Mucormycosis co-infection in COVID-19 patients: An update

Abstract Mucormycosis (MCM) is a rare fungal disorder that has recently been increased in parallel with novel COVID-19 infection. MCM with COVID-19 is extremely lethal, particularly in immunocompromised individuals. The collection of available scientific information helps in the management of this co-infection, but still, the main question on COVID-19, whether it is occasional, participatory, concurrent, or coincidental needs to be addressed. Several case reports of these co-infections have been explained as causal associations, but the direct contribution in immunocompromised individuals remains to be explored completely. This review aims to provide an update that serves as a guide for the diagnosis and treatment of MCM patients’ co-infection with COVID-19. The initial report has suggested that COVID-19 patients might be susceptible to developing invasive fungal infections by different species, including MCM as a co-infection. In spite of this, co-infection has been explored only in severe cases with common triangles: diabetes, diabetes ketoacidosis, and corticosteroids. Pathogenic mechanisms in the aggressiveness of MCM infection involves the reduction of phagocytic activity, attainable quantities of ferritin attributed with transferrin in diabetic ketoacidosis, and fungal heme oxygenase, which enhances iron absorption for its metabolism. Therefore, severe COVID-19 cases are associated with increased risk factors of invasive fungal co-infections. In addition, COVID-19 infection leads to reduction in cluster of differentiation, especially CD4+ and CD8+ T cell counts, which may be highly implicated in fungal co-infections. Thus, the progress in MCM management is dependent on a different strategy, including reduction or stopping of implicit predisposing factors, early intake of active antifungal drugs at appropriate doses, and complete elimination via surgical debridement of infected tissues.


Introduction
Mucormycosis (MCM) is described as a rare invasive fungal infection with high morbidity and mortality. Order Mucorales belong to the class Mucormycetes, subsequently to the subphylum Mucoromycotina [1]. It is associated with immunocompromised patients. However, several immunocompromised with various conditions including those with uncontrolled diabetes mellitus (DM), diabetic ketoacidosis, open wound following trauma, prolonged neutropenia, HIV or AIDS infection, iron overload or hemochromatosis, malignancies, corticosteroid therapy, organ transplant, and severe burns predispose this infection [2]. Although Mucorales belongs to nonpathologic fungus, the prevalence in immunocompetent individuals is a result of the existence of an intact immunity through neutrophils, which eliminate their spores [2,3]. Recently, severe COVID-19 infection was added as a co-factor that might cause a significant and sustained lymphopenia, leading to developing opportunistic infections, either bacterial or fungal. The first case was reported with COVID-19, which developed pulmonary cavitary lesions due to Mucorales fungi co-infection with harmful complications [3]. In fact, Mucorales fungi can invade blood vessels, which causes ischemic necrosis, and can potentially invades various systems such as the lung, central nervous system (CNS), nose, sinuses, skin, orbit, jaw bones, joints, heart, and kidney and can be classified into the following forms: pulmonary, rhino-cerebral, sino-nasal, cutaneous, oral mycosis, gastrointestinal, and disseminated [4]. This review article aimed to update the MCM information involving fungus classification, epidemiology, etiology, diagnostic tools, clinical settings, and treatment recommendations for invasive mycosis, which were found via the available original articles, original case reports, or published case series. Thus, this effort benefits to analyze and understand MCM via the available literature.

