Fusarium Solani in a Case of Mixed Invasive Fungal Disease Due to Mucorales and Fusarium Solani

Bone Marrow Transplantation (2020) 55:873–876 https://doi.org/10.1038/s41409-020-0819-3

TECHNICAL REPORT

Positive quantitative PCR detecting Fusarium solani in a case of mixed invasive fungal disease due to Mucorales and Fusarium solani

1,2 1,2 3 1,2 4 1,2 Anne-Pauline Bellanger ● Stefﬁ Rocchi ● Ana Berceanu ● Emeline Scherer ● Fabrice Larosa ● Laurence Millon

Received: 3 September 2019 / Revised: 21 January 2020 / Accepted: 28 January 2020 / Published online: 5 February 2020 © Springer Nature Limited 2020

Abstract We present a case of invasive fungal co-infection in a young patient treated for an acute myeloid leukemia and having undergone a twice-haploid matched unrelated donor hematopoietic stem cell transplantation (HSCT) with two different donors. A mucormycosis diagnosis was made shortly after the patient’s admission using imagery and specific Mucorales qPCR which was treated with liposomal amphotericin B and posaconazole. Twenty days later, a blood culture was positive for Fusarium solani, and disseminated cutaneous lesions appeared. The antifungal treatment was changed to liposomal amphotericin B and voriconazole. Thanks to a complete hematological reconstitution and despite a co-infection with two aggressive filamentous opportunistic fungi, the patient recovered. We took advantage of this clinical case to test a specific Fusarium solani

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Introduction developed considerably over the last 10 years and has proved to be very contributive to identify mucormycosis Allogenic hematopoietic stem cell transplantation (HSCT) promptly and to detect mixed Aspergillus–Mucorales is the only curative option for several hematological infections [3]. malignancies but is associated with prolonged periods of Mucormycosis and fusariosis are both severe and neutropenia and risk of graft-versus-host disease, which is aggressive opportunistic mold infections that are associated treated by high dosages of steroids. Patients undergoing with high-mortality rates in immunocompromised patients allogenic HSCT are at the high risk of developing invasive [4–6]. The association of both infections in a patient is rare. fungal disease (IFD). For the past several years, our center The antifungal treatment for mucormycosis is based on has been applying a strategy of systematic IFD screening liposomal amphotericin B, often combined with posacona- combining a panel of fungal biomarkers: galactomannan zole [4, 5], while the management of invasive fusariosis (IF) (GM) antigen detection, Aspergillus mitochondrial, and is based on voriconazole [6, 7]. IF was reported as the ribosomal qPCR and Mucorales qPCR [1, 2]. This strategy second most common form of fungal disease, the frequency has proven useful for the early detection of invasive of which exceeds only that of IA, according to an Italian aspergillosis (IA) and mucormycosis [1, 2]. The use of registry [8]. molecular strategies to diagnose mucormycosis has The diagnosis of IF is often based on culture diagnostic tools with positive fongemia and positive cutaneous lesions [9]. Compared with other IFD, such as IA and mucormycosis, few nonculture diagnostic tools are avail- * Anne-Pauline Bellanger able for IF. Only the antigen GM assay, primarily used in [email protected] IA screening, has been shown to cross-react with Fusarium 1 Chrono-Environnement CNRS 6249 Research Team, Franche- species, and has even been proposed as a biomarker of Comté University, Besançon, France IF [10]. 2 Parasitology-Mycology Department, Besançon University We report here a case of mixed Mucormycosis and IF Hospital, Besançon, France infection in an immunocompromised patient, resulting in a 3 Hematology Department, Besançon University Hospital, favorable outcome. This clinical case provided the oppor- Besançon, France tunity to collect biological samples, which allowed us to test 4 Hematology Department, Dijon University Hospital, Dijon, France a speciﬁc qPCR targeting Fusarium solani subspecies. 874 A.-P. Bellanger et al.

L-AmB & posaconazole treatment Fig. 1 Evolution of events for Positive mucor / rhizopus qPCR D9-D55 L-AmB & voriconazole treatment the patient. Positive fusarium qPCR -D38 Isavuconazole treatment

D0 D8 D9 D30 D34 D38 D40 D55 D58 D65 D135

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Cq 41 Complete hematological qPCR fusarium reconstitution Positive blood culture F. solani gram staining Cutaneous lesions Biopsy of cutaneous lesions Chest CT D8 positive direct examination

