A case of Magnusiomyces capitatus isolated during monitoring in an antimicrobial diagnostic stewardship context.

Magnusiomyces capitatus (M. capitatus) is an emerging opportunistic yeast in the Mediterranean region typically isolated from immunocompromised patients, usually affected by blood malignancies. We reported a rare case of M. capitatus infection, isolated from a drainage fluid in a patient affected by lung cancer recovered in the University Hospital of Campania “Luigi Vanvitelli”, Naples, Italy. The isolate was identified by phenotypic methods, i.e., Gram and Lactophenol cotton blue (LCB) staining, and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis. We identified M. capitatus on the third day from Sabouraud Dextrose Agar supplemented with chloramphenicol and gentamicin. Antifungal susceptibility test revealed that 5-fluorocytosine was the most active drug against M. capitatus, followed by itraconazole and voriconazole, micafungin, amphotericin B and fluconazole, posaconazole, anidulafungin, and caspofungin. Our data showed the importance of an early cultural and fast microbiology diagnosis based on the characteristic morphologic features observed in Gram-stained smears of blood culture positive bottles, and the validation via MALDI-TOF MS. This dual approach has significant impact in the clinical management of infectious diseases and antibiotic stewardship, by integrating sample processing, fluid handling, and detection for rapid bacterial diagnosis.


Introduction
In recent years, fungal infections represent a huge and emergent worldwide public health problem with high financial costs for diagnosis, treatment, and prevention [1,2].The severity of the disease varies from bland or asymptomatic skin to appendages or lethal systemic infections [3].Numerous cases are not correctly diagnosed, and turnaround time of conventional diagnostic methods limit the detection of superficial fungal infections probably underestimated.In addition, changes of pathogen distribution, demographics, and antimicrobial resistance in according with antimicrobial diagnostic stewardship program, may affect the epidemiology.The development and optimization of rapid diagnostic approaches for the identification of bacteria represents one of the priorities not only to combat antimicrobial resistance, but also to lead a more accurate detection of pathogens that typically occur at low concentrations in biological samples or are uncommon.Therefore, an accurate etiological diagnosis that combines phenotypic methods, including Gram staining, culture and biochemical test, to molecular analyses, is important both for the prescription of the correct therapeutic treatment [4,5] and for prevention programs [6].
Here we report a case of isolation of M. capitatum in an immunocompromised patient with a solid cancer and resection of the upper pulmonary lobe recovered in the University Hospital of Campania "Luigi Vanvitelli", Naples, Italy.
We describe the important role of antimicrobial diagnostic stewardship program used in our hospital which can support correct treatment strategies.

