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|Year : 2014 | Volume
| Issue : 4 | Page : 383-386
Rhinocerebral mucormycosis: Pathology revisited with emphasis on perineural spread
Tumuluri Sravani, Shantveer Gurulingappa Uppin, Megha Shantveer Uppin, Challa Sundaram
Department of Pathology, Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad, Andhra Pradesh, India
|Date of Submission||01-Jul-2014|
|Date of Decision||08-Jul-2014|
|Date of Acceptance||12-Aug-2014|
|Date of Web Publication||19-Sep-2014|
Nizam's Institute of Medical Sciences, Punjagutta, Hyderabad - 500 082, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Aims and objectives: To study the histopathological features with particular emphasis on perineural invasion in invasive rhinocerebral mucormycosis. Materials and Methods: Tissue sections from 30 patients with invasive rhinocerebral mucormycosis were included in the study. Demographic features, predisposing conditions, and clinical features were obtained from medical records. Tissue sections were reviewed with hematoxylin and eosin (H and E), Gomori's methenamine silver (GMS), and periodic acid Schiff (PAS) stains for (i) the presence and type of inflammation (suppurative/granulomatous; sparse/absent), (ii) invasion into soft tissues, and (iii) type of spread (angio/perineural) and presence of infarction/necrosis and fungal morphology. Results: The study material included 20 males and 10 females with age ranging from 15-84 years. The clinical syndromes included rhino-orbital in 15, rhinocerebral in 6, and rhino-orbito-cerebral in 9 patients. On histopathological examination, inflammation was suppurative with predominance of neutrophils in 25 biopsies. Suppurating granuloma with neutrophils, lymphocytes, and foreign body giant cells was seen in 3 biopsies. Invasion into soft tissues, muscles, and adipose tissues was seen in 20 biopsies. Angioinvasion was noted in 25 and soft tissue invasion in 20 biopsies. Peripheral nerves were identified in 19 and perineural spread was identified in 15 biopsies. In all, biopsies with perineural invasion, angioinvasion, and soft tissue invasion were seen. Conclusions: Perineural invasion is one of the important histological features of invasive rhinocerebral mucormycosis and it indicates advanced the extent of invasion.
Keywords: Angioinvasion, mucormycosis, perineural invasion, rhinocerebral
|How to cite this article:|
Sravani T, Uppin SG, Uppin MS, Sundaram C. Rhinocerebral mucormycosis: Pathology revisited with emphasis on perineural spread. Neurol India 2014;62:383-6
| » Introduction|| |
Mucormycosis is an emerging infection associated with high mortality.  In developing countries such as India, most of the cases occur in patients with uncontrolled diabetes mellitus (DM) or trauma. , Rhinocerebral mucormycosis is the most common form of the disease. ,,,,, The infection spreads rapidly from sinus to orbit, cavernous sinus, and cranium, and if not diagnosed early and treated promptly, results in death.  The identification of risk factors, clinical features, and radiological findings increases the possibility of an early diagnosis, which may prevent progressive tissue invasion, reduce the need and/or extent of surgical resection, and improve survival.  However, clinical features and imaging studies are non-specific. Definite diagnosis requires demonstration of characteristic hyphae in tissue or recovery of the organism in the culture. Yield of organisms in culture is sub-optimal. Hence, histopathological examination plays a critical role in establishing the diagnosis and provides evidence of tissue invasion.
Rhinocerebral mucormycosis is rapidly progressive with marked increase in mortality when fungus penetrates the cranium and invades the intracranial vasculature.  Vascular invasion and neurotropism are considered the common pathologic features of invasive mucormycosis.  Perineural spread of fungus was considered unusual. ,, Recent studies on imaging of rhinocerebral mucormycosis demonstrated spread of Mucorales species for considerable distance from its primary focus of infection along peripheral nerves.  Hence, we aim to study the histopathological features with particular emphasis on perineural invasion in invasive rhinocerebral mucormycosis.