Etiology
When healthy individuals with immunocompetent inhale or ingest the fungal spores or lacerations of the mucosa via the nasal passage or oral cavity, they will not be implicated in immediate or potential harm as the phagocytic response will limit its prevalence. In contrast to the individual with immunocompromised and low polymorphonuclear leukocytes, the fungi become pathogenic when individuals invade by the fungal spores through lacerations of the mucosa in the oral or nasal cavity, may develop hyphae, and reach the paranasal sinuses. Subsequently, the disease can develop and diffuse to the cavernous sinus, the orbits, and the brain. It can also invade the arterial lamina and lead to tissue necrosis or thromboembolisms and infarctions of involved tissues. Then, the patient might suffer from orbital cellulitis, orbital apex syndrome, cerebritis or brain abscess, and a high mortality rate [15]. Mucorales and Entomophthorales orders are the most common etiological agents of MCM in humans. Several co-factors have been reported for a steady increase in MCM risk, such as: 1. Immunosuppression following solid organ, stem cells, and bone marrow transplants [16]. 2. Severe neutropenia consequence of aggressive chemotherapy for hematological and solid malignancies since neutrophils are essential for phagocytosis of the fungus [17]. 3. Rate of breakthrough invasive fungal infections is estimated between 4 and 10% of patients who are taking antifungal prophylaxis or treatment [18,19].
Uncontrolled DM and insufficient healthcare access can lead to an increase in MCM risk factors and hyperglycemia, which contributes to impaired neutrophil function and may cause MCM [20]. Therefore, DM is the most commonly known risk factor for MCM, especially during ketoacidosis. This is because ketones facilitate the fungi to utilize and produce ketoreductase, which facilitates its growth. In addition, hyperglycemia with ketoacidosis is also directly associated with the risk of MCM in different ways: (a) incidence of iron imprisonment because of hyperglycation of iron, which reduces the host protective system; (b) enabling tissue breakthrough by expressing the glucose-regulated protein 78 (GRP78) of cell receptor, which binds to Mucorales fungal species through the direct effect of hyperglycemia and indirectly by raising free iron levels; (c) deteriorating phagocytic functions and diminution the efficiency of chemotaxis; and (d) enhancing fungal existence via iron dissociation from breakthrough proteins [21]. GRP78 was involved within the cell receptor of endothelial vascular tissue [22].

Environmental factors and distribution of MCM
From an environmental perspective, these fungi are distributed in the soil, compost, animal feces, and decaying materials. In France, the analysis of collected soil samples from different geographical zones demonstrated that the most common species of Mucoralean fungi are Mucor circinelloides, Rhizopus arrhizus (synonym: Rhizopus oryzae), Lichtheimia corymbifera, Rhizopus microsporus, and Cunninghamella bertholletiae [8,9]. In comparison, a novel species have been discovered in Mexico (Apophysomyces mexicanus) [23]. Thus, the most common species of Mucorales found in the European countries are Rhizopus spp.
(34%), Mucor spp. (19%), and Lichtheimia spp. (19%) [24], whereas in the United States, Rhizopus species were followed by Mucor species (19%), Rhizomucor species (7%), Cunninghamella species (9%), and Lichtheimia species (formerly Absidia species, 3%) [5]. Of note, MCM incidence among geographical regions is possibly due to the different natural habitats [24]. Conversely, the peak of MCM incidence has been investigated from September to November (autumn season) in some Middle East countries [8,25,26], while in European countries, most incidences are recorded in autumn and winter [27]. As well as in tropical and subtropical zones, the distribution of MCM was higher in autumn [28], suggesting that climate conditions may play a critical role in the prevalence of MCM and might have contributed to increasing airborne spore concentrations in autumn, whereas the fewer concentrations occur in summer. MCM incidence varies considerably from zone to zone regarding the susceptibility of co-factors. Several studies from Europe [24,29,30]

Diagnosis
