IPM are a serious problem in contemporary medicine, associated with high morbidity and mortality, especially in immunocompromised hosts.¹ Epidemiological scenarios of IPM have changed dramatically in the last decade. Although Aspergillus spp. remains the most common cause of infection, infections due to Mucorales and emerging non-Aspergillus hyaline and dematiaceous mold species, including Fusarium spp., Scedosporium spp., and Lomentospora spp., have increased significantly.^2,3

This change is thought to be driven by the use of broad-spectrum antibiotics, by the longer-term use of high doses of corticosteroids, and by the growing number of patients with haematological cancer and solid organ transplantation.⁴ The clinical features of IPM are very non-specific and frequently confused with other forms of pneumonia, including bacterial pneumonia or other lung diseases, and can often result in the delay of effective, life-saving antifungal drugs.⁵ Therefore, the laboratory diagnosis of IPM is of great value, and must be fast and accurate to improve the patient's outcome. The diagnostic approach to IPM is based on a combination of radiological imaging, serum markers (galactomannan and beta-D-glucan), direct microscopy, fungal culture and molecular methods.⁶

However, each modality has limitations. Radiological findings are suggestive but not etiological specific. Growth of serum biomarkers have proven to be very useful in the case of Aspergillus, but not sensitive for Mucorales or many emerging molds.⁷ Fungal culture that is classically regarded as the gold standard method for differentiating species and testing antifungal susceptibility is rarely sensitive, is time consuming, and is subject to a high percentage of false negatives because the disease is focal and antifungal drugs are typically administered empirically.⁸ Histopathological evaluation of biopsies and/or resection specimens of the lung becomes a key and sometimes essential cornerstone of diagnosis in this context. Histopathology can confirm the invasion of tissues, differentiate true infection from colonization and/or contamination, and yield immediate, actionable morphological information that can direct empirical therapy pending culture results.⁹ The hyphae of Mucorales, in contrast, are broad (6-25 µm), irregular in outline and pauciseptate or aseptate and branch at right angles or at random.

The morphological appearance of emerging hyaline molds such as Fusarium can resemble that of Aspergillus but can also be characterized by adventitious sporulation (phialides and microconidia) within the tissue, which is a rare but very specific diagnostic feature.¹⁰ Dematiaceous fungi like Curvularia or Bipolaris have melanin in their cell walls and will stain darkly with Fontana-Masson staining, but can easily be seen as darkly pigmented hyphae on GMS.¹¹ The morphology of the fungi is not the limiting factor and the host's inflammatory and tissue-destructive response will give a second level of diagnostic information. Immune status of the host and the virulence factors of the invading fungus play a major role in determining the nature of the tissue reaction. Tissue reaction is usually poor, and in very neutropenic cases there is mainly extensive coagulative necrosis and hemorrhage, with little inflammatory infiltrate.¹²

Although histopathology has been proven to be a crucial tool in these investigations, the direct, statistically significant linkage of a particular fungal species with definite, measurable histopathological features has not been fully clarified. The subtle differences in tissue reaction patterns, the degree of angioinvasion, and the topography of necrosis with emerging non-Aspergillus molds are less well described in more recent literature.¹³