Case presentation
An 80-year-old male with an anamnesis of juvenile exanthematous diseases, hypertension (in treatment), bronchopneumopathie chronique obstructive (BPCO) and hyperthyroidism (in treatment), hematemesis episodes treated with transfusions, and a malignant tumor which led to the segmental resection of the upper pulmonary lobe in 2019, was successively subjected to duodenocephalopancreasectomy.His postoperative course was complicated by fever (38,5 • C), sinking of the pancreatic stump and anastomosis of gastrojejunum and hepatojejunum.Therefore, he has been hospitalized a month later in July 2022 showing a duodenal heteroplasia in gastroscopy.The patient was subjected on screening for Hepatitis B surface antigen (HbsAg) and Hepatitis C virus antibody (HCVAb) on the first day of hospitalization and both showed negative results.However, capillary serum protein electrophoresis reported increased values of alpha-1, alpha-2 and gamma-globulins, and also decreased levels of albumin, albumin/globulin ratio (A/G) and total proteins.On the contrary, beta-1 and beta-2 globulins showed normal parameters.A month later the patient showed symptoms of infection and drainage was performed, and a part of the drainage liquid was collected on day 28 of hospitalization.
It was rapidly received in our Complex Operative Unit (UOC) of Virology and Microbiology, where it was seeded both on agar plates and in Brain Heart Infusion (BHI) broth, then incubated at 37 • C overnight.On the first day, Escherichia coli (E.coli) colonies grew up, while M. capitatus appeared on the third day in Sabouraud Dextrose Agar supplemented with chloramphenicol and gentamicin (Sabouraud CAF GEN) (Fig. 1).In detail, three different sets, each consisting of two flasks collected in three different days, were performed; all were positive for E. coli, and two out of the three were positive for M. capitatus.
In addition, Gram staining from positive drainage cultures showed typical arthroconidial forms with purple color (Fig. 2 A).Then, a Lactophenol cotton blue (LCB) staining was performed identifying the presence of fungi with characteristic filamentous form (Fig. 2B).
Microorganisms identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) by using Bruker Microflex instrument, Biotyper software (Version 3.0), and database (Version 3.1.66;Bruker Daltonics).The identification species-related data generated by MALDI-TOF MS were classified following the manufacturer's instructions.The score values ≥ 2.0, 1.7-1.99,and < 1.7, indicated species identification, genus level detection, and Non-Reliable Identification (NRI), respectively.The score provided by MALDI-TOF in both isolations, and repeated up to 4 times, was > 2, thus confirming the species as M. capitatus (>99% confidence rate).Spectra were baseline corrected and normalized to total positive ion current [10].Antibiogram was performed in automatized manner showing an Extended-Spectrum Beta-Lactamases (ESBL) producing E. coli sensible for piperacillin-tazobactam and cephalosporins with Beta-Lactamases inhibitors and the patient was treated for thisinfection.However, no treatment was decided to be applied for the M. capitatus infection.As reported in Fig. 3, in vitro antifungal susceptibility of M. capitatus was tested by Sensititre microdiluition (Thermo Fisher Scientific) and in Table 1 the relative minimal inhibitory concentration (MIC) values were indicated.
5-fluorocytosine, fluconazole, itraconazole, posaconazole, voriconazole, caspofungin, amphotericin B, anidulafungin, and micafungin, were used according to the US Clinical and Laboratory Standards Institute (CLSI M27-A4, 2008), with Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 used as quality control strains.Aliquots of 100 μl of the diluted sample and the positive controls were inoculated into the wells with antifungals.The plate was incubated at 28 • C until a change in color from blue (indicative of no growth) to red (indicative of growth).When a purple color remained during a change from red to blue (indicative of partial growth inhibition), the MIC was defined as the lowest drug concentration which resulted in a purple color.

Discussion
Currently, blood culture represents the gold standard for diagnosis of fungemia or candidemia.It has the advantage of isolating the etiologic agent at species level and performing susceptibility testing.Nevertheless, blood culture is characterized by a long turnaround time since on average, it takes around 24-48 h from positive blood culture to the identification of the species.
M. capitatus is an ascomycetous yeast known to cause life-threatening invasive infections in immunocompromised patients, particularly in the case of hemato-oncological malignancies [11][12][13] In detail, infections with these species are associated with a high mortality rate ranged from 40 to 80% [14,15].In recent years, the numbers of published cases of infections with Magnesiomyces clavatus (M.clavatus) and M. capitatus have increased, and both species are considered as emerging pathogens [16,17].
Evaluating the patient's outcome, the isolation of M. capitatus from the drainage liquid was incidental more than suggestive for a coinfection, however considering the sample from which it has been isolated and the relative rarity of the strain itself, we considered significant to highlight this isolation considering the emerging status of M. capitatus as a potential pathogen.Currently, no clinical breakpoints or therapeutic guidelines are available for treating M. capitatus infection.By MIC analysis, we observed a sensibility to azol and 5-flucytosine, but resistance to anidulafungin, micafungin, and caspofungin.
In this case, we highlighted the value of direct microscopy in diagnosing Magnusiomyces bloodstream infection, since we observed the characteristic microscopic feature characterized by arthroconidial forms.It can offer a distinct advantage in achieving rapid diagnosis and may help clinicians to choose an appropriate antifungal therapy.

Conclusions
This case remarks the importance of a careful surveillance of  uncommon fungal infections and the necessity to elaborate and establish valuable breakpoints to determine both sensibility and resistance of antifungals.It has to be strongly associated with the requirement of an Antimicrobial Diagnostic Stewardship among labs and hospitals in order to improve both therapeutic treatments and outcomes.In addition, this case describes the value of Gram-stained smear from positive blood cultures in the early presumptive diagnosis of M. capitatus fungemia.

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