Fast treatment of MCM disease is considered a big challenge. Therefore, the accuracy of diagnosis is very important for helping the treatment and reducing the high mortality (85%, concerned with late or incorrect diagnosis) [2]. Continuous development and discovering an accurate diagnosis are still essential because of the indirect clinical markers/symptoms for confirming MCM. Update of studies is required to understand the epidemiology and diagnosis of this disease among various regions worldwide. In spite of this, multiple studies reported the difficulty in establishing a rapid diagnosis [36]. The first step in the diagnosis begins with suspicion of the occurrence of MCM infection, especially the candidate diseases as co-factors. Hence, progress in diagnosis has affected the increasing reports on invasive MCM in susceptible patients like those with diabetic ketoacidosis secondary to uncontrolled DM, HMs, solid organ transplant, chronic respiratory diseases, and corticosteroid therapy [37]. Although, a conventional clinical path for diagnosis lacks specificity and sensitivity. It is still a basic requirement for the initial diagnosis of MCM when a high indicator of suspicion, via estimate of host factors and fast evaluation of clinical appearances. For example, the initial investigation of gastric MCM by endoscopic examination usually reveals an ulcer with necrosis, ultimately presenting an adherent, thick, green exudate [38]. Histopathology and culture are the fundamentals of diagnosis [13,39]. Prompt microscopy examination of clinical biopsy specimens is carried out, perfectly using optical brighteners such as Blankophor and Calcofluor [40][41][42]. White in clinical specimens gives a fast visualization of the characteristic fungal hyphae of MCM, in this case requiring a fluorescent microscope [2,43]. Also, in the case of diplopia in diabetic patients, pleuritic pain may be an indicator of this infection, and it is essential to obtain a specimen for traditional examination by histopathological and microbiological examinations or by advanced molecular techniques. The markers and symptoms that involve diplopia are cranial nerve palsy, proptosis, sinus pain, orbital apex syndrome, periorbital swelling, and ulcers of the palate [42]. In addition, radiologically, various (≥10) nodules and pleural emissions can reveal the association with pulmonary MCM [44]. Other pathways are computerized tomography (CT) scans with high-resolution and magnetic resonance imaging (MRI), which indicate the existence of MCM [2,13,45]. When studying the CT features of COVID-19-associated pulmonary mucormycosis situations, it was discovered that consolidation and cavitation were the most common computed tomography imaging findings (69%), probably because of delayed disease diagnosis [46]. According to another CT imaging study, almost all the patients (95.7%) displayed signs of pansinusitis. The infection spread beyond the paranasal sinuses was seen in 78.7% of cases, with orbital invasion 40.4% being the most prevalent [47]. On the contrary, MRI is useful for soft tissue imaging and determining the severity of disease [48].
Conversely, identifying different fungal species is important for the best epidemiological understanding of MCM and may be of significance for outbreak determinations. Mucorales fungi can be easily identified in culture. One study described the morphological features alone, and when distinguished by specialists in the fungal investigation, a high level of accuracy can be obtained. [49,50] However, identifying species is still difficult and may face failures in speciation, whereas immunohistochemistry using monoclonal antibodies against R. arrhizus can differentiate aspergillosis from MCM (100% high sensitivity and 100% high specificity for MCM) [51,52]. Recently, a molecular diagnosis is required either for the identification or detection of MCM [53]. It involves conventional polymerase chain reaction (PCR), [54][55][56], restriction fragment length polymorphism (RFLP) [56,57], and DNA sequencing [2,58]. Indeed, several PCR-based techniques have been developed, such as nested PCR, real-time PCR [59,60] (quantitative PCR (qPCR)), nested PCR combined with RFLP [61], PCR coupled with electrospray ionization mass spectrometry [62], and PCR/high-resolution melt analysis [63]. All assays mentioned earlier can be applied for either the identification or the detection of Mucorales. Most of the molecular assays are ribosomal targets (18S, 28S, and internal transcribed spacer) or other DNA targets (the high-affinity iron permease I gene FTRI or cytochrome b) [53,[64][65][66][67]. Several studies have been performed by using either fresh tissue specimens or formalin-fixed paraffin-embedded [68][69][70], yet resulting in different performances. Recent tools directed at molecular assays from blood serum [68,[71][72][73] have yielded promising clinical data with the advantage of early diagnosis in comparison with culture. Moreover, qPCR has been performed to identify circulating Mucor/Rhizopus, Lichtheimia, and Rhizomucor DNA in the serum [74]. However, some studies recommended that molecular diagnosis can be conducted in addition to conventional diagnosis tools [75].

