Approach Considerations
Timely diagnosis is paramount in cases of mucormycosis. [63] Diagnosis of mucormycosis requires positive culture and histopathological changes. However, there are certain instances where culture may not be available or results may be negative; in such instances, diagnosis is made by histopathology alone. [64]
Patients with suspected or confirmed mucormycosis should be treated as a medical emergency; they should be referred to institutions that can perform highest level of care. [39] The European Confederation Medical Mycology (ECMM) society lays out a diagnostic approach based on symptoms and underlying risk factors [39] :
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Among patients with respiratory symptoms (fever, cough) who are neutropenic, chest computed tomography (CT) should be obtained; CT guided biopsy or bronchoalveolar lavage (BAL) with biopsy should be obtained and the specimen should be sent for histopathology, culture with susceptibility testing and molecular identification with PCR, and multiplex target (18s, ITS, 28s, or ribosomal DNA). [39]
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Among patients with facial pain, sinusitis, and underlying diabetes, cranial CT or magnetic resonance imaging should be obtained, followed by biopsy; the specimen should be sent for: histopathology (GMS and PAS stain), culture with susceptibility testing and molecular identification with PCR testing, multiplex target (18s, ITS, 28s, or ribosomal DNA). [39]
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Among patients presenting with history of trauma and cutaneous manifestation, biopsy of the lesion (escher) should be obtained and sent for histopathology, culture with susceptibility testing and molecular identification with PCR testing, multiplex target (18s, ITS, 28s, or ribosomal DNA). [39]
Laboratory Tests
A complete blood cell (CBC) count should be obtained to assess for neutropenia. A chemistry panel that includes blood glucose, bicarbonate, and electrolytes is useful to monitor homeostasis and correction of acidosis. An arterial blood gas (ABG) study can help determine the degree of acidosis and guide corrective therapy. Iron studies may be indicated to assess the presence of iron overload as shown by high ferritin levels and a low total iron-binding capacity. In cases of central nervous system (CNS) involvement, cerebrospinal fluid (CSF) findings may include elevated protein levels and a modest mononuclear pleocytosis. CSF fungal stain and cultures are typically sterile. A CT scan should precede a lumbar puncture to assess for evidence of space occupying lesions, which could lead to herniation.
Blood cultures can be obtained; however, they are usually negative despite the angioinvasive nature of the organism. Blood cultures may be useful to detect bacteremia in addition to Mucorales infection. One study of pulmonary mucormycosis identified concurrent bacteremia as an independent predictor of 28-day mortality. [59] There are no specific biomarkers to identify mucormycosis. Bronchoalveolar lavage (BAL) of fluid culture has a low yield, with a sensitivity of 20%-50%. Antigen tests (beta-D-glucan or galactomannan) are not useful for detecting this infection. [28] ECMM guidelines strongly suggest obtaining a culture of the specimen to identify the organism. [39] Cultures should be incubated at 30°C and 37°C; direct microscopy should be done mainly to identify septation, branching angle, and hyphal width. [39] ECMM guidelines strongly recommend susceptibility testing to expand epidemiological knowledge, although general use of standard methods for susceptibility testing for Mucorales are marginally reinforced as there are no cut-off values established by the European Committee on Antimicrobial Susceptibility testing (EUCAST) or Clinical and Laboratory Standards Institute (CLSI). [39]
Molecular based testing is moderately supported; fresh tissue is preferred over paraffin tissue, as formalin damages DNA. [39] The use of molecular based testing has moderate support only, due to lack of standardization. The use of quantitative polymerase chain reaction (qPCR) for detection of circulating DNA from common Mucorales species (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species), although not yet commercially available, has been described and appears promising for the early diagnosis of mucormycosis in high-risk patients. [65, 66] In a retrospective analysis of 44 cases, qPCR identification was fully concordant with that of culture. Assay positivity was observed at an average of 9 days, at least 2 days before positive imaging findings. Development of PCR negativity after treatment was associated with higher survival rates (48% vs 4%), suggesting that this modality could eventually be used for treatment monitoring. [67] DNA detection can be looked for in CSF or BAL clinical samples. [68, 69, 70, 71]
Radiologic Studies
Imaging should be used to investigate areas of suspected mucormycosis. Because subclinical disease may be present, a thorough history and physical examination are recommended in addition to imaging (CT) of the brain, sinuses, chest, and abdomen. [72]
Rhinocerebral infections
Plain films may show sinus involvement with mucosal thickening, air-fluid levels, and/or bony erosions. [58]
Head and facial CT imaging should be used as the initial investigation in rhinocerebral infections. CT scans may show sinusitis of the ethmoid and sphenoid sinuses, as well as orbital and intracranial extension. As the disease progresses, bony erosion may occur and the infection may spread into the brain or orbits. Additionally, because mucormycosis organisms have a predilection for vascular involvement, thromboses of the cavernous sinus or internal carotid artery may occur. [73] All areas of involvement must be understood, to plan the extent of surgical debridement.
Magnetic resonance imaging (MRI) of the facial sinuses and brain is superior to a CT scan in assessing the degree of tissue invasion and need for ongoing surgery.
