IMC Journal of Medical Science

Abstract

Introduction: Mucormycosis is a rare but deadly fungal infection that often affects those with weakened immune systems. With the rise in predisposing factors such as diabetes, use of steroids, rise in cases of cancer, among others, cases of mucormycosis are increasingly being observed. A surge of cases was noted due to the situation arising out of the COVID-19 pandemic.

Materials and methods: This study analyzed various histo-morphological tissue reaction patterns associated with mucormycosis and explored the utility of crush smear and imprint smear cytology in confirming the presence of fungi. A total of 63 samples were taken. Meticulous history and clinical examination were done. History of COVID-19 infection, diabetes mellitus, hospitalization, intensive care stay, and steroid therapy was taken into account. Biopsy specimens (rhino-orbital, sino-nasal, rhino-cerebral and bone) received in normal saline were first subjected to cytopathological examination using both crush smears and imprint smears and further processed for histopathological examination.

Results: The mean age of the patients was 48.76 ± 13.24 years. Male preponderance was seen with male to female ratio of 1.65:1. An overwhelming majority (92.6%) of patients had a history of COVID-19 infection. Pre-existing diabetes mellitus was found in 83.3% of patients, steroid intake in 72% of patients, and medical oxygen administration in 46.3% of patients. Out of 63 clinically suspected patients, 54 (85.7%) cases were diagnosed with mucormycosis on histopathology. The most common site involved was rhino-orbital (62.9%), followed by sino-nasal (25.9%) and rhino-cerebral (7.4%). Five histo-morphological patterns were identified namely infarct-like necrosis with or without angio-invasion (50%), exudative pattern (24%), mixed pattern (11%), granulomatous (9%) and predominantly histiocytic pattern (6%). With histopathology as gold standard, crush smear cytology yielded a sensitivity of 72.2% (95% confidence interval/CI: 58.4-83.5%), specificity of 77.8% (95% CI: 40.0-97.2%), positive predictive value (PPV) of 95.1% (95% CI: 83.5-99.4%) and negative predictive value (NPV) of 31.8% (95% CI: 13.9-54.9%), with overall diagnostic accuracy of 73.0%. Imprint smear cytology showed marginally better performance with sensitivity of 75.9% (95% CI: 62.4-86.5%), specificity of 77.8% (95% CI: 40.0-97.2%), PPV of 95.3% (95% CI: 84.2-99.4%) and NPV of 31.8% (95% CI: 13.9-54.9%), with overall diagnostic accuracy of 76.2%.

Conclusion: Various histo-morphological patterns encountered on histopathological examination help us keep the suspicion index high and warrant extensive examination for fungi. Histopathology remains the gold standard, providing prompt and definitive diagnosis, essential for establishing surgical and antifungal therapy, prognostication and evaluation of treatment response. Both crush smear and imprint cytology demonstrate high sensitivities (72-76%) and excellent PPVs (>95%), making them valuable rapid diagnostic tools for confirming mucormycosis when positive results are obtained. However, their low NPVs (31.8%) indicate that negative cytology results cannot reliably exclude mucormycosis, and histopathological examination remains mandatory in clinically suspected cases with negative cytological findings.

January 2026; Vol. 20(1):001, DOI: https://doi.org/10.55010/imcjms.20.001

*Correspondence: Mohd. Yasir Zubair, Department of Community Medicine, VALASMC, Etah, UP, India. Email: yasmuhsin@gmail.com.

© 2025 The Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License(CC BY 4.0).

*Abbreviations: COVID- Coronavirus Disease, SARS CoV 2- Severe Acute Respiratory Syndrome Corona-Virus 2, AIDS- Acquired Immuno-Deficiency Syndrome, DM- Diabetes Mellitus, CECT- Contrast Enhanced Computerized Tomography, PAS- Periodic Acid Schiff, H & E- Hematoxylin and Eosin, ACE- Angiotensin-Converting Enzyme

Introduction

Since its outbreak in Wuhan, China, in December 2019, Coronavirus Disease (COVID-19) caused by Severe Acute Respiratory Syndrome Corona-Virus 2 (SARS CoV-2) has spread rapidly across the world and led to a major pandemic. The evolution of the virus into different strains over the following few years led to subsequent waves and local outbreaks. India experienced a deadly second wave beginning in March 2021.Adding more burden to such a challenging situation, mucormycosis, an invasive fungal disease, exhibited a significant surge in patients with COVID-19 [1].

