Deciphering Unexpected Vascular Locations of Scedosporium spp. and Lomentospora prolificans Fungal Infections, France.

Scedosporium spp. and Lomentospora prolificans are emerging non-Aspergillus filamentous fungi. The Scedosporiosis/lomentosporiosis Observational Study we previously conducted reported frequent fungal vascular involvement, including aortitis and peripheral arteritis. For this article, we reviewed 7 cases of Scedosporium spp. and L. prolificans arteritis from the Scedosporiosis/lomentosporiosis Observational Study and 13 cases from published literature. Underlying immunosuppression was reported in 70% (14/20) of case-patients, mainly those who had solid organ transplants (10/14). Osteoarticular localization of infection was observed in 50% (10/20) of cases; infections were frequently (7/10) contiguous with vascular infection sites. Scedosporium spp./Lomentospora prolificans infections were diagnosed in 9 of 20 patients ≈3 months after completing treatment for nonvascular scedosporiosis/lomentosporiosis. Aneurysms were found in 8/11 aortitis and 6/10 peripheral arteritis cases. Invasive fungal disease--related deaths were high (12/18 [67%]). The vascular tropism of Scedosporium spp. and L. prolificans indicates vascular imaging, such as computed tomography angiography, is needed to manage infections, especially for osteoarticular locations.

Stability and Qualification of a Legacy Fungal Collection.

Background: Microbial culture collections are valuable repositories for qualified and diverse microorganisms, playing a pivotal role in research, education, innovation, as well as in our response to current and emerging public health and societal challenges. However, such precious holdings, when not integrated in professional biobank infrastructures, may be vulnerable to major risks such as staff retirement, changes in the institutional strategy, or natural disasters. The process of preserving and rescuing "historical" collections can be long and treacherous with a loss of a part of the collection. At the Biological Resource Center of Institut Pasteur, we undertook the challenge of rescuing the dormant legacy fungal collection. Materials and Methods: A total of 64 freeze-dried strains, including yeasts and filamentous fungi, were characterized by using a polyphasic approach combining morphological features and molecular data. We assessed the viability, purity, and authenticity of selected strains isolated from multiple sources and stored for more than 20 years. Results: Our preliminary results show long-term stability of the selected strains and successful qualification in terms of purity and authentication. Moreover, based on the most recent taxonomic revisions, we updated and revised the nomenclature, where applicable. Conclusion: Our findings demonstrated the potential value of reviving historical microbial collections for biobanking and research activities and reassure us about the collection's future reopening.

Refractory Microascus Bronchopulmonary Infection Treated with Olorofim, France.

We report 3 cases of successful treatment of Microascus spp. bronchopulmonary infection in a multiple-traumatized patient and 2 lung transplant recipients in France. We emphasize the promising use of olorofim antifungal therapy in a rising context of intrinsically less-susceptible respiratory infections caused by mold.

Dynamic full-field optical coherence tomography for live-cell imaging and growth-phase monitoring in Aspergillus fumigatus.

Introduction: The diagnosis of cutaneous manifestations of deep mycoses relies on both histopathological and direct examinations. Yet, the current diagnostic criteria cannot prevent missed cases, including invasive aspergillosis, which requires the development of a novel diagnostic approach and imaging tools. We recently introduced the use of dynamic full-field optical coherence tomography (D-FF-OCT) in fungal diagnostics with a definition approaching that of conventional microscopy and the ability to return metabolic information regarding different fungal species. The present work focuses on subcellular dynamics and live-cell imaging of Aspergillus fumigatus with D-FF-OCT to follow the fungal growth stages. Methods: The A. fumigatus ATCC 204305 quality-control strain was used for all imaging experiments, following incubation times varying between 24 and 72 h at 30°C in a humidified chamber on Sabouraud dextrose agar. Fungal growth was subsequently monitored with D-FF-OCT for up to 5 h at room temperature and following the pharmacological stress of either voriconazole, amphotericin B, or caspofungin gradient concentration. Results: D-FF-OCT images allow not only the visualization of intracellular trafficking of vacuoles but also an evolving dynamic segmentation of conidiophores depending on the chronological development and aging of the hyphae or the effect of antifungal treatment. The same applies to conidial heads, with the most intense D-FF-OCT signal coming from vesicles, revealing a changing dynamic within a few hours only, as well as complete extinction following subsequent drying of the Sabouraud dextrose agar. Discussion: These results provide additional data on the ability of D-FF-OCT to monitor some of the main life cycle processes, dynamics, and intracellular trafficking of vacuoles in A. fumigatus, with or without the effect of pharmacological stress. Such complementary metabolic information could help both clinicians and microbiologists in either mechanistic studies toward experimental mycology or the development of a potential D-FF-OCT-guided diagnosis of superficial fungal infections.