COVID-19 and mucormycosis coinfections
Based on clinical manifestations, invasive MCM can be classified by the anatomic site affected into six main forms: pulmonary, rhino-orbital-cerebral, cutaneous, sinus, gastrointestinal, disseminated, and rare forms, such as endocarditis, osteomyelitis, peritonitis, and renal infection [76][77][78]. Any of Mucorales species can infect any of these sites. Fungal infection can invade these sites by the incorporation or implantation of the fungal spores through oral, nasal, and conjunctival mucosa (rhino-orbital-cerebral), by inhalation (pulmonary), or by the ingestion of contaminated food (gastrointestinal), as they rapidly colonize nutriments rich of glucose for its principal energy source [79].
Then the common ratio of invasive MCM was evaluated as follows: sinuses (39%), pulmonary (24%), and cutaneous (19%) [80]. Dissemination developed in 23% of this infection [81,82]. However, these values may be changeable depending on the incidence of another co-factor such as COVID-19. The first report has suggested that COVID-19 patients might be susceptible to developing invasive fungal infections by different species such as candidiasis, invasive aspergillosis, and pneumocystis jiroveci [83]. However, this report did not include MCM as a co-infection with the COVID-19 pandemic. Subsequently, the case report presented a patient with MCM without any conventional risk factor. The infection was confirmed after being diagnosed with COVID-19 and has been treated with broad-spectrum antibiotics and corticosteroids (which are critical factors for invasive fungal infection) [78,84]. Despite co-infection among patients with COVID-19 has been shown only in severe cases [82,85,86], superinfections in viral pneumonia remain unclear. The first reason is due to challenges confused by the conventional diagnosis of Mucorales. The second is due to the fact that many centers minimized direct testing of at-risk respiratory samples during the current COVID-19 pandemic to diminish exposure to the virus [87][88][89][90]. Subsequently, several reports confirmed increasing cases of MCM in different countries, particularly in India, after COVID-19. Of their findings of an even larger case series of MCM cases in COVID-19, about 80% of cases had DM and more than 75% of cases received a course of corticosteroids. Therefore, these findings indicated that the common triangle associated with COVID-19 and MCM co-infection are DM, diabetic ketoacidosis (DKA), and corticosteroids ( Table 2) [3,. However, some finding in case of rhino-orbital MCM was associated with ketoacidosis DM and infection with severe acquired respiratory syndrome coronavirus 2 [134]. Song et al. have reported an algorithm for the early diagnosis and management of common invasive fungal infections such as MCM, aspergillosis, candidiasis, and cryptococcosis during the COVID-19 pandemic [135]. However, factors leading to increased incidence of opportunistic fungal infections during the COVID-19 pandemic may lead to immunosuppression due to high-dose corticosteroids in the management of the pandemic, thus leading to immunosuppression [136]. Corticosteroid intake during COVID-19 infection often leads to uncontrolled diabetes and precipitation of ketoacidosis. Thus, acidosis (low pH) is considered a favorite media for Mucor spores to grow. In addition, corticosteroid intake also reduces the phagocytic activity of white blood cell (WBC) and causes deterioration of bronchoalveolar macrophages migration, ingestion, and phagolysosome fusion, leading to exposure risk of diabetic people to MCM [89]. Consequently, a report described one case with SARS-CoV-2 infection who developed a cavitary pulmonary MCM [137].
Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection might develop coronavirus disease (COVID- 19), which can be associated with significant and sustained lymphopenia compromising the immune system, especially in the most severe cases [3,[138][139][140][141]. Recent findings described that a significant decrease in lymphocyte count and an increase in neutrophil count together with an inflammatory storm occur more frequently in patients who developed severe COVID-19 and co-infections [141]. Pathogenic mechanisms in aggressive MCM involve reduced phagocytic activity, attainable quantities of ferritin attributed to the displacement of protons by transferrin in diabetic ketoacidosis, and fungal heme oxygenase, which enhances iron absorption for its metabolism. Therefore, severe COVID-19 cases with hemato-lymphohistiocytosis syndrome are associated with the increasing risk factors of invasive fungal coinfections [90]. Clinically, cell counts exhibited that there was an increase in neutrophils and WBC count, but a decrease in lymphocytes progressively [3]. In addition, corticosteroid intake also reduces the phagocytic activity of WBC and causes deterioration of bronchoalveolar macrophages migration, ingestion, and phagolysosome fusion, leading to a diabetic people exposure risk to MCM. Conversely, the presence of free iron is an ideal co-factor for MCM. Hyperglycemia leads to glycosylation of transferrin and ferritin, and minimizing iron-binding leads to a raised ratio of free iron. Moreover, an increase in interleukin (IL)-6 among COVID-19 patients also causes an increase in free iron by elevating ferritin levels, and acidosis increases free iron by reducing the ability of transferrin to chelate iron [89]. In addition, COVID-19 infection drastically affects the immune system via hyperexpression of booth proinflammatory (IL-1, IL-2, IL-6, and tumor necrosis factor (TNF-α)) and anti-inflammatory (IL-4, IL-10, and IL-17) cytokines [4,139] and hypoexpression of interferon (IFN)-γ. With an increase in neutrophiles, impaired lymphocyte-mediated immunity (Th1 and Th2) occurs. Subsequently, COVID-19 infection reduces the cluster of differentiation, especially CD4+ and CD8+ T cell counts, which may be highly implicated in fungal co-infections [141]. Critical cases, especially those admitted to intensive care units and those requiring longer hospital stays might be more prospective to evolve fungal co-infections [142]. Therefore, it is to be noted that severe Covid-19 susceptible to opportunistic fungal coinfections, especially MCM.

Molecular mechanism of mucormycosis in COVID-19 patients
The invasion of fungi usually starts by inhalation of spores during respiration (sometimes from ears), ingesting contaminated food, or invading through injuries/abrasion in the epidermis. Endothelialitis is the one factor of SARS-CoV-2 that might reveal the risk of MCM co-infection in COVID-19 patients. [143,144] Damage to endothelium promotes angio-invasion and prevalence of MCM. In addition, low pH, hyperglycemia, hyper-ferritin, and hyper-ketone due to COVID enhance an appropriate environmental condition for GRP78 of endothelial cells and fungal ligand spore coating homolog (CotH) protein. Molecular explanation between fungal CotH protein and nasal GRP78 enhances adherence in the early stage of infection ( Figure 2). Then, the CotH surface proteins might act as invasion enzymes to cause the infection [145]. In the sequent step of infection, patients with the high levels of CotH and GRP78 lead to trapping of fungi inside sinus cavities ( Figure 2) [146,147]. Then, fungal cells elongate into tube-like hyphal strands and disseminate into the sinuses, lungs, skin, soft tissues, and bloodstream, causing tissue and epithelium penetration ( Figure 2). The second current pathway, corticosteroid intake, is the mainstay therapy in severe COVID-19 patients [148]. However, corticosteroid intake reduces the phagocytic capacity of WBCs, displaying patients' fungal infections. In addition, new-onset corticosteroid-induced DM or exacerbated chronic DM with or without DKA might promote the replication of MCM. The COVID-19 itself launches a chain of events that might render patients prone to MCM infection. Thus, there are expected mechanisms through which SARS-CoV-2 infection might lead to MCM susceptibility. Dramatic elimination in the total number of T-cells involving both CD4+ and CD8+ groups in severe cases of COVID-19 causes significant lymphopenia successive an