifungal treatment was changed for liposomal amphotericin B (3 mg/kg/day) because of fever recurrence and worsening of the lesions on chest CT: GM and mqPCR were still positives in serum (GM >3.5, Cq 36; Figure 1). On D35 the patient was still in aplasia and was transferred in Intensive Care Unit (ICU) for acute respiratory symptoms.

Unfortunately, the patient was refractory to allogenic HSCT and relapsed from the leukemia. She died on D40.

Aspergillus flavus qPCR

DNA extraction from 1 mL of serum (D21) was performed using a Large Volume MagNa Pure Nucleic Acid Isolation Kit on a MagNa Pure Compact apparatus (Roche Diagnostics, Meylan, France).

The different sets of primers and probe targeting A. flavus designed by Environnemental Protection Agency (EPA) of Unites States (available on https://irpcdn.multiscreensite.com/c4e267ab/files/uploaded/gCQnkBNWQuSD96fPIikY_EPA_Technol ogy%20for%20Mold%20Identification%20and%20Enumeration.pdf) were used as a specific qPCR assay [START_REF]EPA Technology for mold identification an enumeration[END_REF]. The qPCR was performed in duplicate in a 20 µL final volume (10 µL of master mix Gene Expression from Applied, 5 µL DNA, 1000 nM primers, 500 nM probe and DNA-free water). Reactions were run using the Applied Biosystems 7500 Fast (Life Technologies Carlsbad, CA, USA) as follows: 3 min at 95°C and 45 cycles (15s at 95°C and 1 min at 60°C). The specificity of the qPCR method was verified using 30 strains of microorganisms.

Quantitative results were expressed using the quantification cycle (Cq) that marked the cycle at which fluorescence of the sample became significantly different from the baseline signal.

The sample collected on D21 was positive with a Cq of 36 (both duplicates were positive). before switching by amphotericin B. The persistence of positive galactomannan antigen and of positive Aspergillus qPCR in serum were poor prognostic signs and argued in favor of a refractory disease [START_REF] Slavin | When to change treatment of acute invasive aspergillosis: an expert viewpoint[END_REF].

Conclusion

This case report illustrate the interest of combining two in-house Aspergillus qPCRs. The mitochondrial target, while less sensitive, allowed suspecting the involvement of another Aspergillus opportunistic species than A. fumigatus. Non fumigatus species, such as A. terreus and A. flavus, are associated with particular susceptibility profiles (low sensitivity to amphotericin B). Current diagnostic strategies focusing specifically on A. fumigatus present the risk to under consider non fumigatus species, which could lead to non-optimal care.

We report here a case of probable invasive pulmonary aspergillosis due to Aspergillus flavus that was diagnosed thanks to our local strategy of combining two in-house Aspergillus qPCRs.

Discussion

In the present clinical case, the patient was diagnosed with a probable IA due to A. flavus that evolved unfavorably. The probable IA diagnosis was based on host factors, chest CT and positive biomarkers in serum (galactomannan antigen and mitochondrial in-house Aspergillus qPCR). Positive Aspergillus qPCR in serum has been included to the microbial criteria allowing the EORTC/MSG classification of IA as probable since 2020 [START_REF] Donnelly | Revision and update of the consensus definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium[END_REF].

We showed previously that the combination of mitochondrial and ribosomal targets increased the sensitivity of qPCR for the diagnosis of invasive aspergillsosis [START_REF] Bellanger | Combining Aspergillus mitochondrial and ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients[END_REF][START_REF] Millon | Ribosomal and mitochondrial DNA target for real-time PCR diagnosis of invasive aspergillosis[END_REF].

The discordance between the negativity of the in-house ribosomal Aspergillus qPCR , specific to A. fumigatus, and the positivity of the in-house mitochondrial Aspergillus qPCR (targeting A. fumigatus and other Aspergillus species) allowed us to suspect that another thermophilic Aspergillus species than A. fumigatus was involved. The involvement of A. flavus was confirmed using a specific A. flavus qPCR. Unfortunately, as no strain was obtained in culture, antifungal susceptibility could not be tested.

Aspergillus flavus is the second most common opportunistic pathogen isolated in case of invasive aspergillosis [START_REF] Taghizadeh-Armaki | Genetic Diversity and In Vitro Antifungal Susceptibility of 200 Clinical and Environmental Aspergillus flavus Isolates[END_REF]. Experimental data suggest that A. flavus may be more virulent than A. fumigatus [START_REF] Ramírez-Camejo | An opportunistic human pathogen on the fly: strains of Aspergillus flavus vary in virulence in Drosophila melanogaster[END_REF]. Several studies investigated the susceptibility to antifungals for A. flavus strains and reported that the A. flavus strains tested in their work displayed reduced susceptibility to amphotericin B, with MIC values ≥2 mg/L [START_REF] Taghizadeh-Armaki | Genetic Diversity and In Vitro Antifungal Susceptibility of 200 Clinical and Environmental Aspergillus flavus Isolates[END_REF][START_REF] Rivero-Menendez | Molecular Identification, Antifungal Susceptibility Testing, and Mechanisms of Azole Resistance in Aspergillus Species Received within a Surveillance Program on Antifungal Resistance in Spain[END_REF][START_REF] Hadrich | Amphotericin B in vitro resistance is associated with fatal Aspergillus flavus infection[END_REF][START_REF] Reichert-Lima | Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates[END_REF][START_REF] Wang | Epidemiology, Drug Susceptibility, and Clinical Risk Factors in Patients With Invasive Aspergillosis[END_REF][START_REF] Slavin | When to change treatment of acute invasive aspergillosis: an expert viewpoint[END_REF]; higher MIC values to isavuconazole for A. flavus strains were also reported [START_REF] Rivero-Menendez | Molecular Identification, Antifungal Susceptibility Testing, and Mechanisms of Azole Resistance in Aspergillus Species Received within a Surveillance Program on Antifungal Resistance in Spain[END_REF].

The patient showed a lack of response while treated by either isavuconazole or amphotericin B: the fungal biomarkers remained positive and aggravation of the lesions was observed by imagery. A recent expert viewpoint recommend to wait until after ≥ 8 or ≥15 days of therapy before changing the antifungal therapy, even if rising galactomannan antigen or radiological increase in size were observed [START_REF] Slavin | When to change treatment of acute invasive aspergillosis: an expert viewpoint[END_REF]. In our case, the patient received 9 days of isavuconazole