Mucormycosis is an opportunistic infection caused by the ubiquitous bread mould fungi belonging to the mucormycetes family. A severe disease spurred on by Rhizopus was initially named phycomycosis or zygomycosisin 1885 by Paltauf [2]. The same entity was later termed as mucormycosis in 1957 by American pathologist Baker [3]. Mucormycosis is a rare but deadly fungal infection that often affects those with weakened immune system. It is the third most common cause of invasive fungal infection, following aspergillosis and candidiasis and causes life-threatening rhino-cerebral disease [4]. Mucor and Rhizopus are the most common causative agents, followed by Lichtheimia, Apophysomyces, Rhizomucor, and Cunninghamella[5,6].

Mucormycosis is generally seen in the immunocompromised population. Individuals with diabetes mellitus, AIDS, organ transplant, and malignancy are especially predisposed to the infection [7]. The common causes attributed to the rise of mucormycosis in patients with COVID-19 are uncontrolled diabetes, hematologic malignancies, solid organ transplant recipients, stem cell transplantation, prolonged neutropenia, excessive use of corticosteroids for immunosuppression, and long-term stays in the intensive care unit [8,9]. Most cases of mucormycosis in patients with history of COVID-19 were detected around a month after the diagnosis of severe or moderate COVID-19 that required oxygen assistance in conjunction with steroid therapy [10]. Glucocorticoids have been used extensively to treat a range of diseases, including COVID-19, influenza, and middle-east respiratory syndrome. Moreover, SARS CoV-2 increases the secretion of hyperglycemic hormones such as glucocorticoids, which abnormally raises blood glucose levels [11]One of the main reasons for the increased production of glycosylation end products, oxidative stress, pro-inflammatory cytokines, etc., is hyperglycemia. Diabetic patients experience tissue inflammation, thus elevating the risk of infection [12]. Nevertheless, prompt diagnosis of mucormycosis is necessary for initiating early life-saving medical and surgical intervention. Based on clinical suspicion, clinicians usually advise contrast-enhanced computerized tomography (CECT), fungal culture and biopsy for fungal infections [13,14]. Since mucormycetes are ubiquitous in nature, culture, which generally takes more than a week, often gives ambiguous results. Hence, a crush and imprint smear by cytology and histopathological examination is helpful in quick and conclusive diagnosis. Due to a lack of population-level data on the Indian population, the prevalence of mucormycosis may be 70 times higher than global estimations [15].

In the present study, we aimed to study crush and imprint smear cytology as a rapid screening tool along with various histo-morphological tissue reaction patterns associated with mucormycosis and analyze their utility in confirming the presence of fungi.

Materials and methods

The study was conducted in the department of pathology, Jawaharlal Nehru Medical College, Aligarh, from May 2021 to November 2021, when a rapid surge of mucormycosis cases started to occur in India. The hospital admitted patients with suspected mucormycosis of all ages and either sex, whose samples were sent to the department of pathology for histological examination. Samples having scant material were excluded.

During the study period, a total of 63 samples were found adequate for inclusion. Detailed history and clinical examination were done. History of COVID-19 infection, diabetes mellitus, hospitalization, intensive care stay and steroid therapy were taken into account. Biopsy specimens received in normal saline were first subjected to cytopathological examination using crush and imprint smear, and then they were further processed for histopathological examination.

For cytological examination, a small tissue with a grossly necrotic and black area was taken from the biopsy. For each biopsy, three to fourcrush smears and one imprint smear were prepared using tissue from necrotic areas. Smears were processed and examined using H and E stain, PAS stain, and papanicolaou stains [16]. Each smear was evaluated for hyphae, spores, necrosis, giant cells, granuloma, and inflammatory infiltration. The slides were prepared for examination in around two hours. All the smears were meticulously searched for fungal hyphae, spores and conidia or fruiting bodies. The presence of necrosis, giant cells, granuloma and inflammatory infiltrate were also noted.