Improving Diagnosis of Pulmonary Mucormycosis: Leads From a Contemporary National Study of 114 Cases.

BACKGROUND: Pulmonary mucormycosis (PM) is a life-threatening invasive mold infection. Diagnosis of mucormycosis is challenging and often delayed, resulting in higher mortality. RESEARCH QUESTION: Are the disease presentation of PM and contribution of diagnosis tools influenced by the patient's underlying condition? STUDY DESIGN AND METHODS: All PM cases from six French teaching hospitals between 2008 and 2019 were retrospectively reviewed. Cases were defined according to updated European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria with the addition of diabetes and trauma as host factors and positive serum or tissue PCR as mycologic evidence. Thoracic CT scans were reviewed centrally. RESULTS: A total of 114 cases of PM were recorded, including 40% with disseminated forms. Main underlying conditions were hematologic malignancy (49%), allogeneic hematopoietic stem cell transplantation (21%), and solid organ transplantation (17%). When disseminated, main dissemination sites were the liver (48%), spleen (48%), brain (44%), and kidneys (37%). Radiologic presentation included consolidation (58%), pleural effusion (52%), reversed halo sign (26%), halo sign (24%), vascular abnormalities (26%), and cavity (23%). Serum quantitative polymerase chain reaction (qPCR) was positive in 42 (79%) of 53 patients and BAL in 46 (50%) of 96 patients. Results of transthoracic lung biopsy were diagnostic in 8 (73%) of 11 patients with noncontributive BAL. Overall 90-day mortality was 59%. Patients with neutropenia more frequently displayed an angioinvasive presentation, including reversed halo sign and disseminated disease (P < .05). Serum qPCR was more contributive in patients with neutropenia (91% vs 62%; P = .02), and BAL was more contributive in patients without neutropenia (69% vs 41%; P = .02). Serum qPCR was more frequently positive in patients with a > 3 cm main lesion (91% vs 62%; P = .02). Overall, positive qPCR was associated with an early diagnosis (P = .03) and treatment onset (P = .01). INTERPRETATION: Neutropenia and radiologic findings influence disease presentation and contribution of diagnostic tools during PM. Serum qPCR is more contributive in patients with neutropenia and BAL examination in patients without neutropenia. Results of lung biopsies are highly contributive in cases of noncontributive BAL.

What Is New in Pulmonary Mucormycosis?

Mucormycosis is a rare but life-threatening fungal infection due to molds of the order Mucorales. The incidence has been increasing over recent decades. Worldwide, pulmonary mucormycosis (PM) presents in the lungs, which are the third main location for the infection after the rhino-orbito-cerebral (ROC) areas and the skin. The main risk factors for PM include hematological malignancies and solid organ transplantation, whereas ROC infections classically are classically favored by diabetes mellitus. The differences between the ROC and pulmonary locations are possibly explained by the activation of different mammalian receptors-GRP78 in nasal epithelial cells and integrin ß1 in alveolar epithelial cells-in response to Mucorales. Alveolar macrophages and neutrophils play a key role in the host defense against Mucorales. The diagnosis of PM relies on CT scans, cultures, PCR tests, and histology. The reversed halo sign is an early, but very suggestive, sign of PM in neutropenic patients. Recently, the serum PCR test showed a very encouraging performance for the diagnosis and follow-up of mucormycosis. Liposomal amphotericin B is the drug of choice for first-line therapy, together with correction of underlying disease and surgery when feasible. After a stable or partial response, the step-down treatment includes oral isavuconazole or posaconazole delayed release tablets until a complete response is achieved. Secondary prophylaxis should be discussed when there is any risk of relapse, such as the persistence of neutropenia or the prolonged use of high-dose immunosuppressive therapy. Despite these novelties, the mortality rate from PM remains higher than 50%. Therefore, future research must define the place for combination therapy and adjunctive treatments, while the development of new treatments is necessary.

