Mucormycosis is an angio-invasive fungal infection, associated with high morbidity and mortality. A change in the epidemiology of mucormycosis has been observed in recent years with the rise in incidence, new causative agents and susceptible population. The rise has been perceived globally, but it is very high in the Asian continent. Though diabetes mellitus overshadow all other risk factors in Asia, post-tuberculosis and chronic renal failure have emerged as new risk groups. The rhino-cerebral form of mucormycosis is most commonly seen in patients with diabetes mellitus, whereas, pulmonary mucormycosis in patients with haematological malignancy and transplant recipients. In immunocompetent hosts, cutaneous mucormycosis is commonly seen following trauma. The intriguing clinical entity, isolated renal mucormycosis in immunocompetent patients is only reported from China and India. A new clinical entity, indolent mucormycosis in nasal sinuses, is recently recognized. The causative agents of mucormycosis vary across different geographic locations. Though Rhizopus arrhizus is the most common agent isolated worldwide, Apophysomyces variabilis is predominant in Asia and Lichtheimia species in Europe. The new causative agents, Rhizopus homothallicus, Mucor irregularis, and Thamnostylum lucknowense are reported from Asia. In conclusion, with the change in epidemiology of mucormycosis country-wise studies are warranted to estimate disease burden in different risk groups, analyse the clinical disease pattern and identify the new etiological agents.

Mucormycosis is an angioinvasive disease caused by saprophytic fungi of the order Mucorales. The exact incidence of mucormycosis in India is unknown due to the lack of population-based studies. The estimated prevalence of mucormycosis is around 70 times higher in India than that in global data. Diabetes mellitus is the most common risk factor, followed by haematological malignancy and solid-organ transplant. Patients with postpulmonary tuberculosis and chronic kidney disease are at additional risk of developing mucormycosis in this country. Trauma is a risk factor for cutaneous mucormycosis. Isolated renal mucormycosis in an immunocompetent host is a unique entity in India. Though Rhizopus arrhizus is the most common etiological agent of mucormycosis in this country, infections due to Rhizopus microsporus, Rhizopus homothallicus, and Apophysomyces variabilis are rising. Occasionally, Saksenaea erythrospora, Mucor irregularis, and Thamnostylum lucknowense are isolated. Though awareness of the disease has increased among treating physicians, disease-associated morbidity and mortality are still high, as patients seek medical attention late in the disease process and given the low affordability for therapy. In conclusion, the rise in the number of cases, the emergence of new risk factors and causative agents, and the challenges in managing the disease are important concerns with mucormycosis in India.

Mucormycosis due to Mucorales is reported at large numbers in uncontrolled diabetics across India, but systematic multicenter epidemiological study has not been published yet. The present prospective study was conducted at four major tertiary care centers of India (two in north and two in south India) during 2013-2015 to compare the epidemiology, treatment strategies and outcome of mucormycosis between the two regions. Molecular techniques were employed to confirm the identity of the isolates or to identify the agent in biopsy samples. A total of 388 proven/probable mucormycosis cases were reported during the study period with overall mortality at 46.7%. Uncontrolled diabetes (n = 172, 56.8%) and trauma (n = 31, 10.2%) were the common risk factors. Overall, Rhizopus arrhizus (n = 124, 51.9%) was the predominant agent identified, followed by Rhizopus microsporus (n = 30, 12.6%), Apophysomyces variabilis (n = 22, 9.2%) and Rhizopus homothallicus (n = 6, 2.5%). On multivariate analysis, the mortality was significantly associated with gastrointestinal (OR: 18.70, P = .005) and pulmonary infections (OR: 3.03, P = .015). While comparing the two regions, majority (82.7%) cases were recorded from north India; uncontrolled diabetes (n = 157, P = .0001) and post-tubercular mucormycosis (n = 21, P = .006) were significantly associated with north Indian cases. No significant difference was noted among the species of Mucorales identified and treatment strategies between the two regions. The mortality rate was significantly higher in north Indian patients (50.5%) compared to 32.1% in south India (P = .016). The study highlights higher number of mucormycosis cases in uncontrolled diabetics of north India and emergence of R. microsporus and R. homothallicus across India causing the disease.

The rare mucoraceous fungus, Apophysomyces species complex ranks second after Rhizopus arrhizus causing mucormycosis in India. The source of this agent in the environment is not clearly known. We conducted an environmental study to find its presence in Indian soil. The soil samples from different geographical locations were analyzed for isolation of Mucorales. Rhizopus arrhizus (24.6%) was most commonly isolated from soil, followed by Lichtheimia spp. (23.2%), Cunninghamella spp. (21.7%), Rhizopus microsporus (14%) and Apophysomyces spp. (4.5%). The isolation of Apophysomyces species complex was significantly associated with low nitrogen content of the soil. Based on sequencing of internal transcribed spacer (ITS) and 28S (D1/D2) regions of ribosomal DNA, the Apophysomyces isolates were identified as Apophysomyces variabilis with 98 to 100% similarity to type strain A. variabilis (CBS658.93). The analysis of amplified fragment length polymorphism (AFLP) fingerprinting data demonstrated genomic diversity of A. variabilis isolates with multiple clades (similarity 40-90%). The minimum inhibitory concentrations (MIC), MIC50 and MIC90 for A. variabilis isolates were 1 and 4 μg/ml for amphotericin B, 0.25 and 0.5 μg/ml for itraconazole, 0.125 and 0.25 μg/ml for posaconazole, 0.06 and 0.12 μg/ml for terbinafine, respectively. The present study revealed abundant presence of A. variabilis in Indian soil with low nitrogen content, its genetic heterogeneity and relatively high MICs for amphotericin B.

