Microascus spp., classified within the family Microascaceae (order Microascales, class Sordariomycetes), represent emerging opportunistic pathogens associated with life-threatening infections globally. These fungi predominantly afflict immunocompromised hosts, including hematopoietic stem cell transplant (HSCT) recipients, critically ill patients, and individuals undergoing prolonged immunosuppressive therapy (Schoeppler et al., 2015). High mortality rates persist due to diagnostic delays, intrinsic antifungal resistance, and limited therapeutic options. Clinical manifestations range from localized cutaneous abscesses to disseminated infections involving lungs, sinuses, cardiac valves, and the central nervous system, often demonstrating poor prognosis despite aggressive interventions (Sandoval-Denis et al., 2016). Recent years have witnessed increased reports of severe pulmonary, hematogenous, and deep-tissue infections, paralleling the growing population of transplant recipients (Baddley et al., 2000; Miossec et al., 2011; Ding et al., 2020; Brasch et al., 2018). However, systematic analyses of clinical features and microbiological characteristics remain scarce, with current literature predominantly limited to case reports.

Ecologically diverse, Microascus spp. typically exhibit saprophytic or weakly parasitic lifestyles, colonizing air, soil, animal feces, plant debris, and humid environments. M. Sandoval-Denis et al. conducted a study on 141 clinical and environmental isolates by integrating morphological, physiological, and multigene sequence analyses of ITS, LSU, EF-1α, and β-tubulin genes. Based on these analyses and applying the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) criterion, they revealed that Microascus and Scopulariopsis belong to two distinct evolutionary lineages. Additionally, they analyzed 54 species from 12 genera within the family Microascaceae, as well as one species from the family Graphiaceae. The results demonstrated that Microascus and Scopulariopsis are polyphyletic genera, with their species distributed across multiple distantly related clades (Sandoval-Denis et al., 2016). According to MycoBank (as of September 2025), 98 species names are currently accepted in Microascus; however, some of these taxa are based on lost specimens or invalid names. To better delineate species boundaries and elucidate evolutionary relationships within the genus, Wei et al. (2024) conducted multi-locus phylogenetic analyses using a combined dataset of four gene regions (ITS, LSU, tef1, and tub2), incorporating expanded taxon sampling and morphological assessments. Their results resolved 49 phylogenetic species in Microascus with high statistical support. Differentiated by ascus morphology (peridium structure) and ascospore characteristics (size, symmetry, surface ornamentation) [drfungus.org/Microascus Species]. Critical gaps persist in morphological identification criteria, antifungal susceptibility patterns, and genomic characterization, contributing to diagnostic and therapeutic challenges.

Our team recently reported China’s first fatal M. cirrosus-associated invasive pulmonary infection in an HSCT recipient, with whole-genome sequencing data deposited in NCBI (PRJNA835605) (Cheng et al., 2023). Subsequent clinical surveillance identified nine additional M. gracilis infections. This study systematically analyzes the clinical and microbiological profiles of Microascus-related severe pneumonia to inform improved management strategies.

The occurrence of severe pneumonia during the stable phase of hematopoietic stem cell transplantation (HSCT) represents a clinically significant challenge. Current evidence implicates multiple risk factors, including environmental distribution of pathogens, host immunocompromised status, and prior antifungal exposure. In our cohort, Microascus ranked third most prevalent fungal genus in HSCT recipients, following Aspergillus and Penicillium spp. While existing literature predominantly documents Microascus infections as sporadic cases or reviews, this pathogen demonstrates concerning clinical versatility, capable of causing cutaneous infections or invasive dissemination to critical organs (heart, lungs, kidneys, and brain) with fatal outcomes (Miossec et al., 2011; Patterson et al., 2016; Eisman and Sinclair, 2014; Skiada et al., 2018; Baddley et al., 2010). A comparative analysis of 26 patients-16 from the literature and 10 from our study-regarding clinical features (age, sex, underlying diseases, infection site), treatment strategies, and outcomes revealed that Microascus infections can occur in immunocompetent individuals, typically presenting as localized infections of the skin, nails, or foot. However, these infections are more prevalent in immunocompromised patients, particularly those with solid organ transplants, mainly lung, or hematopoietic stem cell transplant recipients, and are frequently associated with pulmonary or disseminated infections that lead to high mortality. Both infection site and baseline immune status were identified as critical prognostic factors. Early intervention, including prompt diagnosis and treatment initiation, and individualized therapy are essential. Localized infections may be managed with surgical treatment combined with antifungal therapy, whereas disseminated or mixed fungal infections require aggressive combination antifungal regimens and, where applicable, reduction of immunosuppressive therapy.

