Multicenter Epidemiologic Study of Coronavirus Disease–Associated Mucormycosis, India

During September–December 2020, we conducted a multicenter retrospective study across India to evaluate epidemiology and outcomes among cases of coronavirus disease (COVID-19)–associated mucormycosis (CAM). Among 287 mucormycosis patients, 187 (65.2%) had CAM; CAM prevalence was 0.27% among hospitalized COVID-19 patients. We noted a 2.1-fold rise in mucormycosis during the study period compared with September–December 2019. Uncontrolled diabetes mellitus was the most common underlying disease among CAM and non-CAM patients. COVID-19 was the only underlying disease in 32.6% of CAM patients. COVID-19–related hypoxemia and improper glucocorticoid use independently were associated with CAM. The mucormycosis case-fatality rate at 12 weeks was 45.7% but was similar for CAM and non-CAM patients. Age, rhino-orbital-cerebral involvement, and intensive care unit admission were associated with increased mortality rates; sequential antifungal drug treatment improved mucormycosis survival. The COVID-19 pandemic has led to increases in mucormycosis in India, partly from inappropriate glucocorticoid use.

During September-December 2020, we conducted a multicenter retrospective study across India to evaluate epidemiology and outcomes among cases of coronavirus disease (COVID-19)-associated mucormycosis (CAM). Among 287 mucormycosis patients, 187 (65.2%) had CAM; CAM prevalence was 0.27% among hospitalized COVID-19 patients. We noted a 2.1-fold rise in mucormycosis during the study period compared with September-December 2019. Uncontrolled diabetes mellitus was the most common underlying disease among CAM and non-CAM patients. COVID-19 was the only underlying disease in 32.6% of CAM patients. COVID-19-related hypoxemia and improper glucocorticoid use independently were associated with CAM. The mucormycosis case-fatality rate at 12 weeks was 45.7% but was similar for CAM and non-CAM patients. Age, rhino-orbitalcerebral involvement, and intensive care unit admission were associated with increased mortality rates; sequential antifungal drug treatment improved mucormycosis survival. The COVID-19 pandemic has led to increases in mucormycosis in India, partly from inappropriate glucocorticoid use.
We collected data for all confirmed mucormycosis cases among patients with and without COVID-19 reported during September 1-December 31, 2020. The ethics committees of the respective centers approved the study protocol.

Study Subjects and Definitions
We defined a case of mucormycosis as compatible clinical and radiologic manifestations and demonstration of fungi in the tissue or sterile body fluids of a patient by either direct microscopic visualization of broad ribbon-like aseptate hyphae or isolation of Mucorales. COVID-19 diagnosis was made in patients who tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the causative agent of COVID-19) RNA in respiratory specimens by reverse transcription PCR (RT-PCR) or a positive rapid antigen test. We defined CAM as the occurrence of proven mucormycosis in COVID-19 patients.
Seven participating centers provided additional data on hospitalized COVID-19 patients and number of diagnosed CAM cases during the study pe-riod. The prevalence of CAM was calculated as the total number of CAM cases divided by the number of COVID-19 patients treated at the 7 participating centers during the study period. Similarly, the prevalence of CAM cases in the intensive care unit (ICU) was calculated as the total number of patients developing mucormycosis among COVID-19 patients who received treatment in the ICU. We classified CAM cases as early when mucormycosis was diagnosed <7 days after COVID-19 diagnosis and late when mucormycosis was diagnosed ≥8 days after COVID-19 diagnosis. We also collected the number of mucormycosis cases reported at the participating centers during the same months (September-December) of 2019. For patients who left the hospital against medical advice, we considered a worst-case scenario for mortality analysis and assumed the patients died.

Study Procedure
We developed a standard case-record form that we circulated to all the centers for data collection. We extracted the following information from the patient Patients with intracranial extension received higher doses of amphotericin B for longer periods. We classified antifungal therapy as combination when the patient received both classes of antifungals in any formulation of amphotericin B and posaconazole or isavuconazole, concurrent when both amphotericin B and triazoles were used simultaneously, and sequential when triazole was used after amphotericin B.

Study Objectives
Our primary objective was to compare the epidemiology of mucormycosis between CAM and non-CAM groups during the study period, including the prevalence, underlying diseases, relationship to COVID-19, site of infection, and outcomes. Our secondary objectives were to compare CAM versus non-CAM and ascertain whether COVID-19 is a risk factor for mucormycosis death.

