Fungi belonging to the order Mucorales are majorly divided into six different families wherein Mucoraceae is the most frequent cause of mucormycosis ^[9][22]. The most important species in order of frequency are Rhizopus arrhizus (Oryzae), Rhizopus microsporus var. rhizopodiformis, Rhizomucor pusillus, Cunninghmella bertholletiae, Apophysomyces elegans, and Saksenaea vasiformis ^{[9][10][11][12]}[22,23,24,25]. The newly emerging species include Rhizopus homothallicus, Thamnostylum lucknowense, Mucor irregularis, and Saksenaea erythrospora ^[13][26] (Table 1). Mucoraceae may be divided into sporangium producers, sporangiolum producers, and merosporangium producers based on morphology. For instance, lactophenol cotton blue-stained elements such as rhizoids, stolons, and columella or hyphae are able to differentiate between the fungal species ^[10][11][23,24].

The identity of the fungal species does not infer the relevant therapy as diseases caused by these species is phenotypically identical. The optimal temperature for the growth of aerobic Mucorales is 28–30 °C for an incubation period of 2–5 days. The clinically relevant Mucorales initiate and facilitate the decay of organic material so that exposure to spores of these fungi is inevitable. Despite their ubiquitous existence, the infection caused by the Mucorales is not common owing to its low virulence but is an indication of a serious underlying predisposition ^[2][4].

The role of immune modulation in mucormycosis has been evident as natural killer (NK) cells produce cytokines and chemokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF-α), and interferon-gamma (IFN-γ), which influence the regulation of other immune cells and have both direct and indirect cytotoxic effects on the fungal growth. Necrotic areas have been evident in tissues with no obvious fungal development suggesting that thrombotic ischemia may occur due to the systemic platelet activation. Moreover, phagocytes release pro-inflammatory cytokines such as TNF-α, interleukin-1 beta (IL-1β), and IFN-γ which activate other immune cells. In this regard, for neutropenic patients with mucormycosis, the use of GM-CSF and granulocyte colony-stimulating factors (G-CSF) is highly recommended ^[15][28]. Nevertheless, the phagocytes of normal hosts kill Mucorales by the generation of oxidative metabolites and the cationic defensins ^[16][29] and therefore remain the major host defense mechanism against mucormycosis. However, evidence of the high prevalence of autoantibodies against immunomodulatory proteins (including cytokines, chemokines, complement components, and cell-surface proteins) in COVID-19 patients further supports the exacerbations associated with mucormycosis ^[17][30].

4. Diagnosis

Since some of the symptoms are shared between different sources of fungal infection, the specific diagnosis of mucormycosis can be evaluated through major pathologic manifestations such as areas of vasculitis with thrombosis, hemorrhage, and infarction. The identification of morphological differences between Mucorales and Aspergillus is also a commonly used laboratory practice to diagnose this fungal condition.

4.1. Microscopic Examination

Phenotypic observation and visualization of characteristic hyphae are one of the most definitive ways to characterize the differences between mucormycosis and aspergillosis owing to the former’s irregularly shaped and right-angled broad hyphae (10–20 µm in diameter) structure. Both can be differentiated by culture and pathological examination of biopsy specimens. In addition, certain Gram-negative bacilli such as Pseudomonas aeruginosa can mimic vasculitic lesions in skin or viscera as produced by Mucorales for which hyphae can be easily visualized in routine hematoxylin-eosin-stained sections or the periodic acid–Schiff reaction and Grocott–Gomori methenamine-silver nitrate stains ^[18][51]. A rapid diagnosis of mucormycosis could be made through direct histopathological examination of the tissue through fluorescence microscopy using brighteners such as Blankophor and Calcofluor White, which bind to chitin and cellulose in the fungal cell wall and fluoresce under ultraviolet light ^[8][10].

4.2. Imaging Methods

CT scans and magnetic resonance imaging (MRI) scans of the head typically reveal only evidence of sinus involvement associated with the opacification of sinuses and thickening of optic muscles, especially the medial rectus muscle as demonstrated by lucencies on a CT scan ^{[18][19][20][21][22]}[51,52,53,54,55]. Proptosis may also be evident in some cases. The results of an MRI scan may also reveal abnormalities in involved structures such as the ocular muscles and sinuses as a byproduct of prominent mucosal thickening and secretions. Another commonly reported abnormality is cavernous sinus thrombosis of vessels due to ischemic changes in the area of distribution by failure in the enhancement of the affected vessels ^[22][55]. In the absence of comparative studies, the benefits of CT vs. MRI are not known; however, MRI scanning could be a preferred measure for a diabetic patient for whom intravenous contrast agents may be contraindicated. In addition to imaging methods, functional and metabolic imaging using PET/CT coupled with [18F]-fluorodeoxyglucose (FDG) has been considered a valuable tool in the diagnosis and management of mucormycosis due to its sensitivity and accuracy in picking up anatomic abnormalities. Advanced biosensor-based diagnosis and detection of CAM are discussed in detail in ^[8][10].

