Mucorales DNA detection in serum specimens for early diagnosis of mucormycosis

Journal Pre-proof Mucorales DNA detection in serum specimens for early diagnosis of mucormycosis

Claire Hoffmann, Gaëlle Guillerm, Patrice Le Pape, Liana Carausu, Rose-Anne Lavergne, Gilles Nevez, Solène Le Gal PII:

S0732-8893(19)31055-7

DOI:

https://doi.org/10.1016/j.diagmicrobio.2020.115004

Reference:

DMB 115004

To appear in:

Diagnostic Microbiology & Infectious Disease

Received date:

23 October 2019

Revised date:

13 January 2020

Accepted date:

26 January 2020

Please cite this article as: C. Hoffmann, G. Guillerm, P. Le Pape, et al., Mucorales DNA detection in serum specimens for early diagnosis of mucormycosis, Diagnostic Microbiology & Infectious Disease(2020), https://doi.org/10.1016/ j.diagmicrobio.2020.115004

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© 2020 Published by Elsevier.

Journal Pre-proof Mucorales DNA detection in serum specimens for early diagnosis of mucormycosis

Claire Hoffmann

a,b

, Gaëlle Guillerm c, Patrice Le Pape

d,e

, Liana Carausu c, Rose-Anne

Lavergne d,e, Gilles Nevez a,b, Solène Le Gal a,b

a

Laboratoire de Parasitologie et Mycologie, Hôpital de La Cavale Blanche, CHU de Brest,

Brest, France Groupe d’Etude des Interactions Hôte-Pathogène (GEIHP; EA 3142), Université de

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b

Bretagne-Occidentale-Université d’Angers, Brest, France

Service d’Hématologie Clinique, Hôpital Morvan, CHU de Brest, Brest, France

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Laboratoire de Parasitologie-Mycologie, Institut de Biologie, CHU de Nantes, France

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Département de Parasitologie et Mycologie Médicale, Université de Nantes, Nantes

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Corresponding author

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Atlantique Universités, EA1155-IICiMed, Faculté de Pharmacie, Nantes, France

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Solène Le Gal, Laboratoire de Parasitologie et Mycologie, Hôpital de La Cavale Blanche, CHU de Brest, boulevard Tanguy Prigent, 29609, Brest, France. Tel: +33 (0)2 98 14 51 02, Fax: +33(0)2 98 14 51 49, e-mail: [email protected] ; [email protected]

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Journal Pre-proof Abstract We report a case of pulmonary mucormycosis in a patient with T-cell acute lymphoblastic leukemia. The diagnosis of mucormycosis was initially based on mycological examination of a pulmonary specimen. However, we describe how it could have been made two months earlier using qPCR assays targeting Mucorales species on serum specimens.

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Keywords

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Mucorales, mucormycosis, qPCR

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Journal Pre-proof A 17-year-old woman presented with asthenia, left basi-thoracic pain and dyspnea (day 0 (d0)). Clinical examination revealed hepatosplenomegaly and left supraclavicular adenopathy. A chest-X-ray and a thoracic Positron-Emission tomography (PET)/computed tomography (CT) scan showed a voluminous mediastinal mass of 12.7x14x14.5 cm associated with pleural and pericardial effusions. She had leukocytosis (26x103/µL) with blasts (1.3x103/µL) and elevated LDH (542 UI/L). Bone marrow aspiration and cerebrospinal fluid examinations were compatible with diagnosis of T-cell acute lymphoblastic leukemia with central nervous

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system involvement. Induction chemotherapy (protocol CAALL-F01group T-HR CNS3) was started on d3. The patient was placed in a protective environment room during the cycles of

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chemotherapy for the prevention of infections caused by environmental fungi.

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On d121, she developed Escherichia coli bacteremia contemporary to febrile neutropenia. A thoracic CT scan revealed bilateral pleural effusion and consolidation surrounded with ground

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glass opacities in the right lower lobe and the upper lingula compatible with bilobar

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pneumonia. She remained febrile despite antibiotic treatment combining imipenem and vancomycin. Iterative serum galactomannan detections were negative. Nonetheless,

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considering possible fungal infection, liposomal amphotericin B (3 mg/kg per day) was

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started on d126 and continued for 3 weeks. The patient improved clinically contemporary to neutropenia resolution on d129 (Figure 1). On d173, leukemia remission was assumed considering the results of bone marrow aspiration examination. On d188, the thoracic CT scan which was performed as part of pre-stem cell transplant workup, revealed opacity persistence in the lingula compatible with aspergillosis. Voriconazole was started (200 mg twice a day) and lingula resection was performed on d195. The lung specimen was sent to the mycology laboratory for direct examination and culture. Microscopic examination using calcofluor-white stain showed non-septate fungal hyphae. Cultures on Sabouraud agar at 27°C and 35°C remained negative after 3-week incubation

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Journal Pre-proof whereas culture on liquid Sabouraud medium led to mycelium growth but without sporulation. Sub-culture on Sabouraud agar medium was unsuccessful. Nonetheless, PCR amplification and sequencing targeting the ITSs and the D1/D2 region of the 28S ribosomal DNA (Garcia-Hermoso et al., 2009) allowed Rhizopus microsporus identification. Considering that this fungus is resistant to voriconazole, this antifungal was switched to amphotericin B on d210. Allogeneic stem cell transplant was performed on d219. On d255, amphotericin B treatment was switched to isavuconazole because of renal toxicity. No relapse

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of mucormycosis was observed during the following 10 months.