immunocompromised state and then susceptibility to MCM [4]. During severe COVID-19 infection, inflammatory cytokines such as IL-6, IL-2R, IL-10, and TNF-alpha elevate and lead to a term called "cytokine storm," resulting in lymphopenia [4]. Consequently, cytokine storm raises iron levels and reduces its export. As a result, iron accumulates inside the cells. Iron overload causes tissue damage and necrosis. In addition, free iron is ideal for growth and spreading MCM ( Figure 2) [149,150]. Moreover, SARSinduced pneumonia results in atelectasis, which results in COVID-19-contributed silent hypoxia. Usually, the hypoxiainducible factor 1 (HIF-1) stays inactive in normal conditions. During hypoxic conditions of COVID-19, the HIF-1α transcription factor subunit has a risky role in the expression of the COVID-19 receptor gene (ACE-2 gene), and hypoxia causes endothelium damage and upregulation of adaptive and innate immunocytes [151,152]. The overall effect of HIF-1 results in raised localized inflammation and tissue damage in COVID-19 patients. In addition, COVID-19 infection has also been demonstrated to impact ferritin metabolism. Therefore, several studies have reported hyperferritinemia syndrome post-COVID-19 infection. Thus, high free iron levels lead also to tissue damage via the production of reactive oxygen species (ROS) [150,[153][154][155][156]. The overall potentials such as corticosteroid therapy, immunocompromised state, DM, DKA, hyperglycemia, hypoxia, cytokine storm, hyperferritinemia, neutropenia, metabolic dysfunction, and ROS make COVID-19-infected patient more susceptible to MCM. These main symptoms increased the body temperature, and altered osmolarity make an ideal environment for the growth and development of MCM ( Figure 2).

Antifungal treatments
Progress in management and treatment of MCM is dependent on a different strategy, including reduction or stopping of implicit predisposing factors, early intake of active antifungal drugs at appropriate doses, complete elimination via surgical debridement of infected tissues, and administration of assistant therapies [57]. The therapy includes available antifungal medicines such as azithromycin, oseltamivir, ceftriaxone, amphotericin B (liposomal), cefepime, meropenem, linezolid, caspofungin, vancomycin, piperacillin, tazobactam, and oseltamivir. Intravenous amphotericin B (a lipid formulation) is the best and most common medicine for initial treatment, especially with COVID-19 co-infection. Despite surgical operations and available antifungal drugs, the prognosis for recovery from MCM is poor [95,106] (Table 2). Kieliszek and Lipinski [157] suggested that the sodium selenite, but not selenate, possibly oxidizes viral thiol groups in the virus protein disulfide isomerase, coating it incapable to penetrate the host cell membrane. In this way, the sodium selenite inhibits invasion of viruses into the healthy cells and diminishes their infectivity [157]. Thus, this simple chemical compound is potentially applied to the recently infected COVID-19 patients all over the globe.

Conclusion
The increase in MCM worldwide due to co-factors, especially diabetes with or without ketoacidosis, leads to outbreaks of corticosteroid intake during the incidence of COVID-19 (increases in blood glucose lead to opportunistic fungal infection). Outbreaks of COVID-19 are accompanied by immunocompromised through hyperexpression of both proinflammatory (IL-1, IL-2, IL-6, and TNF-α) and antiinflammatory (IL-4, IL-10, and IL-17) cytokines, and vice versa with IFN-γ. With an increase in neutrophils, impaired lymphocyte-mediated immunity (Th1 and Th2) occurs. Subsequently, COVID-19 infection leads to a reduction in the cluster of differentiation, especially CD4+ and CD8+ T cell counts, which may be highly implicated in fungal coinfections. Therefore, it is to be noted that severe COVID-19 susceptible to opportunistic fungal co-infections, especially MCM. Every effort should be made to progress in the management and treatment of MCM is dependent on a different strategy, including reduction or stopping of implicit predisposing factors, early intake of active antifungal medicines. Corticosteroids were used only in cases co-infected with COVID-19 to reduce the load of mortal MCM.
Acknowledgments: The researchers would like to thank the Deanship of Scientific Research, Qassim University, for funding the publication of this project.
Funding information: Authors received funding from the Qassim University.