Invasive bone and joint infections from the French Scedosporiosis/lomentosporiosis Observational Study (SOS) cohort: no mortality with long-term antifungal treatment and surgery.

Little is known about localized osteoarticular Scedosporiosis (LOS). Most data come from case reports and small case series. Here we present an ancillary study of the nationwide French Scedosporiosis Observational Study (SOS), describing 15 consecutive cases of LOS diagnosed between January 2005 and March 2017. Adult patients diagnosed with LOS defined by osteoarticular involvement without distant foci reported in SOS were included. Fifteen LOS were analyzed. Seven patients had underlying disease. Fourteen patients had prior trauma as potential inoculation. Clinical presentation was arthritis (n = 8), osteitis (n = 5), and thoracic wall infection (n = 2). The most common clinical manifestation was pain (n = 9), followed by localized swelling (n = 7), cutaneous fistulization (n = 7), and fever (n = 5). The species involved were Scedosporium apiospermum (n = 8), S. boydii (n = 3), S. dehoogii (n = 1), and Lomentospora prolificans (n = 3). The species distribution was unremarkable except for S. boydii, which was associated with healthcare-related inoculations. Management was based on medical and surgical treatment for 13 patients. Fourteen patients received antifungal treatment for a median duration of 7 months. No patients died during follow-up. LOS exclusively occurred in the context of inoculation or systemic predisposing factors. It has a non-specific clinical presentation and is associated with an overall good clinical outcome, provided there is a prolonged course of antifungal therapy and adequate surgical management.

Localized osteoarticular scedosporiosis mostly occurs following direct inoculation. Management was most often based on voriconazole therapy and concomitant surgery. Unlike other invasive scedosporiosis, no patient died during follow-up.

First case of subcutaneous cystic phaeohyphomycosis due to Phialophora chinensis in a kidney transplant recipient in Martinique.

We report a case of subcutaneous mycosis in the form of a subcutaneous cyst of the index finger, successfully treated by surgery and posaconazole in an 84-year-old female kidney transplant patient. Intra-operative mycological analysis enabled the diagnosis of Phialophora chinensis phaeohyphomycosis. Phialophora chinensis is an environmental mold recently described in human pathology in cases of chromoblastomycosis. This is the first case of subcutaneous phaeohyphomycosis due to Phialophora chinensis in an immunocompromised patient.

Detection of circulating DNA for the diagnosis of invasive fusariosis: retrospective analysis of 15 proven cases.

Fusarium spp. are plant pathogens and opportunistic pathogens in severely immunocompromised (hematological malignancy, neutropenia, solid organ transplantation, etc.) and severely burned patients. Invasive fusariosis often disseminates and mortality remains high partly due to delayed diagnosis in the absence of a positive culture. The aim of our study is to design a quantitative PCR (qPCR) assay and evaluate the detection of Fusarium spp. DNA for early diagnosis of invasive infection. A qPCR assay was designed and optimized to identify all Fusarium species complex and secondarily evaluated on patient samples. A total of 81 blood samples from 15 patients diagnosed with proven invasive fusariosis from 9 centers in France were retrospectively tested. Circulating DNA was detected in 14 patients out of 15 (sensitivity of 93% [95% Confidence Interval (CI95), 70.1-99.7]). Detection was possible up to 18 days (median 6 days) before the diagnosis was confirmed by positive blood culture or biopsy. By comparison serum galactomannan and ß-D-glucan were positive in 7.1 and 58.3% of patients respectively. qPCR was negative for all patients with other invasive fungal diseases (IFD) tested (n = 12) and IFD-free control patients (n = 40). No cross-reactions were detected using DNA extracted from 81 other opportunistic fungi. We developed and validated a pan-Fusarium qPCR assay in serum/plasma with high sensitivity, specificity, and reproducibility that could facilitate early diagnosis and treatment monitoring of invasive fusariosis. LAY ABSTRACT: Fusariosis ranks third among invasive mould infections. It is frequently diagnosed late due to the lack of specific tools. We designed and evaluated a new qPCR assay with high sensitivity and specificity allowing detection of Fusarium DNA in serum samples up to 18 days before conventional diagnosis.

Dynamic full-field optical coherence tomography as complementary tool in fungal diagnostics.