Considering the diagnosis of pulmonary mucormycosis due to Rhizopus microsporus, we

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retrospectively performed qPCR assays targeting Mucorales species, as previously described

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(Millon et al., 2016), on 10 archival serum specimens that were collected from d125 through d210. Interestingly, the Mucor/Rhizopus qPCR assay was positive on 3 out of the 10

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specimens, specifically those collected on d125, d127, and d131, the period during which the

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patient presented with febrile neutropenia and pneumonia (Figure 1).

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Mucormycosis is an invasive fungal infection (IFI) caused by filamentous fungi belonging to

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the Mucorales order. Common underlying conditions are haematological malignancies (HM), diabetes mellitus, and trauma. In France, mucormycosis is the second most frequent IFI due to filamentous fungi after invasive aspergillosis (IA), with an overall incidence of 0.09/100,000 per year versus 1.4 for IA (Bitar et al., 2014). According to the RetroZygo study, the mortality rate of mucormycosis in patients with HM is 60% (Lanternier et al., 2012). For these reasons, an early diagnosis is required. In this case-report, considering the results of the MucorRhizopus qPCR assay, which was retrospectively performed, the specific diagnosis was delayed at least two months, i.e. from d125 the date of first positive serum, to d195, the date of lingula resection. This delay was due to the difficulties to obtain earlier pulmonary

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Journal Pre-proof specimens for mycological examination and the absence of prescription of Mucorales DNA detection in serum specimens. CT scan initially showed bilateral pleural effusion and focal alveolar condensations surrounded with ground glass opacities compatible with halo sign. These observations were compatible with IA diagnosis whereas the reversed halo sign, compatible with mucormycosis was not clearly observed (Georgiadou et al., 2011). Nonetheless, these signs are not specific and their interpretation is difficult (Georgiadou et al., 2011).

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Biological diagnosis of mucormycosis is based on direct microscopic examination showing non-septate, irregular, ribbon-like hyphae with angles of branching 45-90° (Guarner and

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Brandt, 2011), combined with mycological culture of clinical specimens. Usually, Mucorales

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can rapidly and extensively grow on standard mycological media. Nonetheless, cultures can be delayed or negative (Cornely et al., 2014). In our case, the fungus grew but, in the absence

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of sporulation, its microscopic identification failed. In this context, genus or species

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identification relies on DNA sequence analysis according to relevant genomic targets as

2006).

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described elsewhere (Garcia-Hermoso et al., 2009 ; Cornely et al., 2014 ; Schwarz et al.,

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Considering Mucorales cell-wall components, ß-(1,3)-D glucan detection in serum and galactomannan detection in serum or bronchoalveolar lavage specimens for mucormycosis diagnosis is not useful. However, if thoracic CT scan is compatible with IFI and galactomannan detection remains negative, mucormycosis must be highly suspected (Cornely et al., 2014). Our case-report may provide data favoring this approach. Mucorales DNA detection in serum using specific PCR assays is an efficient biomarker for mucormycosis diagnosis. Indeed, Millon and colleagues have developed qPCR assays based on 18S rDNA sequence analysis allowing detection of Mucor/Rhizopus, Rhizomucor and Lichtheimia (Millon et al., 2013, 2016). These assays have a sensitivity rate from 81 to 92%

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Journal Pre-proof (Millon et al., 2013, 2016). Springer and colleagues have also developed a qPCR assay targeting 18S rDNA but requiring an additional step of DNA sequencing, which is time consuming (Springer et al., 2016). This assay has a sensitivity of 100% in patients with probable/proven invasive mucormycosis (Springer et al., 2016). We performed Millon and colleagues’ PCR assays for retrospective DNA detection in serum specimens. Beyond their sensitivity, these qPCR assays permit an earlier diagnosis of invasive mucormycosis, from 0 to 68 days before mycological diagnosis (Millon et al., 2013, 2016 ; Legrand et al., 2016), and

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from 0 to 24 days before the first CT scan signs (Millon et al., 2016 ; Caillot et al., 2016). In our case, the qPCR assays should have permit mucormycosis diagnosis two months before the

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mycological diagnosis if they had been prospectively performed.

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The results of qPCR assays may also be a prognostic marker. The survival rate over 84 days was significantly higher for patients with an initially positive PCR that became negative after

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antifungal treatment than in patients with persistent positive results of Mucorales DNA

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detection (48% vs. 4%, p<10-6) (Millon et al., 2016). It is noteworthy that our patient’s clinical outcome was improvement when Mucorales DNA detection in serum specimens

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turned negative (from d134 to d210) (Figure 1).

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To sum up, this case report brings additional strong arguments favoring Mucorales DNA detection in serum as an efficient biomarker for early diagnosis of mucormycosis in neutropenic patients with leukemia. This biomarker should be included in flow diagrams for diagnosis and treatment of mucormycosis.

Conflict of interests The authors do not have any conflicts of interest to disclose.

Acknowledgements

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The authors thank Professor Laurence Millon for helpful critical review of the manuscript.

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Journal Pre-proof References

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Millon L, Herbrecht R, Grenouillet F, et al. Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF). Clin Microbiol Infect 2016;22(9):810.e1-810.e8. Schwarz P, Bretagne S, Gantier J-C, et al. Molecular Identification of Zygomycetes from Culture and Experimentally Infected Tissues. J Clin Microbiol 2006;44(2):340-9 Springer J, Lackner M, Ensinger C, et al. Clinical evaluation of a Mucorales-specific real-

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Journal Pre-proof Figure 1. Results of Mucor/Rhizopus DNA detection using PCR assays on 10 iterative serum

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specimens and their relationship with neutrophil cell counts in blood.

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Figure 1