Rhino-orbital-cerebral mucormycosis in a patient with SARS-CoV-2 infection. Courtesy of Dr Sujata Rege.
Pulmonary disease
Chest radiography is often the initial test performed; however, its sensitivity and specificity for mucormycosis are low. Nonenhanced high-resolution CT scanning is the imaging modality of choice. [28] The most common findings include pleural effusion, nodules, consolidation, and ground-glass opacities. [59] With disease progression, consolidation can become multilobar. The reverse halo sign (ie, a nodule with central ground-glass opacity and a ring of peripheral consolidation) strongly suggests pulmonary mucormycosis and is rarely seen in invasive aspergillosis. [74] The halo sign (ie, a nodule surrounded by ground-glass opacity) represents a lung infarct surrounded by alveolar hemorrhage; it is associated with invasive mold infections but can be present in bacterial or viral infections and noninfectious lung disease (eg, Wegener granulomatosis, sarcoidosis, malignancy). [28] Other findings such as the air crescent sign and hypodense sign are less specific and may occur in later stages.
Chest computed tomography (CT) scan showing pulmonary mucormycosis with left basal consolidation and widespread nodules due to Rhizopus oryzae infection. The patient was receiving cytotoxic chemotherapy for myelodysplastic syndrome and had iron overload from numerous blood transfusions.
Pulmonary mucormycosis in a patient with SARS-CoV-2 infection. Courtesy of Dr Sujata Rege.
Gastrointestinal disease
In gastrointestinal (GI) disease, abdominal CT scans may show a mass associated with the GI tract. Esophagogastroduodenoscopy (EGD) may show areas of tissue necrosis amenable to biopsy.
Central nervous system
CT scanning or MRI of the central nervous system may reveal abscesses (especially in the setting of intravenous drug use) or extension of rhinocerebral disease into the brain. Cavernous and, less commonly, sagittal sinus thrombosis may also be seen.
Brain MRI (sagittal view) in a patient with uncontrolled diabetes who presented with progressive right eye pain and facial swelling. He underwent multiple surgeries to control rhinocerebral Mucor infection, including partial right frontal lobectomy and right orbital exenteration. He was treated with amphotericin B and his diabetes mellitus was controlled. The disease did not progress, and long-term isavuconazole therapy was initiated for salvage/maintenance therapy.
Biopsy and Histologic Features
Biopsy of the involved tissue is the most definitive means of establishing a diagnosis of mucormycosis. A rapid histologic assessment of a frozen tissue section should be performed to promptly institute surgical and medical management for the infection.
Biopsy of necrotic tissue
Biopsy of necrotic tissue may be obtained from nasal, palatine, lung, cutaneous, gastrointestinal (GI), or abscess wall site.
Stains of fixed tissues with hematoxylin and eosin (H&E) or specialized fungal stains, such as Grocott methenamine-silver (GMS) or periodic acid-Schiff (PAS) stains, show pathognomonic broad (typically 6- to 25-µm diameter), irregular, ribbonlike, nonseptate (or sparsely septate) hyphae with irregular branching occurring at 45-90º. [75] Vascular invasion and necrosis are the characteristic consequences of the infective process. Thus, neutrophil infiltration, vessel invasion, and tissue infarction are often observed. A granulomatous reaction may also be seen.
Histologic findings from an immunocompetent man who sustained a high-pressure water jet injury, resulting in rhinocerebral mucormycosis. Traumatic inoculation of Apophysomyces elegans was the pathogenetic mechanism. Findings show the typical Mucorales hyphae on Grocott methenamine-silver staining. The hyphae are the dark structures with budlike, right-angle hyphae. Courtesy of A Allworth, MD, Brisbane, Australia.
Culture of biopsy samples is generally required to determine the species of Mucorales. Do not crush or grind the specimen, because the nonseptate hyphae are prone to damage. Growth usually occurs in 2-3 days. The genus and species are determined via examination of the fungal morphology (ie, the presence and location of the rhizoids). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) can also provide rapid and accurate identification at the species level but requires a reference database. 18s ribosomal RNA (rRNA) sequencing may provide genus-level identification, even if tissue damage precludes fungal growth. [28]
Laboratory Studies
A complete blood cell (CBC) count should be obtained to assess for neutropenia. A chemistry panel that includes blood glucose, bicarbonate, and electrolytes is useful to monitor homeostasis and correction of acidosis. An arterial blood gas (ABG) study can help determine the degree of acidosis and guide corrective therapy. Iron studies may be indicated to assess the presence of iron overload as shown by high ferritin levels and a low total iron-binding capacity. In cases of central nervous system (CNS) involvement, cerebrospinal fluid (CSF) findings may include elevated protein levels and a modest mononuclear pleocytosis. CSF fungal stain and cultures are typically sterile. A CT scan should precede a lumbar puncture to assess for evidence of space occupying lesions, which could lead to herniation.
