| Abstract|| |
Rhino-orbito-cerebral zygomycosis is a rapidly progressive opportunistic fungal infection characterized by a set of clinical and radiological findings that help in prognostication. The present study is aimed to evaluate its histopathologic features as an aid to prognosis in order to guide the physician at the stage of tissue diagnosis to optimize surgery, chemotherapy and immunosuppression. The study comprises of a microscopic analysis of specific histopathologic variables on 33 cases of zygomycosis that were diagnosed and treated in a seven-year period. Fungal load in the tissue (graded as mild, moderate and marked), mean diameter of fungus, degree of neutrophilic and granulomatous response, tissue invasion and necrosis were graded and assessed for their prognostic significance. Seasonal variation, signs and symptoms, extent of involvement and laboratory parameters were also analyzed to examine the trend of occurrence of the disease and to associate these with patient's outcome, which was categorized as either survived or expired. The follow-up ranged from 1 month to 7 years. Of all the histological variables, angioinvasion was directly related to the mortality rate. Diameter of the fungal hyphae and its intraorbital or intracranial invasion also proved to be significant indicators of poor prognosis. (P = 0.04 and 0.0037, respectively) though angioinvasion was directly related to the mortality rate. Thus, histopathology could assist the clinician in assessing patient's progress and thus optimize the treatment in such patients.
Keywords: Histopathology, mucormycosis, rhino-orbito-cerebral zygomycosis, zygomycosis
|How to cite this article:|
Goel A, Kini U, Shetty S. Role of histopathology as an aid to prognosis in rhino-orbito-cerebral zygomycosis. Indian J Pathol Microbiol 2010;53:253-7
|How to cite this URL:|
Goel A, Kini U, Shetty S. Role of histopathology as an aid to prognosis in rhino-orbito-cerebral zygomycosis. Indian J Pathol Microbiol [serial online] 2010 [cited 2020 Sep 21];53:253-7. Available from: http://www.ijpmonline.org/text.asp?2010/53/2/253/64342
| Introduction|| |
Invasive zygomycosis is a rare but aggressive opportunistic fungal infection associated with angioinvasion and infarction. It is being reported with increasing frequency in recent years. ,, The number of patients at risk for zygomycosis are also increasing as more and more patients seem to live longer with relative immunocompromisation.  The disfigurement caused by radical surgery, which is the primary mode of treatment has a great deal of physical and psychological ill-effects on the patient. Amphotericin B, the drug of choice for zygomycosis has also a very high nephrotoxicity. In this scenario, if only the histological features could forecast patient's progress, the extent of surgery, the dose and duration of administration of amphotericin B and discontinuation of the immunosuppressive therapy etc. can be optimally practiced. Such studies in histopathology to prognosticate zygomycosis have not been carried out so far. The relative deficiency of this literature has prompted this present study over a seven-year period in a tertiary health centre so as to aid in the treatment of patients with zygomycosis.
Although most of the cases of zygomycosis of sinonasal area are caused by Mucorales, we still prefer the term zygomycosis to mucormycosis, as fungal culture is not always possible to confirm Mucorales infection.
| Materials and Methods|| |
The study included all cases of zygomycosis that were diagnosed and treated in a seven-year period. The clinical data and laboratory findings were retrieved from medical records department and analysed. Frozen sections and histopathology sections with their special stains of all the selected cases were also studied.
Only those cases with clinical features suggestive/suspicious of zygomycosis and exhibiting characteristic broad, pauci-septate or aseptate, ribbon-like hyphae with wide-angle branching diagnostic of zygomycosis at histology were taken for the study.
Those patients with clinical features suspected of zygomycosis but could not be confirmed at histopathology or those diagnosed but not treated at the hospital or those cases diagnosed only at post-mortem were excluded from the study. The patients who were lost to follow-up or those whose follow-up data was not available were also excluded
Seasonal variation, signs and symptoms, site of involvement, radiological data and laboratory parameters (blood and urine sugar values, ketonuria and renal function tests) were also analyzed to examine the trend of occurrence of the disease and associate these with outcome of patients. The follow-up ranged from 1 month to 7 years. The outcome was categorized as either survived or expired.
The tissue samples sent for diagnosis were examined grossly and processed as for routine paraffin sections. These tissue preparations were stained with Hematoxylin and Eosin (H and E), Periodic acid - Schiff (PAS), Gomori's methenamine Silver (GMS) and Ziehl-Neelsen stain (in cases of granulomatous inflammation). Cytologic material where available, was sent in the form of fine needle aspiration cytology, squash/imprint or scrape smears.