The same biopsy tissue was then fixed in 10% formalin for about 18 hours and subjected to routine histological processing. A gross examination was performed, noting the presence of necrosis, black crusting, thrombotic vessels, and bony erosion in maxillectomy or mandibulectomy specimens. This was followed by the preparation of paraffin-embedded sections. The tissue samples were stained with routine H and E and PASstains. Histopathological examination included an assessment of fungal morphology (aseptate or pauci-septate, broad [3–25 μm], ribbon-like, hyaline hyphae with irregular or right-angle branching), fungal load (classified as mild, moderate, or severe), and histomorphological patterns such as tissue necrosis, composition of the inflammatory infiltrate, and tissue invasion. Angioinvasion was identified by the presence of fungal hyphae infiltrating the endothelium or lying within the vascular lumen. Co-infection with other fungi, such as Aspergillus or Candida, was also evaluated. For diagnostic confirmation, fungal culture was performed on a subset of cases using protocols described by Skiada et al. [13], while PCR identification could not be carried out due to resource limitations.

On histopathology, the fungi were identified by broad-based, pauci-septate hyphae with right to obtuse angle branching, which was PAS positive. Tissue necrosis, the composition of inflammatory infiltrate, and tissue invasion were taken into account to study the histo-morphological patterns. Based on these, five main patterns were recognized: infarct-like necrosis pattern, exudative pattern, mixed pattern (necrotic and exudative), granulomatous pattern and predominantly histiocytic pattern.

All participants (or primary caretakers when needed) received a full explanation of the study, including its voluntary nature, confidentiality, and data use. They could ask questions and withdraw at any time. Written informed consent was obtained, and all institutional ethical guidelines for human research were followed.

The data was entered in MS Excel (2010) and was imported to IBMSPSS version 20.0 for analysis.

Results

The mean age of the patients was 48.76 ± 13.24 years, with ages ranging from 21-78 years. Male preponderance was seen in our study with male to female ratio of 1.65:1. Out of 63 suspected patients, 54 (85.7%) cases were diagnosed with mucormycosis on histopathology. Of these 54, an overwhelming majority (92.6%, n=50) of patients had a history of COVID-19 infection. A history of pre-existing diabetes mellitus was found in 83.3% (n=45) patients. It was found that 72% (n=39) of the patient had a history of steroid intake, and a history of medical oxygen administration was found in 46.3% (n=25) of patients. No other predisposing condition was found in our patients.

The analysis of clinical presentation revealed that necrosis and black crusting of turbinates was seen in 59.2% (n=32) cases, facial swelling and ophthalmoplegia were seen in 44.4% (n=24) and 53.7% (n=29) cases, respectively and only 3.7% (n=2) of them presented with gingivitis and loosening of teeth. Symptoms associated with cerebral involvement, such as headache, hemiplegia and altered sensorium, were found in 5.5% (n=3) cases (Figure-1).

Figure-1: Clinical presentation of patients

Figure-2: Sites involving mucormycosis infection

The most common site involved was rhino-orbital (62.9%, n=34), followed by sino-nasal (25.9%, n=14) and rhino-cerebral (7.4%, n=4). Unusual involvement of maxillary bone was found in 2 cases (Figure-2).

The most common histopathological pattern encountered was infarct-like necrosis in 40.7% (n=22) cases with or without angio-invasion, followed by an exudative pattern found in 24% (n=13) of cases. Subsequently, mixed pattern (11%, n=6), granulomatous (9%, n=5) and predominantly histiocytic pattern (6%, n=3) were found (Figures-3a-3f). Notably, Splendore- Hoeppli phenomenon was a frequent finding in the exudative pattern (Figure-3g). The fungal load was highest in the necrotic tissue.

Figure-3: (a) Infarct-like necrosis: Fragmented broad based aseptate fungal hyphae in necrotic background. (b) Infarction with angioinvasion (see arrow). (c) Exudative pattern with the neutrophilic response with abscess (see arrow). (d) Mixed pattern showing both infarct type as well as exudative pattern. Fungal hyphae were found in an exudative pattern (inset). (e) Granulomatous response. (f) Histiocytic pattern. (g) Splendore–Hoeppli phenomenon.