| » Materials and Methods|| |
This was a retrospective observational study. Thirty patients diagnosed with invasive rhinocerebral mucormycosis on tissue sections in the department of Pathology of our institute were included in the study. The demographic features, predisposing conditions, and clinical features were obtained from medical records. The clinical syndromes were classified as rhino-orbital, rhinocerebral or rhino-orbito-cerebral based on the presenting features such as unilateral periorbital facial pain, numbness, visual impairment/loss, eyelid edema, chemosis, proptosis, opthalmoplegia, multiple cranial nerve palsies, and focal neurological deficits. Bone destruction and invasion into soft tissues of the orbit or intracranial spread were confirmed on computed tomography (CT)/magnetic resonance imaging (MRI). Tissue sections were reviewed with hematoxylin and eosin (H and E), Gomori's methenamine silver (GMS), and periodic acid Schiff (PAS) stains. The tissue sections were examined for (i) the presence and type of inflammation (suppurative/granulomatous; sparse/absent), (ii) invasion into soft tissues (muscle, adipose tissue), (iii) type of spread (angio/perineural) and presence of infarction/necrosis. Fungal morphology delineated by H and E, GMS, and PAS as broad pauci/aseptate hyphae with irregular/right angle branching were identified as Mucorales species. Presence of mixed infection, if any, was noted. Culture reports wherever tissue was submitted were collected.
| » Results|| |
The study included 20 males and 10 females with age ranging from 15 to 84 years (median, 50 years). Predisposing factors were identified in 25 patients, which included DM in 23, trauma with maceration of face in one, and acute myeloid leukemia on treatment in one. The clinical syndromes included rhino-orbital in 15, rhinocerebral in 6, and rhino-orbito-cerebral in 9 patients. Tissue for culture was submitted in 10 patients. Culture was sterile in 2 and yielded Rhizopus oryzae in 8 patients.
The tissue submitted for histopathological examination included biopsy in 20, and debridement in 10. Inflammation was present in 28 biopsies, and it was sparse to absent in two biopsies. Inflammation was suppurative with predominance of neutrophils in 25 biopsies. Suppurating granuloma with neutrophils, lymphocytes, and foreign body giant cells was seen in 3 biopsies. Necrosis was present in all biopsies, and areas of infarction along with necrosis were seen in 25 biopsies. Invasion into soft tissues, muscles, and adipose tissue was seen in 20 biopsies. Angioinvasion was noted in 25 biopsies. Peripheral nerves were identified in 19 biopsies only and perineural spread was identified in 15 biopsies. The nerves involved did not show any congestion and were negative for iron pigment. In all biopsies with perineural invasion [Figure 1] and [Figure 2], angioinvasion and soft tissue invasion [Figure 3] were seen. Mixed infection of Mucorales with Candida species was seen in one patient.
|Figure 1: Section shows segment of nerve (black arrows) with perineural invasion by broad aseptate hyphae branching at right angle (black arrowheads) consistent with mucor species (H and E ×100)|
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|Figure 2: Section show segment of nerves (black arrows) with perineural invasion by broad aseptate hyphae (black arrowheads) consistent with mucor species (SM stain; ×100)|
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|Figure 3: Sections show (a) Infracted soft tissue with scattered fungal hyphae (black arrows) (H and E stain; ×100); (b) show angioinvasion by broad aseptate hyphae (black arrows) consistent with mucor species (SM stain; ×100)|
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| » Discussion|| |
Mucormycosis remains a threat for patients with DM as the incidence of DM reached epidemic proportions in countries such as India.  DM was identified as the most common risk factor for rhinocerebral mucormycosis in earlier studies from India as well as in the present study. ,,, After inhalation of the spores, the infection spreads rapidly into the adjacent tissues. It extends into cavernous sinus, orbit, and cranium.  Extensive angioinvasion with resultant vessel thrombosis and tissue necrosis is the pathological hallmark of mucormycosis infections. , Angioinvasion with subsequent infarction of the surrounding tissues was noted in all cases of severe disseminated/invasive mucormycosis. , Necrosis with or without infarction was seen in all samples in the present study, whereas angioinvasion was demonstrated in only 83.33% samples. This may be due to limitation of sampling in a biopsy. Angioinvasion facilitates dissemination of fungus from site of infection to distant sites.  Hyphae of Mucorales species are broad, ribbon-like, and aseptate/pauci septate with wide angle bifurcation. The broad hyphae invade and occlude intermediate/large-sized arteries/veins resulting in pale/hemorrhagic infarcts. 