The sample preparations were studied for the following microscopic details and each of them were associated with patient outcome as survived and expired to evaluate their prognostic importance.
- Fungal load (graded as mild, moderate and marked).
- Morphometry of fungal elements
Mean diameter of the fungus in each case was calculated by taking the average of diameters of five randomly selected hyphae in the area of maximum fungal load under high power objective (x400). The diameter were measured using Leitz Wetzler ocular micrometer.
- Composition of inflammatory infiltrate
The presence of neutrophils and granulomatous response was graded (mild and moderate to marked) depending on the intensity of the infiltrate.
- Degree of tissue necrosis
Tissue necrosis was semi-quantitated as percentage of total area of the tissue sampled.
- Tissue invasion by fungus
Invasive fungal sinusitis was diagnosed  when hyphal forms were identified within sinus mucosa or submucosa, blood vessel or bone and was graded as mild and moderate to marked depending on the load of concentration of fungal hyphae within these tissues.
Invasive rhino-orbito-cerebral zygomycosis was further categorized as acute 4 when the duration of signs and symptoms was equal to or less than 4 weeks and when vascular invasion was prominent; and chronic when the duration of signs and symptoms was more than 4 weeks and vascular invasion was absent or minimal. 
The survival rates were analysed with the Chi-square test with Yates' correction. Inferences were made at the 0.05 level of significance. When the expected cell value was less than 5, Fisher exact results were employed.
| Results|| |
A total of 33 patients of zygomycosis were clinically diagnosed and confirmed at histology during the seven-year study period.
Seasonal trend of the disease was prominently noted with maximum number of cases 61%, (n = 20)) occurring during the months, June to September, 21% (n = 7) cases from October to January and 18% ( n = 6) from February to May.
Males predominated (70%, n = 23), with the male : female ratio being 2.3 : 1. The patients age ranged from 15 to 75 years with the larger incidence in the 41-60 year (48%) age group. Approximately 94% ( n = 31) were immunocompromised with diabetes mellitus (85%,( n = 28) as the commonest factor among these patients. One patient had steroid-induced diabetes mellitus, one had renal allograft and one had chronic alcoholism as the predisposing factor. Two patients (6%) had no significant positive history for predisposition.
Of the total 33 cases of invasive type, 8 cases were of chronic rhino-orbito-cerebral type and all of them survived. Twenty five cases were of acute rhino-orbito-cerebral and only 48% ( n = 12) cases survived ( P = 0.01, significant).
All the cases presented with various signs and symptoms. Mucosal necrosis was seen in 48% ( n = 16) of cases and external ophthalmoplegia in 59% ( n = 19) of the cases. [Table 1] shows frequency of signs and symptoms of rhino-orbito-cerebral zygomycosis in 33 patients. The most serious complication of the disease was involvement of the brain, which occurred in 18% of the cases ( n = 6) and were characterized by hemiplegia, altered mental status, stupor and coma. Thirteen of 14 patients survived with limited sinonasal disease, while only 7 of 19 patients survived with intraorbital or intracranial spread ( P = 0.0037, significant). [Table 2] shows the significance of various histopathologic variables as prognostic indicators in these cases of rhino-orbito-cerebral zygomycosis
The plain sinus radiographs where available, showed mucosal thickening, multiple sinus involvement, absence of air fluid levels and spotty destruction of bony walls. The computerized tomography (CT) pictures showed pansinus mucosal thickening, destruction of bone, thickening of extraocular muscles and evidence of intracranial extension such as bifrontal lucencies without mass, abscess formation and destructive changes in the floor of anterior cranial fossa. Magnetic resonance imaging done in occasional cases was helpful in identifying early intracranial extension.
Most of the patients with diabetes had uncontrolled blood sugar levels at presentation with glycosuria from 1+ to 4+. Ketonuria was present in 10 of the total 33 cases of rhino-orbito-cerebral mucormycosis. Survival rate for these 10 patients was 40% (4/10) and of those without ketonuria was 74% (17/23) ( P = 0.1, not significant , Odd's ratio = 4.25).
Renal function tests were done in all the patients to monitor the side-effects of amphotericin B therapy. Reduced nephrotoxicity was observed in patients who were on liposomal amphotericin B and the latter was given if they could not tolerate amphotericin B.
The tissue samples sent for frozen and paraffin sections were studied along with their special stains. The tissue samples were chiefly mushy, friable and grey-white to black in color.