Two cases with bony involvement were subjected to extensive examination. Of these, one case which initially showed only extensive infarct-like necrosis and mixed necrotic and inflammatory patterns later demonstrated fungal hyphae in the follow-up bony resection specimens. A thorough search of fungal filament in the other case led to the identification of fragmented fungal hyphae in a blood vessel. PAS stain played a pivotal role in identifying fungal elements in these cases.

Also, there were three cases which showed co-infection with other fungi. Co-infection with Candida was identified in two cases, and one case showed Aspergillus like hyphaealong with mucormycosis. Candida was identified by the presence of pseudo-hyphae. Interestingly, a single case with squamous cell carcinoma having both Mucor and Candida infection was found.

Among the remaining nine cases that were negative for mucormycosis, the histomorphological findings in two cases were consistent with an inflammatory nasal polyp, while the other seven cases showed mildly hypertrophic mucosal epithelium with a mild chronic inflammatory infiltrate in the stroma.

For crushed smear cytopathology (Table-1), of the 54 histopathologically confirmed positive cases, 39 were correctly identified as positive, while 15 yielded false negative results. Among the 9 histopathologically negative cases, 7 were correctly identified as negative and 2 showed false positive results. Thus, crushed smear demonstrated a sensitivity of 72.2% (95% CI: 58.4-83.5%), specificity of 77.8% (95% CI: 40.0-97.2%), PPVof 95.1% (95% CI: 83.5-99.4%), and NPV of 31.8% (95% CI: 13.9-54.9%). The overall diagnostic accuracy was 73.0% (95% CI: 60.3-83.4%).

Table-1: Performance of cytopathology (crushed smear) against histopathology as gold standard

Imprint smear cytopathology (Table-2) showed marginally better performance. Of the 54 histopathologically positive cases, 41 were correctly identified as positive with 13 false negatives, while 7 of 9 negative cases were correctly identified with 2 false positives. Thus, imprint smear yielded a sensitivity of 75.9% (95% CI: 62.4-86.5%), specificity of 77.8% (95% CI: 40.0-97.2%), PPV of 95.3% (95% CI: 84.2-99.4%), and NPV of 31.8% (95% CI: 13.9-54.9%). The overall diagnostic accuracy was 76.2% (95% CI: 63.8-86.0%).

Table-2: Performance of cytopathology (imprint smear) against histopathology as gold standard

Table-3 demonstrates the site-specific diagnostic performance of crush smear and imprint cytology against histopathology as the gold standard among the 54 histopathologically confirmed mucormycosis cases. Both cytological methods showed variable detection rates across different anatomical sites.

Rhino-orbital samples constituted the majority of cases (n=34, 63.0%), with crush smear cytology detecting mucormycosis in 82.4% (28/34) of cases and imprint cytology showing slightly better performance at 85.3% (29/34). For sino-nasal specimens (n=14, 25.9%), the detection rates were lower, with crush smears positive in 64.3% (9/14) and imprint smears in 71.4% (10/14) of cases. Rhino-cerebral involvement (n=4, 7.4%) showed identical detection rates for both methods at 75.0% (3/4). The two cases with bone involvement (3.7%) demonstrated the lowest detection rates, with both crush and imprint cytology identifying mucormycosis in only 50% (1/2) of cases.

Overall, across all anatomical sites, imprint cytology demonstrated a higher cumulative detection rate of 79.6% (43/54) compared to 75.9% (41/54) for crush smear cytology when evaluated against histopathology-confirmed cases.

Table-3: Crushed and Imprint smear against gold standard histology across anatomical sites

Discussion

Mucormycosis is predominantly seen in immune-compromised individuals. It is a fulminant disease with high rates of morbidity and mortality. In the background of the COVID-19 pandemic, an increasing number of cases of mucormycosis started to occur [17]. Primary factors attributed to the increased incidence of COVID-19-associated mucormycosis are hypoxia associated with involvement of the lungs by SARS COV-2 virus, endocrine disturbances leading to hyperglycemia and acidosis, and altered host immunity caused by leucopenia, phagocytic dysfunction and irrational use of the steroid [9,18]. The post-COVID-19 fungal infections were almost misdiagnosed based on the data from the retrospective analysis of SARS and influenza from different countries, particularly China [19]. In this context its crucial to be vigilant for fungal infection probability particularly where any of predisposing factors are present.