This was a multicenter retrospective cross-sectional analytical study carried out at the Department of Pathology and Department of Microbiology of tertiary care academic medical centers. The study protocol was approved by the Institutional Ethics Committee. The period under study was from January 1, 2021 to December 31, 2025. Selection criteria included the presence of fungal hyphae or yeast forms in pulmonary parenchyma, blood vessels or bronchi by histopathology, and/or the fungus was confirmed by positive fungal culture and/or molecular sequencing from the same tissue specimen or from a specimen collected at the same time from a different site within the respiratory tract (such as bronchoalveolar lavage). The exclusion criteria were: (1) superficial fungal colonisation and no tissue invasion; (2) only a positive culture result, and no evidence of tissue invasion; (3) poor quality of tissue sample (crushed biopsies, or insufficient tissue for special stains); (4) incomplete medical records, as regards the concurrent presence of bacterial and/or viral pneumonias, which precluded accurate histopathological assessment of the fungal tissue reaction; and (5) incomplete medical records as regards underlying co-morbidities and/or immune status. All tissue samples were fixed in 10% neutral buffered formalin for at least 6 hours and for a maximum of 24 hours to preserve the fungal morphology and tissue architecture optimally. Tissues were routinely fixed in an automated tissue processor, embedded in paraffin wax and cut on a rotary microtome at 4-5 micrometers (µm). Hematoxylin and Eosin (H&E) was routinely stained. Serial sectioning was stained with Grocott's methenamine silver (GMS), and Periodic Acid-Schiff (PAS) to best visualise fungal elements. The GMS staining procedure was performed by immersing the slides in 5% chromic acid for 15 minutes followed by incubation in a working solution (hexamethylenetetramine and 5% silver nitrate) at 60°C for 45 minutes with light green SF yellowish as a counterstain. The staining was done by PAS staining, which included the application of 0.5% periodic acid for five minutes and then the application of Schiff's reagent for 15 minutes. Histopathological assessment was done independently by two senior consultant Pathologists with special interest in infectious disease who were blinded to the microbiological and molecular evaluation. Discrepancies were resolved by consensus review which was performed with a multi-head microscope. The evaluation was conducted on four main domains: The fungal morphology includes hyphal width (narrow <5 µm vs. broad ≥5 µm), hyphal septation (septate vs. aseptate/pauciseptate), and branching pattern (acute/dichotomous vs. right-angle/random). The presence of tissue sporulation (e.g., phialides, conidia) was also noted. 2. Inflammatory Tissue Reaction: There are four patterns: (a) Acute suppurative (mainly neutrophils with microabscesses); (b) Granulomatous (epithelioid histiocytes and multinucleated giant cells); (c) Mixed (suppurative and granulomatous); and (d) Necrotizing with little inflammation (extensive necrosis with few inflammatory cells). 3. Angioinvasion (+/-). If present, the degree of involvement was semi-quantitatively assessed as mild ( < 25% of the sampled area with focal vessel involvement), moderate (25-50% involvement) or severe ( > 50% involvement with fungal invasion, thrombosis or mural destruction). 4. Tissue Necrosis: Type (coagulative versus liquefactive), and extent (focal <10%, moderate 10-30%, or extensive >30% of the parenchymal area). In addition to histopathology, fungal cultures were done on several fungal culture media including Sabouraud Dextrose Agar (SDA) with chloramphenicol, Potato Dextrose Agar (PDA), and Brain Heart Infusion (BHI) agar with both gentamicin and chloramphenicol. Plates were incubated at 25°C and 37°C and observed daily for 4 weeks. A commercial fungal genomic DNA extraction kit (Qiagen, Germany) was used for genomic DNA extraction. The PCR products were sequenced on an automated capillary sequencer (Applied Biosytem) and used BLAST algorithm to sequence the products to the NCBI GenBank and ISHAM databases. Species level identification was required at a sequence similarity of ≥99%. The clinical and demographic data such as age, sex, underlying comorbidities (diabetes mellitus, hematological malignancies, solid organ transplantation, chronic corticosteroid use), and neutrophil counts were retrieved from the electronic medical records. IBM SPSS Statistics version 28.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Continuous variables were shown as mean ± standard deviation (SD) or median (interquartile range) and compared by the Student's t-test or Mann-Whitney U test depending on the variables. Frequencies and percentages were used to describe categorical variables and the Chi-square test or Fisher's exact test was used for comparisons between groups. A multivariate logistic regression analysis with adjustment for potentially confounding factors (age, diabetes status, and neutropenia) was used to determine independent histopathologic predictors of specific fungal groups (e.g., Mucorales vs. Aspergillus). All analyses were performed for p-values<0.05, which were deemed to be statistically significant.