Histopathology and microscopic examination of infected tissue are the gold standards to prove the diagnosis of invasive fungal infection (IFI). Yet, they suffer from essential limitations that hamper rapid diagnosis and require the future development of new imaging tools dedicated to fungal diagnostics. To this end, the present work introduces the first use of dynamic full-field optical coherence tomography (D-FF-OCT) for the visualization of microscopic filamentous fungi. Data collected from the observation of three different fungal species (Nannizzia gypsea, Aspergillus fumigatus and Rhizopus arrhizus) confirm the ability of D-FF-OCT to visualize not only the main structures of all selected fungal species (hyphae, spores, conidia, sporulating structures), but also the metabolic activity of the organisms, which could provide additional help in the future to better characterize the signature of each fungal structure. These results demonstrate how D-FF-OCT could serve as potential complementary tool for rapid diagnosis of IFI in both intensive and non-intensive care units.

Active Surveillance Program to Increase Awareness on Invasive Fungal Diseases: the French RESSIF Network (2012 to 2018).

The French National Reference Center for Invasive Mycoses and Antifungals leads an active and sustained nationwide surveillance program on probable and proven invasive fungal diseases (IFDs) to determine their epidemiology in France. Between 2012 and 2018, a total of 10,886 IFDs were recorded. The incidence increased slightly over time (2.16 to 2.36/10,000 hospitalization days, P = 0.0562) in relation with an increase of fungemia incidence (1.03 to 1.19/10,000, P = 0.0023), while that of other IFDs remained stable. The proportion of ≥65-year-old patients increased from 38.4% to 45.3% (P < 0.0001). Yeast fungemia (n = 5,444) was due mainly to Candida albicans (55.6%) with stable proportions of species over time. Echinocandins became the main drug prescribed (46.7% to 61.8%), but global mortality rate remained unchanged (36.3% at 1 month). Pneumocystis jirovecii pneumonia (n = 2,106) was diagnosed mostly in HIV-negative patients (80.7%) with a significantly higher mortality than in HIV-positive patients (21.9% versus 5.4% at 1 month, P < 0.0001). Invasive aspergillosis (n = 1,661) and mucormycosis (n = 314) were diagnosed mostly in hematology (>60% of the cases) with a global mortality rate of 42.5% and 59.3%, respectively, at 3 months and significant changes in diagnosis procedure over time. More concurrent infections were also diagnosed over time (from 5.4% to 9.4% for mold IFDs, P = 0.0115). In conclusion, we observed an aging of patients with IFD with a significant increase in incidence only for yeast fungemia, a trend toward more concurrent infections, which raises diagnostic and therapeutic issues. Overall, global survival associated with IFDs has not improved despite updated guidelines and new diagnostic tools. IMPORTANCE The epidemiology of invasive fungal diseases (IFDs) is hard to delineate given the difficulties in ascertaining the diagnosis that is often based on the confrontation of clinical and microbiological criteria. The present report underlines the interest of active surveillance involving mycologists and clinicians to describe the global incidence and that of the main IFDs. Globally, although the incidence of Pneumocystis pneumonia, invasive aspergillosis, and mucormycosis remained stable over the study period (2012 to 2018), that of yeast fungemia increased slightly. We also show here that IFDs seem to affect older people more frequently. The most worrisome observation is the lack of improvement in the global survival rate associated with IFDs despite the increasing use of more sensitive diagnostic tools, the availability of new antifungal drugs very active in clinical trials, and a still low/marginal rate of acquired in vitro resistance in France. Therefore, other tracks of improvement should be investigated actively.

Morocco as a possible source for acquisition of Rhinocladiella mackenziei.

Rhinocladiella mackenziei cerebral phaeohyphomycosis is a rare severe disease that has been typically described in the Middle East. Here, we report 2 cases of R. mackenziei cerebral phaeohyphomycosis in patients from Morocco, diagnosed and treated in France, and raise a concern about the ever-going extension of the area at risk for this devastating invasive fungal infection.

Different repartition of the cryptic species of black aspergilli according to the anatomical sites in human infections, in a French University hospital.