Mucormycosis is an angio-invasive infection, predominantly acquired by inhalation of sporangiospores from the environment. However, the burden of Mucormycetes sporangiospores in the air is not well studied. We aimed to estimate the burden of Mucormycetes spores in the outdoor and indoor (hospital) environment across different seasons in north India. A total of 380 air samples from outdoor (n = 180) and indoor (n = 200) environment were included in the study. Air samples were suctioned using air sampler (100 l/min) and cultured on Dichloran Rose Bengal Chloramphenicol (DRBC) with benomyl for selective isolation of Mucormycetes. The isolates were identified by phenotypic and genotypic methods. The mean spore count (±SD) of Mucormycetes (cfu/m3) in outdoor samples varied from 0.73 (±0.96) to 8.60 (±5.70) across different seasons. In hospital, the mean spore count varied from 0.68 (±1.07) to 1.12 (±1.07) and 0.88 (±1.01) to 1.72 (±2.17) for air-conditioned wards and non-air-conditioned wards, respectively. Rhizopus arrhizus was the predominant agent isolated from both indoor and outdoor environment followed by Cunninghamella species. We also report a single isolate of the rare mucormycete agent, Apophysomyces variabilis from outdoor environment. The present study highlights the presence of low spore burden of Mucormycetes in outdoor and hospital settings in north India. This study also reports the first isolation of A. variabilis from air samples in the Indian subcontinent.

Coronavirus disease (COVID-19)-associated mucormycosis (CAM) is an emerging threat globally, especially in India. More than 40,000 CAM cases have been reported in India. The emergence of CAM cases in India has been attributed to environmental, host, and iatrogenic factors. Mucorales spore burden has been reported globally; however, their presence is higher in tropical countries such as India, contributing to the emergence of CAM. Before the COVID-19 pandemic, patients with diabetes mellitus, haematological malignancies, solid organ transplants, corticosteroid therapy and neutropenia were more prone to mucormycosis, whereas in COVID-19 patients, virus-induced endothelial dysfunction, hyperglycaemia, and immune dysfunction following corticosteroid use increase the risk of acquiring mucormycosis. The interaction of Mucorales spores with the epithelial cells, followed by endothelial invasion, is a crucial step in the pathogenesis of mucormycosis. Endothelial damage and increased endothelial receptor expression induced by COVID-19 infection may predispose patients to CAM. COVID-19 infection may directly induce hyperglycaemia by damaging beta cells of the pancreas or by corticosteroid therapy, which may contribute to CAM pathogenesis. Iron acquisition from the host, especially in diabetic ketoacidosis (DKA) or deferoxamine therapy, is an important virulence trait of Mucorales. Similarly, the hyperferritinaemia caused by COVID-19 may act as a source of iron for Mucorales growth and invasion. In addition, corticosteroid treatment reduces or abolishes the innate immune functions of phagocytic cells contributing to the pathogenesis of CAM. This review aims to discuss primarily the host and iatrogenic factors shared between COVID-19 and mucormycosis that could explain the emergence of CAM.

Apophysomyces elegans species complex is an important cause of cutaneous mucormycosis in India. However, majority of those cases are reported as case reports only. We desired to analyze our patients with Apophysomyces infection reported over 25 years (1992–2017) to understand the epidemiology, management, and outcome of the disease. During the study period 24 cases were reported, and the majority (95.8%) of them presented with necrotizing fasciitis following accidental/surgical/iatrogenic trauma. One patient presented with continuous ambulatory peritoneal dialysis (CAPD) related peritonitis. Healthcare related Apophysomyces infection was noted in 29.2% patients. In addition to trauma, comorbidities were noted in 37.5% patients (type 2diabetes mellitus-6, chronic alcoholism-2, and chronic kidney disease-1). Of the 24 isolates, 11 isolates starting from year 2014 were identified as Apophysomyces variabilis by molecular methods. Majority (95.8%) of the patients were managed surgically with or without amphotericin B deoxycholate therapy, while one patient was treated with amphotericin B deoxycholate alone. Among 24 patients, seven (29.1%) recovered, six (25%) patients could not afford antifungal management and left the hospital against medical advice, and 11 (45.9%) patients died.The present case series highlights that necrotizing fasciitis caused by A. variabilis is prevalent in India, and the disease may be healthcare related. Although diagnosis is not difficult, awareness among surgeons is still limited about the infection, leading to a delay in sending samples to the mycology laboratory. Apophysomyces infection must be considered in the differential diagnosis in apatient with progressive necrosis of a wound who is not responding to antibacterial therapy.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species level Gap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation. Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues. Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae. Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues. Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.