Table 1 illustrates the clinical demographics of the cohort across the four major fungal groups. While age showed a statistically significant difference across groups (p=0.042), with Mucorales patients being slightly older, the most striking clinical correlations were observed in the underlying comorbidities. Patients with hematological malignancies were significantly more likely to harbor Aspergillus species (47.2%) compared to Mucorales (29.5%) or dematiaceous fungi (13.6%) (p=0.008). Conversely, diabetes mellitus was overwhelmingly associated with Mucorales infections (61.1%), a finding that was highly statistically significant compared to the other groups (p<0.001).

Table 1: Baseline demographic and clinical characteristics of the study cohort (N=342) stratified by major fungal groups.

Table 2 details the specific etiological agents identified within the broader fungal groups. Among the Aspergillus species, A. fumigatus was the most prevalent (54.4%), followed by A. flavus (25.0%). Within the Mucorales order, Rhizopus arrhizus was the predominant species, accounting for over half (54.7%) of the cases. The "Other Hyaline Molds" group was primarily composed of the Fusarium solani species complex (40.0%) and Scedosporium apiospermum (26.7%). The dematiaceous fungi were largely represented by Curvularia lunata and Bipolaris spicifera.

Table 2: Distribution of specific fungal species identified by culture and molecular methods (N=342).

Table 3 demonstrates a highly significant correlation between the fungal group and the type of host inflammatory tissue reaction (overall p<0.001). Mucorales were overwhelmingly associated with a "necrotizing with minimal inflammation" pattern (52.6%), which was significantly higher than in the other groups (p<0.001). This reflects the rapid, destructive nature of mucormycosis, often occurring in patients with impaired neutrophil function (e.g., diabetic ketoacidosis). In contrast, dematiaceous fungi and other hyaline molds (Fusarium, Scedosporium) elicited a significantly higher rate of pure "granulomatous" inflammation (36.4% and 26.7%, respectively) compared to Mucorales (5.3%) (p<0.001).

Table 3: Correlation between fungal groups and histopathological inflammatory tissue reaction patterns.

Table 4 highlights the profound differences in vascular invasion and tissue destruction among the fungal groups. Angioinvasion was highly prevalent across all groups but was significantly more common in Mucorales (86.3%) compared to dematiaceous fungi (54.5%) (p=0.003). More importantly, when angioinvasion was present, Mucorales exhibited a significantly higher degree of "severe" vascular involvement (54.9%) compared to Aspergillus (24.0%) and other molds (p=0.018). Regarding tissue necrosis, nearly all Mucorales cases (95.8%) exhibited necrosis, which was significantly higher than the other groups (p<0.001).

Table 4: Association between fungal groups and the presence/degree of angioinvasion and tissue necrosis.

Table 5 presents the results of the multivariate logistic regression analysis, which aimed to identify independent histopathological predictors for Mucorales infection when compared to the most common alternative, Aspergillus. After adjusting for clinical confounders (age, diabetes, malignancy, neutropenia), specific morphological and tissue-reaction features remained highly significant independent predictors. The presence of broad, aseptate hyphae (≥5 µm) was the strongest predictor, yielding an adjusted odds ratio (aOR) of 14.52 (p<0.001). Right-angle branching (aOR 8.76, p<0.001), a necrotizing tissue reaction with minimal inflammation (aOR 5.34, p<0.001), severe angioinvasion (aOR 3.85, p=0.001), and extensive coagulative necrosis (aOR 4.12, p<0.001) were all independently associated with Mucorales.

Table 5: Multivariate logistic regression analysis of histopathological predictors for Mucorales versus Aspergillus infections.