Black aspergilli of the section Nigri are rarely differentiated at the species level when originating from human specimens. We wondered whether some cryptic species could be more frequently observed in some clinical entities. We analyzed the 198 black isolates consecutively collected from the external ear canal (EEC; n = 66), respiratory specimens (n = 99), and environment (n = 33). DNA was extracted and species identification was performed upon the partial calmodulin gene. We identified by decreasing frequency: Aspergillus welwitschiae (35.3%), Aspergillus tubingensis (34.3%), Aspergillus niger (17.2%), Aspergillus luchuensis (4%), Aspergillus aff. welwitschiae (3%), Aspergillus neoniger (2%), Aspergillus piperis (1.5%), Aspergillus japonicus (1.0%), Aspergillus vadensis (0.5%), and two Aspergillus tubingensis clade (1%). The distribution of the three main cryptic species was different between EEC and respiratory samples (P < 0.001) but not different between respiratory and environment samples (P = 0.264). Aspergillus welwitschiae was more often associated with EEC (54.5%), whereas A. tubingensis and A. niger were predominant in respiratory samples (39.4 and 26.3%, respectively). Among the 99 respiratory isolates, only 10 were deemed responsible for probable invasive aspergillosis, of which six were mixed with other pathogenic moulds. This study shows the interest to pursue the identification of clinical isolates in the Aspergillus section Nigri to unravel some specific associations with clinical entities. The association of A. welwitschiae with otomycosis suggests a better fitness to infect/colonize the ear canal. Also, members of the Aspergillus section Nigri alone are rarely responsible for invasive aspergillosis. LAY SUMMARY: We analyzed 198 black aspergilli isolates collected from different samples type to determine their species identification. We observe a different distribution of species between ear canal and respiratory samples (P < 0.001), suggesting a better fitness of A. welwitschiae to infect the ear canal.

Evaluation of a New Histoplasma spp. Quantitative RT-PCR Assay.

Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H. capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.

Scedosporiosis/lomentosporiosis observational study (SOS): Clinical significance of Scedosporium species identification.

Scedosporiosis/lomentosporiosis is a devastating emerging fungal infection. Our objective was to describe the clinical pattern and to analyze whether taxonomic grouping of the species involved was supported by differences in terms of clinical presentations or outcomes. We retrospectively studied cases of invasive scedosporiosis in France from 2005 through 2017 based on isolates characterized by polyphasic approach. We recorded 90 cases, mainly related to Scedosporium apiospermum (n = 48), S. boydii/S. ellipsoideum (n = 20), and Lomentospora prolificans (n = 14). One-third of infections were disseminated, with unexpectedly high rates of cerebral (41%) and cardiovascular (31%) involvement. In light of recent Scedosporium taxonomic revisions, we aimed to study the clinical significance of Scedosporium species identification and report for the first time contrasting clinical presentations between infections caused S. apiospermum, which were associated with malignancies and cutaneous involvement in disseminated infections, and infections caused by S. boydii, which were associated with solid organ transplantation, cerebral infections, fungemia, and early death. The clinical presentation of L. prolificans also differed from that of other species, involving more neutropenic patients, breakthrough infections, fungemia, and disseminated infections. Neutropenia, dissemination, and lack of antifungal prescription were all associated with 3-month mortality. Our data support the distinction between S. apiospermum and S. boydii and between L. prolificans and Scedosporium sp. Our results also underline the importance of the workup to assess dissemination, including cardiovascular system and brain.

Scedosporiosis/lomentosporiosis is a devastating emerging fungal infection. Our objective was to describe the clinical pattern and to analyze whether taxonomic grouping of the species involved was supported by differences in terms of clinical presentations or outcomes.

Invasive Infections with Nannizziopsis obscura Species Complex in 9 Patients from West Africa, France, 2004-20201.

Nine new human invasive infections caused by the keratinophilic fungi Nannizziopsis obscura have been reported in France since 2004. The patients had variable clinical manifestations, had frequent dissemination, were mainly T-cell immunocompromised, and all originated from sub-Saharan West Africa. Before collection of the isolates, the etiologies of these infections were often misidentified, underscoring the extent of microscopic and cultural polymorphisms. All isolates but 1 had low MICs for the 8 antifungal drugs tested. When treated, patients received mainly azole therapy. Two of 7 patients with a known outcome died. We performed multilocus sequence analysis of N. obscura clinical strains and several strains of Nannizziopsis spp. isolated from reptiles. The human strains were clearly differentiated from the animal strains. N. obscura might be endemic to West Africa and responsible for undetected infections, which might become reactivated when immunosuppression occurs. N. obscura infection is probably underestimated because only sequencing enables proper identification.