Diabetes mellitus, which was the single most important predisposing factor in our study, was found in 83.3% of cases, followed by steroid intake in 72% of cases. A previous study by Prakash et al. in 2019 showed 57% of patients with uncontrolled diabetes had mucormycosis in the pre-COVID era [15]. A recent study conducted by John et al. in 2021 on confirmed cases of mucormycosis in people with COVID-19 confirmed that DM was seen in 93% of cases while 88% had received steroid therapy [20]. In 2021, Al-Tawfiq et al. discovered that mucormycosis occurred in 19 patients with uncontrolled diabetes mellitus and also had corticosteroid administration [21]. These findings are consistent with our study, which also showed a high association of mucormycosis in patients with COVID-19 who had DM and had received steroid therapy. India is considered the diabetes capital of the world, with an alarming increase in diabetic patients. With this alarming increase in diabetes, cases of mucormycosis may also be speculated to increase thus underscoring the importance of being vigilant regarding this severe complication.

With regards to clinical presentation, in this study, necrosis and black crusting of turbinates was seen in 59% of cases, facial swelling and ophthalmoplegia were seen in 44% and 53% of cases respectively, 3% of them presented with gingivitis and loosening of teeth and symptoms associated with cerebral involvement such as headache, hemiplegia and altered sensorium, were found in 5.5% of cases. This is similar to Goel et al. who reported that mucosal necrosis was seen in 48% of cases and external ophthalmoplegia in 59%, while signs of brain involvement occurred in 18% of the cases which were characterized by hemiplegia, altered mental status, stupor and coma [22].

Mucormycosis can involve the sinuses (sino-nasal) and progressively involve the orbits (rhino-orbital) and central nervous system (rhino-orbito-cerbral). It can also involve the lung, gastrointestinal tract, skin and jaw bones. Even though, lung is the most common organ to be affected in disseminated disease [20], no case of pulmonary involvement was found. Rhino-orbital mucormycosis has been seen to be more common in patients with poorly controlled diabetes mellitus. In contrast, patients with haematological malignancies, neutropenia or organ transplant are more prone to get pulmonary mucormycosis [10]. It has been speculated that before pulmonary progression, the increased propensity of SARS CoV-2 for ACE-2 receptors in nasal mucosalead to breach in the integrity of the mucosa which may in turn lead to the colonization of fungi in the nasal mucosa and its rhino-orbital presentation. In the current study, the most common site involved was rhino-orbital (63%), followed by sino-nasal (25.9%) and rhino-cerebral (7%). Similar findings were depicted by Goel et al., who concluded that extent of involvement determined the prognosis significantly [22]. Singh et al., in their study of mucormycosis in patients with COVID-19 from India and abroad, found that the most specific organ involved with mucormycosis was the nose and sinus (88.9%), followed by rhino-orbital (56.7%) and rhino-orbital-cerebral (22.2%) type [14].

Microscopically, the most common histopathological pattern encountered was infarct-like necrosis in 40.7% (n=22) cases with or without angio-invasion, followed by an exudative pattern found in 24% (n=13) of cases. Subsequently, mixed pattern (11%, n=6), granulomatous (9%, n=5) and predominantly histiocytic pattern (6%, n=3) were found. Ganesan et al. in their study on mucormycosis cases during the pandemic reported acute type of inflammation in 73.33% and granulomatous inflammation in 23.33% cases. Bony invasion and perineural invasion were observed in 8.33% and 91.67% cases, respectively [23]. The study done by Goel et al. in 33 cases found the granulomatous pattern to be the most common pattern. However, some amount of tissue necrosis was seen in all the cases, but no association of the extent of necrosis was found to the outcome of patients [22].

Cytopathology offers the critical advantage of yielding results within a day, making it invaluable for rapid clinical decision-making in suspected mucormycosis cases. In our study, the overall diagnostic accuracy of imprint and crush smear cytology (76.2% vs 73.0%) suggests that imprint smears may better preserve fungal morphology, enhancing detection [13,22], though the clinical significance of this difference requires validation with larger sample sizes.