The morphology of the fungus was delineated on H and E, GMS, and PAS in all our samples, and co-infection by Candida species was identified in one biopsy. Histopathiogical examination of tissue allows differentiation of Mucorales species from other angioinvasive fungi such as Aspergillus spp., Scedosporidium spp., and Fusarium spp.  Histological examination showed predominantly neutrophilic infiltrate in 25/30 (83.33%), suppurating granulomatous in 3/30 (10%), and sparse/absent inflammation in 2/30 samples.
Apart from angioinvasion in rhinocerebral mucormycosis, direct spread through cribriform plate into the anterior cranial fossa can occur, and it was suggested that this represents perineural spread.  Histologically proven perineural extension of disease from cavernous sinus to pons along the trigeminal nerve with no apparent meningeal or intraparenchymal brain involvement was reported by Mc Lean et al. (1996).  Perineural invasion was considered unusual but contrast-enhanced MRI studies have documented perineural invasion via the trigeminal nerve. ,,, Cornley et al. (2014) reported perineural invasion in 90% of biopsies that contained peripheral nerves.  In our series of invasive rhinocerebral mucormycosis, perineural invasion was histologically identified in 15/19 (72.1%) samples, where peripheral nerves were present in the biopsy. Frater et al. (2001) reported high percentage of perineural invasion in patients with invasive mucormycosis and hypothesized that pereineural invasion was another possible mechanism involving extension of the fungi into central nervous system.  In our series, perineural invasion was associated with angioinvasion and soft tissue extension (into muscles, adipose tissue, and fibrous connective tissue) in all samples, indicating the advanced extent of invasion. However, perineural invasion was not reported on MRI in our patients.
Culture studies were available in 10 patients and R. oryzae was isolated in 8 samples. Culture was sterile in 2 patients. Limitations of culture studies were stressed in earlier studies. , Parsi et al. (2013) reported rhino-orbito-cerebral mucormycosis caused by Apophysomyces elegans with perineural spread of infection in an immunocompetent individual.  Implantation of spores by trauma, especially with A. elegans is known.  One of our patients had rhinocerebral mucormycosis following trauma with maceration of face, and culture yielded R. oryzae. Perineural invasion on histology had no correlation with the organism in our study.
Mucorales species are able to produce various toxic proteins and metabolic products but the pathogenic role of these potential virulence factors is unknown. Availability of free iron in plasma and tissues is believed to be crucial for the pathogenesis of vascular invasion. , Nerve microenvironment and neurotropic factors secreted in a gradient along nerves may play a pivotal role in the pathogenesis of perineural invasion. ,
Perineural invasion is a frequent pathological finding in head and neck, pancreatic, prostate, and other malignancies, and is a marker of poor outcome with increased locoregional recurrence rates and decreased survival.  The various hypotheses for this mechanism include neural sheath as low-resistance conduit for tumor cell growth, signaling amongst tumors, nerve, and stromal cells through paracrine mechanisms, upregulation of neurotropic and axonal guidance molecules, and activation of cancer cell chemotaxis, directional cell migration towards and along nerves. 
Availability of free iron in plasma and tissues in acidotic conditions leading to angioinvasion and neurotropism along with limited activity of antifungal agents are implicated in high mortality seen in mucormycosis. , Understanding the pathogenesis is needed for newer therapeutic interventions. Baskt et al. (2012) reported that radiotherapy might impair perineural invasion in malignancies by direct effect on cancer cells and also by potentially altering the nerve microenvironment through disruption of neurotropic agents. 
In conclusion, perineural invasion is one of the important histological features of invasive rhinocerebral mucormycosis and it indicates the advanced extent of invasion. Awareness and efforts to identify perineural invasion on histology are warranted for early diagnosis.
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[Figure 1], [Figure 2], [Figure 3]