Blood cultures can be obtained; however, they are usually negative despite the angioinvasive nature of the organism. Blood cultures may be useful to detect bacteremia in addition to Mucorales infection. One study of pulmonary mucormycosis identified concurrent bacteremia as an independent predictor of 28-day mortality. [59] There are no specific biomarkers to identify mucormycosis. Bronchoalveolar lavage (BAL) of fluid culture has a low yield, with a sensitivity of 20%-50%. Antigen tests (beta-D-glucan or galactomannan) are not useful for detecting this infection. [28] ECMM guidelines strongly recommend obtaining a culture of the specimen to identify the organism. [39] Cultures should be incubated at 30°C and 37°C; direct microscopy should be done mainly to identify septation, branching angle, and hyphal width. [39] ECMM guidelines strongly recommend susceptibility testing to expand epidemiological knowledge, although generally use of standard method for susceptibility testing for Mucorales is marginally reinforced as there are no cut-off values established by European Committee on Antimicrobial Susceptibility testing (EUCAST) or Clinical and Laboratory Standards Institute (CLSI). [39]
Molecular based testing is moderately supported; fresh tissue is preferred over paraffin tissue, as formalin damages DNA. [39] The use of molecular based testing has only moderate support due to lack of standardization. The use of quantitative polymerase chain reaction (qPCR) for detection of circulating DNA from common Mucorales species (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species), while not yet commercially available, has been described and appears promising for the early diagnosis of mucormycosis in high-risk patients. [65, 66] In a retrospective analysis of 44 cases, qPCR identification was fully concordant with that of culture. Assay positivity was observed at an average of 9 days, at least 2 days prior to positive imaging findings. Development of PCR negativity after treatment was associated with higher survival rates (48% vs 4%), suggesting that this modality could eventually be used for treatment monitoring. [67] DNA detection can be looked for in CSF or BAL clinical samples. [68, 69, 70, 71]
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Postmortem photograph of a woman with diabetes and left rhinocerebral mucormycosis complicating ketoacidosis. Rhizopus oryzae was the causative organism. Note the orbital and facial cellulitis and the black nasal discharge. Courtesy of A Allworth, MD, Brisbane, Australia.
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The right eye of an immunocompetent man who sustained a high-pressure water jet injury, resulting in rhinocerebral mucormycosis. Traumatic inoculation of Apophysomyces elegans was the pathogenetic mechanism. Note the proptosis. Courtesy of A Allworth, MD, Brisbane, Australia.
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The right eye of an immunocompetent man who sustained a high-pressure water jet injury, resulting in rhinocerebral mucormycosis. Traumatic inoculation of Apophysomyces elegans was the pathogenetic mechanism. Chemosis is shown in this photograph. Internal and external ophthalmoplegia, no light perception, and afferent pupil defect were present, which is consistent with orbital apex syndrome. Courtesy of A Allworth, MD, Brisbane, Australia.
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An immunocompetent man who sustained a high-pressure water jet injury, resulting in rhinocerebral mucormycosis. Traumatic inoculation of Apophysomyces elegans was the pathogenetic mechanism. A surgical field of this patient is shown. Excision of the right orbit, maxillary antrum, nasal cavity, sphenoid sinus, and infratemporal fossa has taken place. The tissue was infarcted. Courtesy of A Allworth, MD, Brisbane, Australia.
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An immunocompetent man who sustained a high-pressure water jet injury, resulting in rhinocerebral mucormycosis. Traumatic inoculation of Apophysomyces elegans was the pathogenetic mechanism. Picture of the patient after successful treatment with repeated surgical debridement and high-dose liposomal amphotericin B. Courtesy of A Allworth, MD, Brisbane, Australia.
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Histologic findings from an immunocompetent man who sustained a high-pressure water jet injury, resulting in rhinocerebral mucormycosis. Traumatic inoculation of Apophysomyces elegans was the pathogenetic mechanism. Findings show the typical Mucorales hyphae on Grocott methenamine-silver staining. The hyphae are the dark structures with budlike, right-angle hyphae. Courtesy of A Allworth, MD, Brisbane, Australia.
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Chest computed tomography (CT) scan showing pulmonary mucormycosis with left basal consolidation and widespread nodules due to Rhizopus oryzae infection. The patient was receiving cytotoxic chemotherapy for myelodysplastic syndrome and had iron overload from numerous blood transfusions.
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Chest computed tomography (CT) scan showing pulmonary mucormycosis with left basal consolidation and widespread nodules due to Rhizopus oryzae infection. The patient was receiving cytotoxic chemotherapy for myelodysplastic syndrome and had iron overload from numerous blood transfusions. This CT scan of the patient shows resolution of pulmonary mucormycosis after 5 months of antifungal treatment.
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Brain MRI (sagittal view) in a patient with uncontrolled diabetes who presented with progressive right eye pain and facial swelling. He underwent multiple surgeries to control rhinocerebral Mucor infection, including partial right frontal lobectomy and right orbital exenteration. He was treated with amphotericin B and his diabetes mellitus was controlled. The disease did not progress, and long-term isavuconazole therapy was initiated for salvage/maintenance therapy.
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Rhino-orbital-cerebral mucormycosis in a patient with SARS-CoV-2 infection. Courtesy of Dr Sujata Rege.
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Pulmonary mucormycosis in a patient with SARS-CoV-2 infection. Courtesy of Dr Sujata Rege.