Sample Processing
Tissue biopsies from mucormycosis-affected anatomical sites were used for conventional microscopy, culture, and histopathology, as appropriate, at the respective health centers. Microscopy was performed by using potassium hydroxide mount with or without calcofluor stain. The samples were inoculated on 2 sets of Sabouraud dextrose agar and incubated at 25°C and 37°C. Positive cultures were identified by macroscopic and microscopic characteristics. Tissue samples submitted for histopathology were examined by using hematoxylin and eosin, periodic acid Schiff, or Gomori methenamine silver stain.

Statistical Methods
We performed data analysis using SPSS Statistics 21.0 (IBM, Inc, https://www.ibm.com). We provide descriptive statistics as frequencies, mean (SD), or median (interquartile range [IQR]), as appropriate. We compared categorical variables by using χ 2 or Fischer exact test and analyzed differences between continuous data by using Mann-Whitney U tests. We performed multivariate logistic regression analyses to identify factors predicting development of late CAM and mucormycosis mortality rates. We considered p<0.05 statistically significant.

Results
During the study period, a total of 295 consecutive mucormycosis cases were diagnosed at the 16 participating centers. We excluded 8 cases because of incomplete data. Of the remaining 287 cases, 187 (65.2%) had CAM. The mean age of the entire study population was 53.4 years (SD 17.1 years); 74.6% were men and 25.4% were women (Table 1). Patients with CAM were older (mean age 56.9 years), and a higher proportion (80.2%) were men than for the non-CAM patients.

CAM Prevalence
Among participating centers, 7 provided information needed to estimate the prevalence of CAM. During the study period, CAM patients accounted for 28/10,517 COVID-19 patients managed in general wards and 25/1,579 in ICUs. The overall prevalence of CAM was 0.27% (range 0.05%-0.57%); prevalence of CAM in ICUs was 1.6% (range 0.65%-2.0%). More mucormycosis cases were identified during the 2020 study period (231 cases) than during the same time range in 2019 (112 cases). The number of mucormycosis cases unrelated to COVID-19 did not differ much during both the study periods (112 cases in 2019 vs. 92 cases in 2020), indicating the increase in 2020 was chiefly attributed to CAM ( Figure 2).

Predisposing Factors
The most common underlying disease among both CAM and non-CAM groups was uncontrolled diabetes mellitus (62.7% whom 48 (78.7%) received glucocorticoid treatment for COVID-19 management. Other risk factors, including hematologic malignancy and solid organ transplantation, were noted in few among the study population (Table 1).

Clinical Manifestations and Site of Involvement
A greater percentage of patients with CAM had hypoxemia requiring ICU admission during hospitalization than the non-CAM group ( Table 1). The rhino-orbital region was the most common mucormycosis site (58.2%), followed by rhino-orbital-cerebral, pulmonary, and other sites (Table 1). However, site of involvement was similar in both the CAM and the non-CAM groups. Toothache, loosening of teeth, and radiologic involvement of the jaw were noted in many CAM patients ( Figure  3) but were not seen in non-CAM patients. One participating center reported jaw involvement in 10/47 (21.3%) contributed CAM cases ( Figure 3). The common form of pulmonary involvement was cavitary lung disease ( Figure 4).

Treatment
Liposomal amphotericin B was the most used antifungal agent in both groups. However, the use of liposomal amphotericin B was much lower in the CAM group (72.7%) compared with the non-CAM group (84%). Posaconazole and isavuconazole were more frequently used in CAM patients than in the non-CAM group. A combination of antifungal therapy, such as amphotericin B plus triazoles, either concurrent or sequential, was used much more often in CAM patients (49.5%) than in non-CAM (12%) patients. Combined medical and surgical management was performed in

Outcomes
Mortality rates were similar between CAM and non-CAM groups; the combined 6-week mortality rate was 38.3% (110/287 patients) and the 12-week mortality rate was 45.7% (117/256 patients) ( Table 1). Univariate analysis showed that combined medical and surgical management improved survival in the rhino-orbital-cerebral group but did not improve outcomes for patients with infections at other sites (Appendix Table 1, https://wwwnc.cdc.gov/EID/ article/27/9/21-0934-App1.pdf). On multivariate logistic regression analysis, we found age, site of involvement (rhino-orbital-cerebral or pulmonary), and ICU admission were associated with increased mortality rates. In contrast, sequential treatment with a combination of antifungal drugs was independently associated with better survival at 6 and 12 weeks (Table 2; Appendix Table 2).