4.3. Species Identification and Antifungal Susceptibility Testing

Species identification using commercially available kits has aided a better epidemiological understanding of mucormycosis and is indeed valuable for quick and reliable outbreak investigations. For instance, the ID32C kit (BioMerieux, Marcy l’E’ toile, France) has been used successfully for the identification of species such as Lichtheimia corymbifera, Lichtheimia ramose, and Rhizomucor pusillus; and API 50CH (BioMerieux) has been used for Mucor species ^[23][24][56,57]. Reproducible techniques for antifungal susceptibility testing are important methods to evaluate uniformity in reporting and to facilitate interlaboratory comparisons. Many factors influence in vitro susceptibility testing results, such as endpoint definition, inoculum size, incubation time, incubation temperature, and the medium used for testing. A range of traditional methods such as broth microdilution, disk diffusion, and agar screening, as well as commercial methods such as YeastOne and VITEK2, that are easy to set up and perform, are useful for this application ^[18][51]. Finally, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) (Bruker Biotyper, Germany, and Vitek MS France), mass spectrometry, and T2MR (T2 biosystems) are FDA-approved platforms involving the complex between particles coated with target-specific agents with respect to the altered microenvironment for the rapid detection of mucormycosis ^[8][25][10,58].

4.4. Molecular Assays

Molecular-biology-based assays, including conventional polymerase chain reaction (PCR) ^[26][59], restriction fragment length polymorphism analyses (RFLP) ^[27][60], DNA sequencing ^[28][61] of defined gene regions, and melt curve analysis of PCR products ^[29][62], can be used either for the detection of Mucorales based on internal transcribed spacer or the 18S rRNA genes ^[30][63]. Nonetheless, the low number of patients in culture-based studies is a limiting factor that results in varied sensitivity and specificity, never approaching 100%. Moreover, studies performed with formalin-fixed, paraffin-embedded, or fresh tissue samples ^[31][64] lead to variable results as well. Considering the lack of evaluation and other limiting factors, none of the culture or phenotypical approaches can be recommended as a standalone approach in clinical routine diagnostics ^[31][64]. In lieu of this, molecular-based diagnosis ^[32][33][65,66] has been gaining attention to yield promising results and confirm the culture-proven cases. Presently, molecular-based diagnostic assays from blood and serum can be recommended as valuable add-on tools that complement conventional diagnostic procedures.

4.4.1. Serology Assays

Serological techniques such as immunohistochemistry (IHC), enzyme-linked immunosorbent assays (ELISA), immunoblots, and immunodiffusion tests have been reported to highlight variable success as ELISA was observed to be more sensitive than double immunodiffusion (DID) tests. SDS-PAGE-based immunoblots responded positively to R. arrhizus antigens but recognized only a few of the 20–30 gel-separated bands. Mucorales-specific T cells versus control were successfully detected by an enzyme-linked immunospot (ELISpot) assay using heat-killed germinated conidia from patients with invasive mucormycosis ^[34][67]. Additionally, serum tests involving the use of a 1,3-beta-D-glucan assay yield promising results for the Mucorales group based on the presence of glucan in their spore cell walls ^[8][10].

4.4.2. Differential Diagnosis Methods

CAM is mostly characterized by histopathology or culture-based assays as broad, irregular, pauci septate hyphae whereas cryptococcosis and endemic mycoses such as fungal infections are identified by encapsulated yeast cells and budding spherules, respectively. However, unlike aspergillosis and candidiasis, CAM can be identified from both blood and BAL samples. Molecular assays such as Genus-NAAT (Nucleic Acid Amplification Test) and pan fungal PCR, other than mass spectrometry, are recommended for the identification of CAM (Figure 2. However, neither enzyme immunoassays nor antigen detection assays are very efficient, as for other non-CAM fungal species ^[35][68].