The excellent positive predictive values (>95%) observed for both cytological methods have important clinical implications; when cytology is positive, clinicians can confidently initiate antifungal therapy and plan surgical debridement without delay, potentially improving patient outcomes in this rapidly progressive and life-threatening infection. This makes cytopathology an invaluable tool for rapid confirmation and therapeutic decision-making.

However, the low negative predictive values (31.8% for both methods) warrant careful interpretation and represent a critical limitation of cytological diagnosis. This means that approximately two-thirds of cases showing negative cytology may harbor mucormycosis on definitive histopathological examination. The low NPV can be attributed to several factors; for example, the patchy and focal distribution of fungal elements within extensively necrotic tissue, sampling variability inherent to cytological preparations where only a small portion of tissue is examined, the inability of cytology to process all submitted tissue comprehensively, fragmentation or paucicellular nature of fungal hyphae in some areas, and the limited capability of cytology to detect angioinvasion or bone invasion features that are crucial for definitive diagnosis and staging. These limitations underscore that negative cytology cannot be used to exclude or rule out mucormycosis in clinically suspected cases, and histopathological examination with extensive tissue sampling remains mandatory.

Findings of the current study differ from some published literature where cytology has been reported with higher sensitivity. This discrepancy likely reflects differences in sampling techniques, the extent of necrosis in submitted specimens, and the experience of cytopathologists in recognizing fragmented fungal elements. Nevertheless, our data align with the general consensus that cytology, while rapid and useful for confirmation, has inherent limitations for exclusion of fungal infections.

Fungal culture, though considered a gold standard for species identification, is not diagnostic of mucormycosis because of the ubiquitous nature of the fungi. Moreover, it usually takes 3-5 days to grow under ideal conditions. Diagnosis of mucormycosis by culture is rather challenging compared to other fungal infections because under normal laboratory conditions failure of sporulation is high and initial processing of hyphal elements may damage the hyphae rendering it non-viable [24]. The presence of non-viable fungal elements in necrotic biopsy tissue can also lead to negative culture results. While fungal culture is usually required for the identification of a genus, it has no implication on initial treatment decisions, which must be based on rapid histopathological or cytological confirmation.

A high index of suspicion for mucormycosis while encountering the typical histomorphological patterns in suspected cases where no evidence of fungal elements was found initially helped in prompt diagnosis and management. Based on detailed observation, a meticulous search for fungal elements in cases with characteristic tissue reaction patterns proved invaluable. After extensive and careful examination, one case showed fragmented broad aseptate fungal hyphae within a blood vessel, and another case showed fragmented fungal hyphae within giant cells. These findings became evident only after serial sectioning of the submitted tissue and application of PAS stain. In two additional cases, fungal elements were identified only in follow-up specimens of resected jawbone, highlighting the importance of repeat sampling when clinical suspicion remains high despite initial negative results.

This experience underscores several critical points- familiarity with various histo-morphological patterns associated with mucormycosis is essential for maintaining diagnostic vigilance; fungal hyphae can be easily missed in routine H & E sections, particularly in areas of extensive necrosis, hence PAS stain plays an indispensable role in diagnosis; serial sectioning and examination of multiple tissue levels significantly improves fungal detection and when clinical suspicion is high, negative initial results should prompt repeat biopsies or more extensive surgical sampling rather than exclusion of the diagnosis. Early and accurate diagnosis is necessary to initiate appropriate management promptly. The extent of surgery, the dose and duration of amphotericin B administration, and modification of immunosuppressive therapy all depend upon the histological confirmation and features of mucormycosis [22]. Therefore, a systematic approach combining rapid cytological screening with comprehensive histopathological examination optimizes diagnostic accuracy and facilitates timely therapeutic intervention.

Conclusion

The findings confirm a strong association between prior SARS CoV-2 infection, diabetes mellitus, and corticosteroid exposure as pivotal predisposing factors for mucormycosis development. The predominance of rhino-orbital involvement with characteristic clinical signs underscores the aggressive and invasive nature of this fungal disease. Histopathological examination, identifying distinctive pauci-septate, broad hyphae with angioinvasion, remains the diagnostic gold standard for confirmation.