Subgroup Analysis of CAM
The median time to CAM diagnosis was 18 (IQR 11-27) days ( Figure 5). Among 187 CAM patients, 158 (84.2%) were classified as late CAM (  (Table 3). Tocilizumab was administered to 5 (2.7%) patients for COVID-19 management. The demographic characteristics, underlying diseases, and site of involvement were similar among patients with early and late CAM. However, we saw diabetic ketoacidosis more often in patients with early CAM (28%) than late CAM (5%). A higher  proportion of patients with late CAM received glucocorticoid treatment (Table 3). Whereas amphotericin B remained the most common antifungal drugs used in both groups, posaconazole, isavuconazole, or a sequential use of antifungal agents (i.e., amphotericin B followed by posaconazole or isavuconazole) was more often seen in patients with late CAM. We saw no statistically significant difference in 6-and 12week mortality rates between the early and late CAM groups (Table 3). We also explored factors associated with late CAM development (Table 4). After adjusting for age, sex, and underlying risk factors, we found hypoxemia due to COVID-19 and inappropriate glucocorticoid administration were associated with development of late CAM.

Discussion
In our study, the prevalence of CAM was 0.27% in patients managed in hospital wards and 1.6% in patients managed in ICUs. We found a 2.1-fold increase in mucormycosis cases during September-December 2020 than the same months of 2019; we attribute the increase to COVID-19. Most CAM cases were diagnosed >8 days after COVID-19 diagnoses. Hypoxemia due to COVID-19 and inappropriate use of glucocorticoid drugs were independently associated with development of late CAM. The mortality rate for CAM patients was high (44%) but was comparable to rates for non-CAM (49%) patients. Older age (>54 years), admission to an ICU, and pulmonary or brain involvement by Mucorales were independently associated with a higher risk for death. The sequential use of antifungal drugs at any site was associated with improved survival at 6 and 12 weeks, irrespective of anatomical site of mucormycosis.
In our study, 74.6% of patients affected by mucormycosis were men, as observed in previous studies (11)(12)(13). We found diabetes mellitus was the most common underlying disease for both CAM and non-CAM patients. SARS-CoV-2 has been shown to affect the beta cells of the pancreas, resulting in metabolic derangement, possibly causing diabetes mellitus (14,15). Whether more frequent diagnosis (20%) of diabetes mellitus during the evaluation for CAM compared with non-CAM (10%) is related to SARS-CoV-2 infection, glucocorticoid therapy, or a chance occurrence remains unclear. Unfortunately, we do not have glycated hemoglobin values taken at admission for all newly detected diabetes cases in our study, so we cannot determine if these patients had diabetes mellitus before CAM developed.
We found inappropriate glucocorticoid use was independently associated with late CAM. Among 187 CAM cases, 61 (32.6%) had COVID-19 as the only underlying disease; 13 of those cases were not treated with glucocorticoid or other immunomodulatory therapies. Whether COVID-19 itself causes immune dysregulation and predisposes patients to invasive mucormycosis remains an unproven possibility (16)(17)(18). We did not find that COVID-19 was an independent predictor of late CAM, possibly because of the lower numbers of patients in our cohort with COVID-19 as the only underlying disease without any other risk factor. Lymphopenia is common in COVID-19, and progressive lymphopenia has been shown to correlate with COVID-19 severity (19). The persisting immune dysregulation during the recovery phase of COVID-19 infection also confers additional risk. Unfortunately, we have not evaluated the effect of lymphopenia on the development or outcome of CAM. Tocilizumab use in COVID-19 has been reported as a risk factor for invasive candidiasis (20). However, only 2.7% of the CAM patients in this study received tocilizumab.
The high mortality rate for CAM is a major concern (7). Patients with CAM were older (56.9 years) than non-CAM patients (46.9 years). Evidence suggests that older age imparts increased risk for hospitalization, respiratory failure, ICU admission, and attendant glucocorticoid therapy in COVID-19 (21,22). Further, age >54 years also was associated with an increased risk for death among our cohort. The site of mucormycosis involvement and the survival at 6 and 12 weeks was similar in CAM and non-CAM groups. We expected a higher proportion of pulmonary mycosis because respiratory viral infections, such as influenza, often are associated with secondary invasive aspergillosis (8). However, we did not observe an increased occurrence of pulmonary mucormycosis compared with infections in other sites among the CAM group. Considering the low rate of pulmonary involvement, we believe that CAM can be attributed to the systemic effects of COVID-19 or its treatment, rather than a sole alteration in the lungs. Several pulmonary mucormycosis cases also might have remained undiagnosed because of challenges in obtaining diagnostic respiratory samples among critically ill COVID-19 patients.