Of particular relevance to our cohort, HSCT recipients were maintained on long-term cyclosporine-based immunosuppression for graft-versus-host disease prophylaxis alongside routine posaconazole/voriconazole prophylaxis. This therapeutic landscape likely selects for resistant Microascus strains, potentially explaining the observed fulminant fungal pneumonias. Radiological findings consistently revealed characteristic infectious patterns, including bronchovascular bundle thickening and cavitary lesions. Meanwhile, we evaluated the impact of the timing of antifungal therapy initiation on patient outcomes. Among the 10 patients studied, 9 were HSCT recipients, of whom 7 received prolonged prophylactic antifungal therapy initiated pre-admission or at admission, resulting in favorable outcomes. Conversely, the three cases with clinical deterioration comprised two HSCT patients who began antifungal treatment within 24 h post-admission and one non-HSCT patient with delayed initiation at 7 days post-admission. This suggests that early or prophylactic initiation correlates with improved outcomes, aligning with causal inference principles regarding exposure and outcome relationships, although this association does not confirm causation.

Notably, Microascus was detected within 10 days of admission in all cases, frequently alongside cytomegalovirus, Pseudomonas aeruginosa, or Aspergillus spp. The three fatal cases demonstrated synergistic morbidity with influenza A virus, 2019-nCoV, or Nocardia co-infections. The poor outcomes observed were primarily influenced by profound immunosuppression due to HSCT-related immune reconstitution failure and chronic comorbidities impairing antifungal efficacy in all three cases; additionally, one patient experienced COPD exacerbations compromising pulmonary defenses, and viral coinfections such as COVID-19 or influenza A triggered cytokine storms and multi-organ failure, further amplifying pathophysiological deterioration. This aligns with evidence that viral pathogens in immunocompromised hosts exacerbate immune dysfunction, creating a permissive environment for polymicrobial infections and poor outcomes (Vanderbeke et al., 2018; Arastehfar et al., 2020; Uhde et al., 2010).

Despite standard posaconazole/voriconazole prophylaxis, breakthrough Microascus infections occurred in our hematopoietic stem cell transplantation (HSCT) cohort, suggesting that conventional azole-based prophylactic regimens may be inadequate against this pathogen. All eight isolates exhibited elevated MICs (>64 μg/mL) to azoles, polyenes, and flucytosine, indicating potential intrinsic resistance mechanisms. In contrast, terbinafine demonstrated excellent in vitro activity (MIC ≤0.125 μg/mL across all strains). Heterogeneous susceptibility to echinocandins was observed, as reflected by variable MIC values to caspofungin among the isolates. These findings are consistent with those reported by Skóra et al. (2015), who also documented strong resistance of Microascus to single antifungal agents such as terbinafine, caspofungin, and posaconazole, with generally high MICs. Notably, their study revealed significant synergistic effects with triple combination therapy (terbinafine, caspofungin, and posaconazole) in vitro. Our clinical observations further support these results: a dose-escalated voriconazole regimen combined with caspofungin showed efficacy in partial responders, underscoring the potential of combination therapy to overcome monotherapeutic resistance. However, suboptimal responses were still observed in some cases even with susceptible agents, which may be attributed to concurrent viral or Aspergillus co-infections. This complexity underscores the need for comprehensive therapeutic strategies that integrate host immune status, co-infecting pathogens, and antifungal resistance profiles. Further genomic studies are warranted to elucidate species-specific resistance determinants in Microascus.

Current fungal infection diagnostics still rely heavily on morphological identification. These methods are time-consuming and often inaccurate, which may result in missed or incorrect pathogen detection. However, the slow growth kinetics of Microascus and technical challenges in rapidly obtaining observable diagnostic structures significantly impede timely detection. Notably, six next-generation sequencing (NGS) analyses in our cohort failed to detect Microascus species, highlighting the technical limitations of metagenomics for rare fungi detection, especially when pathogen-specific genomic signatures are underrepresented in reference databases. These diagnostic difficulties enhance the clinical significance of our findings, as better characterization of Microascus epidemiology and resistance patterns directly guides therapeutic decision-making for HSCT recipients during the stabilization phase. Our data underscore the necessity for developing rapid molecular diagnostics targeting conserved genomic regions.

The Microascus isolates in our study exhibited grayish-brown to black pigmentation with conidial morphological features consistent with established descriptions of the Microascus morphology (Liu et al., 2021). The observed morphological plasticity (melanized conidia and cleistothecial structures) suggests evolutionary adaptations related to environmental persistence or pathogenic regulation, though the underlying mechanisms remain unclear (Skiada et al., 2018). MALDI-TOF mass spectrometry proved highly effective for fungal identification, showing 100% concordance (7/7 strains) in genus-level classification based on characteristic protein fingerprints (3400–3,600 m/z spectral peaks) (Patel, 2015; Wei et al., 2022). However, species-level discrimination required supplementary phylogenetic analysis using multilocus sequence data. This highlights the necessity of combined approaches, whose diagnostic complexity warrants careful consideration. Notably, the phenotypic similarities among Microascus, Aspergillus, and Penicillium species necessitate implementing a tiered diagnostic algorithm in clinical laboratories. We propose a hierarchical identification framework incorporating: Morphological screening + MALDI-TOF + Multilocus sequence typing (MLST).

