Mucormycosis in Post Novel Corona Virus Patients: An Institutional Experience

Abstract

Mucormycosis is an emerging angioinvasive fungal infection caused by ubiquitous saprophytic filamentous fungus that belongs to the class Zygomycosis. Mucormycosis or black fungus infections in patients with recent COVID-19 infections has given rise to a new epidemic during the second wave of this current COVID-19 pandemic in India. Steroid misuse, poor glycemic control, use of industrial grade oxygen, improper humidification and specific variants strains have resulted in profound immunosuppression and are attributed for the high incidence of COVID associated Mucormycosis (CAM) in India. The aims of the present study were to assess the age and gender specific incidence of CAM, the utility of special stains (PAS and GMS) for diagnosing mucormycosis (false negatives and sensitivity). In the present study emphasis was laid on identifying if there exists a correlation between host response, angioinvasion and bone invasion with mortality. The Present study is a retrospective, cross sectional analytical study taken up in the Department of Pathology, Guntur Medical College for a period of 2 months between 1st of May 2021 to 30th of June 2021. Results were tabulated in Microsoft excel 2016 and SPSS software version 14 was used for calculation of odds ratio and for performing multivariate analysis. All samples were routinely fixed using 10% buffered formalin, processed and sectioned. All the sections were stained with Haematoxylin and Eosin and also special stains for fungus like periodic acid Schiff (PAS) and Gomori Methenamine silver (GMS) were used wherever required. CAM is a serious complication after recovery from COVID-19 infection due to its high morbidity and mortality (13.7% in this series). CAM epidemiology and presentation is mostly similar to non-COVID mucormycosis seen in pre-COVID era.

Introduction

Mucormycosis or black fungus infections in patients suffering from COVID-19 infections has given rise to a new epidemic during the COVID-19 pandemic in India [1]. There has been a heightened prevalence of mucormycosis infections in India compared to western world even before the COVID-19 pandemic. COVID associated Mucormycosis (CAM) has been the term which has been coined specifically for the spectrum of mucormycosis infections seen in post COVID-19 patients.

Steroid misuse, poor glycemic control [2], use of industrial grade oxygen [1], improper humidification and sanitation [3] and specific variant strains of the virus [1] have resulted in profound immunosuppression and are attributed for the high incidence of CAM in India.

CAM presents usually around 3rd week from onset of symptoms of COVID-19. Mucormycosis ravages patients undergoing treatment for active disease as well as those recovering from COVID-19 disease. CAM often results in re-admission and long hospital stay. It is associated with high morbidity and mortality and places a heavy burden on the existing medical resources in our country. Dedicated centers with multidisciplinary teams have been set up for uniform and early treatment of CAM. Treatment of CAM involves combination of surgical debridement and antifungal therapy.

Early diagnosis of CAM is crucial for early specialist referral and best outcomes. Histopathology and culture are conventional methods used for diagnosis of fungal infections using material obtained during surgical debridement. Tissue culture though is gold standard for diagnosis of mucormycosis has its draw backs, and hence histopathological evaluation is currently the mainstay in diagnosing CAM.

At present there is limited data available regarding CAM, as it is an emerging disease. We undertook this study at our institute to look at the spectrum of CAM, risk factors, pathological features and host response from tissue specimens which were sent a with clinical suspicion of CAM.

Material and Methods

The study is a retrospective cross sectional analysis which was undertaken in the Department of Pathology, Guntur Medical College and Government General Hospital. Samples that were received between May 2021 and July 2021, during which we had the peak of second wave of COVID-19 and subsequent CAM cases, were included. Samples from patients who were clinically suspected to have Mucormycosis with confirmed COVID-19 infection (either by RT-PCR or CT chest), in the current or recent past hospitalisation were included in the study. Patients with clinical suspicion of CAM but samples negative for fungal elements by histopathology were excluded from the study.