Importantly, the data demonstrate that both crush smear and imprint cytology offer rapid, reliable, and minimally invasive diagnostic alternatives with reasonably good sensitivities (72.2% and 75.9% respectively) and excellent positive predictive values (>95%). These high positive predictive values indicate that when cytology is positive, clinicians can confidently proceed with antifungal treatment and surgical intervention without delay, enabling earlier therapeutic decision-making in suspected cases.

However, both cytological methods demonstrated identical low negative predictive values meaning that approximately two-thirds of cases with negative cytology may actually harbor mucormycosis on histopathological examination. This critically important finding indicates that negative cytology results cannot be used to exclude or rule out mucormycosis in clinically suspected cases. The low NPV likely reflects the patchy distribution of fungal elements in necrotic tissue, sampling variability inherent to cytological preparations, and the limited tissue volume examined in cytological smears compared to comprehensive histopathological sectioning.

The identification of diverse histo-morphological patterns in this study where infarct-like necrosis was the most common (50%), followed by exudative (24%), mixed (11%), granulomatous (9%), and histiocytic patterns (6%) further enriches the understanding of host-pathogen interactions and highlights the importance of recognizing these patterns to maintain high clinical suspicion. These patterns may also inform prognosis and treatment response evaluation.

The findings advocate a complementary diagnostic approach: cytological methods (crush smear or imprint smear) serve as valuable frontline rapid tools that, when positive, can expedite treatment initiation within hours. However, when cytology is negative in clinically suspected cases, confirmatory histopathological examination with extensive tissue sampling and serial sectioning remains absolutely essential. The application of PAS stain plays a crucial role in identifying fungal elements, particularly in cases with extensive necrosis where fungi may be fragmented or scarce.

In summary, crush smear and imprint cytology are excellent for rapid confirmation of mucormycosis (ruling in disease when positive) but inadequate for exclusion (ruling out disease when negative). Integrating rapid cytological screening with mandatory confirmatory histopathology in negative cases optimizes diagnostic accuracy and helps mitigate the significant morbidity and mortality associated with this aggressive fungal infection. Future research should focus on expanding molecular diagnostic capabilities, evaluating the cost-effectiveness of various diagnostic strategies, and assessing long-term outcomes in mucormycosis patients to refine clinical management protocols further.

Conflict of interest

Authors have no conflict of interest to declare.

Ethical statement

This study was approved by the Institutional Ethics Committee, Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, (Reg. No. ECR/1418/Inst/UP/2020) bearing the file no. IECJNMC/691 dated 27/04/2021.

References

1.     Aranjani JM, Manuel A, Abdul Razack HI, Mathew ST. COVID-19-associated mucormycosis: evidence-based critical review of an emerging infection burden during the pandemic's second wave in India. PLoS Negl Trop Dis. 2021; 15(11): e0009921. doi:10.1371/journal.pntd.0009921.

2.     Singh K, Kumar S, Shastri S, Sudershan A, Mansotra V. Black fungus immunosuppressive epidemic with Covid-19 associated mucormycosis (zygomycosis): a clinical and diagnostic perspective from India. Immunogenetics. 2022; 74(2): 197-206. doi:10.1007/s00251-021-01226-5.

3.     Baker RD. Mucormycosis; a new disease?.J Am Med Assoc. 1957; 163(10): 805-808. doi:10.1001/jama.1957.02970450007003.

4.     Dignani MC. Epidemiology of invasive fungal diseases on the basis of autopsy reports. F1000Prime Rep. 2014; 6:81. doi:10.12703/P6-81.

5.     Park HR, Voigt K. Interaction of Zygomycetes with innate immune cells reconsidered with respect to ecology, morphology, evolution and infection biology: a mini-review. Mycoses. 2014; 57 Suppl 3: 31-39. doi:10.1111/myc.12235.

6.     Farmakiotis D, Kontoyiannis DP. Mucormycoses. Infect Dis Clin North Am. 2016; 30(1): 143-163. doi:10.1016/j.idc.2015.10.011.

7.     Baldin C, Ibrahim AS. Molecular mechanisms of mucormycosis-the bitter and the sweet. PLoS Pathog. 2017; 13(8): e1006408. doi:10.1371/journal.ppat.1006408.

8.     Raut A, Huy NT. Rising incidence of mucormycosis in patients with COVID-19: another challenge for India amidst the second wave?. Lancet Respir Med. 2021; 9(8): e77. doi:10.1016/S2213-2600(21)00265-4.