Appropriate and timely antifungal therapy and surgical resection, when feasible, are considered es-sential in mucormycosis management. Liposomal amphotericin B is the drug of choice, but isavuconazole also is recommended in primary therapy. Triazoles, including posaconazole and isavuconazole, commonly are used in the consolidation phase or as salvage therapy (23). The role of combination antifungal treatment in mucormycosis is not clearly supported by evidence (24). The combination of surgery and antifungal therapy was associated with better survival in the rhino-orbital-cerebral group in this study, conforming with previous experiences (6,11,25). However, the same was not true for mucormycosis in other anatomic sites. Early diagnosis of mucormycosis and the more frequent use of consolidation therapy or combination of antifungals in this study could be one explanation; another could be fewer surgeries performed in patients with other than rhino-orbital mucormycosis. We found the sequential use of antifungal drugs, amphotericin B then posaconazole or isavuconazole, was independently associated with improved survival among mucormycosis patients. However, the lack of randomization, possibility of case selection, and chance survival are potential biases. In addition, the optimal duration and dose of amphotericin B and posaconazole are not clear. The usefulness of antifungal combination administered simultaneously could not be ascertained due to the small number of patients receiving concurrent therapy in our study. A randomized controlled trial could affirm the role of a combination of antifungals or maintenance therapy in mucormycosis.
We expected better survival for the CAM patients in this study. Contrary to the prevailing practices (11,24), a combination of antifungal agents was more frequently used (50%) in CAM patients than in non-CAM patients (12%). Also, hospitalized CAM patients were closely monitored. The treatment practices used for the CAM group, especially those with late CAM, were distinct from those for the non-CAM group and those for patients with early CAM. The occurrence of a mold infection and the apprehension associated with the COVID-19 pandemic could have resulted in more frequent use of combination therapy in CAM. However, we saw no difference in mortality rates between CAM and non-CAM patients. Of course, increased risk for death due to COVID-19 itself cannot be ruled out for these CAM patients.
Our study's first limitation is that we collected data from a single country. The predominant risk factor for mucormycosis in our study was diabetes, which is also the case in some countries, including Bangladesh, China, Iran, Mexico, and Pakistan, from which data on mucormycosis are still limited (26). Further studies should compare data from countries with high rates of diabetes and mucormycosis with that of data from the United States and Europe, where mucormycosis predominantly is encountered in hematological malignancies and organ transplantation. Given the large number of late CAM cases, healthcare-associated mucormycosis remains a distinct possibility (27,28). Contaminated ventilation systems, air conditioners, and ongoing construction in hospitals have been reported to cause outbreaks of mucormycosis in the past (28). However, we did not estimate the burden of Mucormycetes spores in the hospital environment (29). We also do not have data on the timing of amphotericin B use, timing of surgery, or duration of sequential antifungal therapy, which are critical factors that have a bearing on mucormycosis outcomes; hence, we could not analyze these factors. Other unexplored factors, including genetic predisposition, might explain the high prevalence of CAM and non-CAM in India. Thus, prospective studies from the rest of the world, especially those severely affected by the COVID-19 pandemic, would be needed to ascertain the epidemiology of CAM. The strength of our study is the large number of patients, which lends credibility to our observations.
In conclusion, mucormycosis is a rare but critical problem complicating the later part of the clinical course of COVID-19 in India, possibly due to improper glucocorticoid usage. We found no difference in the risk factors, site of involvement, and outcome of mucormycosis complicating COVID-19 cases compared with non-COVID-19 cases. Nevertheless, the prevalence of mucormycosis has increased greatly in India, coinciding with the country's COVID-19 epidemic. Clinicians should be vigilant for mucormycosis in the patients recovering from COVID-19 illness, especially among patients with new or previously diagnosed diabetes mellitus and clinical manifestations of facial or orbital pain or black or blood-stained nasal discharge. In addition, we found improper glucocorticoid use for the COVID-19 treatment to be an additional risk factor in CAM. Therefore, treating physicians should ensure they use appropriate drugs and doses in treating COVID-19 patients.