After application of the inclusion and exclusion criteria clinical data was collected from case sheets and from patients through telephonic contact, wherever required, and necessary data was obtained. Samples were routinely fixed using 10% buffered formalin, processed and sectioned. All the sections were stained with Haematoxylin and eosin, periodic acid Schiff (PAS) and Gomori methenamine silver (GMS) as per our departmental protocol for diagnosis of fungal infection. Histopathologic examination was done on all the samples and specifically tissue response was identified using light microcopy.

Results were tabulated in Microsoft excel and SPSS software version 14 was used for univariate and multivariate analysis of various risk factors, morphological variants, host response and mortality.

Results

Of the 349 cases we received with clinical suspicion of CAM, we could ascertain fungal elements of mucor by histopathological examination in 254 cases only, hence rest of the cases were excluded from the study. Rest of cases needed further evaluation with fungal culture and RTPCR techniques which were not feasible at our center and hence excluded from the study.

Commonest disease manifestation is rhino orbito cerebral followed by pulmonary mucor. We received 37 hemi-maxillectomy specimens for rhinocerebral cases and four orbital enucleations for generalized rhinorbitocerebral cases. The overall mortality is 13.7% in the current series of cases.

Tables 1, 2, 3 summarize the results of the present study.

Table 1.

Depicting age distribution, gender distribution, risk factors and disease manifestation in relation to mortality in CAM in the present study

Demographic data	Number of patients	Mortality
Age (years)
< 40 years	58	5
> 40 years	196	30
Gender
Males	173	26
Females	81	9
Risk factors
Diabetes mellitus	194	28
Steroid usage	162	27
Oxygen therapy	90	23
Immunocompromised states (Organ transplant, HIV, post cancer therapy)	8	4
Disease manifestation
Rhino-orbito-cerebral mucormycosis	248	35
Localized sinus	206	16
Sino-orbital	1	0
Sino-cerebral	37	16
Generalized rhinoorbitocerebral	4	2
Pulmonary mucormycosis	4	0
Gastrointestinal mucormycosis	1	1
Cutaneous mucormycosis	1	0

Table 2.

Histopathological findings predictive of mortality in patients with mucormycosis in the present study

	Survivors	Non survivors	p value
Host response
Acute suppurative inflammation	162	27	0.690
Granulomatous response	83	18	0.129
Predominantly necrotizing response	112	26	0.011
Histopathological findings
Mixed infection	6	3	0.083
Angioinvasion	48	29	0.0001
Bone invasion	25	20	0.0001
Fruiting bodies	8	4	0.044

p value less than 0.05 was taken as significant and is highlighted in bold

Table 3.

Table depicting host response and association with risk factors in the present study

Host response	Diabetes mellitus (p value)	Steroid usage (p value)	Oxygen therapy (p value)
Acute suppurative inflammation	147 (0.37)	113 (0.024)	68 (0.756)
Granulomatous response	78 (0.796)	61 (0.362)	40 (0.259)
Predominantly necrotizing response	106 (0.859)	94 (0.117)	54 (0.179)

p value less than 0.05 was taken as significant and is highlighted in bold

Discussion

Mucormycosis is a saprophytic, often fatal, opportunistic fungal infection seen mostly in immunocompromised patients. Even before the COVID-19 pandemic incidence of mucormycosis varies from 12% to 14% among ICU patients in various studies [4, 5]. Mucormycosis has a worldwide prevalence and has been documented in various countries [6] and is on the rise globally [7]. Mucormycosis cases in India are snowballing and are nearly 70 times higher in incidence as compared to western countries [6, 8]. Unlike the western world, mucormycosis epidemiology in India is significantly different and is mainly seen in patients with poorly controlled type II diabetes [9]. India having world’s second highest number of diabetic patients and with steady increase in people living with diabetes, there has been concordant rise in mucormycosis cases. Large diabetic population, poor diabetic control and late diagnosis have all been attributed to this unique epidemiology of mucormycosis in India as compared to other developed nations.

In the present study, the emphasis was COVID associated mucormycosis [CAM] specifically in order to compare and contrast with non- COVID mucormycosis.