All the cases of rhino-orbito-cerebral zygomycosis, who were histologically analysed were of invasive type. The characteristic hyphae of Zygomycetes, which were broad, ribbon-like and predominantly aseptate with wide-angle branching, were better visualized with H and E stains than with special stains. They were predominantly stained by Hematoxylin in H and E, while a few were eosinophilic [Figure 1]a. Occasional cross-walls were observed in a few hyphae. Similar features were observed in frozen sections and cytological smears but invasion could not be usually determined in the latter. Of 33 cases, four showed mild fungal load, eight moderate and 21 marked load. The load was found to be more in the areas of necrosis. In cases of granulomatous inflammation, the number of fungal filaments within the granulomata were few and were sometimes well appreciated only with PAS and GMS stains [Figure 1]b When the fungal load was mild, the survival rate was 75% ( n = 3/4) and when it was moderate, the survival rate was 63% ( n = 5/8) and when marked, it was 57% ( n = 12/21).
The average diameter of five randomly selected fungal hyphae ranged from 5 to 12 microns; 16 cases had an average diameters ranging from 5 to 8 microns and in 17 cases it ranged from 9 to 12 microns; the survival rate was 81% (13/16) and 41% (7/17), respectively ( P = 0.04, significant). Broader hyphae were seen in association with high fungal load.
Granulomatous inflammation noted in 11/33 cases (33%) were composed of macrophages, multinucleate giant cells and lymphocytes with central necrosis. The fungal hyphae were identified in the necrotic areas and within the multinucleate giant cells at foci. Eight of the 25 cases of acute rhino-orbito-cerebral zygomycosis and three of the 8 cases of chronic rhino-orbito-cerebral zygomycosis showed granulomatous inflammation. Five of the 11 cases with moderate to marked ( n = 5/11) granulomatous inflammation had 100% survival rate, and those who had mild ( n = 6/11) granulomatous inflammation, the survival rate was only 67% ( n = 4/6) ( P = 0.4, not significant).
Neutrophilic infiltration was mild in 6 cases and moderate to marked in 27 cases. No definite pattern of association of neutrophilic inflammation was seen with the outcome of the patients.
All the 33 (100%) cases of zygomycosis showed tissue necrosis. Necrosis was classically pale basophilic with minimal inflammatory response and fungal hyphae were distinctly seen in it. The percentage of necrosis varied from 2 to 95% and the density of fungal organisms was highest in necrotic tissues. Although necrosis was found at microscopy in all the patients of zygomycosis, interestingly, no association of the extent of necrosis was found with the outcome of patients.
Angioinvasion was seen in 17 (51%) of the 33 patients. Those blood vessels showed either viable or part of the necrotic focus. The fungal hyphae were identified in the wall of the blood vessels [Figure 2]a or forming thrombi at foci with necrosis of adjacent tissue. Both veins and arteries were involved by the process. The degree of angioinvasion was inversely related to the survival rate. Cases with mild vascular invasion had a survival rate of 100% (4/4), those with moderate to marked degree of vascular invasion had a survival rate of 46% (6/13) ( P = 0.102).
Perineural/neural invasion [Figure 2]b seen in 9 (27%) cases showed no consistent pattern of association with the outcome of patients.
Bone marrow invasion was noted in 7 (21%) cases with hyphae localized in the bone marrow. Cases with mild invasion had a survival rate of 100% ( n = 2/2), while with moderate to marked invasion had a survival rate of 60% ( n = 3/5) ( P = 1, not significant).
Fungal cultures could be carried out in 10 cases. Six (60%) showed distinct growth for Mucorales, while 4 showed no growth. The Mucorales produced fluffy white/gray, or brownish colonies.
| Discussion|| |
Zygomycosis is a term used to refer to fungal infections caused by order Mucorales, which belong to the class Zygomycetes.  In majority of the cases till today, the infection had been identified as Mucormycosis based on the morphological findings alone and not on culture as it could comprise infection caused not only by genera Mucor but also by Absidia, Rhizopus and Rhizomucor. This led physicians to identify the broad fungal class Zygomycetes found in tissue sections as Zygomycosis which is the currently accepted terminology.
The term rhino-orbito-cerebral zygomycosis (ROCM) refers to the entire spectrum of the disease, which usually starts in the sino-nasal tissue (limited sino-nasal disease), progresses to the orbits (limited rhino-orbital disease) and finally affects central nervous system (rhino-cerebral disease). ,,
There are two basic types of fungal rhinosinusitis: invasive and noninvasive. The invasive form may be of acute invasive and chronic invasive (both granulomatous and non-granulomatous forms). The noninvasive fungal rhinosinusitis can be of three types: fungus balls (occasionally referred to as mycetomas), saprophytic colonization and allergic fungal rhinosinusitis. , Zygomycosis commonly presents as either acute invasive or chronic invasive form. Cases reported as fungus balls, saprophytic colonization and allergic fungal sinusitis associated with zygomycosis are quite rare.  All the cases in the present study were of invasive type.