The epidemiological distribution that we found is similar to the one recently described worldwide with Aspergillus fumigatus as the most common cause of IPM, with an increasing awareness of Mucorales and emerging molds.14 In our cohort, a particular clinical observation was the high prevalence of Mucorales infection in patients with diabetes mellitus, and the very strong association of hematological malignancies and profound neutropenia with Aspergillus infection. This also supports recent publications that have identified the special pathophysiological niches of these fungi and how the acidic, hyperglycemic and iron-rich environment associated with diabetic ketoacidosis adversely affects neutrophil chemotaxis and phagocytosis, particularly making hosts vulnerable to infection with Mucorales.15 In contrast, profound neutropenia associated with hematological malignancies makes it easier for angioinvasion by Aspergillus hyphae.16 But although clinical risk factors are important, they are not always immediately available or definitive at the time of initial biopsy assessment. Thus, the histopathological features are the main point of reference. Our histopathological analysis revealed profound differences in the host tissue reaction patterns. A “necrotizing with minimal inflammation” pattern and large coagulative necrosis were overwhelmingly associated with Mucorales. This is similar to the rapid, destructive pathogenesis of mucormycosis, in which fungal hyphae produce proteases that may destroy tissue architecture even before an adequate host inflammatory reaction can be mounted.17 The fact that 54.9% of Mucorales with vascular invasion demonstrated severe angioinvasion is an additional explanation of the widespread ischemic coagulative necrosis, since the broad hyphae physically occlude and destroy elastic lamina of blood vessels.18 Aspergillus and emerging hyaline moulds such as Fusarium and Scedosporium often produced an acute suppurative or mixed granulomatous reaction, however. The presence of a granulomatous response in dematiaceous fungi and in Scedosporium points to a more chronic, indolent course in which there is partial persistence of the cell-mediated immunity of the host, which attempts to wall off the fungus with macrophages and multinucleated giant cells.19 The primary diagnostic tool available to the pathologist is morphological evaluation. The concept of the classic morphological dichotomy between Aspergillus (narrow, septate, acute angle branching) and Mucorales (broad, aseptate, right angle branching) was confirmed in our study. The most difficult diagnosis, however, occurs when it's necessary to distinguish Aspergillus from emerging hyaline molds such as Fusarium and Scedosporium, which can exhibit almost identical morphologies on both H&E and GMS stains.20 Adventitious sporulation (phialides and microconidia in tissue) was a rare but very characteristic feature of Fusarium in our cohort, and in cavitary lesions, Scedosporium occasionally had the dematiaceous elements or specific conidial structures. For emerging molds, special stains such as Fontana-Masson may be helpful to show melanin in dematiaceous fungi, but morphology alone is not enough. This underscores the critical importance of the combined pathology-microbiology strategy we adopted in this study, in which histopathology serves as the "rule-in" of invasion and as a broad etiological grouping with MALDI-TOF MS and ITS sequencing used for definitive species-level identification, a step needed for targeted antifungal susceptibility testing.21,22 The presence of broad, aseptate hyphae in a pathologist's report, branching at right angles, with massive coagulative necrosis and significant angioinvasion, can confidently alert the clinical team to the high probability of mucormycosis. This enables immediate administration of liposomal amphotericin B and the urgent surgery needed for mucormycosis without waiting for the culture to result negative or the initiation of empirical voriconazole therapy, which is not active against Mucorales.23,24 Our study presents a powerful, statistically sound model for associating fungal species with the histopathological characteristics of IPM that further underscores the critical role of the pathologist in a multidisciplinary approach to invasive fungal diseases.

Histopathological pattern of invasive pulmonary mycoses features specific pattern features that strongly relate to the underlying fungal disease. Aspergillus and emerging hyaline and dematiaceous molds often cause a suppurative or granulomatous tissue reaction, in contrast. These particular patterns can be identified at the morphological and tissue destructive level and provides valuable morphological clues that facilitate early presumptive diagnosis. This integrated approach to both pathogens and microbiology may facilitate earlier clinical decision-making regarding targeted empirical antifungal therapy, and has the potential to enhance clinical outcomes in the treatment of these dreaded infections.

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