Demographical Analysis

In the present series, CAM is found to be more common in males, with a male to female ratio of 2.1. The most common age group is 6th decade of life, with a mean age of 50.1 ± 11.6 years. Our findings are similar to that seen in the review analysis by Prakash H et al. [10]. It is unclear for higher male preponderance as per current literature. The median time interval between COVID-19 diagnosis and occurrence of mucormycosis symptoms in the present study is 25 days. This is a little higher than that observed by Pal R et al. (15 days) [11]. There is literature stating onset of mucor as late as 42 days and 90 days post COVID-19 [12, 13]. Hence, it is emphasized, based on our study that patients require longer vigilance periods in order to identify them early in the disease course.

Underlying Risk Factors for CAM

Uncontrolled diabetes, malignancies and organ transplantation were the most common predisposing factors for mucormycosis in preCOVID era [10]. The most common risk factor in CAM in our study is hyperglycemia at the time of diagnosis (including cases of preexisting diabetes mellitus, new onset hyperglycemia and denovo diabetes mellitus), seen in 76% of cases.

Other risk factors association with CAM as per our observations include steroid usage and inhalational oxygen usage. 63% cases had usage of steroids and 35% cases were on inhalational oxygen during the course of treatment of COVID 19. These findings point towards the familiar link between the predisposing factors in non COVID mucormycosis in pre-COVID era. Our results are concordant with those of John et al. [2] and Singh et al. [14].

Clinical Spectrum /Organ Involvement in CAM

The most common presentation in the present study is Localised nasal or sinus (81.1%) followed by sinocerebral (14.5%), followed by generalized rhinoorbitocerebral(1.5%), for which we received 37 hemimaxillectomy specimens and 4 orbital enucelations (Fig. 1) and pulmonary type (1.5%). This finding is similar to the findings of Pal et al. [11] whose study is based a systematic review of 99 cases of COVID 19 associated Mucormycosis. Similar findings were also seen in the study by Patel et al. [15], which was based on a cohort of 465 cases mucormycosis in pre COVID 19 era. We herewith conclude that there is no significant difference in clinical spectrum of CAM and mucormycosis in pre COVID era.

Fig. 1.

Gross photographs ofhemimaxillectomy (a) and enucleation (b) specimen received from a case of Rhinorbitocerebralmucormycosis

Histopathology Versus Fungal Culture

As depicted in Table 4, both histopathological identification of fungal infections and fungal culture have their own advantages and drawbacks. It is common that there can be disagreement in identification of fungal species by histomorphology and fungal culture. The accuracy of microscopic identification of fungal species can vary from 20 to 80% [16, 17].

Table 4.

Table contrasting histopathological diagnosis and fungal culture in identification of fungal infections

Limitations of histopathological diagnosis [16, 17]	Limitations of fungal culture [18, 19]
Missing the diagnosis due to scanty fungal elements  Misidentification of fungal species due to swelling, kinking, folding and fragmentation	Time consuming process  High chances of cross contamination  Inability to differentiate colonization and true infection
Advantages of Histopathological diagnosis [16, 17]	Advantages of Fungal culture [18, 19]
Differentiate colonization from infection   Ascertain invasion and spread	Gold standard  Accurate identification of fungal species

Staining for Diagnosis of Mucormycosis

In the present study it was found that use of special stains like GMS can increase the sensitivity of diagnosing fungal infections on histopathology, as depicted in the Table 5. The findings in the present study also established that routine H & E staining is adequately sensitive to identify fungal infections and also has additional advantage like identifying host response, especially in resource poor settings. Our results are concordant with the findings of Heaton et al. [20]. Wherever available use of special stains is justified as we can increase sensitivity of fungal infections identification. In cases where mixed or dual infections are suspected special stains can be used as morphology can be better analyzed [16].

Table 5.