The probable reason for the seasonal distribution, namely, the higher incidence of 61% ( n = 20) of zygomycosis from June to September can be the high degree of humidity with an optimal temperature range of 28-30°C during these months in tropical areas, which is well suited for the growth of Mucorales. Earlier studies reflect a survival rate of 66.5% among 18 patients with rhino-orbito-cerebral  and 60% among 145 review cases.  Our results also show a survival rate of 61% in rhino-orbito-cerebral zygomycosis with the conventional management of rhino-orbito-cerebral zygomycosis, which includes control of metabolic abnormality, administration of amphotericin B and surgery. The survival rate for the sinonasal form was 93% (13/14), while 37% (7/19) for rhino-orbital disease or rhino-orbito-cerebral disease ( P = 0.0037). Thus the extent of involvement by zygomycosis determines the prognosis of the disease significantly.
When the fungal load in the tissue was categorized as mild, moderate and marked, the survival rate was 75, 63 and 57%, respectively ( P = 0.7). So we can conclude that as the fungal load in the tissue increases, the survival rate decreases. It is assumed that the patients who have a high fungal load have a state that favors fungal growth and are relatively more immunocompromised. The patients who had fungal hyphal diameter ranging from 5 to 8 microns had 81% survival rate as compared to 41% in patients who had fungal hyphal diameter ranging from 9 to 12 microns ( P = 0.04). If those patients with broader hyphae have had a worse prognosis than those with thinner hyphae, it is probable that the in vivo conditions must be favoring their florid growth resulting in broader hyphae.
The cases where there was a moderate to marked ( n = 5/11) granulomatous inflammation, the survival rate was 100% and those who had mild ( n = 6/11) granulomatous host response, the survival rate was 67% ( n = 4/6). So the patients who had florid granulomatous inflammation had a better prognosis although the results have not reached significant levels. Castillo et al. have found that multinucleate giant cell granulomas may be correlated with the disease with better prognosis. Whether the difference between the granulomatous and nongranulomatous forms is caused by immunologic dysfunction is not clear at this time.
Microscopically, all the cases of zygomycosis showed varying amounts of necrosis and the density of fungal organisms was higher in necrotic tissues. So it can be suggested that necrotic tissue must be well sampled to identify the fungal elements. Although necrosis was found in all the 33 cases of zygomycosis, no consistent pattern of association was noted between the amount of necrosis and the outcome of patients. The percentage of necrosis varied from 2 to 95% and the density of fungal organisms was highest in necrotic tissues. Angioinvasion was seen in 17 (51%) of total of 33 patients. Cases with mild vascular invasion had a survival rate of 100% (4/4) and those with moderate to marked degree of vascular invasion had a survival rate of 46% (6/13). Thus, as the degree of angioinvasion increases, the survival decreases ( P = 0.102). No consistent pattern of association of perineural or neural invasion was noted in this series with the outcome of patients and its importance in the spread of disease is still unknown.  But as the degree of bone marrow invasion increased, the survival rate of patients decreased although it did not reach significant levels.
Several reports of negative culture results, on both autopsy and premortem cultures as noted in English literature were observed in this study too. The negative cultures is assumed to be due to aggressive processing of the specimen that occurs before plating. Better recovery have been observed if slices of minimally manipulated tissue are placed onto the culture medium.  Despite aggressive invasion of vessels by the Zygomycetes, blood cultures have been rarely positive. 
Of all the histological variables, diameter of the fungus has also proved to be a significant ( P = 0.04) finding in this series. The prognosis of the patients with chronic rhino-orbito-cerebral zygomycosis was significantly ( P = 0.01) better than that of acute rhino-orbito-cerebral zygomycosis. The extent of involvement by zygomycosis also determined the prognosis significantly ( P = 0.0037). Using the above variables, the histopathologists can help the clinician in assessing prognosis at the time of tissue diagnosis, and thus considering the risk/benefit, the clinicians could optimize the treatment accordingly. The limitation of this aspect of fungal diameter is that the size of the fungus, which is a variable, depends to certain extent on the plane of section and when and how soon the biopsy tissue was fixed. These variables, however, need to be considered too at the time of evaluation.
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Professor & Head of Pathology, St. John's Medical College, Bangalore - 560 034
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]
[Table 1], [Table 2]