Our data suggest Microascus spp. as emerging opportunistic pathogens in post-hematopoietic stem cell transplantation (HSCT) invasive fungal infections, demonstrating a secondary prevalence rate to Aspergillus and Penicillium spp. This epidemiological pattern warrants heightened clinical vigilance, particularly because therapeutic complexities emerge from both frequent polymicrobial coinfections and intrinsic antifungal resistance. The technical challenges in species identification necessitate a multidimensional diagnostic protocol: (1) Prolonged incubation (5–14 days) for cleistothecia development; (2) MALDI-TOF MS spectral analysis focusing on the characteristic range; (3) Multilocus sequence typing (MLST) targeting β-tubulin, ITS, and CAL loci for definitive speciation. Antifungal susceptibility profiles revealed universal resistance to triazoles and echinocandins, while terbinafine showed the lowest MIC values (≤0.125 μg/mL). These data underscore the urgency to establish species-specific interpretive criteria for Microascus, distinct from existing guidelines for Aspergillus or Candida.

To investigate potential nosocomial sources of Microascus spp., targeted environmental sampling of air and surfaces in bronchoscopy suites and patient wards was performed. Although these fungi are ubiquitously distributed in natural environments—including soil, decaying plant materials, animal feces, and moist habitats—all environmental specimens tested negative for Microascus spp. by culture. This finding suggests an absence of detectable contamination within the examined healthcare settings during the study period. However, temporal and spatial variations in fungal load may occur, and the inherent limitations of conventional culturing in detecting low-abundance spores must be considered. These negative culture results, while not fully defining transmission dynamics, support excluding hospital-based environmental exposure as a source of these infections. If consecutive cases of Microascus spp. detection occur in the same clinical unit, surveillance involving multiple sampling locations and time points in high-risk areas, combined with molecular genotyping techniques for source attribution, should be prioritized.

The novel contributions of this study include the development of a tiered diagnostic strategy integrating morphological, MALDI-TOF MS, and MLST for accurate identification of Microascus; evidence-based recommendations for prophylaxis adjustment informed by in vitro susceptibility and literature review; and environmental surveillance data supporting targeted infection control. These findings provide critical insights for early detection and tailored management of Microascus infections in high-risk populations. Nevertheless, our study has three main limitations. First, its single-center design and small sample size (10 patients) limit the generalizability of the findings, particularly to non-HSCT populations. Second, the lack of molecular resistance data (e.g., ERG11 mutations, efflux pump expression) constrains the mechanistic interpretation of antifungal resistance. Third, the absence of fungal burden quantification and autopsy data impedes accurate correlation of pathogen load with clinical outcomes and precise attribution of mortality. To address these issues, future studies should incorporate molecular profiling (e.g., whole-genome sequencing), standardize specimen processing, and implement fungal quantification methods (e.g., qPCR). Furthermore, enhanced autopsy efforts with ethical compliance are needed to clarify mortality causes. Prioritized research directions include developing rapid molecular diagnostics for high-risk groups, establishing multicenter surveillance to monitor resistance, and exploring combination therapies that leverage unique antifungal mechanisms such as those of terbinafine.

The increasing incidence of severe pulmonary Microascus infections, particularly among immunocompromised patients, is accompanied by high treatment failure rates and significant mortality. This is further compounded by the pathogen’s pan-resistance to conventional antifungal agents, underscoring an urgent need for improved early detection, elucidation of resistance mechanisms, and development of novel therapeutic strategies. In response, our future perspectives emphasize several key directions: First, diagnostic accuracy must be enhanced through the integration of Microascus-specific protein fingerprints into MALDI-TOF-MS databases and standardization of mNGS reporting protocols to facilitate early and reliable identification. Second, due to the high in vitro resistance observed, although resistance patterns are not fully defined, empirical antifungal susceptibility testing remains critical for tailoring targeted therapy. This is especially important for high-risk groups, such as transplant recipients receiving prophylaxis with azoles (e.g., posaconazole, voriconazole). Clinical vigilance should be heightened in these populations, incorporating multimodal data to differentiate colonization from invasive infection. Finally, strengthened environmental surveillance in high-risk hospital areas is essential to control potential nosocomial transmission. Collectively, these strategies aim to improve clinical outcomes through earlier intervention, individualized treatment, and evidence-based infection control in the face of this emerging and challenging pathogen.