Table depicting sensitivity and false negativity rate in detection of mucormycosis in the present study

Stain	Sensitivity (%)	False negativity rate(%)
Hematoxylin and eosin	93.7	6.3
Periodic acid Schiff	88.9	11.1
Gomori Methenamine silver	97.6	2.4

Host Response in CAM

Innate immunity plays an important role in combat with fungal infections [21, 23]. Profound immune suppression after COVID-19 is attributed to hyperglycemia, steroid use, and specific strains of COVID-19. Host response is crucial and determines the outcome in most invasive fungal infections. Histopathology when compared to other techniques has this advantage as it helps to determine host response in fungal infections. Common host reactions that can be identified include acute suppurative inflammatory response, granulomatous inflammatory response, predominantly necrotizing response and mixed patterns.

In the present series, the most common host response was acute suppurative inflammation, followed by necrotizing response and granulomatous response (Fig. 2). Our findings are different from that of Sri Devi et al. [24], who reported granulomatous inflammation as the most common host response. When risk factors and host response were analysed (Table 3), it was identified that patients who had received steroid therapy (either oral, parenteral or both) during COVID 19 treatment, showed acute suppurative inflammatory response to CAM (p value = 0.024). Another interesting finding (Table 2) that was observed in the present study was that patients who had predominantly necrotizing response showed significantly higher mortality (p value = 0.011). Thus, it is emphasized that identification of host immune response early in the course of disease may help in choice of treatment options.

Fig. 2.

Host response a Acute suppurative inflammatory response, b. Necrosis infiltrated by broad aseptate fungal hyphae, c Granulomatous inflammation with foreign body giant cell reaction

Histopathological Features in CAM

In the present series, nine cases had mixed fungal infections. The most common fungus in mixed infections was aspergillus and mucormycosis followed by candida and mucormycosis (Fig. 3c). However, on statistical analysis, there was no significant association between mixed infections and risk factors or mortality.

Fig. 3.

a H&E stained section showing angioinvasion by fungal hyphae, b H&E stained section showing invasion of fungal hyphae into the intertrabecular spaces of bone, c GMS stained section showing mixed fungal infection (mucormycosis and aspergillus), d PAS stained section showing sporangium (fruiting body)

Presence of fruiting bodies (sporangium) in surgical debridement specimens is a rare histopathological finding. In the present series fruiting bodies were identified in twelve cases (Fig. 3d). It was found that there was a significant association between presence of fruiting bodies and mortality (p value = 0.04).

In the present series, 29 cases had angioinvasion and 20 cases had bone invasion by mucormycosis on microscopy (Fig. 3a and b). On univariate analysis, presence of bone invasion and angioinvasion by microscopy had significant association with mortality in the present study (p value = 0.0001 for both).

Mortality

CAM is associated with high mortality rates unless diagnosed early and treated aggressively. 13.7% of cases in the present study died during the treatment for CAM. Our mortality rate is low compared to that of other studies which have observed a mortality rate of 34% [25]. Pulmonary or systemic mucormycosis is associated with highest mortality. In our series the most common disease manifestation was localized type of mucormycosis and this may be the reason for the lower mortality. In the present study, when various factors affecting mortality were analysed, it was found that presence of predominantly necrotizing host response, presence of fruiting bodies on tissue sections, presence of angioinvasion and bone invasion had significant association with mortality.

Conclusion

CAM is a serious complication seen in patients admitted with COVID-19 and also after their recovery from active infection due to its high morbidity and mortality (13.7% in this series). CAM epidemiology and presentation is mostly similar to non-COVID mucormycosis seen in pre-COVID era. Hyperglycemia is the single most important risk factor of CAM, other risk factors are steroid use and O2 therapy in minority cases. Rhinoorbitocerebral manifestation predominantly localised mucor is the most common type of mucor in post COVID-19 patients. Microscopic examination of tissue is still the most cost effective and best modality currently available for early and reasonably accurate diagnosis of fungal infections. Larger studies from multiple centers are further needed to better understand CAM and host response and its impact on mortality.

Declarations

Conflict of interest

There is no potential conflict of interest.

Ethical approval

Institutional ethical clearance and approval was taken before conduction the study (IEC Application No: GMC/IEC/179/2021).