9.     Khatri A, Chang KM, Berlinrut I, Wallach F. Mucormycosis after Coronavirus disease 2019 infection in a heart transplant recipient - case report and review of literature. J Mycol Med. 2021; 31(2): 101125. doi:10.1016/j.mycmed.2021.101125.

10.  Jeong W, Keighley C, Wolfe R, Lee WL, Slavin MA, Chen SCA, et al. Contemporary management and clinical outcomes of mucormycosis: a systematic review and meta-analysis of case reports. Int J Antimicrob Agents. 2019; 53(5): 589-597. doi:10.1016/j.ijantimicag.2019.01.002.

11.  Wang A, Zhao W, Xu Z, Gu J. Timely blood glucose management for the outbreak of 2019 novel coronavirus disease (COVID-19) is urgently needed. Diabetes Res Clin Pract. 2020; 162: 108118. doi:10.1016/j.diabres.2020.108118.

12.  Knapp S. Diabetes and infection: is there a link?--A mini-review. Gerontology. 2013; 59(2): 99-104. doi:10.1159/000345107.

13.  Skiada A, Pavleas I, Drogari-Apiranthitou M. Epidemiology and diagnosis of mucormycosis: an update. J Fungi (Basel). 2020; 6(4): 265. doi:10.3390/jof6040265.

14.  Singh AK, Singh R, Joshi SR, Misra A. Mucormycosis in COVID-19: a systematic review of cases reported worldwide and in India. Diabetes Metab Syndr. 2021; 15(4): 102146. doi:10.1016/j.dsx.2021.05.019.

15.  Prakash H, Chakrabarti A. Global epidemiology of mucormycosis. J Fungi (Basel). 2019; 5(1): 26. doi:10.3390/jof5010026.

16.  Suvarna SK, Layton C, Bancroft JD. Bancroft’s Theory and Practice of Histological Techniques. 8th ed. Elsevier; 2018. doi: 10.1016/C2015-0-00143-5.

17.  Parfrey NA. Improved diagnosis and prognosis of mucormycosis. a clinicopathologic study of 33 cases. Medicine (Baltimore). 1986; 65(2): 113-123. doi:10.1097/00005792-198603000-00004.

18.  Challa S. Mucormycosis: pathogenesis and pathology. Curr Fungal Infect Rep. 2019; 13(8): 11–20. doi:10.1007/s12281-019-0337-1.

19.  Song G, Liang G, Liu W. Fungal co-infections associated with global COVID-19 pandemic: a clinical and diagnostic perspective from China. Mycopathologia. 2020; 185(4): 599-606. doi:10.1007/s11046-020-00462-9.

20.  John TM, Jacob CN, Kontoyiannis DP. When uncontrolled diabetes mellitus and severe COVID-19 converge: the perfect storm for mucormycosis. J Fungi (Basel). 2021; 7(4): 298. doi:10.3390/jof7040298.

21.  Al-Tawfiq JA, Alhumaid S, Alshukairi AN, Temsah MH, Barry M, Al Mutair A, et al. COVID-19 and mucormycosis superinfection: the perfect storm. Infection. 2021; 49(5): 833-853. doi:10.1007/s15010-021-01670-1.

22.  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(2): 253-257. doi:10.4103/0377-4929.64342.

23.  Ganesan N, Sivanandam S. Histomorphological features of mucormycosis with rise and fall of COVID-19 pandemic. Pathol Res Pract. 2022; 236: 153981. doi:10.1016/j.prp.2022.153981.

24.  Spellberg B, Edwards J Jr, Ibrahim A. Novel perspectives on mucormycosis: pathophysiology, presentation, and management. Clin Microbiol Rev. 2005; 18(3): 556-569. doi:10.1128/CMR.18.3.556-569.2005.

Cite this article as:

Afrose R, Fatima ZH, Zubair MY, Hasan M, Arif SH, Aftab M, et al.Histomorphological patterns and diagnostic utility of crush and imprint smear cytology in mucormycosis: a prospective study.IMC J Med Sci. 2026; 20(1):001. DOI:https://doi.org/10.55010/imcjms.20.001.