Case report testing using the European Committee on Antibiotic Sus- ceptibility Testing (EUCAST) methods. The following A 16-year-old boy of northeastern France was diagnosed MICs were obtained: amphotericin B ≥ 4 mg/L (resistant), with acute myeloid leukemia (AML). He received a first itraconazole ≥ 8 mg/L (resistant), isavuconazole ≥ 4 mg/L allogenic HSCT 1 year after the AML diagnosis. A secind (resistant), voriconazole ≥ 8 mg/L (resistant), posacona- allogenic HSCT was performed 3 years after the initial zole ≥ 8 mg/L (resistant), caspofungine ≥ 4 mg/L (resistant), AML diagnosis. Four years after the second allo-HSCT, the and micafungine ≥ 4 mg/L (resistant). patient was 23 years old and a truck driver. He presented a recurrence of his AML and was hospitalized with fever, hypoxia, pancytopenia, and thoracic pain (D0). The chest- Fusarium solani qPCR computed tomography showed a large pulmonary lesion covering both the upper and median lobes (D8) (Fig. 1). The different sets of primers and probes targeting Fusarium Systematic fungal surveillance testing on sera showed solani, designed by EPA were used as a Fusarium solani repeated positive in-house Mucor/Rhizopus qPCRs (from specific qPCR assay [11]. Primers and probes designed by D9 to D55 with Cq from 32 to 38 Fig. 1). From D9, based EPA consist of four couples of primers and one hydrolysis on the imagery and the positive Mucor/Rhizopus qPCRs, probe all targeting F. solani but different subspecies the patient received liposomal amphotericin B (5 mg/kg) (batatas, cucurbitae, mori, xanthocyli, pisi, and robiniae) combined with oral posaconazole (300 mg/day). In parallel, [11]. The qPCR was performed in duplicate in a 20 µL final the induction treatment of the AML recurrence began on volume (10 µL of master mix Gene Expression from D21. On D30, the patient experienced fever again and one Applied, 5 µL DNA, 1000 nM primers, 500 nM probe, and blood culture was positive for Fusarium solani. From D31, DNA-free water). Reactions were run using the Applied the antifungal treatment was changed to liposomal ampho- Biosystems 7500 Fast (Life Technologies Carlsbad, tericin B (5 mg/kg) combined with oral voriconazole (400 CA, USA) as follows: 3 min at 95° C and 45 cycles (15 s at mg/day). On D34, disseminated cutaneous lesions appeared 95° C and 1 min at 60° C). The specificity of the qPCR (Fig. 1). Biopsies performed on D35 were also positive for method was verified using 50 strains of microorganisms F. solani (direct examination and culture). From D0, the (mold, yeast, and bacteria). DNA extract of four sera sam- patient was screened weekly for fungal biomarkers includ- ples from the patient in this case study were retrospectively ing the GM antigen assay, which remained negative analyzed by qPCR (D27, D31, D38, and D43). Quantitative throughout the entire period of hospitalization. On D58, the results were expressed using the quantification cycle (Cq) patient had a complete hematological reconstitution. The that marked the cycle at which fluorescence of the evolution of the patient was very favorable then, and, on sample became significantly different from the baseline D65, he was authorized to return home with isavuconazole signal. The sample collected on D38 was the only positive (200 mg/day). The French National Reference Center for one with the Fusarium solani, specific qPCR with a Cq of Invasive Mycoses and Antifungals (Paris, France) con- 41 (both duplicates were positive). In a timely manner, the firmed the identification of F. solani and performed MIC positive sample (D38) occurred between the positive blood Positive quantitative PCR detecting Fusarium solani in a case of mixed invasive fungal disease due to. . . 875 culture (D30) and before the biopsy of the cutaneous species are common occurrences and were even proposed as lesions (D40). a possible biomarker of IF [9, 10].

Discussion Conclusion

In the present clinical case, the patient was diagnosed with a The Fusarium solani qPCR presented here needs to be mixed Mucorales–Fusarium solani infection that evolved tested on a larger cohort to specify its timing with regard to favorably. This rare association of aggressive mold infec- the development of the infection, but it could be a pro- tions gave us the opportunity to collect biological samples mising tool to screen for IFD in severe immunocompro- and to first verify that DNA from Fusarium solani sub- mised patients, along with Aspergillus and species was not detected by the Mucorales qPCR, which Mucorales qPCRs. was positive from D9 to D55 (while a proven IF was diagnosed by culture tools from blood culture and a cuta- Acknowledgements We thank Pamela Albert for her editorial assis- neous lesion biopsy). tance. We thank Dr. Olivier Lortholary for his advice on antifungal therapeutic management regarding the MIC result obtained from the Secondly, these biological samples were tested using a National Reference Center (Paris, France). Fusarium solani qPCR, and one sample (D38) out of the four tested (D27, D31, D38, and D43) was positive with a Compliance with ethical standards high Cq (41), corresponding to a low fungal load. The positive sample tested positive after the positive blood Conflict of interest The authors declare that they have no conflict of culture (D30) and before the appearance of the cutaneous interest. lesions (D34). Of course, this Fusarium solani qPCR must Publisher’s note Springer Nature remains neutral with regard to be tested further, ideally on a large cohort of biological jurisdictional claims in published maps and institutional affiliations. samples positive for IF, to better understand the kinetics of DNA release during the infection. We may hypothesize that in the present case the concomitant antifungal treatment References with liposomal amphotericin B may have limited the per- formance of the assay. 1. Bellanger AP, Millon L, Berceanu A, Grenouillet F, Grenouillet IF is usually associated with a high-fatality rate, espe- FE, Larosa F, et al. Combining Aspergillus mitochondrial and cially if combined with another IFD such as mucormycosis. ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients. Med In the present case, the patient luckily responded well to the Mycol. 2015;53:760–4. induction treatment initiated at D21, despite the post- 2. Millon L, Herbrecht R, Grenouillet F, Morio F, Alanio A, transplantation relapse. The patient benefited from effective Letscher-Bru V. French Mycosis Study Group et al. 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